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33 New Methods for the Isolation of Mutagenic Components of Organic Residuals in Sludges M. Wilson Tabor and John C. Loper Downloaded by FUDAN UNIV on January 24, 2017 | http://pubs.acs.org Publication Date: December 15, 1986 | doi: 10.1021/ba-1987-0214.ch033

1

1,2

Department of Environmental Health and Department of Microbiology and Molecular Genetics , University of Cincinnati Medical Center, Cincinnati, OH 45267 1

2

A general procedure has been developed for the isolation of residue organics from sewage treatment plant sludges for mutagenic assessment. This procedure features milling the sludge with anhydrous sodium sulfate to a homogeneous powder, sequential extractions of the powder with a solvent series from nonpolar to polar, and bioassay of the extracted organics via the Salmonella microsomal mutagenicity assay. This new method was compared to published U.S. Environmental Protection Agency procedures and solvent extraction procedures for isolating organics from sludges. Results of this study suggested that both published procedures either destroyed labile mutagens in sludges or caused the formation of mutagenic artifacts during the isolations. The new procedure features gentle isolation conditions and produces a homogeneous sample that is easily manipulated for further studies to isolate mutagens for chemical and biological characterization.

SLUDGES, RESULTING FROM THE TREATMENT of municipal sewage, are complex mixtures of organic and inorganic chemicals, some of which may have biological origins but many of which come from anthropogenic or industrial sources. Many of the residue organics isolated from these sludges have been characterized as toxic and/or mutagenic in bacterial, animal, and plant tester systems (1-5). Although some of the chemical constituents of sludges have been identified and partially quantified (2-4, 6-9), not much is known about the chemical 0065-2393/87/0214/0675$06.00/0 © 1987 American Chemical Society

Suffet and Malaiyandi; Organic Pollutants in Water Advances in Chemistry; American Chemical Society: Washington, DC, 1986.

Downloaded by FUDAN UNIV on January 24, 2017 | http://pubs.acs.org Publication Date: December 15, 1986 | doi: 10.1021/ba-1987-0214.ch033

676

O R G A N I C P O L L U T A N T S IN W A T E R

identity and source of the vast majority of the mutagenic compounds (5). Such information is needed to assess the importance of these compounds as potential human health hazards and to develop methods to limit their concentrations i n sludges and their release to the environment. Knowledge of mutagenic constituents i n sludges is important for human health because of the scale of r a w sludge production: 7 million dry tons annually in the United States (JO, J J ) . Progress toward the identification of the mutagens has been slow partly because of the lack of reliable isolation methods for these compounds. T h e objectives of this study were to isolate residue organic mutagens f r o m municipal sewage treatment plant sludges for chemical and biological characterization and to determine their origin. This chapter describes our first step i n that effort: the investigation of methods to extract organic mutagens from sludges.

Experimental Chemicals. American Society for Testing Materials Type I water and Type IV water (12) were generated in our laboratory by using a Continental-Millipore Water Conditioning System, as described previously (13). Organic solvents used for extraction were nanograde acetone, benzene, hexane, methanol, methylene chloride, and isopropyl alcohol obtained from Mallinckrodt and high-performance liquid chromatographic (HPLC) grade 1,1,2-trichloro1,2,2-trifluoroethane (Freon 113) obtained from Fisher Scientific. Sodium sulfate (reagent grade, low in nitrogen and suitable for Kjeldahl analyses, Fisher Scientific) was muffled at 500 °C for 6 h prior to use. All other chemicals were reagent grade or better and were used as supplied. Samples. Sludge samples were obtained from a municipal sewage treatment plant of the Metropolitan Sewer District in Cincinnati, Ohio. This facility, the U.S. Environmental Protection Agency/Municipal Environmental Research Laboratory—Cincinnati ( USEPA/MERL-CIN) Technical and Evaluation Facility at the Mill Creek Sewage Treatment Plant, receives an influent sewage composed primarily (>703>) of industrial discharges. A general outline of the treatment process stream for this facility is shown in Figure 1. Not all effluent discharges from this facility are disinfected via chlorination, as indicated in Figure 1. Primary sludge and secondary sludge samples were taken for the purposes of this study. Primary sludge is the solids and particulate matter settled from treatment plant influent waste water by clarification. The aqueous portion from this treatment process is called primary effluent waste water. Secondary sludge is the solids and particulate matter settled after aerobic activated sludge treatment of the primary effluent waste water. Samples were collected in 4-kg amber glass jars fitted with Teflon-lined caps and were frozen at —20 °C until processing. At the time of workup, the samples were thawed and allowed to settle overnight at 4 °C, and the supernatant fluids, about 75% of the volume of the parent samples, were decanted. For each sample, the remaining slurry was centrifuged for 20 min at 8000 X g at 6 ° C This supernatant fluid was decanted and combined with the previous supernatant fluid and then stored for processing as an aqueous sample (14, 15). The


33.

TABOR A N D LOPER

677

Organic Residuals in Sludges

screen Influent Wastewater*

Sewage-

Primary Sludge*


Primary Effluent

Activated Sludge Process (Secondary Sludge*)

recycle stream

dewatering

i— Secondary • Sludge Return*

Secondary

Effluent*

chlorination

Chlorinated Secondary Effluent*

Discharge

Figure 1. Schematic of municipal sewage treatment of the EPA/MERL-CIN Technical and Evaluation Facility at the MM Creek Sewage Treatment Plant, Cincinnati, Ohio.

remaining moist pellet was processed as outlined in Figure 2 for the preparation of residue organics for bioassay and further separations. A weighed aliquot of each sludge pellet was oven dried at 125 °C for 24 h at a negative pressure of 30 mmHg and then reweighed for the calculation of dry weight. Biological Analysis. Tester strains TA90 and TA100 for the Salmonella microsomal mutagenicity tests were provided by B. Ames. Mutagenesis assays were conducted as described previously (14-16). Characteristic properties of the bacterial tester strains were verified for each fresh stock, and their mutagenicity properties were verified again by using positive and negative controls as part of each experiment as recommended (16, 17). Tests requiring metabolic activation used polychlorinated biphenyl mixture (Aroclor 1254) induced rat liver 9000 X g supernatant fraction, S9, from Litton Bionetics. Duplicate bioassays were made for each dose. The bioassay of each extract was repeated within 1 week of the original bioassay. Additionally, each extraction


Suffet and Malaiyandi; Organic Pollutants in Water Advances in Chemistry; American Chemical Society: Washington, DC, 1986. WEIGHT

PROCEDURE

PROCEDURE

Τ

HlTES

SOLIDS

EPA

«R~M0IST

CENTRIFUGE

DECANT

OVERNIGHT

FLUID

PROCEDURE

CONCENTRATION

MILLING

1

XAD

SUPERNATANT

Figure 2. Schematic of the processing of sludge samples to produce uniform samples of solids.

ANALYSIS

A L I Q U O T FOR

SLURRY

SETTLE

1) 2)

SLUDGE


33.

TABOR A N D LOPER

Organic Residuals in Sludges

679


experiment was repeated. The mutagenesis results are averages of these four bioassays, each in duplicate, for each sample extract. The detection of mutagenic activity in experimental samples was based upon a dose-dependent response exceeding the zero-dose spontaneous control value by at least twofold; that is, the ratio of total revertant colonies per plate to spontaneous colonies per plate was >2. All recoveries of bioactivity from concentrated residue organic samples were based upon an expression of mutagenesis in terms of net revertant colonies per gram equivalent dry weight of sludge, representative of the original sludge sample. Typical mean revertant colony counts ± standard error, obtained from each group of spontaneous plates and positive control plates from our laboratory for the time period of the experiments described herein, were recently reported (14). Preparation of Residue Organics from Sludges. Three isolation methods were investigated to prepare residue organic samples from the sludges for bioassays and fractionations. These methods are outlined in Figures 3-5. SOXHLET EXTRACTION. The procedure was based upon that described by Hites and co-workers (18, 19). A 50-g aliquot of a moist pellet of a sludge sample was added to a borosilicate glass thimble, which was then placed into a standard Soxhlet extraction apparatus. The sample was extracted for 7 h with 300 mL of isopropyl alcohol, followed by a 7-h extraction with 300 mL of benzene. These extracts were dried separately via passage through a 1- X 25-cm column of anhydrous sodium sulfate. Then each drying column was washed with 100 mL of the respective solvent followed by 50 mL of a hexane:acetone (85:15 by volume) solvent system. These eluates were combined with the respective extracts, and the two sample extracts were concentrated 100-fold via rotary evaporation at 50 ° C while a pressure of 30 mmHg was maintained. The samples were concentrated to 10 mL via evaporation at 50 °C under a stream of dry nitrogen. Immediately prior to bioassay, an aliquot of the concentrate was returned to a nitrogen stream and evaporated to dryness; the residue was dissolved in dimethyl sulfoxide. M E T H Y L E N E CHLORIDE EXTRACTION.

This procedure was a modified

version of the U SEP A/Environmental Monitoring Support Laboratory—Cin cinnati (USEPA/EMSL-CIN) Method 624S/625S (20). By using a Sorvall Omnimixer (Du Pont) at the high-speed setting, a 50-g aliquot of a moist pellet of a sludge sample was homogenized for 1 min in 300 mL of methylene chloride. During all homogenization operations, the sample container was immersed in an ice bath to avoid overheating. The ρ H of the homogenate was adjusted to >11 by the addition of 1.0 Ν sodium hydroxide, the homogenization was repeated, and the phases were separated by centrifugation at 1500 X g for 10 min. The organic layer was removed, and the residual basic aqueous layer was extracted two additional times with methylene chloride. The combined methylene chlo ride base-neutral extracts were dried via passage through a column of anhydrous sodium sulfate and then concentrated by using the procedures described in the previous section except that temperatures for evaporation were maintained at 30 °C rather than 50 °C. The second series of methylene chloride extractions was accomplished by the addition of 300 mL of solvent to the aqueous layer, homogenization for 1 min, then pH adjustment to

New Methods for the Isolation of Mutagenic ... - ACS Publications

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