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AR226-3356 Effects of Fluorotelomer Products on the Bacterial Growth, Degradation of Organic Substances, and Microbial Population from Activated Sludge of a Domestic Sewage Treatment Plant Wana N.1. Folsom P.W. \ Decarolis, J. \ Fallon, R.1, Berti W.R.1, Buck R.C.2 and Gannon J.T.1 1 DuPont Central Research and Development, Newark, DE, ' USA: 2 DuPont Chemical Solutions Enterprise, Wilmington, DE, USA BACKGROUND INFORMATION The backbones of fluorotelomers are made of fluorinated monomers with a -(CF2)- chain length of approx. 4-12. The unique structure of fluorotelomers give their surface protection properties (oil, water, and dirt repellent). The fluorotelomer products also contain some additives plus ~80% water to increase their dispersity to finish products. 1 EXPERIMENTAL METHODS (Continued) If. D egradation o f u C.-aniline h v activated sludge 250-mL volume Pyrex glass bottles were used. Each loose-capped bottle contained 50 mL o f fresh activated sludge from a POTW (Elkton, MD, USA) in mineral medium plus a test chemical (10 mg glucose Equiv. L'1) a n d - 1.2 mg I / 1o f l4C-labeled aniline (-4 .5 pCi 14C per bottle). The degradation (mineralization) of the ,4C-aniline by the activated sludge was monitored periodically by measuring 14C counts remained in the sample solution; this was done by transferring 0.1 mL sample solution to a scintillation vial containing 0.2 mL o f 1 N HC1 to remove 14C 0 2and subsequently 5 mL of a liquid scintillation cocktail was added to the vials for UC counting by a liquid scintillation counter (Beckman LS 5000 TD). The difference in 14C counts between day 0 and a particular sampling time point approximates the level o f ,JC-amline that have been mineralized. #4 STUDY OBJECTIVES To assess whether fluorotelomer products have any adverse effect on microbial growth. To assess the ability of microbes to degrade pollutants at the presence of these products. To assess whether these products have any impact on the microbial communities over time. 2 EXPERIMENTAL METHODS (Continued) ..... ....... ........ I I I Analysis o f M icrobial C om m unities in activated sludge The experiment set up is very similar to the Experiment II, except that no ,4C-aniline was used. 250 mL volume Pyrex glass bottles were used. Each loose-capped bottle contained 50 mL o f fresh activated sludge from a POTW (Elkton, MD, USA) in mineral medium plus a test chemical (10 mg glucose Equiv. L). The test medium was sampled periodically to extract DNA for DGGE (denatured gradient gel electrophoresis) analysis of possible microbial community changes. Universal bacterial PCR primers (Forward Primer 5' CGC CCG CCG CGC CCC GCC CCG CCG CCC CCG CCC GCC TAC GGG AGG CAG CAG 3' and Reverse Primer: 5' GTA TTA CCG CGG CTG CTG GC 3') containing 40 bp of GC clamp were used to amplify the variable V6 region of bacterial 16S ribosomal DNA. The amplified DNA fragment was used to perform DGGE and the agarose gel was stained with SYBR Gold to visualize the separated DNAs. 5 EXPERIMENTAL METHODS S tu d y M aterials: Fluorotelomer A ciylate Polym er, Fluorotelom er Urethane Polym er Fluorotelom er Ethoxylate, and Fluorotelom er Phosphate. I. M icro b ia l G row th 1. 24-well cell culture plates(the volume ofeachw eli = 3.75mL)were used. Each well contained 1 mL o f the growth medium (0.1% yeast extract in mineral medium) plus a test chemical (0 - lg TOC Equiv. L*1or 0 - 2.5 g glucose Equiv. L'1) and an inoculum from a mixed bacterial culture originated-from activated sludge of a POTW (Elkton, MD, USA). The growth was monitored automatically at 30 C every 30 min by measuring the absorbance at 600 nm with a Tecan Genres plate reader set at kinetics mode. (The optical pathiength o f the 1 mL medium =0.5 cm.) 2. 250 mL culture flasks were used. Each loose-capped flask contained 50 mL o f the growth medium plus a test chemical (1 g TOC Equiv. L-') and the inoculum. The growth (at room temperature) was monitored periodically by transferring 1 mL o f the medium to a 24-well culture plate and the absoibance at 600 nm was monitored with a Tecan Genios plate reader. (Thooptical pathiengthofthe I mLm e d iu m e ta ). ^ RESULTS AND DISCUSSION 1. At up to 2.5 g glucose Equiv. L'1concentration, an extremely high concentration unlikely to he found in any environmental compartment, no any adverse effect on bacterial growth was observed for any of the fluorotelomer products (Figures 1-5). 2. In general, the presence of the fluorotelomer products in the bacterial culture slightly enhanced the bacterial growth in comparison with the growth medium only (Figures 1-5), possibly due the additional carbon from the products serving as an energy source for the microorganisms. 6 The miracles o f science' ' ' `SSaoS**'" Effects of Fluorotelomer Products on the Bacterial Growth, Degradation of Organic Substances, and Microbial Population from Activated Sludge of a Domestic Sewage Treatment Plant (Continued) - 9 - FluorattlcmrUraUumPo(jnir - 9 - Fluofou lomifEftoKyaa RvorotlemrPlmifttt FluofotlomrAe>yttiPoiymr -S - GrowthiMdtfn Inoculumonfc 3. 75% of 14C-aniline has been mineralized in the presence of fluorotelomer products in the activated sludge over 3 weeks, compared with 64% in the sludge only control. H ^ S uOjoWH -#* SudJoanuereUrtmarFhoapfut aMsa6btn> -a-SudgonucRMenurAajWaiaasin suSgoFhaMefomwUraMiiaPctymw AUceeCenRf SK**Fljdnrtomerarrai)<o Figure 1. Effect of Fluorotelomer Acrylate Polymer on the bacterial growth. Figure 2. Effect of Fluorotelomer Urethaac Polymer on the bacterial growth. Figure 5. Effect of fluorotelomer products on the bacterial growth. 8 Figure 6. Degradation of MC-aniline by the activated sludge amended with fluorotelomer products. Figure 3. Effect of Fluorotelomer Ethoxylate on the bacterial growth. Figure 4. Effect of Fluorotelomer Phosphate on the bacterial growth 7 4. The presence offluorotelomer products in the activated sludge seemed to have no effect on the microbial communities. ' g r.-i" T i r 13- '1' 11 t^ . :x i n tt ? I t' j l - Sludge only. n: - IO mg glucose L l sludge ni: - 1 0 mg glucose Eqnlvof Floorolckimor U rethane Polymer L'1 sludge : IV - 1 0 mg glucose Equiv of Flaorotelomer Ethoxylate L'1 sludge ^ V-lOm gglucoseEqtdvofFluorotelom er i: Phosphate L 1 sludge V I - 1 0 mg glucose Equiv of Flaorotelomer : Acrylate Polymer I / 1 sludge Numeric numbers indicate the days alter the : initiation of the experiment. ': Each blue arrow represents a possible bacterial population. 10 CONCLUSIONS At up to 2.5 g glucose Equiv. L`l concentration, no any adverse effect on bacterial growth was observed for any of the fluorotelomer products. The presence o f fluorotelomer products in the activated sludge did not affect the sludge to degrade 14C-aniline over 3 weeks. The presence of fluorotelomer products in die activated sludge did not induce or dimmish any major bacterial population in die activated sludge, in comparison with sludge only control. Because the concentration of die fluorotelomer products in a domestic sewage treatment plant is likely much lower dial 10 mg glucose Equiv. L*`, it is apparent that none of these products would have any adverse physiological impact on the microbial communities to degrade organic substances. 11 The mmeles o f science'