An Immunoaffinity Purification Method for the Simultaneous Analysis of Triclocarban and Triclosan in Foodstuffs by Liquid Chromatography Tandem Mass Spectrometry.

Triclocarban (TCC) and triclosan (TCS) have been simultaneously detected in five kinds of foodstuffs using an immunoaffinity purification method coupled with ultrahigh-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) for the first time. Two highly specific monoclonal antibodies against TCC and TCS were produced and coupled to N-hydroxysuccinimide-activated Sepharose 6B gel to prepare the immunosorbent. Under the optimal conditions, mean recoveries from spiked samples by the IAC-UHPLC-MS/MS method were 70.1-92.8% for TCC and 76.6-102.5% for TCS. Intraday relative standard deviations were below 14.5%. The limits of quantification (LOQs) of TCC were 1 ng/L for beverage samples and 0.01-0.02 µg/kg for food samples. The LOQs of TCS were 0.03 µg/L for beverage samples and 0.2-0.3 µg/kg for food samples. The applicability of the method has been proven by analyzing TCC and TCS in different samples from supermarkets in Beijing. The proposed method is sufficiently sensitive and reliable for monitoring trace concentrations of TCC and TCS in food samples.

Performance and kinetics of triclocarban removal by entrapped Pseudomonas fluorescens strain MC46.

This study investigated removal of triclocarban (TCC) from contaminated wastewater by Pseudomonas fluorescens strain MC46 entrapped in barium alginate. Appropriate entrapped cell preparation conditions (cell-to-entrapment material ratio and cell loading) for removing TCC were examined. The highest TCC removal by the entrapped and free cell systems at the initial TCC concentration of 10 mg/L was 72 and 45%, respectively. TCC was degraded to less toxic compounds. Self-substrate inhibition was found at TCC concentration of 30 mg/L. The kinetics of TCC removal by entrapped and free cells fitted well with Edwards model. Scanning and transmission electron microscopic observations revealed that entrapment matrices reduced TCC-microbe contact, which lessened TCC inhibition. A live/dead cell assay also confirmed reduced microbial cell damage in the entrapped cell system compared to the free cell system. This study reveals the potential of entrapment technology to improve antibiotic removal from the environment.

Using mass struvite precipitation to remove recalcitrant nutrients and micropollutants from anaerobic digestion dewatering centrate.

The primary objective of this research was to remove recalcitrant nutrients from anaerobically digested sludge dewatering centrate. A struvite precipitation methodology is proposed where salt crystals are encouraged to ballast colloidal particles through heterogeneous nucleation and subsequent crystal growth. The secondary objective was to assess presence of micropollutants in precipitates. Four biologically unique dewatering centrates were used to test the precipitation methodology on the variety of anaerobic digester configurations that can be expected from municipal wastewater treatment plant. The effect of digestion sludge retention time (2 day, 20 day) and digestion temperature (35 °C, 55 °C) on the removal of dissolved unreactive phosphorus (P) and nitrogen (N) was monitored. Averaged across all four centrates, the precipitation methodology resulted in dissolved unreactive P and N removal of 82.4% and 66.6%, respectively. Antimicrobial contaminants (triclosan, triclocarban) were observed in the precipitates at minute concentrations (<18 ng/g-dry solids). Therefore, mass struvite precipitation can provide a means of recalcitrant nutrient treatment and reactive nutrient recovery without the micropollutant burden of biosolids land application.

Determination of selected parabens, benzophenones, triclosan and triclocarban in agricultural soils after and before treatment with compost from sewage sludge: A lixiviation study.

An accurate and sensitive method for the determination of selected EDCs in soil and compost from wastewater treatment plants is developed and validated. Five parabens, six benzophenone-UV filters and the antibacterials triclosan and triclocarban were selected as target analytes. The parameters for ultrasound-assisted extraction were thoroughly optimized. After extraction, the analytes were detected and quantified using ultra-high performance liquid chromatography tandem mass spectrometry. Ethylparaben (ring-(13)C6 labelled) and deuterated benzophenone (BP-d10) were used as internal standards. The method was validated using matrix-matched calibration and recovery assays with spiked samples. The limits of detection ranged from 0.03 to 0.40 ng g(-1) and the limits of quantification from 0.1 to 1.0 ng g(-1), while precision in terms of relative standard deviation was between 9% and 21%. Recovery rates ranged from 83% to 107%. The validated method was applied for the study of the behavior of the selected compounds in agricultural soils treated and un-treated with compost from WWTP. A lixiviation study was developed in both agricultural soil and treated soil and first order kinetic models of their disappearance at different depths are proposed. The application of organic composts in the soil leads to an increase of the disappearance rate of the studied compounds. The lixiviation study also shows the risk of pollution of groundwater aquifers after disposal or waste of these EDCs in agricultural soils is not high.

Extraction of 3,4,4'-Trichlorocarbanilide from Rat Fecal Samples for Determination by High Pressure Liquid Chromatography-Tandem Mass Spectrometry.

Triclocarban (3,4,4'-Trichlorocarbanilide; TCC) in the environment has been well documented. Methods have been developed to monitor TCC levels from various matrices including water, sediment, biosolids, plants, blood and urine; however, no method has been developed to document the concentration of TCC in fecal content after oral exposure in animal studies. In the present study, we developed and validated a method that uses liquid extraction coupled with HPLC-MS/MS determination to measure TCC in feces. The limit of detection and limit of quantitation in control rats without TCC exposure was 69.0 ng/g and 92.9 ng/g of feces, respectively. The base levels of TCC in feces were lower than LOD. At 12 days of treatment, the fecal TCC concentration increased to 2220 µg/g among 0.2% w/w exposed animals. The concentration in fecal samples decreased over the washout period in 0.2% w/w treated animals to 0.399 µ/g feces after exposure was removed for 28 days. This method required a small amount of sample (0.1 g) with simple sample preparation. Given its sensitivity and efficiency, this method may be useful for monitoring TCC exposure in toxicological studies of animals.

Transformation products and human metabolites of triclocarban and triclosan in sewage sludge across the United States.

Removal of triclocarban (TCC) and triclosan (TCS) from wastewater is a function of adsorption, abiotic degradation, and microbial mineralization or transformation, reactions that are not currently controlled or optimized in the pollution control infrastructure of standard wastewater treatment. Here, we report on the levels of eight transformation products, human metabolites, and manufacturing byproducts of TCC and TCS in raw and treated sewage sludge. Two sample sets were studied: samples collected once from 14 wastewater treatment plants (WWTPs) representing nine states, and multiple samples collected from one WWTP monitored for 12 months. Time-course analysis of significant mass fluxes (α=0.01) indicate that transformation of TCC (dechlorination) and TCS (methylation) occurred during sewage conveyance and treatment. Strong linear correlations were found between TCC and the human metabolite 2'-hydroxy-TCC (r=0.84), and between the TCC-dechlorination products dichlorocarbanilide (DCC) and monochlorocarbanilide (r=0.99). Mass ratios of DCC-to-TCC and of methyl-triclosan (MeTCS)-to-TCS, serving as indicators of transformation activity, revealed that transformation was widespread under different treatment regimes across the WWTPs sampled, though the degree of transformation varied significantly among study sites (α=0.01). The analysis of sludge sampled before and after different unit operation steps (i.e., anaerobic digestion, sludge heat treatment, and sludge drying) yielded insights into the extent and location of TCC and TCS transformation. Results showed anaerobic digestion to be important for MeTCS transformation (37-74%), whereas its contribution to partial TCC dechlorination was limited (0.4-2.1%). This longitudinal and nationwide survey is the first to report the occurrence of transformation products, human metabolites, and manufacturing byproducts of TCC and TCS in sewage sludge.

Dispersive liquid-liquid microextraction for the determination of three cytokinin compounds in fruits and vegetables by liquid chromatography with time-of-flight mass spectrometry.

The paper presents a novel approach for the determination of three cytokinin compounds, thidiazuron (TDZ), 1,3-diphenylurea (1,3-DPU) and forchlorfenuron (CPPU), in fruit and vegetables samples using liquid chromatography with electrospray ionization and time-of-flight mass spectrometry (LC-ESI-TOFMS). Analytes were extracted from the sample matrix with ethanol, and the extract, after dilution with water, was submitted to dispersive liquid-liquid microextraction (DLLME). Once acetonitrile and 1,2-dichloroethane had been selected as extraction and disperser solvents, respectively, the influence of the following experimental parameters was studied using a Plackett-Burman design: volume of extraction and disperser solvents, sample mass and time and speed of centrifugation. The best analytical conditions were 250 µL 1,2-dichloroethane, 1.5 mL acetonitrile, 5 g sample mass, and centrifugation at 3000 rpm for 3 min. The optimized method provided DLs in the range 0.02-0.05 ng g(-1), depending on the compound. Satisfactory recovery values between 89 and 106% were obtained for spiked samples (kiwifruit, watermelon, grape and tomato) in the 0.2-1.0 ng g(-1) concentration range, depending on the compound. None of the target analytes was detected in any of the samples analyzed.

Fate of Triclocarban, Triclosan and Methyltriclosan during wastewater and biosolids treatment processes.

Triclocarban (TCC) and Triclosan (TCS) are two antibacterial chemicals present in household and personal care products. Methyltriclosan is a biodegradation product of TCS formed under aerobic conditions. TCC and TCS are discharged to Waste Water Treatment Plants (WWTP) where they are removed from the liquid phase mainly by concentrating in the solids. This study presents a thorough investigation of TCC, TCS and MeTCS concentrations in the liquid phase (dissolved + particulate) as well as solid phases within a single, large WWTP in the U.S. Total TCC and TCS concentrations decreased by >97% with about 79% of TCC and 64% of TCS transferred to the solids. The highest TCC and TCS removal rates from the liquid phase were reached in the primary treatment mainly though sorption and settling of solids. The TCC mass balances showed that TCC levels remain unchanged through the secondary treatment (activated sludge process) and about an 18% decrease was observed through the nitrification-denitrification process. On the other hand, TCS levels decreased in both processes (secondary and nitrification-denitrification) by 10.4 and 22.6%, respectively. The decrease in TCS levels associated with observed increased levels of MeTCS in secondary and nitrification-denitrification processes providing evidence of TCS biotransformation. Dissolved-phase concentrations of TCC and TCS remained constant during filtration and disinfection. TCC and TCS highest sludge concentrations were analyzed in the primary sludge (13.1 ± 0.9 µg g(-1) dry wt. for TCC and 20.3 ± 0.9 µg g(-1) dry wt. for TCS) but for MeTCS the highest concentrations were analyzed in the secondary sludge (0.25 ± 0.04 µg g(-1) dry wt.). Respective TCC, TCS and MeTCS concentrations of 4.15 ± 0.77; 5.37 ± 0.97 and 0.058 ± 0.003 kg d(-1) are leaving the WWTP with the sludge and 0.13 ± 0.01; 0.24 ± 0.07 and 0.021 ± 0.002 kg d(-1) with the effluent that is discharged.

Phase distribution and removal of pharmaceuticals and personal care products during anaerobic sludge digestion.

The fate and removal of 48 pharmaceuticals and personal care products (PPCPs) during anaerobic digestion of sewage sludge were investigated in four full-scale sewage treatment plants (STPs). We measured concentrations in both the liquid and solid phases of the sludge to compare the distribution ratios (Kd) between phases before and after digestion. The results showed changes in Kd values of PPCPs with carboxyl or amino functional groups, probably due to a shift of dissociation equilibrium with the increase in pH. Sulfamethoxazole and trimethoprim were almost completely degraded (>90%); triclosan, triclocarban, and ofloxacin were moderately degraded (around 30-50%); but carbamazepine was not eliminated. To our knowledge, this is the first report that shows (i) the occurrence and removal of several tens of PPCPs by anaerobic sludge digestion in full-scale municipal STPs and (ii) the change of distribution between the liquid and solid phases during digestion.

Simultaneous determination of triclosan, triclocarban, and transformation products of triclocarban in aqueous samples using solid-phase micro-extraction-HPLC-MS/MS.

The presence of triclosan and triclocarban, two endocrine-disrupting chemicals and antimicrobial agents, and transformation products of triclocarban, 1,3-di(phenyl)urea, 1,3-bis(4-chlorophenyl)urea and 1,3-bis(3,4-dichlorophenyl)urea, in tap water, treated household drinking water, bottled water, and river water samples were investigated using solid-phase micro-extraction coupled with-HPLC-MS/MS, a rapid, green, and sensitive method. Factors influencing the quantity of the analytes extracted onto the solid-phase micro-extraction fiber, such as addition of salt, sample pH, extraction time, desorption time, and sample volume, were optimized using solid-phase micro-extraction-HPLC-MS/MS. The results showed that the method gave satisfactory sensitivities and precisions for analyzing sub-part-per-trillion levels of triclosan, triclocarban, and transformation products of triclocarban in samples collected locally. The recoveries of analytes ranged from 97 to 107% for deionized water samples, and 99 to 110% for river water samples, and limits of detection were in the range of 0.32-3.44 and 0.38-4.67 ng/L for deionized water and river water samples, respectively. On average, the daily consumption of triclosan and triclocarban by an adult by consuming 2 liters of different types of drinking water were estimated to be in the range of 6.13-425 ng/day as a result of the concentrations of triclosan and triclocarban measured in this study.

Bioconcentration of triclosan, methyl-triclosan, and triclocarban in the plants and sediments of a constructed wetland.

Constructed wetlands are a potential method for the removal of two pharmaceutical and personal care products from wastewater effluent. Triclosan (TCS; 5-chloro-2-[2,4-dichlorophenoxy]phenol) and triclocarban (TCC; 3,4,4'-trichlorocarbanillide) are antimicrobial agents added to a variety of consumer products whose accumulation patterns in constructed wetlands are poorly understood. Here, we report the accumulation of TCS, its metabolite methyl-triclosan (MTCS; 5-chloro-2-[2,4-dichlorophenoxy]), and TCC in wetland plant tissues and sediments. Three wetland macrophytes: Typha latifolia, Pontederia cordata, and Sagittaria graminea were sampled from a constructed wetland in Denton, Texas, USA. MTCS concentrations were below the method detection limit (MDL) for all species. TCS root tissue concentrations in T. latifolia were significantly greater than root concentrations in P. cordata (mean±SE in ng g(-1): 40.3±11.3 vs. 15.0±1.9, respectively), while for TCC, shoot tissue concentrations in S. graminea were significantly greater than in T. latifolia (22.8±9.3 vs. 9.0 (MDL), respectively). For both TCS and TCC, T. latifolia root tissue concentrations were significantly greater than shoot concentrations (TCS: 40.3±11.3 vs. 17.2±0.2, TCC: 26.0±3.6 vs. 9.0, (MDL)). TCC concentrations in P. cordata roots were significantly greater than in shoots (34.4±5.3 vs. 15.4±2.8, respectively). TCS concentrations in T. latifolia roots and sediments and TCC concentrations in sediments generally decreased from wetland inflow to outflow. To our knowledge, this is the first study documenting species and tissue specific differences in the accumulation of TCS and TCC in plants from an operational constructed wetland. The species specific differences in bioaccumulation suggest TCS and TCC removal from constructed wetlands could be enhanced through targeted plantings.

High-performance liquid chromatographic method for the determination of the dinitrocarbanilide component of nicarbazin in eggs with on-line clean-up.

A high-performance liquid chromatographic method for the determination of the dinitrocarbanilide component of the anti-coccidial drug nicarbazin has been developed. The drug was extracted from egg with acetonitrile. The extract was evaporated to dryness and taken up in water-acetonitrile (80:20, v/v). The extract was then injected onto a reversed-phase precolumn. After clean-up with 20% aqueous acetonitrile for 5 min, the precolumn was eluted onto a Chromspher C18 cartridge column with 0.01 M potassium dihydrogen-phosphate pH 4.0-acetonitrile (50:50, v/v). Detection was by ultraviolet at 343 nm. Average recoveries at five levels from 0.005 to 0.500 mg kg-1 were > 80%. The limit of determination was 0.005 mg kg-1.

A method for the unambiguous identification of tryptophan in automated protein sequence analysis.

Some widely used standard protocols for the separation of phenylthiohydantoin amino acid derivatives by reverse-phase gradient HPLC do not provide separation of the phenylthiohydantoin derivative of tryptophan (PTH-Trp) from diphenylurea (DPU), a by-product generated during Edman degradation of proteins in variable amounts. Furthermore, PTH-Trp is usually recovered in low yield under typical experimental conditions used with automated sequencing equipment. These factors may compromise the unambiguous assignment of tryptophan residues in automated protein sequence analysis, especially when sequencing is performed at high sensitivity. We devised a reverse-phase HPLC method which allows the separation of DPU and PTH-Trp and therefore the correct assignment of PTH-Trp. The method is based on a modification of the HPLC gradient used to elute and separate all PTH amino acids of interest. With Applied Biosystems Model 477A protein sequencers with on-line PTH amino acid identification, the correct assignment of tryptophan was consistent and reproducible even when sequencing at very high sensitivity (5 pmol).