Determination of bisphenol A in river water and body fluid samples by stir bar sorptive extraction with in situ derivatization and thermal desorption-gas chromatography-mass spectrometry

A new method, based on stir bar sorptive extraction (SBSE) with in situ derivatization and thermal desorption (TD)-gas chromatography-mass spectrometry (GC-MS) is described for the determination of trace amounts of bisphenol A (BPA) in river water, urine, plasma, and saliva samples. The derivatization conditions with acetic acid anhydride and the SBSE conditions such as sample volumes and extraction time are investigated. Then, the stir bar is subjected to TD followed by GC-MS. The detection limits of BPA in river water, urine, plasma, and saliva samples are 1-5, 20, 100, and 20pgml(-1) (ppt), respectively. Calibration for BPA was shown to be linear with a correlation coefficient of >0.99. The average recoveries of BPA in all samples are higher than 95% (R.S.D. < 10%) with correction using an added surrogate standard, 13C12-bisphenol A. This simple, accurate, sensitive, and selective analytical method may be applicable to the determination of trace amounts of BPA in liquid samples.     

Stir bar sorptive extraction and thermal desorption-gas chromatography-mass spectrometry for the measurement of 4-nonylphenol and 4-tert-octylphenol in human biological samples

Alkylphenols, 4-nonylphenol (NP) and 4-tert-octylphenol (OP), in human urine and plasma samples were analyzed using stir bar sorptive extraction (SBSE) in combination with thermal desorption-gas chromatography-mass spectrometry (TD-GC-MS). The method involved correction by stable isotopically labeled surrogate standards, 4-(1-methyl)octylphenol-d5 (m-OP-d5) and deuterium 4-tert-octylphenol (OP-d). A biological sample was extracted for 60 min at room temperature (25 degrees C) using a stir bar coated with a 500 microm thick polydimethylsiloxane (PDMS) layer. Then, the stir bar was analyzed by TD-GC-MS in the selected ion monitoring (SIM) mode without any derivatization step. The average recoveries in human urine and plasma samples spiked with NP and OP at levels of 0.5 and 10 ng ml-1 were between 95.8 and 99.8% with correction using the added surrogate standards. The limits of quantitation were 0.2 ng ml-1 for NP and 0.02 ng ml-1 for OP. We measured the background levels of NP and OP in five human urine and three human plasma samples from healthy volunteers. NP and OP were not detected in all human urine samples (N.D. < 0.2 ng ml-1 for NP, and N.D. < 0.02 ng ml-1 for OP). However, 0.2-0.3 ng ml-1 for NP and 0.1-0.2 ng ml-1 for OP in human plasma samples were observed by this method.     

Hollow-fiber-supported liquid phase microextraction with in situ derivatization and gas chromatography-mass spectrometry for determination of chlorophenols in human urine samples

A simple and highly sensitive method that involves hollow-fiber-supported liquid phase microextraction (HF-LPME) with in situ derivatization and gas chromatography-mass spectrometry (GC-MS) was developed for the determination of chlorophenols (CPs) such as 2,4-dichlorophenol (DCP), 2,4,6-trichlorophenol (TrCP), 2,3,4,6-tetrachlorophenol (TeCP) and pentachlorophenol (PCP) in human urine samples. Human urine samples were enzymatically de-conjugated with beta-glucuronidase and sulfatase. After de-conjugation, HF-LPME with in situ derivatization was performed. After extraction, 2mul of extract was carefully withdrawn into a syringe and injected into the GC-MS system. The limits of detection (S/N=3) and quantification (S/N>10) of CPs in the human urine samples are 0.1-0.2ngml(-1) and 0.5-1ngml(-1), respectively. The calibration curve for CPs is linear with a correlation coefficient of >0.99 in the range of 0.5-500ngml(-1) for DCP and TrCP, and of 1-500ngml(-1) for TeCP and PCP, respectively. The average recoveries of CPs (n=6) in human urine samples are 81.0-104.0% (R.S.D.: 1.9-6.6%) with correction using added surrogate standards. When the proposed method was applied to human urine samples, CPs were detected at sub-ngml(-1) level.     

Determination of 4-nonylphenol, nonylphenol monoethoxylate, nonylphenol diethoxylate and other alkylphenols in fish and shellfish by high-performance liquid chromatography with fluorescence detection

A simple and highly sensitive method is described for the HPLC determination of 4-nonylphenol (NP), 4-nonylphenol mono- (NP1EO) and diethoxylates (NP2EO) in fish and shellfish together with bisphenol A (BPA), 4-tert.-butylphenol (BP) and 4-tert.-octylphenol (OP). The NP, NP1EO, NP2EO and other alkylphenols in the samples are extracted with acetonitrile and the lipid in the sample extract is eliminated by partitioning between hexane and acetonitrile. After Florisil PR clean-up the sample extract is analyzed by HPLC with a fluorescence detection. Recoveries in Japanese smelt, carp and corbicura are 81.8–84.3% for NP, 83.5–84.3% for NP1EO, 90.5–96.2% for NP2EO, 70.7–72.9% for BPA, 71.0–73.4% for BP and 77.1–83.2% for OP spiked at 0.5 μg each chemical per 5 g of the fish and shellfish samples. The detection limits are 2 ng/g for NP, NP1EO and NP2EO, and 1ng/g for BPA, BP and OP.     

Measurement of Phenolic Environmental Estrogens in Women with Uterine Leiomyoma

Objectives

To investigate the effect of phenolic environmental estrogens on uterine leiomyoma from the perspective of clinical epidemiology.

Methods

Urine and blood samples were collected from Han women with uterine leiomyoma and women without uterine leiomyoma, living in Nanjing, China, between September 2011 and February 2013. A total of 156 urine samples and 214 blood samples were collected from the uterine leiomyoma group and 106 urine samples and 126 blood plasma samples from the control group. Bisphenol A (BPA), nonylphenol (NP) and octylphenol (OP) concentrations were determined by solid-phase extraction (SPE) coupled with liquid chromatography-tandem mass spectrometry (HPLC-MS/MS).

Results

Phenolic environmental estrogens in the uterine leiomyoma and control groups were compared based on: gravida>3 and gravida ≤ 3. In participants with gravida>3, urine OP concentration was significantly (P<0.05) higher in the uterine leiomyoma group than in the control group. In participants with gravida ≤ 3, urine NP concentration was significantly (P<0.05) higher in the uterine leiomyoma group compared to controls. Despite obstetric history, urine BPA mean exposure concentration was significantly (P<0.05) different between uterine leiomyoma group and control group. The urine BPA concentration was not significantly (P>0.05) different between gravida>3 and gravida ≤ 3 patients. There was no significant (P>0.05) difference in plasma concentrations of BPA, OP and NP between the leiomyoma group and control group. Mean exposure concentration and range of distribution of BPA, OP and NP plasma concentration differed between the uterine leiomyoma and control group.

Conclusion

Exposure level of phenolic environmental estrogens in human was related with leiomyoma tumorigenesis.     

Urinalysis of 4-hydroxynonenal, a marker of oxidative stress, using stir bar sorptive extraction-thermal desorption-gas chromatography/mass spectrometry

A simple and fast method for the measurement of 4-hydroxynonenal (4HNE), a highly toxic end-product of lipid peroxidation, in urine samples is described. The method combines stir bar sorptive extraction (SBSE) with two derivatization steps, followed by thermal desorption and GC/MS. 4HNE is derivatized in situ with O-(2,3,4,5,6-pentafluorobenzyl) hydroxylamine and the oxime is extracted from the aqueous phase with SBSE. The 4HNE-oxime is further acylated by headspace derivatization prior to thermal desorption. Derivatization reactions and extraction were optimized in terms of reagent quantities, temperature and time. The method is linear over a concentration range of 0.5-5 ng mL(-1) with a correlation coefficient of 0.997. The limit of detection and limit of quantitation are 22 and 75 pg mL(-1) urine, respectively. The high sensitivity of the method allows the measurement of physiological concentrations of 4HNE in urine samples.     

Analysis of alkylphenol and bisphenol A in eggs and milk by matrix solid phase dispersion extraction and liquid chromatography with tandem mass spectrometry

A method based on matrix solid phase dispersion (MSPD) using C18 as dispersant, and a subsequent cleanup step with amino-propyl solid phase extraction cartridges and liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) has been developed for the simultaneous determination of nonylphenol (NP), octylphenol (OP) and bisphenol A (BPA) in eggs and milk. Recovery studies were performed at different fortification levels. Average recoveries by MSPD varied from 79% of BPA to 98% of NP and relative standard deviations were equal or lower than 15% for egg samples. The average recoveries in milk ranged from 86 to 84% for BPA, 90 to 99% for NP and 82 to 103% for OP and relative standard deviations were equal to or lower than 8%. The limits of detection (LODs) in eggs were 0.10, 0.10 and 0.25 microg/kg for BPA, NP and OP, respectively and LODs for milk were 0.10, 0.05 and 0.10 microg/kg for BPA, NP and OP, respectively. Investigation of the levels in commercial samples indicated that NP was ubiquitous in milk and eggs at levels ranging from 4.24 to 17.60 microg/kg, and the milk samples were more heavily contaminated by NP than were the egg samples.     

Miniaturized hollow fiber assisted liquid-phase microextraction with in situ derivatization and gas chromatography-mass spectrometry for analysis of bisphenol A in human urine sample

A new method that involves miniaturized hollow fiber assisted liquid-phase microextraction (HF-LPME) with in situ derivatization and gas chromatography-mass spectrometry (GC-MS) is described for the determination of trace amounts of bisphenol A (BPA) in human urine samples. The detection limit and the quantification limit of BPA in human urine sample are 0.02 and 0.1 ng ml(-1) (ppb), respectively. The calibration curve for BPA is linear with a correlation coefficient of >0.999 in the range of 0.1-50 ng ml(-1). The average recoveries of BPA in human urine samples spiked with 1 and 5 ng ml(-1) BPA are 101.0 (R.S.D.: 6.7%) and 98.8 (R.S.D.: 1.8%), respectively, with correction using the added surrogate standard, bisphenol A-(13)C12. This simple, accurate, sensitive and selective analytical method can be applicable to the determination of trace amounts of BPA in human urine samples.     

Genotoxic effects of environmental estrogen-like compounds in CHO-K1 cells

Some environmental estrogen-like compounds, such as bisphenol A (BPA), 4-nonylphenol (NP), 4-octylphenol (OP), propyl p-hydroxybenzoate (P-PHBA), and butyl p-hydroxybenzoate (B-PHBA), synthetic estrogen, diethylstilbestrol (DES), and natural estrogen, 17beta-estradiol (E2), were studied for their genotoxicity in CHO-K1 cells using sister-chromatid exchange (SCE), chromosome aberration (CA), and DNA strand break (comet) assays. Six of the chemicals, excluding E2, caused DNA migration in the comet assay and induced SCEs at one or more of the highest doses. Among the chemicals, OP produced an especially high incidence of SCEs. Structural CA was induced by five of the chemicals, excluding OP and NP, and BPA, E2, and DES also induced aneuploid cells. E2 and DES particularly increased the rate of polyploidy at high doses. The incidence of colchicine-mitosis-like (c-mitotic) figures suggesting spindle disrupting effects was also detected with five of the chemicals, excluding OP and NP, and six of the chemicals, excluding E2, caused endoreduplication (ERD), a form of nuclear polyploidization induced by block of cell cycle at G2 phase, at one or more high doses. Our present results suggest that OP and NP cause repairable DNA damage, including SCEs, and do not result in CA, while the damage caused by DES, BPA, P-PHBA, and B-PHBA results in the induction of CAs together with SCEs probably because of imperfect repair. We are unable to explain the observation that the DNA damage caused by E2 resulted in CA induction but not DNA migration or SCE induction, except for speculating that the DNA damage is different from that caused by DES and the estrogen-like chemicals. Our findings also suggest that E2, DES and BPA have aneuploidogenic properties, and that the former two of chemicals also are polyploidy-inducing agents.     

The analysis of estrone and 17beta-estradiol by stir bar sorptive extraction-thermal desorption-gas chromatography/mass spectrometry: application to urine samples after oral administration of conjugated equine estrogens

The development of a sensitive and solvent-free method for the measurement of estrone (E(1)) and 17beta-estradiol (17beta-E(2)) in human urine samples is described. The deconjugated estrogens were derivatized in situ with acetic acid anhydride and the derivatives were extracted directly from the aqueous samples using stir bar sorptive extraction (SBSE). The compounds containing a secondary alcohol function are further derivatized by headspace acylation prior to thermal desorption and gas chromatography/mass spectrometry (GC/MS). A number of experimental parameters, including salt addition, temperature and time, were optimized to increase the recovery of E(1) and 17beta-E(2) by SBSE. The derivatization reactions were also optimized to obtain the highest yields of the acylated estrogens. Detection limits of 0.02 and 0.03 ng mL(-1) were obtained for E(1) and 17beta-E(2), respectively. The method was applied to determine the effect of conjugated equine estrogen intake on the excretion of E(1) and 17beta-E(2) in human urine samples. Increased levels of the endogenous estrogens were detected after administering a standard dose of Premarin to a female volunteer. Routine monitoring of estrogen levels is recommended to avoid a high urinary excretion of E(1) and 17beta-E(2), nowadays enlisted as endocrine disrupting chemicals (EDCs), during hormone replacement therapy.     

Characterization of a cytochrome P450 gene (CYP4G) and modulation under different exposures to xenobiotics (tributyltin, nonylphenol, bisphenol A) in Chironomus riparius aquatic larvae

Cytochrome P450 family members participate in xenobiotic transformation as a detoxification mechanism. We have characterized a CYP gene, assigned to the 4G family, in Chironomus riparius, a reference organism in aquatic toxicology. Due to the potential interest of CYP genes and P450 proteins for monitoring pollution effects at the molecular level, the alterations in the pattern of expression of this gene, induced by different xenobiotics, were analyzed. Different compounds, such as the biocide tributyltin (TBTO) and two other well-known endocrine disruptors, nonylphenol (NP) and bisphenol A (BPA), were tested at different concentrations and acute exposures. Upregulation of the CrCYP4G gene was found after exposures to TBTO (1 ng/L 24h-0.1 ng/L 96 h) and, as measured by RT-PCR mRNA quantification, its level was up to twofold that of controls. However, in contrast, NP (1, 10, 100 μg/L, 24h) and BPA (0.5mg/L 24h-3mg/L 96 h) downregulated the gene (by around a half of the control level) suggesting that this gene responds specifically to particular chemicals in the environment. Glutathione-S-transferase (GST) enzymatic activity was also evaluated for each condition. A fairly good correlation was found with CYP4G gene behavior, as it was activated by TBTO (96 h), but inhibited by NP and BPA (24h). Only the higher concentration of BPA tested activated GST, whereas it inhibited CYP4G activity. The results show that different xenobiotics can induce distinct responses in the detoxification pathway, suggesting multiple xenobiotic transduction mechanisms. This work confirms that specific P450 codifying genes, as well as GST enzyme activities, could be suitable biomarkers for ecotoxicological studies.     

Application of a novel mass spectrometric (MS) method to examine exposure to Bisphenol-A and common substitutes in a maternal fetal cohort

The use of Bisphenol A (BPA) has widely been replaced in consumer products by analogs BPB, BPE, BPF, BPS, and BPAF. Recent studies have linked these substitutes to similar adverse health outcomes as BPA, including disruption of endocrine pathways in animal and human studies. We designed a novel MS method, developed specifically for this study, to capture the most relevant BPA alternatives, BPB, BPE, BPF, BPS, BPAF and 4-NP in human blood and urine to quantify potential in utero exposures. To our knowledge, this is the first study to explore in utero exposure to these BPA analogs and the first U.S. study to test for BPA in maternal/fetal pairs. The method was run on 30 paired maternal urine and fetal cord blood samples from mothers undergoing elective Caesarean sections. 90% of mothers and 77% of babies tested positive for at least one BP analog. 83% of mothers tested positive for BPAF, 60% for BPS, 57% for BPB, 17% for BPF and 7% for BPA. 57% of babies tested positive for BPAF and 50% for BPF. BPA and BPB were detected in one cord blood sample each. BPS was not detected in cord blood. BPE was not detected in any fetal cord blood or maternal urine samples. These findings demonstrate the pervasiveness of some BP analogs in pregnant women and their babies at birth.     

Vitellogenin-inducing activities of natural, synthetic, and environmental estrogens in primary cultured Xenopus laevis hepatocytes

Vitellogenin (VTG)-inducing activities of natural estrogens (E1: estrone, E2:17beta-estradiol, E3: estriol, alpha-E2: 17alpha-estradiol), synthetic estrogens (EE2: 17alpha-ethynyl estradiol, DES: diethylstilbestrol,), phytoestrogen (GEN: genistein), and xeno-estrogens (BPA: bisphenol A, NP: nonylphenol, OP: octylphenol) were investigated by an assay system using primary-cultured hepatocytes of Xenopus laevis. An enzyme-linked immunoabsorbent assay (ELISA) was able to detect VTG at a minimum detection limit of 0.06 ng/mL. Relative estrogenic activities of the compounds were determined from their dose-response curves. The activities relative to E2 activity were 138% for DES, 121% for EE2, 6.1% for E3, 0.33% for E1, 0.29% for alpha-E2, 0.037% for GEN, 0.008% for BPA, 0.005% for NP, and 0.002% for OP. Comparison with data reported for other bioassay systems revealed that there were significant interspecies-and cell-type-differences in the activities of DES, E3, E1 and alpha-E2. BPA was found to have a substantial antagonistic activity (approximately 0.8% of tamoxifen activity) under the influence of physiological concentrations of E2. Complex-effects of endocrine disrupters on aquatic animals will be discussed.     

GC-FPD measurement of urinary dialkylphosphate metabolites of organophosphorous pesticides as pentafluorobenzyl derivatives in occupationally exposed workers and in a general population in Shanghai (China)

Measurement of organophosphorus (OP) pesticide metabolites in human biological fluids is an important biomarker of pesticides exposure. We measured the urinary excretion of OP pesticide metabolites to evaluate occupational and non-occupational exposure to OP pesticides in the Chinese population in Shanghai (Eastern China). We collected urine samples from 30 exposed workers in a dimethoate emulsion packing division and from 60 healthy adults without any report of occupational exposure. DMP, DMTP, DMDTP, DEP, DEDP and DEDTP were measured by GC-FPD after derivatization with pentafluorobenzyl bromide. The LOQ values (1 mL urine) were 2.0 μg/L for DMP and DETP, 4.0 μg/L for DEP and DEDTP, 8.0 μg/L for DMDTP, and 10.0 μg/L for DMTP. Dimethyl phosphates were detected in the majority of the urine samples, i.e., 90–100% in the exposed group and 80–87% in the control group. The concentration of the urinary diethyl phosphates DEP and DETP was above the LOQ values in 40 and 20% of samples for the exposed group, and in 50 and 30% of the samples for the control group, respectively. DEDTP was not detectable in the urine samples except for a post-shift exposed worker (detection frequency, 6.7%). Median creatinine-adjusted values (μg/g cr.) for DAP in Chinese with pre-shift, post-shift and without occupational exposure to OP were 316, 584 and 170 for DMP, below LOQ, 115 and 114 for DEP, 840, 1730 and 693 for DMTP, and 255, 756 and 135 for DMDTP, respectively. In all subjects, the highest excretion levels were found for DMTP. Several OP pesticide metabolites were frequently detected in urine samples of both populations studied.     

A Mechanism of Male Germ Cell Apoptosis Induced by Bisphenol-A and Nonylphenol Involving ADAM17 and p38 MAPK Activation

Germ cell apoptosis regulation is pivotal in order to maintain proper daily sperm production. Several reports have shown that endocrine disruptors such as Bisphenol-A (BPA) and Nonylphenol (NP) induce germ cell apoptosis along with a decrease in sperm production. Given their ubiquitous distribution in plastic products used by humans it is important to clarify their mechanism of action. TACE/ADAM17 is a widely distributed extracellular metalloprotease and participates in the physiological apoptosis of germ cells during spermatogenesis. The aims of this work were: 1) to determine whether BPA and NP induce ADAM17 activation; and 2) to study whether ADAM17 and/or ADAM10 are involved in germ cell apoptosis induced by BPA and NP in the pubertal rat testis. A single dose of BPA or NP (50 mg/kg) induces germ cell apoptosis in 21-day-old male rats, which was prevented by a pharmacological inhibitor of ADAM17, but not by an inhibitor of ADAM10. In vitro, we showed that BPA and NP, at similar concentrations to those found in human samples, induce the shedding of exogenous and endogenous (TNF-α) ADAM17 substrates in primary rat Sertoli cell cultures and TM4 cell line. In addition, pharmacological inhibitors of metalloproteases and genetic silencing of ADAM17 prevent the shedding induced in vitro by BPA and NP. Finally, we showed that in vivo BPA and NP induced early activation (phosphorylation) of p38 MAPK and translocation of ADAM17 to the cell surface. Interestingly, the inhibition of p38 MAPK prevents germ cell apoptosis and translocation of ADAM17 to the cell surface. These results show for the first time that xenoestrogens can induce activation of ADAM17 at concentrations similar to those found in human samples, suggesting a mechanism by which they could imbalance para/juxtacrine cell-to-cell-communication and induce germ cell apoptosis.     

Application of the OECD 301F respirometric test for the biodegradability assessment of various potential endocrine disrupting chemicals

The biodegradability of several potential endocrine disrupting compounds, namely 4-n-nonylphenol (4-n-NP), nonylphenol monoethoxylate (NP1EO), nonylphenol diethoxylate (NP2EO), bisphenol A (BPA), triclosan (TCS), di-(2-ethylhexyl)-phthalate (DEHP), perfluorooctanoate (PFOA) and perfluorononanoate (PFNA) was evaluated in this study, using OECD method 301F (manometric respirometry test) and activated sludge as inoculum. According to the results, 4-n-NP and BPA meet the strict definition of ready biodegradability and they are not expected to be persistent during the activated sludge process. Partial biodegradation was observed for DEHP (58.7+/-5.7%, n=3), TCS (52.1+/-8.5%, n=3) and NP1EO (25.9+/-8.1%, n=3), indicating their possible biodegradation in wastewater treatment systems, while no biodegradation was observed for NP2EO, PFOA and PFNA. Experiments in the co-presence of a readily biodegradable compound showed the absence of co-metabolic phenomena during 4-n-NP, BPA and TCS biodegradation. Using first order kinetics to describe biodegradation of the target compounds, half-lives of 4.3+/-0.6, 1.3+/-0.2, 1.8+/-0.5, 6.9+/-2.6 days were calculated for 4-n-NP, BPA, TCS and DEHP, respectively. Toxicity tests using marine bacterium Vibrio fischeri showed that biodegradation of 4-n-NP, NP1EO, BPA and TCS is a simultaneous detoxification process, while possible abiotic or biotic transformations of NP2EO, DEHP, PFOA and PFNA during respirometric test resulted to significant increase of their toxicities.     

GC-MS analysis of bisphenol A in human placental and fetal liver samples

A method based on extraction with acetonitrile, followed by solid-phase extraction, derivatization with acetic anhydride, and isotope dilution gas chromatography-mass spectrometry (GC-MS) analysis was applied to determine levels of free and conjugated BPA in human tissues. β-Glucuronidase was used to de-conjugate the glucuronized BPA in the samples. The method was validated using various animal organ meat samples including pork liver and kidney, beef and calf liver, chicken liver and heart; recoveries were from 85% to 112% at two spiking levels. The average method limit of quantification (LOQ) was estimated at 0.77 ng/g for placenta samples and 1.2 ng/g for fetal liver samples based on 10 times the signal to noise ratio. BPA was detected in all animal tissue samples, with concentrations ranging from 1.8 ng/g in beef and calf livers to 17.1 ng/g in pork kidney. The method was used successfully to determine both free and conjugated BPA levels in human placental and fetal liver tissue samples. BPA was detected in 86% of the placental samples; concentrations of free BPA in the positive samples ranged from 0.60 ng/g to as high as 64 ng/g with an average of 9.5 ng/g and a median of 3.0 ng/g, and conjugated BPA was as high as 7.8 ng/g. BPA was also detected in most of the fetal liver samples (57%); concentrations of free BPA in the positive samples ranged from 1.3 to 27 ng/g with an average of 8.5 ng/g and a median of 3.2 ng/g. Conjugated BPA was also detected in most of the liver samples analysed for total BPA, ranging from 0.64 to 20 ng/g with an average of 3.9 ng/g and a median of 1.5 ng/g. This study, while primarily designed as a method validation, has demonstrated that BPA can be detected in human fetal liver samples as early as the third month of fetal life. Further work will be conducted to validate these preliminary findings.     

Effects of p-cresol and chlorophenols on pentachlorophenol biodegradation

The effects of three aromatic compounds, p-cresol, 2,4-dichlorophenol (DCP), and 2,4,6-trichlorophenol (TCP), on cell growth and pentachlorophenol (PCP) degradation bya Flavobacterium species were investigated. While p-cresol was not degraded by this bacterium, DCP and TCP were simultaneously degraded with PCP. Both DCP and TCP lowered cell growth and PCP degradation rate. Cell growth was modeled by cell death, because p-cresol, DCP, and TCP were toxic to the organism. A new model was used to predict cell death rate in a mixture of two toxic compounds from the cell death kinetics for each individual compound. PCP degradation rates were modeled by conventional inhibition models, but only over a small concentration range for the secondary toxic compound. However, a new empirical model described PCP degradation over a wider concentration range of the secondary toxic compound. (c) 1995 John Wiley & Sons Inc.