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Jeq50282 695.700

Published online April 3, 2006
Determination of Steroidal Estrogens in Flushed Dairy Manure Wastewater by Gas Travis A. Hanselman,* Donald A. Graetz, Ann C. Wilkie, Nancy J. Szabo, and Carolyn S. Diaz portant to know the types and amounts of steroidal There is a critical need to accurately measure the concentrations estrogens that occur in dairy wastes so that endocrine of natural steroidal estrogens in flushed dairy manure wastewater disruption risks can be minimized or avoided.
(FDMW) to assess any potential risk of waterway contamination Freshly voided dairy manure is a mixture of undigest- resulting from land application. Estrogens are a concern because low ed dietary residues, gut microflora, and their metabolic concentrations (10–100 ng L21) in water can adversely affect aquatic end-products. As collected, the manure may include vertebrate species such as fish, turtles, and frogs by disrupting the spilled feed, bedding materials, and water, in addition to normal function of their endocrine systems. The objective of this study feces and urine. Typically, manure is either scraped or was to develop a sample preparation method that permits the quan- flushed from the barns (Wilkie, 2005). Many dairies use tification of four natural steroidal estrogens (17a-estradiol, 17b- hydraulic flushing for manure management, followed by estradiol, estrone, and estriol) in FDMW by gas chromatography– primary treatment (mechanical screening or sedimenta- mass spectrometry (GC–MS). Solid-phase extraction with graphitizedcarbon black was used for the bulk extraction of estrogens from tion, or both) to remove coarse solids. The liquid frac- FDMW and additional sample purification was accomplished with tion of flushed dairy manure after settleable solids are C-18. The sample preparation method allowed estrogens to be detected removed is referred to as flushed dairy manure waste- accurately by GC–MS in FDMW. Spiked recovery experiments in- water (FDMW) (Wilkie et al., 2004). Gauging the ste- dicated that the method is satisfactory for measuring the estrogens roidal estrogen profile of FDMW or other livestock of interest in FDMW with average recovery of .90%. As expected waste is not a trivial task, however, due to the low con- in FDMW, characterization of the estrogen profile revealed a large centrations that must be measured, the difficulties as- abundance of 17a-estradiol relative to 17b-estradiol and estrone.
sociated with extracting estrogens from the waste, the Estriol was not detected in FDMW. The methodology developed in this chemical complexity of the resulting extract matrix, and research helps provide an analytical foundation for the quantification the potential for degradation losses to occur during sam- of steroidal estrogens in FDMW by GC–MS.
Quantitative enzyme immunoassays have been used in LIVESTOCK MANURE contains appreciable amounts of previous studies for measuring estrogens in dairy waste natural steroidal estrogen hormones, such as estra- (Raman et al., 2001). However, previous work in our diol, estrone, and estriol, that can potentially con- laboratory showed that enzyme immunoassays were taminate surface and ground water (Shore et al., 1993; prone to matrix interference which resulted in inaccurate Finlay-Moore et al., 2000; Hanselman et al., 2003; quantification of estrogen in dairy waste (Hanselman Raman et al., 2004). Estrogen contamination of water et al., 2004). Gas chromatography–mass spectrometry resources is a concern because low part per trillion (GC–MS) or liquid chromatography–mass spectrometry concentrations (10–100 ng L21) of these chemicals may (LC–MS) based methods are more conclusive than immu- adversely affect the reproductive biology of aquatic noassays, but few of these techniques have been devel- vertebrates by disrupting the normal function of their oped for measuring estrogens in livestock waste (Raman endocrine systems (Jobling et al., 1998; Tyler et al., 1998; et al., 2001; Fine et al., 2003). Raman et al. (2001) Panter et al., 1998; Irwin et al., 2001).
developed a procedure for measuring 17a-estradiol, 17b- The ecological hazard, if any, posed by steroidal estradiol, and estrone in dairy waste by GC–MS. The estrogens resulting from dairy production is not clearly sample preparation involved liquid–liquid extraction of known. Nevertheless, based on the amount of estrogens estrogens from the waste sample using diethyl ether excreted in urine and feces, Lange et al. (2002) es- followed by BSTFA [N,O-bis(trimethylsilyl)fluoro- timated that pregnant and cycling cows (Bos taurus) are acetamide] derivatization in DMF (dimethylformamide) responsible for about 90% of the steroidal estrogen (Raman et al., 2001). However, the detection limits re- input to the environment by domestic livestock in the ported by Raman et al. (2001) are poor (10 mg L21) rela- United States and Europe. Therefore, it is critically im- tive to the endogenous concentrations of steroidalestrogens (ng L21) likely to be found in dilute dairywastes such as FDMW (Kolodziej et al., 2004). Regardless T.A. Hanselman, D.A. Graetz, and A.C. Wilkie, Soil and Water ScienceDepartment, P.O. Box 110510, University of Florida, Gainesville, FL of detection limit restrictions, liquid–liquid ether extrac- 32611-0510. N.J. Szabo and C.S. Diaz, Analytical Toxicology Core tion of FDMW gives a sample that is not of sufficient Laboratory, P.O. Box 110885, University of Florida, Gainesville, FL quality for quantitative derivatization and introduction 32611-0885. Received 22 July 2005. *Corresponding author (travish@ into the GC–MS (Hanselman et al., 2004). Thus, a more extensive extraction and purification technique is re- Published in J. Environ. Qual. 35:695–700 (2006).
quired for the analysis of wastes such as FDMW. The Short Communicationsdoi:10.2134/jeq2005.0282ª ASA, CSSA, SSSA Abbreviations: FDMW, flushed dairy manure wastewater; GC–MS, gas chromatography–mass spectrometry; SPE, solid-phase extraction.
J. ENVIRON. QUAL., VOL. 35, MAY–JUNE 2006 objective of this study was to develop a sensitive and were prepared and analyzed with their corresponding unspiked reliable sample preparation method that permits the quantification of four natural steroidal estrogens (17a- Extraction efficiency was calculated by dividing the mea- estradiol, 17b-estradiol, estrone, and estriol) in FDMW sured concentration of estrogens in the spiked sample by thetheoretically expected concentration in spiked samples and the result was multiplied by 100. To assess the extraction efficiencyat different levels, an additional set (n 5 4) of sample replicates were prepared from the FDMW 5 sample for each of fourspiking amounts (20, 40, 60, and 80 ng each of 17a-estradiol, 17b-estradiol, estrone, and estriol).
Estrone, 17a-estradiol, 17b-estradiol, estriol, and N,N- dimethylformamide (high performance liquid chromatography [HPLC]-grade), were purchased from Sigma-Aldrich (St. Louis,MO). Methanol (HPLC-grade), methylene chloride (HPLC- Estrogens were extracted from the replicate samples using grade), acetone (Optima-grade), water (HPLC-grade), ethyl Carbograph solid-phase extraction (Andreolini et al., 1987; acetate (HPLC-grade), acetonitrile (Optima-grade), hexane Baronti et al., 2000; Lagana et al., 2000, 2001; Gentili et al., (Optima-grade), and formic acid (ACS-grade) were purchased 2002). The samples were poured into fritted reservoirs and from Fisher Scientific (Hampton, NH). The derivatizing re- passed through Carbograph SPE columns sequentially pre- agent, BSTFA, was purchased from Supelco (Bellefonte, PA).
conditioned with 10 mL methylene chloride–methanol (80:20 Internal standard mixture US-108 containing d v/v), 5 mL methanol, and 10 mL water acidified to pH 2. The purchased from Ultra Scientific (Kingston, RI). Sample res- samples were passed through the columns with the aid of vac- ervoirs (75 mL), filtration frits (approximately 20 mm), 500 mg uum at 5 to 10 mL min21. Once the sample passed, the flasks Carbograph (graphitized carbon) solid-phase extraction (SPE) were rinsed with 50 mL of water and the rinse was applied to columns, 1000 mg C-18 (octadecylsiloxane-bonded silica) high- the columns. After rinsing, the reservoir was removed and the flow SPE columns, and nylon syringe filters (13 mm, 0.2 mm) Carbograph column was washed sequentially with 5 mL of 75% were purchased from Alltech Associates (Deerfield, IL).
methanol acidified with 100 mmol L21 formic acid and 5 mL Accubond II C-18 SPE cartridges (500 mg) were purchased of 75% methanol. The base/neutral fraction of retained or- from Agilent Technologies (Wilmington, DE).
ganics that included the target estrogens was eluted with 2 mLmethanol and 15 mL of methylene chloride–methanol (80:20 v/v) into 50-mL flasks. The captured eluant was heated at 708C under a gentle stream of N2 until the methylene chloride evap-orated. After cooling, 50 mL of HPLC-grade water was added A single 1-L grab sample of fresh FDMW was collected on to the residual methanol and mixed by swirling.
each of five consecutive days (19–23 Jan. 2004) from the Uni-versity of Florida Dairy Research Unit located at Hague, FL, transported on ice within 1 h to the laboratory in Gainesville, FL, and immediately extracted. The total solids content of the To improve sample purity, C-18 SPE was performed. The FDMW sample collected each day was determined by stan- aqueous-solvent sample mixtures resulting from Carbograph dard methods (American Public Health Association, 1998) to extraction were poured into reservoirs and passed with the aid be 0.79, 1.04, 0.66, 1.31, and 0.91%, respectively.
of vacuum at 5 to 10 mL min21 through C-18 columns pre-conditioned with 5 mL acetone and 5 mL water. After thesamples passed, the flasks were rinsed with 50 mL of water and Initial Preparation of Samples and Matrix Spikes the rinse was applied to the C-18 column. When the rinse From each of the five bulk daily samples (designated as passed through, vacuum was applied to the columns for an FDMW 1 through 5), eight replicate aliquots (40 mL each) additional approximately 15 min to remove excess water. A were measured into separate 50-mL Teflon tubes and cen- nylon syringe filter was attached to the bottom of each C-18 trifuged in an Eppendorf 5810 (Brinkmann Instruments, column and estrogens were eluted with 4 mL of acetone.
Westbury, NY) at 15 000 3 g for 15 min to pelletize sus- Sample volumes were adjusted to a final volume of 4.0 mL in pended solids. Each clarified supernatant was transferred into acetone, capped tightly, and stored at 2208C.
a 125-mL flask without disturbing the pellet and set aside.
In preparation for GC–MS analysis, an additional SPE step Estrogens adsorbed to pelletized solids were extracted with was required to reduce matrix background. The samples were 10 mL methanol in a 408C ultrasonic bath for 30 min. After taken to dryness under nitrogen. After resuspending each centrifugation at 4000 3 g for 15 min, the methanol extract was residue in 0.2 mL of methanol with vortexing, 4 mL of water combined with the aqueous portion of the sample and set was added to each vial. The samples were then applied to aside. The pellet was extracted once more with 10 mL of 500 mg C-18 cartridges (preconditioned sequentially with 4 mL methanol for 30 min in a 408C ultrasonic bath and, after cen- methanol and 4 mL water) and allowed to percolate through trifuging at 4000 3 g for 15 min, the methanol extract was the columns by gravity. The cartridges were then washed with added to the previous supernatant and mixed thoroughly.
4 mL of water, dried under vacuum for 3 min, washed with 4 mL At this point, four replicates of each sample were selected to of hexane, and eluted with two 4-mL portions of ethyl acetate.
continue processing as samples. To monitor the efficiency of The resulting extracts were taken to dryness under nitrogen the solid-phase extraction series when applied to the complex in preparation for derivatization. To each vial, 400 mL of DMF matrix left from bulk-extracted FDMW samples, the remain- and 100 mL of BSTFA were added, after which the solutions ing replicates were fortified with known amounts (40 ng each) were vortexed, capped, and allowed to incubate for 16 h at room of 17a-estradiol, 17b-estradiol, estrone, and estriol from a temperature. The derivatized products were then taken to dry- 1000 ng mL21 stock solution in acetone. Samples were spiked ness under nitrogen, redissolved in 500 mL of acetonitrile, after centrifugation and methanol extraction to minimize mi- spiked with internal standard d12–pyrelene (from US-108) to a crobial degradation of the target analytes. All spiked samples final concentration of 1 mg mL21, and analyzed.
HANSELMAN ET AL.: STEROIDAL ESTROGENS IN DAIRY MANURE WASTEWATER Gas Chromatography–Mass Spectrometry Analysis Estrogens were separated using an HP-6890 gas chromato- graph (Hewlett-Packard, Palo Alto, CA) with split/splitless inlet operated in splitless mode at 2608C. The sample wasintroduced in a 1-mL injection and separated across an HP- 5MS column (30 m 3 0.25 mm; 0.25-mm film thickness; J & W Scientific, Folsom, CA) under a temperature program thatbegan at 2008C, held for 2 min, then increased at 108C min21 to 3008C with a 10-min hold. The He flow rate was 1 mL min21.
Estrogen detection was accomplished using an HP 5973 mass selective detector operated in electron impact mode with a source temperature of 2308C and a quadrupole temperature of 1508C. Identification of all analytes was conducted in full scan mode, where all ions are monitored. For improved sensitivity, selected ion monitoring was used for quantitation. The ionsmonitored for 17a-estradiol and 17b-estradiol were m/z 5 285 and 416, for estriol, m/z 5 504 and 345, and for estrone, m/z 5 For quantitation, a standard curve containing at least six points was prepared for each analyte (R2 $ 0.995). Calibration standards were prepared in acetonitrile and fresh curves were analyzed with each set of samples. Like each sample, each standard was fortified to contain deuterated internal standard, d12–pyrelene, at a final concentration of 1 mg mL21. Standard Fig. 1. Net amount of 17a-estradiol, estrone, 17b-estradiol, and estriol curves ranged from 10 pg mL21 to 400 pg mL21 with R2 $ 0.995 extracted from flushed dairy manure wastewater (FDMW) (n 5 4) for all analytes but estriol, which ranged from 20 pg mL21 to after spiking with 20, 40, 60, and 80 ng of target analytes.
400 pg mL21 with R2 $ 0.995. The calibration curves for allanalytes were consistent throughout the course of the study.
Water blanks, prepared and analyzed alongside standards and 17a-estradiol, 17b-estradiol, estrone, and estriol aver- samples, were found to contain no detectable levels of any aged 100, 122, 99, and 92%, respectively (n 5 4). Preci- analyte. Method detection limits were based on the lowest sion (RSD # 12%) was also acceptable for each analyte concentrations of the analytes that could be accurately and re- at the four spiking levels evaluated.
peatedly identified against the matrix background. The values Overall, the spiked recovery experiment demon- were estimated against low level standards. Quantitation limits strates that Carbograph SPE and C-18 purification is a were based on the lowest concentrations of the analytes thatcould be accurately and precisely quantified from the matrix.
reliable sample preparation method for the sensitive At the lowest standard on the calibration curve, RSD precision determination of estrogens in FDMW by GC–MS. The Carbograph/C-18 extraction and purification method used in this study compares favorably with other re-search involving SPE of estrogens from environmental matrices. For example, Baronti et al. (2000) reported $86% recovery of added 17b-estradiol, estrone, andestriol from aerobic sewage treatment plant influent and Spiked recovery of 40 ng 17a-estradiol, 17b-estradiol, effluent, and river water when using Carbograph SPE, estrone, and estriol averaged 96, 125, 101, and 99%, and Lee and Peart (1998) reported $98% recovery of respectively (n 5 4 for each of five samples; Table 1).
added 17b-estradiol, estrone, and estriol from sewage Method precision (RSD # 12%) was also acceptable for all target analytes (Table 1). As shown in Fig. 1, the netamount of each estrogen extracted from FDMW after spiking with 20, 40, 60, and 80 ng was linear within the range evaluated. Recovery of the four spiking levels of Using the methodology developed in this study, the limit of detection for each analyte was determined to be Table 1. Percentage recovery of spiked estrogens from five sam- 75 ng L21 FDMW with a limit of quantitation of 125 ng ples of flushed dairy manure wastewater.
L21 for 17a-estradiol, 17b-estradiol, and estrone, and250 ng L21 for estriol. These parameters represent a significant improvement in sensitivity for the analysis of dairy waste samples as compared with the method of Raman et al. (2001) who reported a GC–MS detec- tion limit of approximately 10 000 ng L21. Furthermore, our work confirmed that direct injection of the DMF/ BSTFA derivative as specified in their paper resultedin the rapid and severe degradation of the GC col- † Flushed dairy manure wastewater.
‡ Mean values with (RSD), n 5 4.
umn (Raman et al., 2001). Drying and resuspending the J. ENVIRON. QUAL., VOL. 35, MAY–JUNE 2006 Fig. 2. Selected ion chromatogram of unspiked flushed dairy manure wastewater (FDMW) showing the retention time of the BSTFA [N,O- bis(trimethylsilyl)fluoroacetamide] derivatives of (a) estrone, (b) 17a-estradiol, (c) 17b-estradiol, and the internal standard (d) d12–pyrelene.
derivatized product in acetonitrile was found to alle- cretion profile of cattle (Mellin et al., 1965; Hoffmann viate column degradation which permitted the analy- sis of all study samples on the same GC column with It is difficult to compare in a meaningful way the es- minimal instrument maintenance. Selected ion chro- trogen concentrations in FDMW with other types of matograms for an unspiked and a corresponding 40-ng dairy manure samples because FDMW is highly dilute spiked FDMW replicate sample are shown in Fig. 2 due to extensive flushwater volumes. Herd size and and 3, respectively. The chromatograms illustrate the storage duration also affect the measured estrogen con- typical retention times of the BSTFA estrogen deriva- centrations significantly. However, compared with other tives and the typical matrix background observed with low solids content dairy wastes such as from holding FDMW samples using the proposed sample prepara- ponds, estrogen concentrations in FDMW appear to be less (Williams, 2002; Raman et al., 2004). For example,Williams (2002) reported GC–MS measured concentra-tions of estrone, 17a-estradiol, and 17b-estradiol in dairy holding ponds from scraped dairy operations averaged 7595, 5185, and 3350 ng L21, respectively. However, as The endogenous concentration of estrogens in five mentioned previously, the detection limits associated samples of FDMW determined by GC–MS is shown with the sample preparation frequently hindered GC– in Table 2. Estrone, 17a-estradiol, and 17b-estradiol MS quantification of estrogens and resulted in a high concentrations averaged 879, 2282, and 643 ng L21, frequency of “below detectable limits” reported for sev- respectively, but estriol was not detected during five eral dairy waste samples (Williams, 2002). For example, consecutive sampling days. The absence of estriol and 87 and 60% of samples collected from dairy holding the abundance of 17a-estradiol relative to 17b-estra- ponds were below the method detection limits for 17b- diol and estrone is consistent with the estrogen ex- estradiol and estrone, respectively (Williams, 2002). The HANSELMAN ET AL.: STEROIDAL ESTROGENS IN DAIRY MANURE WASTEWATER Fig. 3. Selected ion chromatogram of spiked flushed dairy manure wastewater (FDMW) showing the retention time of the BSTFA [N,O- bis(trimethylsilyl)fluoroacetamide] derivatives of (a) estrone, (b) 17a-estradiol, (c) 17b-estradiol, the internal standard (d) d12–pyrelene, and(e) estriol after spiking with 40 ng of each estrogen analyte.
estrone concentrations reported in the present study appear to be consistent with concentrations reported by A new sample preparation method involving liquid Kolodziej et al. (2004) for FDMW using GC–MS–MS and solid-phase extraction was developed for the mea- technology. They found estrone concentrations up to surement of 17a-estradiol, 17b-estradiol, estrone, and 650 ng L21 in a dairy waste lagoon from a flushed estriol in FDMW by GC–MS. Results of spiking experi- dairy operation (Kolodziej et al., 2004). 17b-Estradiol ments indicated that the method is satisfactory for was not detected, which may be a result of degradation determining four estrogens in FDMW with recovery during storage. Unfortunately, 17a-estradiol—the primary of .90%. Characterization of the estrogen profile of endogenous estrogen excreted by cattle (Mellin and Erb, FDMW revealed a large abundance of 17a-estradiol 1966; Hoffmann et al., 1997)—concentrations of the relative to 17b-estradiol and estrone. Estriol was not manure cannot be compared since they were not mea- detected in FDMW. The methodology developed in this research appears useful for the analysis of steroidal estrogens in dairy wastewater and provides some an-alytical foundation for future research involving the Table 2. Estrogen concentrations in five samples of flushed dairy determination of estrogens in livestock wastes.
manure wastewater measured by gas chromatography–massspectrometry (GC–MS).
This research was supported by the University of Florida School of Natural Resources and Environment Mini-Grants American Public Health Association. 1998. Standard methods for the † Flushed dairy manure wastewater.
‡ Mean values 6 SE, n 5 4.
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Necrotizing soft tissue infections: a primary care review - american family physician

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