Fluorescent whitening agents in Baiyangdian Lake in North China: Analysis, occurrence, distribution and ecological risk assessment.

Fluorescent whitening agents (FWAs) are very important chemical additives that are widely applied in the industrial production field. The history of global FWA production and use spans more than 60 years, but the environmental fate of FWAs has been less reported in the public literature and most studies predate 2000; in addition, the studied FWAs were still limited to FWA71 and FWA351. In this study, the occurrence and distribution of 9 commonly used FWAs in a lake in North China were reported for the first time. We found that 6 target FWAs were prevalent in the lake, and the concentration levels were usually at the ng L-1 level. Decreasing FWA levels with increasing distance from the estuary area were observed in summer. FWA135, FWA185, and FWA367, the most detected 3 FWAs, with the ecological risk at high levels, and ΣRQ >10 were obtained from all the investigated samples, suggesting that all the sampling sites could be considered with certain ecological risk for aquatic life. As a category of heavily and widely used dyes, FWAs in environmental media have been ignored for a long time. Substantial additional research needs to be conducted to determine the environmental behavior and ecological toxicology of FWAs.

Quaternary ammonium salt ion pair reagent sensitizing for determination of fluorescence whitening agent 85 in paper food packaging.

Fluorescent whitening agents (FWAs) are added to food packaging during manufacture to improve the whiteness and brightness. It is of great significance to develop a fast and sensitive FWAs detection method because their potential hazards to human health. Herein, we found that the quaternary ammonium salt ion pair reagents could enhance the fluorescence of FWAs, and then developed a fluorescent determination method for FWA 85 in paper food packaging. Under the optimized conditions, the linear range for the determination of FWA 85 was 5-200 ng/mL (R2 = 0.9996). The limit of detection was 1.20 ng/mL (S/N = 3) and the limit of quantitation was 3.99 ng/mL. The analytical feasibility was investigated further via determination of FWA 85 in paper food packing, which shows the recoveries were varied from 83.84% to 115.29% with RSDs from 1.64% to 7.22%. The method provides a new quaternary ammonium salt ion pair reagent sensitization for the detection of FWAs in paper food packaging.

Relationships between chemical and microbial faecal source tracking markers in urban river water and sediments during and post-discharge of human sewage.

This study explores the relationships between faecal source tracking (FST) markers (quantitative Polymerase Chain Reaction (qPCR) markers and steroids), microbial indicators, the faecal ageing ratio of atypical colonies/total coliforms (AC/TC) and potential human pathogens (Giardia, Cryptosporidium and Campylobacter). Faecal source PCR markers tested were GenBac3, HumM3, HumBac (HF183-Bac708R); Bifidobacterium adolescentis, wildfowl and canine-associated markers. Sediment and water samples from the Avon River were collected during and post-discharge of untreated human sewage inputs, following a series of earthquakes, which severely damaged the Christchurch sewerage system. Significant, positive Spearman Ranks (rs) correlations were observed between human-associated qPCR markers and steroid FST markers and Escherichia coli and F-specific RNA bacteriophage (rs 0.57 to 0.84, p < 0.001) in water samples. These human source indicative FST markers demonstrated that they were also effective predictors of potentially pathogenic protozoa in water (rs 0.43-0.74, p ≤ 0.002), but correlated less well with Campylobacter. Human-associated qPCR and steroid markers showed significant, substantial agreement between the two FST methods (Cohen's kappa, 0.78, p = 0.023), suggesting that water managers could be confident in the results using either method under these contamination conditions. Low levels of fluorescent whitening agents (FWA) (mean 0.06 µg/L, range 0.01-0.40 µg/L) were observed in water throughout the study, but steroids and FWA appeared to be retained in river sediments, months after continuous sewage discharges had ceased. No relationship was observed between chemical FST markers in sediments and the overlying water, and few correlations in sediment between chemical FST markers and target microorganisms. The low values observed for the faecal ageing ratio, AC/TC in water, were significantly, negatively correlated with increasing pathogen detection. This study provides support for the use of the AC/TC ratio, and qPCR and steroid FST markers as indicators of health risks associated with the discharge of raw human sewage into a freshwater system.

Determination of Fluorescent Whitening Agents in Cosmetics and Liquid Detergent by High-Performance Liquid Chromatography with Diode Array Detector in Tandem with Fluorescence Detector.

Five distyryl-type fluorescent whitening agents (FWA85, 210, 220, 351, and 353) were determined in cosmetics and liquid detergent by high-performance liquid chromatography with diode array detector in tandem with fluorescence detector. The samples were extracted with ultrasound in 33% acetonitrile for 10 minutes and the components were determined by ion-pair chromatography on an MG C18 column. The limits of detection were from 0.01 to 0.1 mg·kg-1 and the limits of quantification were from 0.04 to 0.4 mg·kg-1. The recovery was from 80.7 to 103.3%. A linear relationship was present from 0.10 to 100 µg·ml-1 of FWAs. The protocol was simple, sensitive, selective, and was successfully applied to analyze distyryl-type FWAs in cosmetics and liquid detergent. FWA351 and FWA85 were detected in several samples with the concentrations of 19.4-1,130 mg·kg-1.

Magnetic-covalent organic polymer solid-phase extraction coupled with high-performance liquid chromatography for the sensitive determination of fluorescent whitening agents in cosmetics.

Magnetic solid-phase extraction is a powerful sample preparation tool. Combined with covalent organic polymers, this technique permits an excellent extraction performance. We present a sensitive, efficient, and easy operating magnetic- covalent organic polymer solid-phase extraction method. By using the magnetic covalent organic polymer nanoparticles yielded by a one-pot reaction, five fluorescent whitening agents were successfully preconcentrated and separated. The saturated extraction amounts of the magnetic covalent organic polymer nanoparticles for the five fluorescent whitening agents were in the range of 97-155 nmol/mg. Coupled with high-performance liquid chromatography, the limit of detection of the fluorescent whitening agent was improved by at least an order of magnitude compared to current literature records. Under the optimal conditions, the linear ranges of these five fluorescent whitening agents were up to 0.5-100 ng/L, the limit of detection was as low as 0.1 ng/L, and the recoveries were in the range of 78.2-105% with relative standard deviations lower than 5.5%. Meanwhile, a trace level of 7-diethylamino-4-methylcoumarin was found in a typical cosmetic sample by using this magnetic-covalent organic polymer solid-phase extraction method when coupling with high-performance liquid chromatography. Moreover, this sensitive, efficient, and easy operating sample preparation method can be extended to analysis of other analytes that have a π-π conjugated structure.

Development and validation of an HPLC method with fluorescence detection for the determination of fluorescent whitening agents migrating from plastic beverage cups.

An HPLC method with fluorescence detection has been developed and validated for the quantification of six fluorescent whitening agents (FWA) in plastic beverage cups after extraction and in food simulants after migration at 70°C for 2 h. The sensitivity of the method was high with LODs ranging from 0.053 to 0.251 µg kg-1 and LOQs from 0.107 to 0.504 µg kg-1. Accuracy and precision were highly acceptable, with recoveries greater than 82% and RSDs (%) below 16%. The expanded combined uncertainty was found to be less than 23% for the measurements of all studied FWAs. In extracting the analytes from food contact materials (FCM), accelerated solvent extraction (ASE) and Soxhlet extraction were applied using ethanol as the extraction solvent. The results obtained for FWA in 10 different food plastic cups, made from different polymers, were compared. The ASE technique proved to be faster, more effective and efficient than Soxhlet extraction. Migration tests with official food simulants from Regulation (EU) No 10/2011 showed that the substances studied could potentially migrate using the selected migration conditions. The most pronounced effect was observed in case of simulant D1 (50% w/v ethanol in water). The analytical method proved to be a simple, fast, sensitive and reliable tool for the simultaneous quantification of six of the most used FWAs in both FCM extracts and food simulants after migration experiments.

Development and validation of a screening and quantification method for simultaneous determination of seven fluorescent whitening agents in commercial flour using UPLC-MS/MS.

A sensitive UPLC-MS/MS method was established for the simultaneous determination of seven fluorescent whitening agents in flour matrix. Samples were ultrasonically extracted by trichloromethane-acetone. Then analytes were separated on a C18 column and detected by MS/MS. The developed method was validated in terms of the linearity, matrix effect, accuracy and precision. The method showed a good linearity (r>0.997) for all analytes in their respective concentration ranges. The LOQ for seven analytes were in the range of 2.5-25.0µg/kg. The matrix effect was not significant and almost negligible. The average recoveries of seven analytes from the negative samples spiked at three different concentrations were in a range from 81.5% to 105%. The intra-day precision and inter-day precision were in the ranges of 2.9-9.0% and 6.7-11.2%. The developed method was successfully applied to analyze forty commercial flours and FWA184 was detected in three samples with the concentrations of 31-60µg/kg.

Near-infrared hyperspectral imaging for detection and quantification of azodicarbonamide in flour.

BACKGROUND: The present study aimed to establish a method for the detection and quantification of azodicarbonamide (ADC) in flour using hyperspectral imaging technology. Hyperspectral images of pure flour, pure ADC and flour-ADC mixtures with different concentrations of ADC were collected. F-values of one-way analysis of variance for all possible wavebands within the spectra of the flour and ADC were calculated, and the maximum value indicated that the two wavebands have more significant differences, i.e. the optimal two wavebands. Threshold segmentation was used for band ratio images of two wavebands to create a binary image. This allowed visual identification of ADC-rich pixels in the mixtures. RESULTS: The two wavebands with the largest difference between flour and ADC were 2039 nm and 1892 nm. Using the binary image construction method, different concentrations of ADC in flour were identified. The minimum detected concentration was 0.2 g kg-1 . In the mixtures, the number of ADC-rich pixels detected had a good linear relationship with the ADC concentrations, with a correlation coefficient of 0.9845. CONCLUSION: This study indicated that the band ratio algorithm combination with threshold segmentation for hyperspectral images provides a non-destructive method for detecting and quantifying of ADC in flour. © 2017 Society of Chemical Industry.

Benzoyl Peroxide Detection in Real Samples and Zebrafish Imaging by a Designed Near-Infrared Fluorescent Probe.

A novel near-infrared fluorescence off-on probe, (E)-3,3-dimethyl-1-propyl-2-(2-(6-(2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyloxy)-2,3-dihydro-1H-xanthen-4-yl)vinyl)-3H-indolium (1), is developed and applied to benzoyl peroxide (BPO) detection in real samples and fluorescence imaging in living cells and zebrafish. When arylboronate as the recognition unit is connected to a stable hemicyanine skeleton, the probe is readily prepared, which exhibits superior analytical performance, such as near-infrared fluorescence emission over 700 nm and high sensitivity with a low detection limit of 47 nM. Upon reaction with BPO, phenylboronic acid pinacol ester is oxidized, followed by hydrolysis and 1,4-elimination of o-quinone methide to release fluorophore. In addtion, the probe displays high selectivity toward BPO over other common substances, which makes it of great potential use in quantitative and simple detection of BPO in wheat flour and antimicrobial agent. More importantly, the probe has been successfully demonstrated for monitoring BPO in living HeLa cells and zebrafish. The probe with superior properties could be of great potential use in other biosystems and in vivo studies.

Acylhydrazone bond dynamic covalent polymer gel monolithic column online coupling to high-performance liquid chromatography for analysis of sulfonamides and fluorescent whitening agents in food.

A new dynamic covalent polymer (DCP) gel was well designed and constructed based on imine chemistry. Polycondensation of 4,4'-biphenyldicarboxaldehyde and 1,3,5-benzenetricarbohydrazide via Schiff-base reaction resulted in an acylhydrazone bond gel (AB-gel) DCP. AB-gel DCP had three-dimensional network of interconnected nanoparticles with hierarchically porous structure. AB-gel DCP was successfully fabricated as a monolithic column by an in-situ chemical bonding method for online enrichment and separation purpose with excellent permeability. AB-gel DCP based monolithic column showed remarkable adsorption affinity towards target analytes including sulfonamides (SAs) and fluorescent whitening agents (FWAs) due to its strong π-π affinity, hydrophobic effect and hydrogen bonding interaction. Then, AB-gel DCP based monolithic column was applied for online separation and analysis of trace SAs and FWAs in food samples coupled with high-performance liquid chromatography (HPLC). Sulfathiazole (ST) and sulfadimidine (SM2) in one positive weever sample were actually found and determined with concentrations of 273.8 and 286.3µg/kg, respectively. 2,5-Bis(5-tert-butyl-2-benzoxazolyl) thiophene (FWA184) was actually quantified in one tea infusion sample with the concentration of 268.5ng/L. The spiked experiments suggested the good recoveries in range of 74.5-110% for SAs in weever and shrimp samples with relative standard deviations (RSDs) less than 9.7% and in range of 74.0-113% for FWAs in milk and tea infusion samples with RSDs less than 9.0%. AB-gel DCP monolithic column was proved to be a promising sample preparation medium for online separation and analysis of trace analytes in food samples with complex matrices.

[Simultaneous determination of six fluorescent whitening agents in plastic and paper packaging materials by high performance liquid chromatography].

A novel analytical method was developed for the simultaneous determination of six fluorescent whitening agents (FWAs:FWA 135, FWA 184, FWA 185, FWA 199, FWA 378 and FWA 393) in paper and plastic food packaging materials by high performance liquid chromatography with fluorescence detection (HPLC-FLD). The sample was extracted with mixed solution of chloroform and acetonitrile (3:7, v/v), then cleaned up by HLB solid phase extraction column. Qualitative and quantitative analyses were carried out by HPLC. The sample was separated on a Phenomenex C18 column using acetonitrile and 5 mmol/L ammonium acetate aqueous solution as mobile phases. The results indicated that the linear range of FWA393 was 15-1500 µg/L and the linear ranges of the other five FWAs were 5-500 µg/L with correlation coefficients greater than 0.999. The recoveries in spiked samples were between 80.4% and 125.0% with RSDs (n=6) of 1%-13%. Furthermore, this method was applied to analyze 12 samples in the market to verify the practicality of the method. The method showed the advantages of simplicity, high recovery and good precision, and is suitable for the detection of the six fluorescent whitening agents in food packaging materials.

Increased detection of smear-negative pulmonary tuberculosis by GeneXpert MTB/RIF® assay after bleach concentration.

OBJECTIVE/BACKGROUND: The GeneXpert MTB/RIF assay (Xpert) was endorsed as the initial diagnostic tool in people suspected of human immunodeficiency virus-associated or drug-resistant tuberculosis (TB). However, information regarding the performance of Xpert for diagnosing smear-negative TB in high burden settings remains limited. We evaluated the diagnostic accuracy of Xpert and the impact of bleach concentration on the performance of Xpert using smear-negative sputum samples from human immunodeficiency virus-negative patients. METHODS: One spot and one morning smear-negative sputum samples per patient were examined using Xpert and culture at the Mycobacteriology Research Center of Jimma University, Ethiopia. The sputum culture on both Löwenstein-Jensen and/or Mycobacteria Growth Indicator Tube was the gold-standard. RESULTS: Of 185 smear-negative presumptive pulmonary TB cases, 19 (10.3%) had culture-proven TB. The sensitivity of Xpert on spot and morning sputum was similar (63.2%). Testing two specimens per patient insignificantly increased the sensitivity of Xpert. Bleach concentration and pelleting improved the sensitivity of Xpert over unprocessed sputum in paired samples (73.8% vs. 63.2%) without affecting the specificity (95%). Bleach concentration and pelleting allowed an additional seven cases of TB (missed on the first and second direct Xperts) to be detected, five of which were from culture-negative cases. CONCLUSION: Testing of a single sputum sample by Xpert can reach reasonable sensitivity and results would be available on the same day, avoiding loss of patients and treatment delay. The sensitivity of Xpert was improved after bleach concentration and pelleting, although its added value needs further study on a larger scale.

[Determination of eleven fluorescent whitening agents in paper food packaging materials by UPLC-FLD/PDA with series double-detector].

OBJECTIVE: To establish a new qualitative and quantitative ultraperformance liquid chromatography-fluorescence detector / photodiode array detector with series double-detector method for the determination of eleven fluorescent whitening agents in paper food packaging materials. METHODS: The sample was extracted with 40%acetonitrile water solution, separated by Waters ACQUITY UPLC BEH C_(18)column( 1. 7µm, 2. 1 mm × 100 mm) and eluted gradient. The excitation wavelength and emission wavelength of fluorescence detector( FLD) were 350 nm and 430 nm, and the wavelength of photodiode array detector( PDA) was 350 nm. The detectors were used in series to achieve qualitative and quantitative detection. RESULTS: In the substrates of paper cups, paper bowls, paper trays and paper boxes, those eleven fluorescent whitening agents were separated properly. For both detectors, in the linear range of 25- 1000 ng / m L, the correlation coefficient was greater than 0. 99, and the recoveries of spiked recoveries were between 82. 2%- 104. 1% with the RSD less than 10%( n = 6). The detection limits ofthose eleven fluorescent whitening agents were 0. 20- 0. 28 mg / kg for FLD and 1. 4- 2. 5mg / kg for PDA. CONCLUSION: The eleven fluorescent whitening agents could be separated properly with complete separation, good shapes and high recovery rate. This method is easy to operate also. Thus it's an effective method to detect the fluorescent whitening agents in paper food packaging materials.

Effect of temperature and concentration on benzoyl peroxide bleaching efficacy and benzoic acid levels in whey protein concentrate.

Much of the fluid whey produced in the United States is a by-product of Cheddar cheese manufacture and must be bleached. Benzoyl peroxide (BP) is currently 1 of only 2 legal chemical bleaching agents for fluid whey in the United States, but benzoic acid is an unavoidable by-product of BP bleaching. Benzoyl peroxide is typically a powder, but new liquid BP dispersions are available. A greater understanding of the bleaching characteristics of BP is necessary. The objective of the study was to compare norbixin destruction, residual benzoic acid, and flavor differences between liquid whey and 80% whey protein concentrates (WPC80) bleached at different temperatures with 2 different benzoyl peroxides (soluble and insoluble). Two experiments were conducted in this study. For experiment 1, 3 factors (temperature, bleach type, bleach concentration) were evaluated for norbixin destruction using a response surface model-central composite design in liquid whey. For experiment 2, norbixin concentration, residual benzoic acid, and flavor differences were explored in WPC80 from whey bleached by the 2 commercially available BP (soluble and insoluble) at 5 mg/kg. In liquid whey, soluble BP bleached more norbixin than insoluble BP, especially at lower concentrations (5 and 10 mg/kg) at both cold (4°C) and hot (50°C) temperatures. The WPC80 from liquid whey bleached with BP at 50°C had lower norbixin concentration, benzoic acid levels, cardboard flavor, and aldehyde levels than WPC80 from liquid whey bleached with BP at 4°C. Regardless of temperature, soluble BP destroyed more norbixin at lower concentrations than insoluble BP. The WPC80 from soluble-BP-bleached wheys had lower cardboard flavor and lower aldehyde levels than WPC80 from insoluble-BP-bleached whey. This study suggests that new, soluble (liquid) BP can be used at lower concentrations than insoluble BP to achieve equivalent bleaching and that less residual benzoic acid remains in WPC80 powder from liquid whey bleached hot (50°C) than cold (4°C), which may provide opportunities to reduce benzoic acid residues in dried whey ingredients, expanding their marketability.

Photodegradation-based detection of fluorescent whitening agents in a mountain river.

Fluorescent whitening agents (FWAs) are highly soluble and poorly biodegradable ingredients used in laundry detergents and in industries (paper, textile, plastic manufacturing). They are likely to pass through biological wastewater treatment systems. The presence of FWAs in a mountain river was detected by monitoring the decay of synchronous fluorescence intensity at λ(ex)=360 nm after exposing samples to ultraviolet (UV) light (365 nm), for mimicking sunlight, for 15 min. The method was first validated on four commercial FWAs (DAS-1, FB28, DMA-X and CBS-X) in different water matrices (deionized water and pristine river water in the presence of humic acid and dyes). A 40% decay was observed after 15 min for the least photosensitive FWA (CBS-X). A field application was then performed on samples collected along a mountain river in which impacts of FWAs from domestic sources (laundry greywater) and industrial sources (paper and textile mills) were suspected. Variations of fluorescence decay at λ(ex)=360 nm could be explained by these potential sources of pollution. It is suggested that the fluorescence decay at λ(ex)=280 nm also be considered as an indicator, as some FWAs can exhibit fluorescence at that excitation wavelength.

[Determination of fluorescent whitening agents in plastic food contact materials by high performance liquid chromatography with fluorescence detector].

A method for the determination of fluorescent whitening agents in plastic food contact materials by high performance liquid chromatography (HPLC) with fluorescence detector was developed. The samples were extracted with trichloromethane by sonication for 30 min at 40 degrees C. The HPLC method was performed on a column of Eclipse XDB-C18 (250 mm x 4.6 mm, 5 microm) by gradient elution using 5 mmol/L ammonium acetate and acetonitrile as the mobile phases, and detected by the fluorescence detector at an excitation wavelength of 350 nm and an emission wavelength of 430 nm. The experimental results indicated that the four fluorescent whitening agents were separated well. The limits of detection (LOD) (S/N = 3) were 0.3, 0.1, 0.05, 0.14 mg/L, and the limits of quantification (LOQ) (S/N = 10) were 1.0, 0.4, 0.2, 0.5 mg/L for 1,4-bis (4-cyanostyryl) benzene (C. I. 199), 1,4-bis (2-benzoxazolyl) naphthalene (C. I. 367), 4,4'-bis(2-methoxystyryl) biphenyl (C. I. 378) and 2,5-thiophenediylbis (5-tert-butyl-1,3-benzoxazole) (C. I. 184), respectively. Good linearities with correlation coefficients (r2) not less than 0.991 were obtained. The proposed method is simple, accurate, sensitive and can meet the requirements of the routine determination of fluorescent whitening agents in entry-exit products.

Formation of organochlorine by-products in bleached laundry.

Laundering fabrics with chlorine bleach plays a role in health and hygiene as well as aesthetics. However, laundry bleaching may create chlorinated by-products with potentially adverse human health effects. Studies have shown that toxic chlorinated gases are produced in the headspace of washing machines when hypochlorite-containing bleach is used. Laundry bleaching has also been implicated in contributing dissolved organochlorine to municipal wastewater. However, there have been no reports of organochlorines produced and retained in fabric as a result of laundry bleaching. We have used a chlorine-specific X-ray spectroscopic analysis to demonstrate the formation of organochlorine by-products in cotton fabrics laundered with chlorine bleach under typical household conditions. Organochlorine formation increases at higher wash temperature. At least two pools of organochlorine are produced in bleached fabric: a labile fraction that diminishes over several months of storage time as well as a more stable fraction that persists after more than 1 year. Our results also suggest that residual hypochlorite remains in fabric after laundering with bleach, presenting the possibility of direct and sustained dermal contact with reactive chlorine. This study provides a first step toward identifying a new risk factor for elevated organochlorine body burdens in humans.

Rapid and selective determination of free chlorine in aqueous solution using electrophilic addition to styrene by gas chromatography/mass spectrometry.

We developed a rapid and selective method for determination of free chlorine in aqueous solution by gas chromatography/mass spectrometry for the first time. Free chlorine was converted to styrene chlorohydrin using electrophilic addition to styrene in sodium acetate buffer solution (pH 5). The chlorine derivative obtained was extracted with chloroform, and then analyzed by GC/MS. The calibration curve showed good linearity from 0.2-100 µg/mL (as available chlorine). The detection limit was 0.1 µg/mL, and the intra- and interday accuracy were measured at concentrations of 10, 50, and 75 µg/mL to be -1.3 to 6.9% (intraday) and 3.8-8.0% (interday) as % Bias. The precision was between 1.4 and 4.5% as % RSD. These results indicate that this method is a superior technique for the identification of free chlorine. This method was successfully applied to quantification in commercial samples and in samples of a criminal case.

Response of white sucker (Catostomus commersoni) to pulp and paper mill effluent in the Androscoggin River, Maine, USA.

Adverse effects of pulp and paper mill effluent on fish populations have been well documented in many countries over the last two decades. Some of the initial studies were at mills with conventional chlorine bleaching and no secondary effluent treatment. Following installation of secondary treatment, changes in bleaching technology to elemental chlorine-free bleaching, and other process changes, adverse effects on fish were reduced or eliminated at some mills. Because no two mills are exactly alike, it is difficult to predict adverse impacts of any given mill on fish populations. In 1994, a study of female white sucker (Catostomus commersoni) in the Androscoggin River, Maine, USA, showed induction of mixed function oxidase, reductions in gonad size and plasma estradiol, and an increase in plasma testosterone in fish downstream of discharges from three large bleached kraft pulp and paper mills, and host community municipal sewage treatment plants (STP). After all three mills switched to elemental chlorine-free bleaching in the late 1990s, studies from 2001 to 2003 found that the pattern of reproductive impacts on white sucker populations measured in 1994 was not repeated. In addition, population estimates of white sucker from 2002 to 2003 using mark-recapture techniques found that densities and biomass were well within the range of those of a reference population, and of those reported in the literature for unimpacted populations. Detailed studies immediately above and below each mill/sewage treatment plant showed no evidence of reproductive effects. However, a clear pattern of eutrophication was noted, which increased cumulatively downstream below each mill/STP.

Tyrosinase immobilized magnetic nanobeads for the amperometric assay of enzyme inhibitors: application to the skin whitening agents.

The immobilization of tyrosinase onto glutaraldehyde activated streptavidine magnetic particles and subsequent retention onto a magnetized carbon paste electrode for the amperometric assay of tyrosinase inhibitors is described. Tyrosine was used as substrate as it is the first substrate in the melanogenesis process. The sensing mode is based on monitoring the decrease of the amperometric signal corresponding to the electrochemical reduction of dopaquinone enzymatically generated. This current decrease is due to the presence of inhibitors acting directly on the enzyme or inhibitors acting on the product of the enzymatic reaction, i.e. dopaquinone. The methodology is designed for the evaluation of the inhibitory potency of the most frequently used active substances in cosmetic marketed products against hyperpigmentation such as kojic acid, azelaic acid and benzoic acid. These compounds bind to the tyrosinase active center. Ascorbic acid is also investigated as it interrupts the synthesis pathway of melanin by reducing the melanin intermediate dopaquinone back to l-dopa. By comparing the obtained IC(50), under the same experimental conditions, the order of their inhibitory potency was: kojic acid (IC(50)=3.7 × 10(-6)M, K(i)=8.6 × 10(-7)M), ascorbic acid (IC(50)=1.2 × 10(-5)M), benzoic acid (IC(50)=7.2 × 10(-5)M, K(i)=2.0 × 10(-5)M) and azelaic acid (IC(50)=1.3 × 10(-4)M, K(i)=4.2 × 10(-5)M) in close agreement with literature spectrophotometric inhibition data using the soluble tyrosinase.