The enzymatic activity of activated sludge was investigated with special emphasis on the localization of the enzymes in the sludge floc matrix. Activated sludge from an advanced activated-sludge treatment plant, performing biological N and P removal, was used. An enzymatic fingerprint was established using a panel of six different enzymes. The fingerprint revealed peptidase as the most dominating specific enzyme tested. By monitoring sludge bulk enzymatic activity over a 3-month period using fluorescein diacetate as an enzyme substrate, considerable variations in activity were observed even over short periods (a few days). The variation in esterase activity was to some extent correlated to the presence of humic compounds in the sludge, but not to the sludge protein content. Comparison of full sludge enzyme activity to the activity of a batch-grown sludge culture indicated that enzymes accumulated in sludge flocs. A large proportion of the exoenzymes were immobilized in the sludge by adsorption in the extracellular polymeric substances (EPS) matrix. This was demonstrated by extraction of EPS from the activated sludge using cation exchange. Contemporary to the release of EPS a very large fraction of the exoenzymes was released into the water. This showed that the exoenzymes should be considered to be an integrated part of the EPS matrix rather than as direct indicators of the microbial activity or biomass.

Enzymatic activity in the activated-sludge floc matrix

More than 2,100 chemically defined organic chemicals are listed in the Research Institute of Fragrance Materials/Flavor and Extract Manufacturers' Association (RIFM/FEMA) Database that are used as ingredients of fragrances for consumer products. An approach was developed for prioritizing these fragrance materials for aquatic risk assessment by first estimating the predicted environmental concentration (PEC) of these fragrance materials in the aquatic environment based upon their physicochemical properties and annual volume of use. Subsequently, an effect level was predicted with a general quantitative structure-activity relationship (QSAR) for aquatic toxicity, and a predicted no-effect concentration (PNEC) was calculated from this effect level by using an assessment factor (AF) that accounts for uncertainty in the toxicity QSAR prediction. A conservative AF of 10(6) was applied to the endpoint predicted by the QSAR to provide an adequate margin of safety in the calculation of the PNEC. The PEC was compared to the PNEC to characterize the risk to freshwater aquatic organisms (e.g., Daphnia magna and Pimephales promelas). If the ratio of PEC to PNEC was below one, the material was considered to have negligible environmental risk and to be acceptable for the aquatic environment at current use levels. If this ratio exceeded one, the PNEC was refined by using more specific QSAR models (Ecological Structure-Activity Relationships [ECOSAR]). If the ratio continued to exceed one, the material became a candidate for further aquatic risk assessment procedures, which involve iterative steps to refine the PEC, the PNEC, or both by using measured ecotoxicological endpoints. Prioritization for this latter process can be based upon the magnitudes of the estimated PEC:PNEC ratios. When using the first tier of this approach, only 568 of 2,141 fragrance materials (26.5%) in the RIFM/FEMA Database had PEC:PNEC ratios greater than one. This percentage decreased to only 164 materials (7.7%) when PNECs were derived with ECOSAR. Comparison of predicted PECs and PNECs with those based upon measured data confirmed the conservatism and low risk for type I errors associated with the framework. These combined exercises demonstrated the ability of this highly precautionary risk-based screening approach to quickly prioritize a large number of materials without benefit of experimental ecotoxicological or fate data.

A framework for prioritizing fragrance materials for aquatic risk assessment.

Sewage treatment plant (STP) effluents with primarily domestic inputs are strongly suspected to be an important source of natural and synthetic estrogens contaminating the aquatic environment. Even a few ng/L of some of these substances can provoke reproductive disturbances in riverine fish. The main purpose of this investigation has been that of ascertaining whether activated sludge STPs (ASSTPs) are able to produce significant amounts of free estrogens. For this purpose, we have monitored monthly estriol (E3), estradiol (E2), estrone (E1) and ethinylestradiol (EE2) in influents and effluents of six Roman ASSTPs for five months. To do this, we have developed an original analytical method involving analyte extraction with a Carbograph 4 cartridge and LC coupled with negative turbo ion spray tandem mass spectrometry in the selected reaction monitoring mode. Analyte recovery ranged between 86 and 91%, and limits of quantification were below 1 ng/L. Over five months, inlet concentrations of E3, E2, E1 and EE...

Monitoring Natural and Synthetic Estrogens at Activated Sludge Sewage Treatment Plants and in a Receiving River Water

http://www.beamreach.org/data/101/Science/processing/Nora/Papers/Fate,%20behavior%20and%20effects%20of%20surfactants%20and%20their%20degradation%20products%20in%20the%20environment.pdf

Fate, behavior and effects of surfactants and their degradation products in the environment

The recent identification of one class of fluorinated surfactants in groundwater impacted by fire-fighting activity has created an awareness of the potential environmental issues resulting from the...

Perfluorinated Surfactants and the Environmental Implications of Their Use in Fire-Fighting Foams

An analytical method was developed and used to measure trace levels of fragrance materials (FMs) in municipal wastewater and treated wastewater. Sixteen FMs were selected as analytes because of their wide range of physical chemical properties. The analytical method included the use of nine perdeuterated FMs as internal standards, a high-flow C18 speed disk for the extraction of FMs from aqueous matrices, and an accelerated solvent extraction system for the extraction of FMs from solid matrices. For aqueous matrices, average FM recoveries (relative to the perdeuterated FM internal standard) were 97−115%, with limits of quantitation ranging from 0.5 to 35 ng/L. For activated sludge solids and primary influent solids, the average FM recovery from the extraction procedure was 81%. Concentrations and removal of FMs at an activated sludge and a trickling filter wastewater treatment plant were determined in the U.S. FM influent concentrations ranged from 0.3 to 154 μg/L, while FM effluent concentrations ranged f...

Trace Analysis of Fragrance Materials in Wastewater and Treated Wastewater

An environmental monitoring study was conducted to assess the removal of alkyl ethoxylate sulfates (AESs), alcohol ethoxylates (AEs), and linear alkylbenzene sulfonates (LASs) during wastewater treatment. Samples were obtained from six trickling filter and four activated sludge treatment plants located in the midwestern United States. The average removal rates for AESs, AEs, and LASs in activated sludge treatment were 98, 97, and >99%, respectively. Lower and more variable removal rates were observed at trickling filter treatment plants with average values of 83, 90, and 82% for AESs, AEs, and LASs, respectively. Using average influent concentrations and removal rates, the predicted exposure concentrations for these surfactants in U.S. receiving waters immediately below treatment plant outfalls were less than their corresponding biological predicted no-effect concentrations in greater than 98% of the locations under low-flow conditions.

Removal of alcohol ethoxylates, alkyl ethoxylate sulfates, and linear alkylbenzene sulfonates in wastewater treatment

Alkyl ethoxylate alcohols (AE) are used in a wide variety of household cleaning products. In order to monitor environmental levels of AE and to determine AE removal during wastewater treatment, an analytical procedure that provides total AE concentration resolved by alkyl chain length for various environmental matrices (influent, effluent, and river water) was developed. The method utilizes a reverse-phase column to extract and concentrate AE from surface waters and wastewaters and utilizes strong anionic and cationic exchange columns to remove potential interferences. AE were reacted with hydrogen bromide to form corresponding alkyl bromide derivatives that were analyzed by capillary gas chromatography with mass selective detection. Recovery of AE from influent, treatment plant effluent, and river water was quantitative (65-102%) over a range of concentrations for all matrices. AE removal was 99% at two activated sludge treatment plants and 92% at two trickling filter plants. Total AE in low dilution (effluent to river water) surface waters downstream from wastewater treatment plants were less than 0.037 mg/L.

Measurement of alkyl ethoxylate surfactants in natural waters.

The biodegradation activity of activated sludge was characterized by the API-ZYM and LRA-ZYM Esterase test systems. These systems provide rapid, semiquantitative information on the activity of hydrolytic enzymes associated with the biodegradation of lipids, proteins, carbohydrates, nucleic acids, and xenobiotic chemicals. Activated sludge was collected from several municipal wastewater treatment plants (WTPs) which varied with respect to their operating parameters and influent wastewater composition. Sludge samples were separated into various fractions (mixed liquor, sonicated mixed liquor, freeze-thaw, and extracellular fractions) and then assayed for enzyme activity relative to inactivated controls

Characterization of enzyme activity in activated sludge using rapid analyses for specific hydrolases

An analytical technique was developed to monitor alkyl sulfate (AS) surfactants in natural water samples and determine AS removal during wastewaster treatment. The method utilizes a reverse-phase extraction column followed by strong anionic and cationic exchange column cleanup steps. AS are then derivatized with N,O-bis(trimethylsilyl)trifluoretimide with 1% trichloromethylsilane and analyzed by GC/FID as trimethylsilyl ethers.

William M. Begley

Papers

Trace Analysis of Fragrance Materials in Wastewater and Treated Wastewater

Removal of alcohol ethoxylates, alkyl ethoxylate sulfates, and linear alkylbenzene sulfonates in wastewater treatment

Measurement of alkyl ethoxylate surfactants in natural waters.

Characterization of enzyme activity in activated sludge using rapid analyses for specific hydrolases

Determination of alkyl sulfate surfactants in natural waters