Thomas W. Federle

Biodegradation of [S,S], [R,R] and mixed stereoisomers of Ethylene Diamine Disuccinic Acid (EDDS), a transition metal chelator

An in-depth biodegradation test program was executed on the hexadentate ligand Ethylene Diamine Di Succinate (EDDS). The EDDS structure contains two chiral carbon atoms, and has three stereoisomers ([R,R], [R,S]/[S,R], [S,S]). Our research has focused on the isomer mixture (i.e. 25%[S,S]; 25%[R,R]; 50%[S,R]/[R,S], as produced from the reaction of ethylene diamine with maleic anhydride) and on the single [S,S]- and [R,R]-isomers. Biodegradation screening of the 14C-labelled EDDS isomer mixture in a Batch Activated Sludge (BAS) test with various inocula revealed incomplete mineralization, up to ca. 65% after 28 days. N-(2-aminoethyl) aspartic acid (AEAA), probably the d-isomer, was identified as the major portion of the 14C-material remaining in solution. Further testing revealed that the [S,S]-isomer is rapidly and completely mineralized in all test systems. By contrast, [R,R]-EDDS remained undegraded in a Sturm (OECD 301B) test, but was very slowly biotransformed into the recalcitrant metabolite AEAA in a BAS test. The [S,R]/[R,S] form undergoes biotransformation to AEAA in both high and low biomass systems. In a sewage treatment simulation test (OECD 303) the steady state DOC removal of mixture-EDDS in a CAS test was limited to 25-35%, even after extensive pre-acclimation, while the [S,S]-isomer achieved nearly complete removal (96%). This study illustrates the importance stereospecificity may have on the biodegradation and metabolite formation of a chemical. A biodegradation scheme for the different EDDS stereoisomers is proposed.

A framework for using structural, reactivity, metabolic and physicochemical similarity to evaluate the suitability of analogs for SAR-based toxicological assessments

A systematic expert-driven process is presented for evaluating analogs for read across in SAR (structure activity relationship) toxicological assessments. The approach involves categorizing potential analogs based upon their degree of structural, reactivity, metabolic and physicochemical similarity to the chemical with missing toxicological data (target chemical). It extends beyond structural similarity, and includes differentiation based upon chemical reactivity and addresses the potential that an analog and target could show toxicologically significant metabolic convergence or divergence. In addition, it identifies differences in physicochemical properties, which could affect bioavailability and consequently biological responses observed in vitro or in vivo. The approach provides a stepwise decision tree for categorizing the suitability of analogs, which qualitatively characterizes the strength of the evidence supporting the hypothesis of similarity and level of uncertainty associated with their use for read across. The result is a comprehensive framework to apply chemical, biochemical and toxicological principles in a systematic manner to identify and evaluate factors that can introduce uncertainty into SAR assessments, while maximizing the appropriate use of all available data.

Environmental fate and effects of DEEDMAC : a new rapidly biodegradable cationic surfactant for use in fabric softeners

This paper introduces the environmental safety database for a new fabric softening cationic surfactant, the di-(tallow fatty acid) ester of di-2-hydroxyethyl dimethyl ammonium chloride, DEEDMAC >Diethyl Ester Dimethyl Ammonium Chloride). The physiochemical properties of DEEDMAC are similar to those for ditallow dimethyl ammonium chloride (DTDMAC), the major cationic surfactant used in fabric softener formulations world-wide for over thirty years. Importantly, however, DEEDMAC differs structurally from DTDMAC by the inclusion of two weak ester linkages between the ethyl and tallow chains. These ester linkages allow DEEDMAC to be rapidly and completely biodegraded in standard laboratory screening tests and a range of environmental compartments. including raw sewage, activated sludge, anaerobic digestor sludge, sludge amended soil, and river waters. Removal of DEEDMAC during sewage treatment is greater than 99%, as determined by computer model predictions and confirmed by laboratory simulation testing (OECD Continuous Activated Sludge confirmatory test). Using estimated tonnages, per capita waste water flows, incidences of sewage treatment for individual countries, measured removal rates, and validated computer models, maximum river water and soil concentrations of DEEDMAC have been estimated for representative usage scenarios. Based upon these maximum predicted environmental concentrations, acute and chronic toxicity testing offish, invertebrates and algae, predicted aquatic safety factors range from 272 to > 1000. Predicted steady state terrestrial safety factors are > 1000, based on EC50 values to earthworms and plants >50 mg/kg. The environmental safety database developed for DEEDMAC indicates that this cationic surfactant is rapidly and completely biodegraded, will be highly removed during sewage treatment. has an ecotoxicity profile similar to broadly used anionic and nonionic surfactants, and is environmentally safe at intended maximum usage volumes.

Mineralization of surfactants in anaerobic sediments of a laundromat wastewater pond

Abstract The mineralization of 14C-labeled linear alkylbenzene sulfonate (LAS), stearyltrimethylammonium chloride (STAC) and linear alcohol ethoxylate (LAE) was examined in the anaerobic sediments of a laundromat wastewater pond and a pristine control pond. The microbial communities in sediments of the wastewater pond had been exposed to high levels of surfactants for over 25 years. Mineralization was determined by measuring the evolution of 14CO2 and 14CH4 from the 14C-test substances. [14C]Glucose and [14C]benzoic acid served as positive controls. LAS and STAC were not mineralized in sediments from either pond: LAE, glucose and benzoic acid were mineralized in both ponds. LAE mineralization was most rapid in sediment from the margin of the wastewater pond, while glucose and benzoic acid mineralization was most rapid in the control pond. With sediment from the center of the wastewater pond, mineralization of LAE, glucose and benzoic acid was preceded by a substantial lag. An experiment with specific metabolic inhibitors suggested that both methanogenic and sulfate reducing bacteria may play a role in LAE mineralization. In summary, this work demonstrates that LAE degrades anaerobically in sediments and indicates that extended acclimation does not relieve the need for molecular oxygen in LAS and STAC mineralization.

Biotransformation of linear alkylbenzene sulfonate (LAS) by Phanerochaete chrysosporium: oxidation of alkyl side-chain.

The white rot fungus Phanerochaete chrysosporium, which generally mineralizes substituted aromatics to CO2, transformed linear alkylbenzene sulfonate (LAS) surfactants mainly at their alkyl side chain. Degradation of LAS was evidenced by a zone of clearing on LAS-containing agar plates and colorimetric analysis of liquid cultures. Disappearance of LAS was virtually complete within 10 days in low nitrogen (2.4 mM N), high nitrogen (24 mM N) and malt extract (ME) liquid media. After 5 days of incubation in ME medium, transformation of LAS was complete at concentrations≤4 mg l-1, but decreased at higher concentrations. The LAS degradation was not dependent on lignin peroxidases (LiPs) and manganese-dependent peroxidases (MnPs). Mineralization of14C-ring-LAS to 14CO2 by P. chrysosporium was <1% regardless of the culture conditions used. Thin layer chromatography and mass spectral analyses indicated that P. chrysosporium transformed LAS to sulfophenyl carboxylates (SPCs) through oxidative shortening of the alkyl side-chains. While LAS disappearance in the cultures was not dependent on LiPs and MnPs, transformation of the parent LAS moieties to SPCs was more extensive in low N medium that favors expression of these enzymes. The SPCs produced in LN cultures were shorter in chain-length than those produced in ME cultures. Also there was a notable shift in the relative abundance of odd and even chain length metabolites compared to the starting LAS particularly in the low N cultures suggesting the possible involvement of processes other than or in addition toβ-oxidation in the chain-shortening process.

Spatial Distribution of Microbial Biomass, Activity, Community Structure, and the Biodegradation of Linear Alkylbenzene Sulfonate (LAS) and Linear Alcohol Ethoxylate (LAE) in the Subsurface

The vertical distribution of microbial biomass, activity, community structure and the mineralization of xenobiotic chemicals was examined in two soil profiles in northern Wisconsin. One profile was impacted by infiltrating wastewater from a laundromat, while the other served as a control. An unconfined aquifer was present 14 meters below the surface at both sites. Biomass and community structure were determined by acridine orange direct counts and measuring concentrations of phospholipid-derived fatty acids (PLFA). Microbial activity was estimated by measuring fluorescein diacetate (FDA) hydrolysis, thymidine incorporation into DNA, and mixed amino acid (MAA) mineralization. Mineralization kinetics of linear alkylbenzene sulfonate (LAS) and linear alcohol ethoxylate (LAE) were determined at each depth. Except for MAA mineralization rates, measures of microbial biomass and activity exhibited similar patterns with depth. PLFA concentration and rates of FDA hydrolysis and thymidine incorporation decreased 10–100 fold below 3 m and then exhibited little variation with depth. Fungal fatty acid markers were found at all depths and represented from 1 to 15% of the total PLFAs. The relative proportion of tuberculostearic acid (TBS), an actinomycete marker, declined with depth and was not detected in the saturated zone. The profile impacted by wastewater exhibited higher levels of PLFA but a lower proportion of TBS than the control profile. This profile also exhibited faster rates of FDA hydrolysis and amino acid mineralization at most depths. LAS was mineralized in the upper 2 m of the vadose zone and in the saturated zone of both profiles. Little or no LAS biodegradation occurred at depths between 2 and 14 m. LAE was mineralized at all depths in both profiles, and the mineralization rate exhibited a similar pattern with depth as biomass and activity measurements. In general, biomass and biodegradative activities were much lower in groundwater than in soil samples obtained from the same depth.

Characterization and distribution of esterase activity in activated sludge

The location and activity of esterase enzymes in activated sludge from three municipal wastewater treatment plants were characterized using model substrates and denaturing and non-denaturing polyacrylamide gel electrophoresis (PAGE) of particulate, freeze-thaw (primarily periplasmic enzymes and those associated with outer cell surfaces) and extracellular fractions of activated sludge bacteria. Particulate and freeze-thaw fractions had a similar spectrum of substrate specificity and contained significant levels of protein and esterase activity against model substrates, C2-C18 monoesters of p-nitrophenol and C2-C8 diesters of fluorescein. Esterase activity was highest with substrates that had short alkyl chains (C4) and decreased as the chain lengths increased beyond C8. Extracellular fractions contained very low levels of protein (<0.1 mg/l) and showed no esterase activity against any of the model substrates tested. Multiple bands were observed upon analysis of particulate and freeze-thaw fractions by non-denaturing PAGE in combination with activity staining using various alpha-naphthol ester substrates (C2-C8). Our results indicate that esterase enzymes in activated sludge are fairly diverse from a structural standpoint but exhibit a high level of functional redundancy, with different enzymes catalyzing the same reactions in different sludges. Extracellular esterase activity was totally absent for the substrates we tested and the esterase activity that we observed was closely linked to a particulate floc or cellular material.

Effect of ethoxylate number and alkyl chain length on the pathway and kinetics of linear alcohol ethoxylate biodegradation in activated sludge

Batch activated-sludge die-away studies were conducted with various pure homologs to determine the effect of ethoxylate number and alkyl chain length on the kinetics of primary and ultimate biodegradation of linear alcohol ethoxylates. The 14C-(ethoxylate) homologs C14E1, C14E3, C14E6, and C14E9 were used to investigate the effect of ethoxylate number, and 14C-(ethoxylate) homologs C12E6, C14E6, and C16E6 were used to examine the effect of chain length. Activated sludge was dosed with a trace concentration (0.2 microM) of each homolog, and the disappearance of parent, formation of metabolites, production of 14CO2, and uptake into solids were monitored with time. Ethoxylate number had little effect on the first-order decay rates for primary biodegradation, which ranged from 61 to 78 h(-1). However, alkyl chain length had a larger effect, with the C16 chain-length homolog exhibiting a slower rate of parent decay (18 h(-1)) compared to its corresponding C12 and C14 homologs (61-69 h(-1)). Ethoxylate number affected the mechanism of biodegradation, with fission of the central ether bond to yield the corresponding fatty alcohol and (poly)ethylene glycol group increasing in dominance with increasing ethoxylate number. Based upon the measured rates of primary biodegradation, removal of parent during activated-sludge treatment was predicted to range between 99.7 and 99.8% for all homologs except C16E6, which had a predicted removal of 98.9%. Based upon the measured rates of ultimate biodegradation, removal of ethoxylate-containing metabolites was predicted to exceed 83% for all homologs. These predictions corresponded closely with previously published removal measurements in laboratory continuous activated-sludge systems and actual treatment plants.

Probabilistic analysis of risks to us drinking water intakes from 1,4‐dioxane in domestic wastewater treatment plant effluents

The risks of 1,4-dioxane (dioxane) concentrations in wastewater treatment plant (WWTP) effluents, receiving primarily domestic wastewater, to downstream drinking water intakes was estimated using distributions of measured dioxane concentrations in effluents from 40 WWTPs and surface water dilution factors of 1323 drinking water intakes across the United States. Effluent samples were spiked with a d8 -1,4-dioxane internal standard in the field immediately after sample collection. Dioxane was extracted with ENVI-CARB-Plus solid phase columns and analyzed by GC/MS/MS, with a limit of quantification of 0.30 μg/L. Measured dioxane concentrations in domestic wastewater effluents ranged from <0.30 to 3.30 μg/L, with a mean concentration of 1.11 ± 0.60 μg/L. Dilution of upstream inputs of effluent were estimated for US drinking water intakes using the iSTREEM model at mean flow conditions, assuming no in-stream loss of dioxane. Dilution factors ranged from 2.6 to 48 113, with a mean of 875. The distributions of dilution factors and dioxane concentration in effluent were then combined using Monte Carlo analysis to estimate dioxane concentrations at drinking water intakes. This analysis showed the probability was negligible (p = 0.0031) that dioxane inputs from upstream WWTPs could result in intake concentrations exceeding the USEPA drinking water advisory concentration of 0.35 μg/L, before any treatment of the water for drinking use.

Inability of the human fecal microflora to metabolize the nonabsorbable fat substitute, olestra

SummaryOlestra is a non-caloric fat substitute under review by the Food and Drug Administration. It consists of a mixture of octa-, hepta- and hexaesters of sucrose formed with long chain fatty acids. Previous studies showed olestra is not hydrolyzed by mammalian lipases and is not absorbed. The objective of this study was to evaluate the potential for colonic microflora to metabolize olestra after continued exposure. Neat and emulsified14C-[fatty acid] olestra was incubated for 72 h in both minimal and organically-enriched anaerobic media inoculated with feces from seven subjects who had consumed olestra (9 g per day) over a 3–4 week period.14C-sucrose and14C-glucose served as positive controls. Production of14CO2,14CH4,14C-volatile fatty acids (VFAs) and14C-long chain fatty acids (LCFAs) was determined. In addition, the ester distribution and fatty acid composition of olestra were examined before and after incubation. Significant quantities of14CO2 and14C-VFAs were generated from the14C-sugars, indicating that the microflora were active under the incubation conditions. Furthermore, free oleic acid was extensively hydroxylated and hydrogenated. In contrast, no degradation products (gas, VFAs, LCFAs) or changes in the olestra resulting from bacterial activity were detected. These results indicate that under anaerobic conditions the colonic microflora of the humans, consuming olestra, did not metabolize olestra.