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
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.
examination of water and wastewater. 20th ed. APHA, Washing-
J. ENVIRON. QUAL., VOL. 35, MAY–JUNE 2006
Andreolini, F., C. Borra, F. Caccamo, A. Di Corcia, and R. Samperi.
Lagana, A., G. Fago, A. Marino, and D. Santarelli. 2001. Liquid
1987. Estrogen conjugates in late-pregnancy fluids: Extraction and
chromatography tandem mass spectrometry applied to the analysis
group separation by a graphitized carbon black cartridge and
of natural and synthetic steroids in environmental waters. Anal.
quantification by high-performance liquid chromatography. Anal.
Lange, I.G., A. Daxenberger, B. Schiffer, H. Witters, D. Ibarreta, and
Baronti, C., R. Curini, G. D’Ascenzo, A. Di Corcia, A. Gentili, and R.
H.H.D. Meyer. 2002. Sex hormones originating from different
Samperi. 2000. Monitoring natural and synthetic estrogens at
livestock production systems: Fate and potential disrupting activity
activated sludge sewage treatment plants and in a receiving river
in the environment. Anal. Chim. Acta 473:27–37.
water. Environ. Sci. Technol. 34:5059–5066.
Lee, H.B., and T.E. Peart. 1998. Determination of 17b-estradiol and its
Fine, D.D., G.P. Breidenbach, T.L. Price, and S.R. Hutchins. 2003.
metabolites in sewage effluent by solid-phase extraction and gas
Quantitation of estrogens in ground water and swine lagoon samples
chromatography mass spectrometry. J. AOAC Int. 81:1209–1216.
using solid-phase extraction, pentafluorobenzyl/trimethylsilyl de-
Mellin, T.N., and R.E. Erb. 1966. Estrogen metabolism and excretion
rivatizations and gas chromatography-negative ion chemical ioniza-
during the bovine estrous cycle. Steroids 7:589–606.
tion tandem mass spectrometry. J. Chromatogr. A. 1017:167–185.
Mellin, T.N., R.E. Erb, and V.L. Estergreen. 1965. Quantitative es-
Finlay-Moore, O., P.G. Hartel, and M.L. Cabrera. 2000. 17b-Estradiol
timation and identification of estrogens in bovine urine. J. Dairy Sci.
and testosterone in soil and runoff from grasslands amended with
broiler litter. J. Environ. Qual. 29:1604–1611.
Panter, G.H., R.S. Thompson, and J.P. Sumpter. 1998. Adverse
Gentili, A., D. Perret, S. Marchese, R. Mastropasqua, R. Curini, and
reproductive effects in male fathead minnows (Pimephales prome-
A. Di Corcia. 2002. Analysis of free estrogens and their conjugates
las) exposed to environmentally relevant concentrations of the
in sewage and river waters by solid-phase extraction then liquid
natural oestrogens, oestradiol and oestrone. Aquat. Toxicol. 42:
chromatography-electrospray-tandem mass spectrometry. Chroma-
Raman, D.R., A.C. Layton, L.B. Moody, J.P. Easter, G.S. Sayler, R.T.
Hanselman, T.A., D.A. Graetz, and A.C. Wilkie. 2003. Manure-borne
Burns, and M.D. Mullen. 2001. Degradation of estrogens in dairy
estrogens as potential environmental contaminants: A review.
waste solids: Effects of acidification and temperature. Trans. ASAE
Environ. Sci. Technol. 37:5471–5478.
Hanselman, T.A., D.A. Graetz, and A.C. Wilkie. 2004. Comparison of
Raman, D.R., E.L. Williams, A.C. Layton, R.T. Burns, J.P. Easter, A.S.
three enzyme immunoassays for measuring 17b-estradiol in flushed
Daugherty, M.D. Mullen, and G.S. Sayler. 2004. Estrogen content of
dairy manure wastewater. J. Environ. Qual. 33:1919–1923.
dairy and swine wastes. Environ. Sci. Technol. 38:3567–3573.
Hoffmann, B., T. Goes de Pinho, and G. Schuler. 1997. Determination
Shore, L.S., M. Gurevitz, and M. Shemesh. 1993. Estrogen as an en-
of free and conjugated oestrogens in peripheral blood plasma, feces
vironmental pollutant. Bull. Environ. Contam. Toxicol. 51:361–366.
and urine of cattle throughout pregnancy. Exp. Clin. Endocrinol.
Tyler, C.R., S. Jobling, and J.P. Sumpter. 1998. Endocrine disruption in
wildlife: A critical review of the evidence. Crit. Rev. Toxicol. 28:
Irwin, L.K., S. Gray, and E. Oberdorster. 2001. Vitellogenin induction
in painted turtle, Chrysemys picta, as a biomarker of exposure to
Wilkie, A.C. 2005. Anaerobic digestion of dairy manure: Design and
environmental levels of estradiol. Aquat. Toxicol. 55:49–60.
process considerations. p. 301–312. In Dairy manure management:
Jobling, S., M. Nolan, C.R. Tyler, G. Brighty, and J.P. Sumpter. 1998.
Treatment, handling, and community relations. NRAES-176. Nat-
Widespread sexual disruption in wild fish. Environ. Sci. Technol.
ural Resource, Agriculture, and Engineering Service, Cornell Univ.,
Kolodziej, E.P., T. Harter, and D.L. Sedlak. 2004. Dairy wastewater,
Wilkie, A.C., H.F. Castro, K.R. Cubinski, J.M. Owens, and S.C. Yan.
aquaculture, and spawning fish as sources of steroid hormones in
2004. Fixed-film anaerobic digestion of flushed dairy manure after
the aquatic environment. Environ. Sci. Technol. 38:6377–6384.
primary treatment: Wastewater production and characterisation.
Lagana, A., A. Bacaloni, G. Fago, and A. Marino. 2000. Trace analysis
of estrogenic chemicals in sewage effluent using liquid chromatog-
Williams, E.L. 2002. Survey of estrogen concentrations in dairy and
raphy combined with tandem mass spectrometry. Rapid Commun.
swine waste holding and treatment structures in and around
Tennessee. M.S. thesis. Univ. of Tennessee, Knoxville.
Necrotizing Soft Tissue Infections: A Primary Care Review ADRIENNE J. HEADLEY, M.D., University of Medicine and Dentistry of New Jersey–Robert Wood Johnson Medical School, New Brunswick, New Jersey Patients with necrotizing soft tissue infections often present initially to family physi- cians. These infections must be detected and treated rapidly to prevent loss of limb or a fatal outcome.
TECHNICAL DATA SHEET MOLYLITH 3 LITHIUM EP GREASE Molylith 3 is formulated for multipurpose use. Molylith 3‘s wide temperature range (-10° to 325°F) eliminates the need for two or more greases in our • MULTI-PURPOSE – allowing for inventory lubricant inventory. Frequent relubrication allows reduction and a wide variety of lubricant Effective anti-wear and extreme p