Mohammad A. Mottaleb

Occurrence of pharmaceuticals and personal care products in fish: Results of a national pilot study in the united states

Pharmaceuticals and personal care products are being increasingly reported in a variety of biological matrices, including fish tissue; however, screening studies have presently not encompassed broad geographical areas. A national pilot study was initiated in the United States to assess the accumulation of pharmaceuticals and personal care products in fish sampled from five effluent-dominated rivers that receive direct discharge from wastewater treatment facilities in Chicago, Illinois; Dallas, Texas; Orlando, Florida; Phoenix, Arizona; and West Chester, Pennsylvania, USA. Fish were also collected from the Gila River, New Mexico, USA, as a reference condition expected to be minimally impacted by anthropogenic influence. High performance liquid chromatography-tandem mass spectrometry analysis of pharmaceuticals revealed the presence of norfluoxetine, sertraline, diphenhydramine, diltiazem, and carbamazepine at nanogram-per-gram concentrations in fillet composites from effluent-dominated sampling locations; the additional presence of fluoxetine and gemfibrozil was confirmed in liver tissue. Sertraline was detected at concentrations as high as 19 and 545 ng/g in fillet and liver tissue, respectively. Gas chromatography-tandem mass spectrometry analysis of personal care products in fillet composites revealed the presence of galaxolide and tonalide at maximum concentrations of 2,100 and 290 ng/g, respectively, and trace levels of triclosan. In general, more pharmaceuticals were detected at higher concentrations and with greater frequency in liver than in fillet tissues. Higher lipid content in liver tissue could not account for this discrepancy as no significant positive correlations were found between accumulated pharmaceutical concentrations and lipid content for either tissue type from any sampling site. In contrast, accumulation of the personal care products galaxolide and tonalide was significantly related to lipid content. Results suggest that the detection of pharmaceuticals and personal care products was dependent on the degree of wastewater treatment employed.

Gas chromatography-mass spectrometry screening methods for select UV filters, synthetic musks, alkylphenols, an antimicrobial agent, and an insect repellent in fish.

Two screening methods have been developed for simultaneous determination of ten extensively used personal care products (PCPs) and two alkylphenol surfactants in fish. The methods consisted of extraction, clean-up, derivatization and analysis by gas chromatography-mass spectrometry with selected ion monitoring (GC-SIM-MS) or gas chromatography-tandem mass spectrometry (GC-MS/MS) techniques. Among solvents tested to assess recovery of target compounds from 1-g tissue homogenates, acetone was selected as optimal for extracting compounds with dissimilar physicochemical properties from fish tissue. Initial experiments confirmed that GC-SIM-MS could be applied for analysis of lean fillet tissue (<1% lipid) without gel-permeation chromatography (GPC), and this approach was applied to assess the presence of target analytes in fish fillets collected from a regional effluent-dominated stream in Texas, USA. Benzophenone, galaxolide, tonalide, and triclosan were detected in 11 of 11 environmental samples at concentrations ranging from; 37 to 90, 234 to 970, 26 to 97, and 17 to 31 ng/g, respectively. However, performance of this analytical approach declined appreciably with increasing lipid content of analyzed tissues. Successful analysis of samples with increased lipid content was enabled by adding GPC to the sample preparation protocol and monitoring analytes with tandem mass spectrometry. Both analytical approaches were validated using fortified fillet tissue collected from locations expected to be minimally impacted by anthropogenic influences. Average analyte recoveries ranged from 87% to 114% with RSDs <11% and from 54% to 107% with RSDs <20% for fish tissue containing <1% and 4.9% lipid, respectively. Statistically derived method detection limits (MDLs) for GC-SIM-MS and GC-MS/MS methodologies ranged from 2.4 to 16 ng/g, and 5.1 to 397 ng/g, respectively.

Enantiospecific toxicity of the β-blocker propranolol to Daphnia magna and Pimephales promelas

Propranolol is a widely prescribed, nonselective beta-adrenergic receptor-blocking agent. Propranolol has been detected in municipal effluents from the ng/L to the low-microg/L range. Like many therapeutics and other aquatic contaminants, propranolol is distributed as a racemic mixture ((R,S)-propranolol hydrochloride). Although the (S)-enantiomer is the most active form in mammals (up to 100-fold difference), no information is available regarding the enantiospecific toxicity of propranolol to aquatic organisms. Acute and chronic studies were conducted with Daphnia magna and Pimephales promelas to determine enantiospecific toxicity of propranolol to a model aquatic invertebrate and vertebrate, respectively. Also, enantiospecific effects of propranolol on D. magna heart rate were examined. Propranolol treatment levels were verified using high-performance liquid chromatography/mass spectrometry. Acute (48-h) responses of both organisms were similar for all enantiomer treatments. Chronic P. promelas responses to propranolol enantiomers followed the hypothesized relationship of (S)-propranolol being more toxic than (R)-propranolol, but chronic D. magna responses did not. This is potentially the result of a lack of beta-type receptors in cladocerans. No enantiospecific effects on daphnid heart rate were observed in acute exposures. Interestingly, some propranolol enantiomer treatments produced significant increases in reproduction before causing reproduction to decrease at higher treatment levels. To our knowledge, this research represents the first study of enantiospecific toxicity of chiral pharmaceutical pollutants.

Simultaneous analysis of select pharmaceuticals and personal care products in fish tissue using pressurized liquid extraction combined with silica gel cleanup

Analytical improvements were developed and validated for measuring select personal care products (PCPs) and two pharmaceuticals in fish tissue. The method was validated using fortified fillet tissue for twelve PCPs including fragrance materials, alkylphenols, photo initiators, and triclosan as well as two pharmaceuticals including carbamazepine (anti-seizure) and diazepam (anti-convulsant). The analytical method utilized pressurized liquid extraction (PLE) combined with silica gel cleanup, gel permeation chromatography, and gas chromatography ion-trap tandem mass spectrometry. Silica gel cleanup was combined with the PLE to produce one automated extraction/cleanup technique. This analytical improvement served to reduce the incurred cost, time, and loss of potential target analytes associated with independent cleanup steps. The combined extraction/cleanup technique resulted in an average increase of 10% in analyte recoveries. Average triplicate recoveries and relative standard deviations for the entire method, using 2.5 g of fish fillet tissue, were 92 ± 9% (recoveries ranged from 64 to 131%). The sensitivity of the analytical methods was improved by optimizing the resonant collision induced dissociation energy to the hundredths place (0.01 V). Improvements in ion production range from 24 to 122% for six of the 12 PCPs. Statistically derived method detection limits (MDLs) were also lowered on average by a factor of 8 and ranged from 1.2 to 38 ng/g wet weight. MDLs for carbamazepine and diazepam were 18 and 3.7 ng/g wet weight, respectively. Galaxolide and tonalide were measured in an environmental sample at concentrations of 81 and 5.5 ng/g wet weight, respectively.

Development of a HPLC Method for Analysis of Linear Alkylbenzene Sulphonates and Detection by UV and FTIR Spectroscopy Using Thermospray Interface

Abstract. A reversed-phase high performance liquid chromatography (RP-HPLC) method, with acetic acid and sodium perchlorate phase modifiers, was developed to separate a mixture of linear alkylbenzene sulphonates (LAS), containing 10 to 13 carbon atoms. The effects of methanol-water compositions and concentrations of used modifiers were investigated and compared. The separation achieved with 50 mg L−1 acetic acid was found satisfactory whereas a concentration of 10 g L−1 sodium perchlorate was preferred. Chromatograms obtained with UV and Fourier transform infrared (FTIR) detection showed almost similar features and HPLC-FTIR interface spectra of LAS components exhibited excellent agreement of absorption features to those of standard FTIR spectrum and no thermal degradation was found to occur.

Accelerated solvent extraction for natural products isolation.

Accelerated solvent extraction (ASE(®)), first introduced in 1995, is an automated rapid extraction technique that utilizes common solvents at elevated temperature and pressure, and thereby increases the efficiency of extraction of organic compounds from solid and semisolid matrices. ASE(®) allows extractions for sample sizes 1-100 g in minutes, reduces solvent uses dramatically, and can be applied to a wide range of matrices, including natural products.

Ecological relationship between floral thermogenesis and pollination in Nelumbo lutea (Nelumbonaceae)

PREMISE OF STUDY Floral thermogenesis is an unusual floral trait with a well-documented physiological process, and yet, there is limited understanding of how this trait influences plant reproduction. The current study was undertaken to gain a better understanding of how floral thermogenesis in Nelumbo lutea impacts pollinator attraction and consequent plant reproduction. METHODS We conducted field studies on floral thermogenesis and thermoregulation, flower sexual development, floral visitation patterns, breeding system, pollen transfer dynamics, and floral scent production. KEY RESULTS The most abundant visitors to the thermoregulatory flowers included the Phoridae (Diptera), Chrysomelidae (Coleoptera), and Hymenoptera. Chrysomelid beetles, particularly Diabrotica, were frequent visitors to both first-day female- and second-day bisexual-phase flowers, while phorid flies were most common in bisexual-phase flowers. Pollen transfer experiments indicated that Diabrotica was equally effective in depositing pollen on stigmas, as were the less frequent, but pollen-loaded halictid bees. CONCLUSIONS Flowers received a taxonomically wide assemblage of floral visitors and appear adapted to attract beetles, primarily Chrysomelidae and medium-sized bees. This study is the first to provide strong support that beetles can comprise the dominant portion of floral visitors and are as effective in pollen transfer as bees. Thermogenesis aids in dispersing the main floral scent component-1,4-dimethoxybenzene-attracting both chrysomelids and bees, while thermoregulation causes chrysomelid beetles to actively seek out new flowers for evening residence. This search behavior likely results in chrysomelids affecting cross-pollination.

Biotransformation of musk xylene in trout haemoglobin: dose–response and toxicokinetics of musk xylene metabolites haemoglobin adducts by gas chromatography-mass spectrometry

Musk xylene (MX) is frequently used as a fragrance in commercial toiletries. Biotransformation of MX into 4-amino-MX (4-AMX) and 2-amino-MX (2-AMX) metabolites in rainbow trout haemoglobin (Hb) has been described. The dose–response relationship and toxicokinetics of the metabolites as adducts in the Hb were determined by gas chromatography (GC)–electron capture negative chemical ionization (NCI)–mass spectrometry (MS), and GC–electron ionization (EI)–MS/MS, using selected ion monitoring (SIM). The trout were subjected to a single exposure of 0.010, 0.030, 0.10, and/or 0.30 mg MX/g of fish. Hb samples were collected from exposed and control fish, and analysed subsequent to exposure at intervals of 24, 72, and 168 h. Alkaline hydrolysis released 4-AMX and 2-AMX metabolites from the Hb, and the solutes were extracted into n-hexane. The extracts were preconcentrated and analysed. The presence of the metabolites in the Hb extracts was confirmed based on agreement of similar mass spectral features from NCI/MS and EI-MS/MS spectra, and retention times of the metabolites with standards. The NCI/MS results were used for dose–response and toxicokinetics measurements. For dose–response, the concentrations of adducts of the metabolites increased with dosage, and a maximum adduct formation was observed at 0.10 mg g−1, beyond which it decreased. The average concentrations of 4-AMX and 2-AMX at a dosage of 0.10 mg g−1 were 700 and 7.4 ng g−1, respectively. For toxicokinetics, the concentration of the metabolites in the Hb reached a maximum in the 3 day sample after administration of MX. Further elimination of the metabolites exhibited kinetics with a half-life estimated to be 1–2 days, assuming first-order kinetics. Quantitations were made based on an internal standard and a calibration plot. In control samples, non-hydrolysed Hb, and reagent blank extracts, the metabolites were not detected. The limits of detection for 4-AMX and 2-AMX in the Hb were approximately 1.7 and 1.4 µg L−1, respectively, based on a signal-to-noise ratio of 3 with NCI/MS.

Biological transformation, kinetics and dose-response assessments of bound musk ketone hemoglobin adducts in rainbow trout as biomarkers of environmental exposure.

Low levels (ng/g) of musk ketone (MK), used as a fragrance additive in the formulation of personal care products, are frequently detected in the water and other environment. Thus, aquatic organisms can be continuously exposed to MK. In this study, kinetics and dose-response assessments of 2-amino-MK (AMK) metabolite, bound to cysteine-hemoglobin (Hb) in rainbow trout, formed by enzymatic nitro-reduction of MK have been demonstrated. Trout were exposed to a single exposure of 0.010, 0.030, 0.10, and 0.30 mg MK/g fish. Twenty-seven Hb samples were collected from exposed- and control fish subsequent to exposure intervals of 1 d (24 h), 3 d (72 h), and 7 d (168 h). Basic hydrolysis released bound AMK metabolite was extracted into n-hexane and then concentrated and analyzed by gas chromatography (GC) electron capture negative ion chemical ionization (NICI) mass spectrometry (MS) using selected ion monitoring (SIM). The presence of the AMK metabolite in Hb extracts was confirmed by agreement of similar mass spectral features and retention time with a standard. In the dose-response study, maximum adduct formation was obtained at the 0.10 mg/g dose with an average AMK metabolite concentration of 2.2 ng/g. For kinetics, the highest concentration of the AMK metabolite was found to be 32.0 ng/g at 0.030 mg/g dose in 3-d sample. Further elimination of the metabolite showed kinetics with a half-life estimated to be 2 d, assuming first-order kinetics. The metabolite was not detected in the control samples, non-hydrolyzed Hb, and reagent blank extracts. The detection limit for AMK in the Hb was approximately 0.30 ng/g, based on a signal to noise ratio of 3 (S/N = 3).

Nitro musk metabolites bound to carp hemoglobin: determination by GC with two MS detection modes: EIMS versus electron capture negative ion MS

Nitroaromatic compounds including synthetic nitro musks are important raw materials and intermediates in the synthesis of explosives, dyes, pesticides, and pharmaceutical and personal-care products (PPCPs). The nitro musks such as musk xylene (MX) and musk ketone (MK) are extensively used as fragrance ingredients in PPCPs and other commercial toiletries. Identification and quantification of a bound 4-amino-MX (4-AMX) metabolite as well as a 2-amino-MK (2-AMK) metabolite were carried out by gas chromatography–mass spectrometry (GCMS), with selected ion monitoring (SIM) in both the electron ionization (EIMS) and electron capture (EC) negative ion chemical ionization (NICIMS) modes. Detection of 4-AMX and 2-AMK occurred after the cysteine adducts in carp hemoglobin, derived from the nitroso metabolites, were released by alkaline hydrolysis. The released metabolites were extracted into n-hexane. The extract was preconcentrated by evaporation, and analyzed by GC-SIM-MS. A comparison between the EI and EC approaches was made. EC NICIMS detected both metabolites whereas only 4-AMX was detected by EIMS. The EC NICIMS approach exhibited fewer matrix responses and provided a lower detection limit. Quantitation in both approaches was based on an internal standard and a calibration plot.