Transcript Hanselman, Travis - Soil and Water Science

Determination of Natural Steroidal Estrogens
in Flushed Dairy Manure Wastewater and
Surface and Ground Water
Travis Hanselman
Ph.D. exit seminar
What are estrogens?
Where do they come from?
Why are they a concern?
What are estrogens?
• A group of natural steroid hormones with an
aromatic A-ring as a distinctive part of their
tetracyclic molecular framework
OH
O
OH
OH
HO
HO
Estradiol
Estrone
HO
Estriol
Physicochemical Properties
Table 1. Estrogen characteristics
Compound
Estradiol
Estrone
Estriol
MW Solubility† log Kow
g mol-1
mg L-1
272.4
<13.0
3.1
270.4
<12.4
3.4
288.4
<13.3
2.6
pKa
VP
Pa
10.5 3E-08
10.3 3E-08
10.4 9E-13
MW=Molecular weight; Kow=Octanol-water partition
coefficient; VP=Vapor pressure; †, Solubility in water
• Estrogens are nonvolatile, moderately
hydrophobic, nonionic compounds with low water
solubility
Where do estrogens come from?
• Estrogens are excreted
naturally in the urine and
feces of humans, wildlife
and livestock; especially
from females
Why are estrogens a concern?
• Endocrine disruption
–Feminization of male
aquatic vertebrate species
–-Estradiol is biologically
active at 10-100 ng L-1
Critical Research Needs
1. Development of methods for measuring
estrogens in livestock wastes and surface and
ground water resources
2. Surveys of estrogens in waterways
3. Characterization of estrogens in livestock
wastes
4. Estrogen fate in manure, soil, and water
5. Wildlife and/or test organisms should be studied
for evidence of reproductive abnormalities
6. Other biologically-active agents in livestock
wastes such as androgens, gestagens, growth
promoters, and antibiotics need to be
characterized
Research Objective
• Measure estrogens in
flushed dairy manure
wastewater (FDMW) and
surface and ground
water
FDMW
UF Dairy Research Unit
FDMW
Few “Ideal” Analytical Options
• Previous researchers have relied heavily on
enzyme immunoassay (EIA) methods due to their
commercial availability, ease of use, pg mL-1
detection limits, and a lack of alternative
quantitation methods
• However, EIAs can suffer from false-positive and
false-negative interferences due to cross-reactivity
and matrix effects associated with humic
substances
• Thus, depending on sample complexity and EIA
reagents, antibodies, and protocol, a potential
exists for different EIAs to yield different or
inaccurate results
Immunoassay Experiment
• Comparison of three commercially-available
ß-estradiol EIAs
• Sample preparation according to a published
method for dairy wastes (Raman et al., 2001)
• Quality control experiment
FDMW (20 mL),n=4
extract w/ ether (20 mL)
OH
Sub sample ether (4 mL)
evaporate to dryness and
reconstitute with buffer
HO
ß-Estradiol
EIA1
EIA2
EIA3
Immunoassay Description
Table 2. Description and cross-reactivity of three immunoassays.
Description
Assay principle
Estradiol Antibody
Matrix
Conjugate/Enzyme
Substrate
Range (pg mL-1)
MDL (pg mL-1)
EIA1
Competitive
Rabbit poly
TBS
E2-ALP
p-NPP
0-30,000
29
EIA2
Competitive
Rabbit poly
Serum
E2-Biotin/SHRP
TMB
0-6,000
7
EIA3
Competitive
Rabbit poly
Serum
E2-HRP
TMB
0-2,000
10
100
0.1
4.6
0.5
100
0.3
1.4
1.1
100
0.3
2.1
1.5
Cross-reactivity (%)
ß-Estradiol
α-Estradiol
Estrone
Estriol
E2, estradiol; ALP, alkaline phosphatase; SHRP, streptavidin horseradish
peroxidase; HRP, horseradish peroxidase; p-NPP, p-nitrophenol phosphate;
TMB, tetramethylbenzidine; MDL, minimum detection limit. TBS, Tris-buffered
saline containing proteins, detergents, and azide
17ß-Estradiol (ng L-1)
Immunoassay Comparison
1000
FDMW 1
750
500
250
a
ab
b
0
A1 A2 A3
17ß-Estradiol (ng L-1)
Immunoassay Comparison
2000
1500
FDMW 2
a
1000
500
b
b
0
A1 A2 A3
Why different and which is right?
• Quality control data (accuracy, precision, linearity)
• Calibration accuracy “pure” ß-estradiol solutions
(all assays reported the same concentrations)
• Duplicate precision was quite good (CV<13%)
• Dilution and spiked recovery (linear deviation)
• The QC results suggested that coextracted humic
substances affected one or more EIAs
• Ideally, EIA disagreement can be reconciled with a
more conclusive technique like gas
chromatography-mass spectrometry (GC-MS),
but……………
Method Development for GC-MS
• A sensitive method for GC-MS analysis of
estrogens in dairy wastes not available
• Raman et al., 2001 reported GC-MS method
detection limits of ~10 μg L-1
• Immunoassay of FDMW showed ng L-1
• Needed to develop a method
for measuring endogenous
estrogen concentrations by
GC-MS
Method Development for GC-MS
A difficult, expensive,
frustrating, trial and error
process……
Method Development
FDMW (40 mL)
centrifuged 15,000 g
Ultrasonic MOH extraction of solids,
2x10 mL; centrifuge 4,000 g
combine supernatants
Filtration 20 μm
Carbograph SPE extraction
MOH:MC elution and evaporation
C18 SPE purification
Acetone elution and filtration 0.2 μm
GC-MS
EIA 1
EIA 2
Method Performance
• GC-MS recovery and reproducibility experiment
• Recovery of estrogens spiked into FDMW over 5days of sampling; 40 ng fixed level and 20, 40, 60,
and 80 ng variable spike
Table 3. Five day recovery of 40 ng from FDMW.
FDMW estrone
α-estradiol ß-estradiol estriol
--------------------recovery, % (RSD)----------------1
92 (5)
96 (6)
116 (5)
90 (9)
2
104 (5)
105 (5)
134 (8)
99 (9 )
3
105 (2)
93 (5)
121 (2)
109 (5)
4
107 (7)
94 (10)
139 (8)
107 (10)
5
98 (7)
94 (9)
114 (8)
90 (12)
101 (5)
96 (7)
125 (6)
99 (9)
Avg.
Method Performance
0
20
40
60
80
100
0
20
40
60
80
100
100
100
α-estradiol
100%
80
estrone
99%
80
60
60
40
40
20
20
0
0
100
100
ß-estradiol
122%
80
estriol
92%
80
60
60
40
40
20
20
0
0
0
20
40
60
80
100
0
20
40
Estrogen added (ng)
60
80
100
GC-MS FDMW Concentrations
Estrogen (ng L-1)
4000
3000
estrone
estrone
a-estradiol
α-estradiol
ß-estradiol
b-estradiol
estriol-ND
estriol-ND
2000
1000
0
1
2
3
4
FDMW
5
GC-MS vs. EIA 1
17ß-estradiol ( ng L-1)
1400
GC-MS
GC-MS
EIA1
A1
EIA2
A2
1200
1000
800
*
600
*
400
200
0
1
2
3
FDMW
4
5
GC-MS vs. EIA 2
17ß-estradiol ( ng L-1)
1400
GC-MS
GC-MS
EIA1
A1
EIA2
A2
1200
1000
800
600
*
400
*
*
200
*
*
0
1
2
3
FDMW
4
5
Are dairy manure estrogens
finding their way into
surface and ground water?
Surface Water
• Preliminary GC-MS method
development and survey experiment
• 4 sampling locations, 1 impacted
Ground Water
• 3 FDEP monitoring wells, 1 “other” well
Method Development
• Method adaptation
• Spiked recovery of 40 ng of estrogens from surface
and ground water
200 mL water samples
filtration 20 μm
Carbograph SPE extraction
MOH:DCM elution and evaporation
C18 SPE purification
Acetone elution and filtration 0.2 μm
GC-MS
Method Performance
• Positive interference of the GC-MS signals was
observed for water samples; particularly for
estrone
Table 4. Estimated recovery of estrogens from water.
surface water
analyte
% recovery RSD
estrone
99
9
17α-estradiol 100
10
17ß-estradiol 99
7
estriol
98
9
ground water
% recovery RSD
88
7
90
12
87
9
77
16
Survey of Estrogens in Water
• No measurable concentrations of α-estradiol, ßestradiol, or estriol in any of the surface or ground
water samples
• Estrone was detected in one surface water sample
and one ground water sample, <60 ng L-1
Summary & Conclusions
• The accurate measurement of estrogens in
environmental samples is a challenging task
• Data acquired from EIAs should be interpreted
cautiously and with strict quality control protocol
• A method was developed which enabled ng L-1
measurements of estrogens in FDMW and surface
and ground water by GC-MS
• The survey of surface and ground water at the
dairy did not reveal any gross contamination by
estrogens at the time of sampling
• The method contributes a foundation for future
research involving estrogen analysis of livestock
wastes and water resources
Acknowledgments
Committee
• Don Graetz, Chair
• Ann Wilkie, CoChair
• Tom Obreza
• Nancy Denslow
Also…
• Nancy Szabo and the ATCL staff
• Dawn Lucas
• Funding provided by the School of Natural
Resources and Environment Mini-grants
Program
Questions?