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Theses and Dissertations
2011
Removal of natural and synthetic steroid hormones
through constructed wetland microcosm
Min Fang
The University of Toledo
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A Thesis
entitled
Removal of Natural and Synthetic Steroid Hormones through Constructed
Wetland Microcosm
by
Min Fang
Submitted to the Graduate Faculty as partial fulfillment of the requirement
for the Master of Science Degree in Geology
__________________________________________
Dr. Alison L. Spongberg, Advisor
__________________________________________
Dr. Mark J. Camp, Committee Member
__________________________________________
Dr. James M. Martin-Hayden, Committee Member
__________________________________________
Dr. Patricia Komuniecki, Dean
College of Graduate Studies
The University of Toledo
May 2011
Copyright 2011, Min Fang
This document is copyrighted material. Under copyright law, no parts of this document
may be reproduced without the expressed permission of the author.
An Abstract of
Removal of Natural and Synthetic Steroid Hormones
through Constructed Wetland Microcosm
by
Min Fang
Submitted to the Graduate Faculty in partial fulfillment of the
requirement for the Master of Science Degree in Geology
The University of Toledo
May 2011
Steroid hormones are natural substances excreted by humans and livestock while
synthetic steroid hormones are extensively used as growth promoters and contraceptives.
Both have been widely detected in environmental matrices. These compounds enter
environmental water bodies dissolved in effluent of municipal wastewater treatment
plants or in runoff of animal waste from livestock operations.
Batch equilibrium sorption studies of progesterone, melengestrol-acetate, estrone,
17β-estradiol, 17α-estradiol, 17α-ethinylestradiol and estriol on a sandy loam soil have
been conducted. Results suggest that the mobility of estrone, 17β-estradiol, 17αethinylestradiol and estriol in this sandy loam soil would be low due to their strong
sorption. Sorption of progesterone and melengestrol-acetate is stronger, thus mobility of
these two would be even lower. However, estriol would be more mobile than any of the
other hormones tested due to the low sorption to the tested soil.
iii
Degradation of progesterone and melengestrol-acetate in this soil was tested and
gave half-lives of 1.06 and 1.94 days, respectively. The five hormones (17-α
ethynylestradiol, 17α-estradiol, 17β-estradiol, estrone and estriol) with lower sorption are
expected to degrade rapidly and be effectively removed from the system.
In the high and low flow rate microcosms, progesterone, melengestrol-acetate,
estrone, 17β-estradiol and estriol in effluent were below the detection limits throughout
the experiment. Only 17α-ethinylestradiol and 17α-estradiol were detected in much lower
concentrations in the effluent than at the input. Residuals of progesterone and
melengestrol-acetate in soil columns from the microcosms showed that degradation
dominated in the microcosm. Overall, the high flow rate tested in this study was favored
due to its good removal efficiency and the higher performance.
iv
Acknowledgements
First, I would like to acknowledge my advisor Dr. Alison L. Spongberg for her
support and advise during my study for the masters degree. Thank you for letting me live
with you. I really enjoy the time we spend together.
Second, I would like to thank our lab manager Jason D. Witter for his help.
Because of you, our lab is well organized. Discussions with you are always helpful, from
which I have learned a lot.
Further, I greatly appreciate the help from my committee members – Drs James M.
Martin-Hayden and Mark J. Camp. I love your classes and your instructions have been
very helpful with my research and thesis.
Finally I would like to thank my families, especially my husband Chenxi Wu, my
friends, and many others from the Department of Environmental Sciences for their help
and support.
v
Table of Contents
Abstract ............................................................................................................................. iii
Acknowledgements ............................................................................................................. v
Table of Contents ............................................................................................................... vi
List of Tables ..................................................................................................................... ix
List of Figures ..................................................................................................................... x
Chapter 1 Introduction of Natural and Synthetic Hormones .............................................. 1
1.1 Introduction ........................................................................................................... 1
1.2 Analytical Methods ............................................................................................... 4
1.3 Transport and Occurrence ..................................................................................... 6
1.4 Environmental Effects .......................................................................................... 8
1.5 Project Description and Hypotheses ................................................................... 10
Chapter 2 Materials and Methods ..................................................................................... 11
2.1 Introduction ......................................................................................................... 11
2.2 Analytical Methods Development ...................................................................... 12
2.2.1 Chemicals and Reagents .............................................................................. 12
2.2.2 Sample Collection and Preparation .............................................................. 12
2.2.3 Accelerated Solvent Extraction (ASE) ........................................................ 13
vi
2.2.4 Solid-Phase Extraction Procedure................................................................ 13
2.2.5 Derivatization ............................................................................................... 14
2.2.6 Liquid Chromatography-tandem Mass Spectrometry (LC-MS/MS) Analysis
............................................................................................................................... 15
2.2.7 Statistic Analysis .......................................................................................... 16
2.3 Sorption Experiments.......................................................................................... 16
2.3.1 Materials and Methods ................................................................................. 16
2.3.2 Data Analysis ............................................................................................... 18
2.4 Degradation Experiments.................................................................................... 20
2.4.1 Materials and Methods ................................................................................. 20
2.4.2 Data Analysis: .............................................................................................. 21
2.5 Constructed Wetland Microcosm Experiments .................................................. 22
2.5.1 Materials and Methods ................................................................................. 22
2.5.2 Data Analysis ............................................................................................... 24
Chapter 3 Results and Discussion ..................................................................................... 26
3.1 Analytical Methods Development ...................................................................... 26
3.1.1 Optimization of ASE Procedure .................................................................. 26
3.1.2 Optimization of SPE Procedure ................................................................... 28
3.1.3 Optimization of Derivatization .................................................................... 33
3.1.3 Method Validation ....................................................................................... 35
vii
3.2.6 Method Application ..................................................................................... 37
3.3 Sorption of Hormones on Soil ............................................................................ 41
3.4 Degradation of Hormones in Soil ....................................................................... 48
3. 5 Removal of Hormones through Constructed Wetland Microcosm.................... 52
3.5.1 Hydraulic Conditions ................................................................................... 52
3.5.2 Fate and Transport of Hormones in Wetland Microcosm ........................... 55
3.5.3 Correlation between Removal and Physico-chemical Parameters............... 61
Chapter 4 Conclusion and Future Research ...................................................................... 63
References ......................................................................................................................... 67
viii
List of Tables
Table 1-1 Properties of the natural and synthetic steroid hormones ................................... 2
Table 2-1 MS/MS breakdown parameters ........................................................................ 16
Table 3-1 Method performance for surface water samples .............................................. 36
Table 3-2 Method performance for soil samples .............................................................. 36
Table 3-3 Water sample sites information (samples collected on 10/16/2009) ................ 37
Table 3-4 Parameters of Freundlich and linear models .................................................... 44
Table 3-5 Sorption data reported in the literature ............................................................. 45
Table 3-6 Model-fitting parameters .................................................................................. 50
Table 3-7 Concentration (ng L-1) of tested hormones in effluents from microcosms ..... 55
Table 3-8 Residues (ng Kg-1) of progesterone and melengestrol-acetate in microcosm
substrate under flow rate 1 ml L-1 ..................................................................................... 57
Table 3-9 Mass (ng) balance of the progesterone and melengestrol-acetate in microcosms
........................................................................................................................................... 57
Table 3-10 Total removed mass (µg) of hormones in microcosms .................................. 61
ix
List of Figures
Figure 2-1 Fiberglass nesting box, A=60.96 cm, B=20.32cm, C=15.24 cm, D=53.66 cm,
E=12.07 cm, F=14.605 cm................................................................................................ 22
Figure 2-2 (a), (b) Wetland microcosm setup ................................................................... 23
Figure 3-1 Recoveries of different ASE methods ............................................................. 28
Figure 3-2 Recoveries of SPE cartridges at different pH ................................................. 29
Figure 3-3 Different matrix effects ................................................................................... 31
Figure 3-4 Recoveries using different percentages of acetone in wash solution .............. 33
Figure 3-5 Derivatization efficiencies for five hormones at two concentrations each with
increasing concentration of deriviting agent (Dansyl chloride). ....................................... 35
Figure 3-6 Water sample sites in Northwest Ohio ............................................................ 39
Figure 3-7 Hormones found in water samples from northwest Ohio ............................... 40
Figure 3-8 Sorption kinetic results of hormones. Data are presented as concentration
remaining in solution over time ........................................................................................ 42
Figure 3-9 Linear sorption isotherms ................................................................................ 43
Figure 3-10 Freundlich sorption isotherms ....................................................................... 43
Figure 3-11 Residuals of progesterone and melengestrol-acetate in soil with time during
the degradation experiment ............................................................................................... 49
Figure 3-12 Model fitting results of progesterone and melengestrol-acetate ................... 49
x
Figure 3-13 Bromide breakthrough curves from two different microcosms under the same
flow rate 1 mL min-1 ......................................................................................................... 53
xi
Chapter 1
Introduction of Natural and Synthetic Hormones
1.1 Introduction
Steroid hormones are a group of compounds with a cyclopentane-o-phenanthrene
ring in their structure. Natural estrogens, such as estrone, estradiol and estriol have
solubilities of approximately 13 mg L-1 (Ying et al., 2002). Synthetic estrogenic steroids
have much lower solubilities, such as 4.8 mg L-1 for 17-α ethynylestradiol (Lai et al.,
2000) and 0.1mg L-1 for melengestrol acetate (Schiffer et al., 2004). All these steroids
have very low vapor pressures, ranging from 2.3×10-10 to 6.7×10-15 mm Hg (Lai et al.,
2000), indicating low volatility of these compounds. The acid dissociation constant (Ka)
is a quantitative measure of the strength of an acid in solution. The larger the value of
pKa, which is equal to –log Ka, of a compound the smaller the extent of dissociation. A
weak acid has a pKa value in the approximate range -2 to 12 in water. Steroid hormones
have pKa values greater than 10 indicating small extent of dissociation of these
compounds. The octanol-water partitioning coefficient (Kow) is used to predict sorption of
dissolved hydrophobic organic compounds in organic rich soil and model the migration
in water. Log Kow is 2.81 for estriol, 3.42 for estrone and 3.94 for estradiol (Lai et al.,
2000). Synthetic steroids have higher log Kow values such as 4.15 for 17-α
1
ethynylestradiol (Lai et al., 2000). High octanol-water partitioning coefficients indicate
strong attraction of these dissolved compounds from water to organic content of soil.
Table 1-1 shows structures and properties of several natural and synthetic steroid
hormones.
Table 1-1 Properties of the natural and synthetic steroid hormones
Natural steroid hormones
MW (g mol-1)
Sw
pKa
log Kow
MW (g mol-1)
Sw
pKa
log Kow
estrone (E1)a
17α-estradiol (αE2)a
17β-estradiol (βE2)a
270.37
12.42
10.3-10.8c
3.1-3.43
272.39
1.3-3.9
10.5-10.7c
3.47-3.62
272.39
12.96
10.5-10.7c
3.94
estriol (E3)a
progesteroneb
288.39
13.25
10.4c
2.6-2.81
314.47
Synthetic steroid hormones
MW (g mol-1)
Sw
pKa
log Kow
17-α ethynylestradiol (EE2)d
melengestrol acetatee
296.41
9.7
10.4c
3.67-4.15
396.5
100µg L-1
Note: MW-molecular weight, Sw-solubility in water, Ka-acidionization constant, Kowoctanol-water partition coefficient, a: Beck et.al., (2008), b: Díaz-Cruz et al., (2003), c:
Hurwitz and Liu, (1977), d: Lai et al.,(2000), e: Schiffer et al., (2004).
2
Steroid hormones are a group of endocrine-disrupting compounds that are derived
from cholesterol, which include natural steroid groups such as progestogens,
glucocorticoids, mineralocorticoids, androgens,and estrogens. Synthetic steroids such as
17-α ethynylestradiol are used as contraceptives while melengestrol acetate is used as a
growth promoter. Natural steroids are secreted by the adrenal cortexes, testes, ovaries and
placentas in humans and other animals (Richardson and Ternes, 2005). After excretion
hormones are introduced to the environment through sewage discharge and animal waste
disposal (Sumpter, 2005; Vos et al., 2000). As a result, steroidal hormones have been
detected in effluent from wastewater treatment plants and surface water (Ingrand et al.,
2003; Sellin et al., 2009).
The bio-activity of these compounds results in their ability to interfere with the
normal function of endocrine systems thus potentially affects the reproduction and
development of aquatic and terrestrial organisms in the environments where these
compounds are introduced. For example, at a few nanograms per liter levels, estrone and
17β-estradiol can affect the normal sexual development of some aquatic species and
could potentially cause yet unknown effects on ecosystems (Tyler et al., 2005). Since the
1990s, with the development of advanced analytical methodology and equipment,
increasing attention has been focused on the identification of steroidal hormones in
environmental matrices (Routledge et al., 1998; Tyler et al., 1998). A majority of this
research has been directed towards their occurrence and fate in wastewaters, soils,
sediments and manures (Campbell et al., 2006; Khanal et al., 2006; Lorenzen et al.,
2004). However, little research has been done to investigate their transport and fate in
3
constructed wetland systems, which are often used to alleviate problems associated with
other types of contaminants.
1.2 Analytical Methods
The presence of estrogenic compounds in surface waters has been noted since the
1980s (Richardson and J.M. Bowron, 1985). Environmental scientists started to look at
these environmental contaminants which had been ignored mostly due to the absence of a
sufficiently sensitive analytical procedure (Belfroid et al., 1999). The analysis of
hormones in environmental samples is challenging because their concentrations are very
low and the inherently complex matrices can affect extraction and instrumental signals.
17β-estradiol, estriol and 17α-ethinylestradiol had been detected in effluents of British
and Israeli waste water treatment plants (WWTPs) and in surface water in English rivers
and an Israeli lake in the ng L-1 range around the early 1990s (Aherne and Briggs, 1989;
Shore et al., 1993). In the late 1990s, the detection limit of 17β-estradiol, 17α-estradiol,
estrone and 17α-ethinylestradiol in surface water and effluent from WWTPs were
achieved below 1 ng L-1 (Belfroid et al., 1999).
Extraction from aqueous samples is usually done using solid phase extraction
(SPE) after filtration of 100-2000 mL samples (Carpinteiro et al., 2004; MouatassimSouali et al., 2003). In most cases, the filtration of aqueous samples is performed by
using a 0.45-1.2 µm filter (Laganà et al., 2004; Salvador et al., 2007). The use of SPE
cartridges such as C18 bonded silica and polymer-based sorbents as the stationary phase
has been widely reported (Ingrand et al., 2003; Liu et al., 2004). Polymer-based SPE
4
products such as Waters Oasis HLB and Phenomenex Strata-X are popular as they are
“water wettable” and often can be used to simultaneously extract many other compounds
together with steroid hormones. However, standard C18 cartridges (i.e. Supelco ENVI18) provide comparable extraction efficiency and are less costly. Tandem SPE extraction
using C18 and Florosil cartridge has been reported to further reduce matrix problems in
aqueous samples (Ingrand et al., 2003).
Extraction of steroid hormones from solid samples or more complex samples such
as sewage sludges, soils and sediments is usually achieved by using ultrasonication
(Ternes et al., 2002), microwave-assisted extraction (Labadie and Budzinski, 2005),
accelerated solvent extraction (ASE) (Takigami et al., 2000) or pressurized liquid
extraction (PLE) (Ternes et al., 2002; Xu et al., 2008). Prior to extraction, sediment,
sludge or soil samples are often freeze-dried or could be dried at 30°C for approximately
16 h (Hájková et al., 2007). The extraction of steroid hormones from solid samples is
usually preformed with polar solvents. After extraction, the samples can be further
cleaned and concentrated using SPE similar to the procedure used for liquid samples.
Hormones in final extracted samples are typically identified and quantified using
gas chromatography-mass spectrometry (GC-MS) (Mouatassim-Souali et al., 2003), gas
chromatography-mass spectrometry (tandem) (GC-MS/MS) (Ternes et al., 2002)or liquid
chromatography-mass spectrometry (tandem) (LC-MS/MS) (Ingrand et al., 2003). GC
methods typically are more sensitive than LC due to the poor ionization efficiency of the
compounds (Díaz-Cruz et al., 2003). However, when GC-MS is used, samples need to be
derivatized before analysis which is time consuming and increases the risk of analyte
losses. When LC-MS is used, derivatization is required as steroid hormones are non-
5
volatile (Quintana et al., 2004). However LC-MS/MS does not necessarily require
derivatization. Nevertheless, to take advantage of the sensitivity of LC-MS/MS
technology some researchers have reported derivatization procedures using dansyl
chloride to enhance ionization of non-volatile (or low-volatile) steroid hormones and thus
increase the signal (Anari et al., 2002; Lin et al., 2007). Current methodology allows the
detection of steroid hormones from liquid matrices to less than 0.1 ng L-1 for water and
less than 0.1 ng g-1 in solid matrices (Gabet et al., 2007).
Many papers have reported analytical methods for various hormones in surface
waters and wastewaters (Alda and Barcelo, 2000; Schlüsener and Bester, 2005;
Stavrakakis et al., 2008). Recoveries of the target compounds are generally in the range
75-100% depending on the analyte. However the methods for hormones in sediment or
sludge have fewer references (Gabet et al., 2007). Generally, recoveries for solid matrices
are more variable than those for aqueous matrices and range between 60-130%.
1.3 Transport and Occurrence
The two major pathways for steroid hormones to enter the environment are human
and animal wastes. Estrone and 17β-estradiol are naturally excreted by women (3-20 µg
person-1 day-1 and 2-12 µg person-1 day-1, respectively) and female animals, but also by
men (estrone 5 µg person-1 day-1) (Belfroid et al., 1999). Municipal wastewater is
typically collected through municipal sewer systems and treated in WWTPs before
discharging into the environment. Previous research has shown that the removal of
steroid hormones depends on the treatment techniques as well as the properties of
6
compounds. The removal efficiency for estradiol can be as high as 85-99% while the
removal of estrone ranges from 25 to 80% (Khanal et al., 2006). Although considerable
amounts of hormones can be removed from the waste stream, trace amounts are still
detected in effluents with typical concentration ranges from the lower ng L-1 to tens of ng
L-1 (Belfroid et al., 1999; Schlüsener and Bester, 2005). The removal of steroid hormones
results from biodegradation and sorption to suspended materials, which is referred to as
sludge during treatment (Khanal et al., 2006). As a result of sorption steroid hormones
are also found in treated sludge, which is termed as biosolids (Braga et al., 2005; Ternes
et al., 2002). Effluent discharge and land application of biosolids can therefore introduce
steroid hormones into the environment. Elevated hormones have been found downstream
of WWTPs (Williams et al., 2003). In river water samples collected downstream from a
WWTP outlet, hormones were detected between 0.04 (17-α ethynylestradiol) and 1.5 ng
L-1 (estrone) (Baronti et al., 2000). In rivers, hormones were also found able to adsorb to
sediment. A combination of sorption and degradation results in a rapid decline of
concentrations downstream in rivers with an estimated decay half-life of only a few days
(Williams et al., 2003). However in the surface sediments from Tokyo Bay, several
steroid estrogens were detected at lower ng g-1 (Isobea et al., 2006), suggesting that
sediments could be a sink for those compounds.
The detection of steroid hormones in soil receiving biosolids application has not
been reported. Concentrations in animal wastes were found to depend on their treatment
as well as their storage time. In dairy and swine wastes, the concentrations of estrone,
17β-estradiol, and estriol vary from a few µg kg-1 to several mg kg-1 (Raman et al., 2004).
The reuse of animal waste could be a significant source of hormones.
7
Previously, sorption and degradation experiments were conducted to understand
the behavior of hormones in soils (Sarmah et al., 2008; Scherr et al., 2009). Steroid
hormones have relatively strong sorption to soil with the reported sorption coefficient Kd
ranging from 14-170 L kg-1 (for estradiol), and 12-40 L kg-1 (for 17-α ethynylestradiol)
(Sarmah et al., 2008). In column studies, steroid hormones present low mobility (Casey
et al., 2005) which increases when preferential pathways exist in soil columns
(Sangsupan et al., 2006). Rapid degradation of hormones in soils was also observed. The
half-life of the studied compounds was only a few days and was affected by moisture,
soil type, temperature and redox conditions (Czajka and Londry, 2006; Xuan et al., 2008)
which suggests that steroid hormones are unlikely to persist in soils. The fast degradation
behavior of steroid hormones in soils highlights the possibility of using constructed
wetlands for the treatment of hormone-contaminated water, assuming that preferable
physical and chemical parameters can be maintained.
1.4 Environmental Effects
Increasing research attention has been focused on the possible health problems
caused by endocrine-disrupting chemicals (EDCs). EDCs are substances that interfere
with hormone biosynthesis, metabolism or action, resulting in a deviation from normal
homeostatic control or reproduction (Diamanti-Kandarakis et al., 2009). The natural
estrogens, 17-β estradiol, estrone, and estriol and the synthetic steroid hormone 17αethynylestradiol, have been shown to act as endocrine disrupters in the aquatic
environment (Desbrow et al., 1998). Laboratory studies have shown that some of these
8
hormones mimic or disrupt reproductive functions. Exposure of male rats to estrogen
during gestation or during the first 21 days of postnatal life decrease testicular size and
spermatogenesis in adulthood (90-95 days of age) (Sharpe et al., 1995). Exposure of male
rainbow trout (Oncorhynchus mykiss) to four different estrogenic alkylphenolic
chemicals caused synthesis of vitellogenin, a process normally dependent on endogenous
estrogens, and a concomitant inhibition of testicular growth (Jobling et al., 2009).
Exposure to estrogens has been implicated to cause sperm count decreases in males
human (Sharpe and Skakkebaek, 1993). A seven year whole-lake study performed at an
experimental lake area in Canada showed that continuous input of 17α-ethynylestradiol at
environmentally relevant concentrations (5-6 ng L-1) could affect the reproduction of
fathead minnows by causing feminization of males and affecting the development of
gonads in males and altering oogenesis in females (Kidd et al., 2007). Chronic exposure
over years has caused the collapse of the fathead minnow population in the study lake.
The assessment on male fishes in English rivers conducted by the Environmental
Agency of England and Wales confirmed that steroid estrogens are capable of eliciting
the effects observed in wild fish at concentrations that have been measured in effluents
and in the environment (Gross-Sorokin et al., 2006). White suckers collected from
downstream of an effluent site showed gonadal intersex, sex ratio shift, reduced gonad
size, disrupted ovarian and testicular histopathology, and vitellogenin induction. These
effects were not found at a corresponding upstream site (Vajda et al., 2008). The
chemical analysis of the associated WWTP effluent revealed the presence of estradiol,
17α-ethynyestradiol, alkyphenols, and bisphenol A. All these studies suggest that steroid
9
hormones can cause potential ecological consequences even at very low concentrations.
Thus it is important to remove hormones from wastewater.
1.5 Project Description and Hypothesis
Constructed wetlands are artificial systems designed to treat wastewater using
natural microbial, biological, physical and chemical processes (García et al., 2005). They
have been successfully used to treat a variety of wastewaters including urban runoff,
municipal sewage, industrial wastewater, agricultural drainage and acid mine drainage
(Cheng et al., 2002; Dordio et al., 2010; Matamoros et al., 2008). Although the transport
and fate of steroid hormones in constructed wetlands have not been characterized, their
potential to treat steroid hormones is promising based on their physicochemical
properties (Song et al., 2009). In this research, I hypothesized that wetland systems can
be designed to eliminate several natural and synthetic steroid hormones from receiving
wastewaters. The target analytes are five natural hormones, estrone, 17β-estradiol, 17αestradiol, estriol and progestrone and the two most commonly used synthetic steroid
hormones, melengestrol-acetate and 17α-ethinylestradiol. Several objectives were
developed to address this hypothesis including: (1) development of analytical methods of
target hormones in water and soil; (2) investigation of sorption of target hormones onto
soil using batch equilibrium studies; (3) examination of degradation of target hormones in
soil; (4) evaluation of remediation of the target hormones in constructed wetland
microcosms.
10
Chapter 2
Materials and Methods
2.1 Introduction
In this chapter, the methods used to fulfill the four objectives addressed in the
hypotheses are detailed. First, compound identification and detection were optimized
using liquid chromatography tandem mass spectrometry (LC-MS/MS). Then, solid-phase
and accelerated solvent extraction methods to isolate the tested compounds from water
and soil matrices, respectively, were developed. Sorption and degradation of target
hormones in a sandy loam soil were investigated using laboratory based batch
experiments. Finally, using the most promising parameters based on previous and
concurrent experiments, constructed microcosm experiments were designed and
conducted to determine the optimal removal efficiencies of target compounds to be
expected if this technique is employed on a larger field scale.
11
2.2 Analytical Methods Development
2.2.1 Chemicals and Reagents
Analytical standards including progesterone, melengestrol-acetate, estrone, 17estradiol, 17α-estradiol, 17α-ethinylestradiol and estriol were purchased in pure powder
form from Sigma-Aldrich (St Louis, MO). Internal standards 17-β-estradiol-D3 and
estriol-D2 were obtained from C/D/N Isotopes Inc. (Pointe-Claire, Quebec, Canada). Deionized water (>18.0 MΩ-cm) was provided by a Barnstead NANOpure Infinity water
system (Dubuque, IA). Dansyl chloride was purchased from Sigma-Aldrich (St Louis,
MO). All other chemicals and solvents were supplied by Fisher Chemicals (Fair Lawn,
NJ).
Dansyl Chloride was dissolved in acetone at 1 mg L-1. Sodium bicarbonate was
dissolved in water at 0.1 M and adjusted to pH 10.5 with sodium hydroxide (NaOH).
Individual stock standard solutions were dissolved in methanol at 100 mg L-1 and 1 mg L1
. Mixed standards were prepared from individually prepared standards and all standards
were stored at -20°C.
2.2.2 Sample Collection and Preparation
Water used for method development was collected from the Ottawa River on the
University of Toledo campus and from the wastewater influent and effluent at the Oregon
WWTP, Oregon, OH. Water samples were collected in 1 L HDPE bottles pre-rinsed with
methanol and de-ionized water, and were transported back to the lab under ice in a
cooler. In the lab, samples were filtered using 0.7 µm glass fiber filters (Fisher
12
Chemicals, Fair Lawn, NJ) and stored in a refrigerator at 4oC. Filtered samples were
subsequently processed using solid-phase extraction (SPE, described below) within 48 h
to minimize degradation. Soil samples were collected from the top layer (0-20cm) of a
sandy loam soil (Lamson series) found at the R.A. Stranahan Arboretum (Toledo, OH).
Soil samples were freeze-dried, sieved to less than 0.2 cm and stored at -20 oC.
2.2.3 Accelerated Solvent Extraction (ASE)
The Dionex ASE 200 accelerated solvent extraction system (Dionex, Sunnyvale,
CA) was used to extract analytes from soil samples. Three different ASE methods with
different extraction solvents (acetone and methanol) and temperature (60 ºC and 100 ºC)
were tested for their extraction efficiency.
The ASE procedures were as follows: 5 g (freeze-dried) soil samples were spiked
with internal standards (50 μL, 500 μg L-1), mixed with Hydromatrix (Varian, Inc.,
Walnut Creek, CA), and placed into 11 mL extraction cells and sealed with glass fiber
filters at both ends. Extraction was performed using tested extraction solvents under
tested temperatures at 1500 psi, 5 min preheat, 5 min static, 2 cycles, 60% flush volume
and 120 sec purge time. The final volume was approximately 20 mL. Extracts were
diluted to a final volume of 200 mL with de-ionized water and further cleaned and
concentrated using the SPE procedure outlined in the following section before analysis.
2.2.4 Solid-Phase Extraction Procedure
The SPE procedure was optimized using spiking experiments with de-ionized
water, surface water and influent and effluent from the Oregon WWTP. Aliquot of 200
mL water samples were spiked with 200μL of 100μg L-1 mixed standards. Two SPE
13
cartridges, LC18 (3 mL, Sigma-Aldrich, St Louis, MO) and ENVI-18 (6 mL, 0.5g,
Sigma-Aldrich, St Louis, MO) were tested for their performance at sample pH 3 and 7.
Different percentages of acetone in water were tested as wash solutions to reduce matrix
effects without affecting the recoveries.
The SPE procedures were as follows: SPE cartridges were pre-conditioned with
6mL acetone then 6 mL de-ionized water. Aliquots of 200 mL of filtered water sample
were adjusted to tested pH using H2SO4 and NH3H
· 2O, spiked with internal standards (40
μL, 500 μg L-1), and loaded onto pre-conditioned tested cartridges at a rate of ~10 mL
min-1. After loading, the cartridges were washed with tested wash solutions. Analytes
were then eluted into glass test tubes with 6 mL acetone. The eluants were brought to
dryness under a gentle nitrogen stream for subsequent derivatization and analysis.
2.2.5 Derivatization
Estrone, 17-estradiol, 17α-estradiol, 17α-ethinylestradiol and estriol need to be
derivatized before (LC) instrumental analysis due to their low-ionization abilities.
Prepared standards and samples were derivatized following a previously published
method by Anari et al., (2002). Briefly, nitrogen-dried standards or samples from SPE
were re-dissolved with 100 μL sodium bicarbonate buffer and vortexed for 1 min. Then,
100 μL dansyl chloride (1 mg L-1) was added into each sample. After vortexing for
another 1 min, samples were incubated at 60 oC for 3 min. Finally, the solution was
evaporated to dryness under a gentle nitrogen stream and re-dissolved in 0.4 mL
methanol, transferred into 2 mL amber glass vials and stored at -20 oC until instrumental
analysis. Target compounds at different concentrations (10, 50, 100 and 200 μg L-1) were
14
derivatized with 100μl and 200μl 1 mg L-1 dansyl chloride to test the efficiency of the
derivatization.
2.2.6 Liquid Chromatography-tandem Mass Spectrometry (LC-MS/MS)
Analysis
Derivatized standards or samples were analyzed using LC-MS/MS. The
instrument consists of a ProStar 210 solvent delivery module with a ProStar 430
autosampler and a Varian 1200L triple-stage quadrupole mass spectrometer with a dual
off-axis ESI interface (Varian Inc., Walnut Creek, CA). Single standards of target
compounds including internal standards which were used to compensate for matrix
effects and instrument variability were directly injected into the mass spectrometer to
determine the optimized instrumental parameters, such as needle, shield and capillary
voltages. Then LC columns along with mobile phases based on polarity and miscibility of
target hormones were tested under different solvent gradients. The combination providing
the best compounds signal and separation was selected.
Analytes were separated using a Nova Pack C18 column (150×4.6 mm, 4 μm)
with guard column (Waters, Milford, MA). Mobile phase A was 0.1% formic acid in
water and mobile phase B was acetonitrile. The flow rate was 0.3 mL min-1. The gradient
started with 40% B, ramped to 73% in 5 min, ramped to 78% in 20 min, increased to
100% in 2 min, held for 5 min, then returned to 60% in 2 min and held 6 min to reach
equilibrium. The instrument was operated under positive ionization mode and the
instrumental settings were: drying gas temperature, 300oC; shield voltage, 150 V; needle
voltage, 6000 V; collision gas pressure, 2.0 mTorr. The MS/MS breakdown parameters
15
are listed in Table 2-1. 17-β-estradiol-D3 and estriol-D2 are internal standards used to
compensate for the matrix effects and instrument variability.
Table 2-1 MS/MS breakdown parameters
Compounds
Progesterone
Melengestrol-acetate
Estrone
17-α-estradiol
17-β-estradiol
17-β-estradiol-D3
Estriol
Estriol-D2
17-α-ethinylestradiol
MW
Parent ion
Mode
314.46
396.5
270.37
272.38
272.38
275.4
288.38
290.4
296.4
315.1
397.2
504.3
506.3
506.3
509.2
522.2
524.2
530.3
positive
positive
positive
positive
positive
positive
positive
positive
positive
Needle Shield
6000
6000
6000
6000
6000
6000
6000
6000
6000
150
150
150
150
150
150
150
150
150
Capillary
PI
CE
PII
CE
55
42
110
110
110
110
115
115
119
96.9
279.1
171
171
171
171
171
171
171
18.5
19
31.5
32.5
32.5
32.5
32
32
33
108.9
337.1
156
156
156
156
156
156
156
24
13.5
45
45
45
45
45
45
45
2.2.7 Statistic Analysis
All experiments in method development did in triplicate. Analysis of variance
(ANOVA) was conducted using SPSS v15.0 software (Chicago, IL). One way ANOVA
Holm-Sidak test was performed to determine statistical differences between treatment
combinations. A probability level of P<0.05 was selected to delineate treatment
differences.
2.3 Sorption Experiments
2.3.1 Materials and Methods
Screw top round bottom glass centrifuge tubes (50mL), purchased from Pyrex
(Lowell, MA) were used for the sorption experiment. This thesis is part of a larger project
evaluating the use of wetlands for remediation of several contaminants. Dennis Topsoil
16
(Toledo, OH) had previously been selected as the wetland substrate for the larger project.
Therefore, our sorption experiments also used Dennis Topsoil soil. Soil samples were airdried, sieved to less than 0.2 cm and stored at room temperature (22-23 °C).
Characterization of the selected soil was determined following “Particle size
distribution analysis” method from USDA NRCS laboratory method manual (USDA and
NRCS, 2004). The selected soil is a sandy loam soil with 66.10% sand, 20.92% silt and
12.99% clay. The organic matter content was measured by loss on ignition and was
3.86%. Soil pH was measured in water (1:1) and in 0.01 M CaCl2 solution and was 7.28
and 7.20, respectively.
Sorption of the seven selected hormones onto the test soil was determined using
batch equilibrium experiments in dark condition at room temperature (22-23 °C)
following Organization for Economic Co-operation and Development (OECD) guidelines
(OECD, 2000). The soil/solution ratio was 1/20 g mL-1. Soil samples were autoclaved
using a Barnstead Harvey autoclave (Dubuque, IA) at 120 oC for 15 min before use to
prevent biodegradation. All the experiments were performed in triplicate to allow
estimation of variance. Adsorption on the surface of the test vessels was tested and was
negligible.
Sorption kinetic experiments at one concentration of the test substances were
conducted to determine equilibrium time (<5% change between samples) to be used in
the subsequent sorption experiment and the degree of adsorption of test substances at
equilibrium. Aliquots of 2 g of soil and 40 mL of 0.01 M CaCl2 were added in 50 mL
glass centrifuge tubes and agitated overnight using a reciprocal shaker. Afterwards 40 µL
of a 10 mg L-1 mixed hormone standards were added to each tube to achieve a solution
17
concentration of 10 µg L-1. Samples were agitated and aliquots were collected at 2, 6, 14,
24, 48, and 72 h. Then samples were centrifuged at 2000 g for 15 min, 20 mL of
supernatant was collected and extracted for analysis using the previously described
method. A blank sample with the same amount of soil and CaCl2 solution but without test
hormones was included as a background control.
Sorption of the test hormones to the selected soil at five different concentrations was
determined using batch equilibrium experiments. Centrifuge tubes were filled with 2 g
Dennis Topsoil and 40 mL 0.01 M CaCl2 and shaken overnight. Afterwards mixed
hormone standards were spiked into tubes to achieve solution concentration of 0.5, 1, 5,
10 and 20 µg L-1. The lowest concentration used was based on the predicted equilibrium
aqueous concentration above the instrumental detection limits. The content of organic
solvent in the aqueous phase was less than 0.1% to minimize the influence of solvent.
Samples were shaken for 48 hours, which was determined by the kinetics experiment for
equilibrium. After agitation, samples were collected and extracted as mentioned above.
Blanks were included for background controls.
In all blank samples no target compounds were detected, hence no background
adjustment was made.
2.3.2 Data Analysis
Adsorption Ati is defined as the percentage of test substance adsorbed on the soil.
Ati was calculated at each time point ti, according to equation (1):
Ati 
Caq ti  100
Co
%
(1)
18
where Caq(ti) is the aqueous phase concentration measured at time point ti, Co is
the aqueous phase concentration at the beginning of the test.
The distribution coefficient Kd is the ratio between the concentration of the test
substance in the soil phase and in the aqueous solution. After sorption equilibrium is
reached, Kd was calculated using equation (2):
Kd 
C s eq 
Caq eq 
(2)
where Cs(eq) is the content of the test substance in the soil phase, Caq(eq) is the
concentration of the test substance in the aqueous solution at equilibrium.
When only aqueous phase concentrations, Caq were measured, solid phase
concentrations, Cs were calculated using equation (3):
Cs 
mtot  Vaq  Caq
(3)
ms
where mtot is the total mass of added analyte, Vaq is the volume of aqueous phase and ms
is the mass of solid phase.
The data from the adsorption experiments were described using a Freundlich
isotherm:
(4)
n
C s  K f  C aq
where Kf is Freundlich distribution coefficient and n is the linearity constant. When n = 1,
equation (4) is converted to the linear distribution equation (5):
Cs  K d  Caq
(5)
where Kd is the linear distribution coefficient.
19
2.4 Degradation Experiments
2.4.1 Materials and Methods
Mason jars (120 mL) purchased from Ball Corporation (Muncie, IN) were used
for the degradation experiments.
Dennis Topsoil was used in the degradation
experiments.
Water-holding capacity of the soil was determined by the method described by
Wang et al. (2001). 50 g soil sample was packed into a PVC pipe (4 cm in internal
diameter), fitted with glass fiber and a screen net at one end. The pipe was immersed in
deionized water for 24 hours, and then freely drained through a funnel for 12 hours. Pipe
with soil was weighed before and after drainage. Water holding capacity was calculated
by equation (6):
H
(Ws  Wd ) 100
%
ms
(6)
where Ws is the mass before drainage, Wd is the mass after drainage and ms is the mass of
the soil used.
Due to instrumental problems, only progesterone and melengestrol-acetate were
tested in degradation experiment. Degradation was performed under aerobic conditions.
Twenty-four mason jars were each filled with 20 g soil. Soil moisture was adjusted to
50% water holding capacity using de-ionized water. Each sample then was spiked with
0.2 mL of 10 mg mL-1 progesterone and melengestrol-acetate standard solution prepared
in methanol, to reach a theoretical concentration of 1 µg g-1. This concentration is
simulating the high concentration that had been reported in soils (Hanselman et al.,
2003). To allow methanol to evaporate, each sample was put under a ventilation hood for
20
15 min. Soil samples were then well mixed with a stainless steel stirring rod; jars were
covered with lids and incubated in the dark at room temperature (22-23 °C). Samples
were collected at specified times (0.5, 3, 8, 21, 45, 69, 93 and 117 h). Collected samples
were freeze-dried and 2 g sub-samples were taken and analyzed for the two compounds
using the previously described method. Concentrations of samples from 0.5 h were used
as initial concentrations.
2.4.2 Data Analysis:
The degradation kinetic data were fit using a first-order model and twocompartment model:
The first-order model is described as follows:
Ct  Co  e  kt
(7)
where Co (mg kg-1) is the initial concentration, Ct (mg kg-1) is the concentration at time t
(hour) and k (h-1) is the rate constant. The half-life, t1/2 (h) was calculated by equation (8):
t1 
2
ln( 2)
k
(8)
The two-compartment model was described in equation (9):
Ct  C1  e  k1t  C2  e  k2 t
(9)
where C1 and k1 are initial concentration and rate constant for compartment one and C2
and k2 are initial concentration and rate constant for compartment two. In this model,
rapid degradation of compounds is used in compartment one with much slower
degradation being used in compartment two due to sorption onto the soil.
Isotherm parameters were estimated using SPSS v15.0 software (Chicago, IL).
21
2.5 Constructed Wetland Microcosm Experiments
2.5.1 Materials and Methods
Sodium bromide was supplied by Fisher Chemicals (Fair Lawn, NJ). Bromide ion
electrode was purchased from Cole-Parmer Instrument Company (Vernon Hills, IL).
Wetland microcosms were packed in fiberglass nesting boxes (cells, shown in figure 2-1)
from U.S. Plastic Corp. (Lima, OH). Carter peristaltic pump was purchased from Thermo
Fisher Scientific Inc. (Waltham, MA). The Dennis Topsoil used in this study was, airdried, sieved to less than 0.2 cm and stored at room temperature (22-23 °C) before use.
Figure 2-1 Fiberglass nesting box, A=60.96 cm, B=20.32cm, C=15.24 cm, D=53.66 cm,
E=12.07 cm, F=14.605 cm
A subsurface flow system was employed in the two constructed wetland
microcosms. The system was built with a hydraulic head difference between the free
surface inlet and a height variable outlet, which in this case was fixed at 9 cm above the
bottom of the cell in both cells. First, both ends of the cell were packed with gravel, held
in place with cardboard during packing, to promote equal flow. Volume of the wetland
22
cell (13 L) was calculated and the mass of the soil particles (10 kg) needed was measured
before packing. Soil was added to the cell incrementally in thirds to minimize uneven
packing. The depth of each layer was recorded as reference for the other cell to get
consistent packing. The cardboard was removed after all three batches of soil were
packed. The completed setup had soil substrate lengths of 45-46 cm and gravel ends of 78 cm widths as shown in Figure 2-2. The microcosms were covered with aluminum foil
during the whole experiment to reduce evaporation of water and to prevent photodegradation of hormones.
(a) Map view
(b) Side view
Figure 2-2 (a), (b) Wetland microcosm setup
Two microcosm setups were tested in this experiment. Sodium bromide solution
at a concentration of 200 mg L-1 was run through each microcosm at a flow rate of 1 mL
min-1 to simulate loading (500 m3 ha-d-1) (EPA, 1988) in a constructed wetland. The
water level was kept below the surface at the inlet. Effluent was collected in 200 mL
beakers during three hour periods and measured using a bromide ion electrode. Due to
the detection accuracy range of the bromide ion electrode, a 25 mL sample was diluted to
23
100 mL with de-ionized water and 2 mL Ionic Strength Adjuster (ISA), 5M NaNO3
before measurement. The bromide ion electrode was calibrated before use and every two
hours during use to avoid interference. Bromide breakthrough curves of the cells were
compared to determine whether the wetland cells were uniformly packed. The average
hydraulic retention time (HRT), theoretical amount of time required for a given flow to
pass through a unit process, for each cell was determined using these breakthrough
curves.
The removal efficiency of seven selected contaminants in the microcosm system
was tested under two different hydraulic loading rates, simulating high (500 m 3 ha-d-1)
and low (150 m3 ha-d-1) (EPA, 1988)loadings in constructed wetlands. De-ionized water
spiked with the seven target hormones was introduced into the inlet of each cell and was
collected at the outlet. The spiked water had a calculated concentration of 1 µg L-1 in both
microcosms. This concentration was at the high end of those reported in effluents from
concentrated animal feeding operations (CAFOs) (Zheng et al., 2008) and represent the
worst case scenario. Inputs were controlled by a peristaltic pump at 0.3 and 1 mL min -1.
Microcosm effluent was collected every 48 hours and 200 ml of each sample were
analyzed for hormone residuals using the previously developed methods. The experiment
lasted 32 days. Afterwards, six soil column samples centrally located and equally spaced
along the length of each microcosm were collected. Soils in each column were well
mixed. Due to the instrumental problem, 5 g soils were only analyzed for progesterone
and melengestrol-acetate residuals.
2.5.2 Data Analysis
24
Microcosm data: Bulk density (ρb) of soil substrate in the microcosm was
calculated by:
b 
ms
V
(10)
where ms is the mass of the soil, and V is the volume of soil after packing.
Average water velocity (V) in each cell was calculated by:
V
Q
A n
(11)
where Q is the volumetric flow rate, A is the cross section area of the saturated
microcosm, and n is the porosity of the soil.
Bromide tracer test: Breakthrough curves were plotted using bromide
concentrations (Ct) at time t vs. time (t). HRT was determined as the time when Ct
reached half the initial concentration (Co).
Retardation factor: Retardation factors (Rf) of target hormones in the microcosm
were calculated using an equation presented by Domenico and Schwartz (1997):
Rf  1
b
n
(12)
 Kd
Removal efficiency: Removal efficiencies (RE) of target hormones in the
microcosm were calculated by:
RE 
Ct
100%
Co
(13)
where Ct is the concentration of the target hormone at time t, and Co is the initial
concentration of the target hormone.
The total removed mass ( mr) was calculated by:
mr  Co  RE  Q  t
(14)
25
Chapter 3
Results and Discussion
The main objective of this thesis was to determine the viability of using
constructed wetlands to remediate steroidal hormones under the environmental conditions
set forth in a larger project being funded through the USDA. To achieve this objective:
(1) analytical methods needed to be developed; (2) these methods were applied to
laboratory sorption and (3) degradation experiments; and finally these data were used to
(4) test a prototype of the microcosm system.
3.1 Analytical Methods Development
3.1.1 Optimization of ASE Procedure
Efficient isolation of hormones from complex matrices is required both to
eliminate analytical inferences caused by competing compounds in the matrices and to
improve detection for these compounds that are expected in very small quantities. Soil
samples were extracted using accelerated solvent extraction. The extraction efficiency
was tested using acetone at 60 and 100 oC and methanol at 60 oC. Results are presented in
Figure 3-1. Recoveries ranged from 13% to 130% for all tested compounds for all
26
methods. For all compounds the recovery was highest using acetone at 60 oC and lowest
for methanol. Recoveries for progesterone and melengestrol-acetate were the highest of
all compounds, averaging around 100% when acetone was used, while recoveries were
below 82% when methanol was used. Significant difference was found between method
using acetone at 60 oC and method using methanol (p=0.03, n=9) for progesterone, while
for melengestrol-acetate no significant difference was found between any of the methods.
For the other five compounds recoveries were above 40% when acetone was used, while
recoveries were below 16% when methanol was used. However, when acetone was used
at 60
o
C, recoveries for estrone, 17β-estradiol, 17α-estradiol, estriol and 17α-
ethinylestradiol were above 62%, but when acetone was used at 100 oC, recoveries were
below 50%. Significant difference was found between the three methods for each of these
five compounds (p<0.001, n=9). The recoveries achieved by the method using acetone at
60 oC for all target compounds in soil ranged from 63% for estriol to 119% for
progesterone, which fall in the range of reported recoveries (60-130%) for hormones in
soil from the literature. The final optimized ASE procedure utilized acetone at 60 oC for
the best recoveries achieved.
27
Figure 3-1 Recoveries of different ASE methods
3.1.2 Optimization of SPE Procedure
Although the ASE extraction was optimized to recover as much of the target
compounds as possible, the extract still contains compounds that can interfere with
analytical detection. Therefore, an intermediate step to remove the unwanted compounds
needed to be optimized as well. For the extraction of hormones from water, two reverse
phase SPE cartridges (LC-18 and ENVI-18) were tested. The reverse phase sorbent can
adsorb non-polar compounds while allowing polar compounds and salts to escape. pH
plays an important role in this procedure with too high or too low pH causing the
bounded phase to hydrolyze or cleave. To maximize retention, the pH should be adjusted
28
to make sure the desired analyte is not charged. Target analytes here are weak acids (pKa
>10) with low polarity. Their ionization in water can be affected by pH, which might
affect their extraction efficiency. Thus SPE recovery was tested using de-ionized water at
pH 3 and 7, which may meet the pH condition that the reversed phase cartridge required.
Recoveries of two tested cartridges under two sample pHs are given in Figure 3-2.
Figure 3-2 Recoveries of SPE cartridges at different pH
Recoveries for most analytes averaged 80% under both pH conditions for both
cartridges. Statistic analyses showed no significant differences between recoveries under
sample pH3 and pH7, and no significant differences between recoveries for the two
cartridges under either pH. This may be attributed to the low polarity of these
29
compounds. Even at pH7, the majority of compounds exist in neutral form. pH7 was
chosen for these analytes since most of the environmental samples have near neutral pH.
ENVI 18 was chosen for further testing based on its low cost compared with LC 18 and
increased flow rate during SPE loading, likely due to the larger diameter of the ENVI-18
cartridge.
Matrix effect: Matrix effects have been identified as one of the main drawbacks of
ESI-MS. Interference caused by the presence of co-extracted constituents, such as salts,
leads to a difference of response (either higher or lower) between a standard solution and
sample extract. In order to assess the matrix effects from different sources, the SPE
extract from environmental samples, including surface water from Ottawa River, and
influent and effluent from Oregon WWTP, were spiked with mixed standards after
having been filtered. Results are presented in Figure 3-3. Matrix effects were calculated
as the ratio of the signal in post-SPE spiked samples with blank sample signals having
been subtracted, to that of standards made in solvent at the same spiked concentration
levels.
30
Figure 3-3 Different matrix effects
In general, effects of different matrices for all target compounds ranged from 29%
to 110%, averaging around 90%. Average value for progesterone, melengestrol-acetate,
17β-estradiol, 17α-estradiol, estriol and 17α-ethinylestradiol were close to 100%
indicating no effect on signal for the matrices tested. No significant differences were
found for these five compounds. Only estrone showed significant effects from matrix
interferences (p<0.001, n=9) differences between the influent sample (30%) and surface
water ditch sample (50%). This suggests that if this protocol is used to evaluate estrone
from influent, effluent and surface waters the data will be „underreported‟ and that
analyses for this compound will necessarily have higher detection limits than the other
compounds included in this study.
31
However, to reduce the matrix effects for estrone, a higher percentage of organic
solvent can be used in the SPE wash step as long as analyte recovery remains acceptable.
Therefore, different percentages of acetone in water (10, 20, 30, and 40%, v/v) were
tested on surface water samples. Results are given in Figure 3-4.
For most compounds recoveries from different wash solutions were statistically
insignificant, with variation in recoveries being no more than 30%, except estriol and
progesterone. Recoveries of estriol under the higher percentage wash solutions were
lower than 15%, while that under the lower percentage wash solution increased to greater
than 86%. Unfortunately, the opposite trend was noticed for progesterone, where
recoveries increased with increasing percentage of acetone in the wash solution
(p<0.001). Recoveries for estrone were around 100% for all concentrations of organic
solvent tested, which are about 100% better than the recoveries reported above for
protocol using no organic solvent (average<50%). Therefore, the final acetone percentage
was chosen as a compromise to optimize the majority of compounds, including estriol,
estrone and progesterone. Since recoveries for progesterone using 20% of acetone were
an acceptable 75%, which was 15% higher than using 10% acetone (p=0.006) and the
20% solution provided insignificant changes or improved results for the other compounds
except estriol, 20% acetone in water was selected for the remainder of this study.
32
Figure 3-4 Recoveries using different percentages of acetone in wash solution
The final optimized SPE procedure utilized ENVI-18 SPE cartridge, auueous
sample adjusted to pH 7 and 20% acetone in water as wash solution.
3.1.3 Optimization of Derivatization
Due to the low polarity of five of the seven tested compounds, estrone, 17βestradiol, 17α-estradiol, estriol and 17α-ethinylestradiol, their ionization efficiency is low
in ESI-LC-MS/MS systems and results in poor detection efficiencies. A derivatization
step to add a more polar functional group to the structure of these compounds was
employed to enhance ionization. Progesterone and melengestrol-acetate cannot be
33
derivatized and their signals were not affected by the derivatization procedure. Analyses
using two concentrations (100 and 200 l) of the derivatizing agent, dansyl chloride,
were evaluated to assess improvement in compound recovery.
Analyses were not
replicated and cannot be used for statistical analysis; however, the data for the trial are
given in Figure 3-5.
Signals for all five compounds appeared to be slightly affected by the volume of
derivatization agent. When standard concentrations were less than 100 μg L-1, higher
signals were obtained by using 100 μL of dansyl chloride. But when standard
concentrations were higher than 100 μg L-1, a higher signal was obtained by using 200 μL
of dansyl chloride. This suggests that more dansyl chloride is needed for complete
derivatization of high concentration samples; while at low concentration excessive dansyl
chloride may suppress the signal. However, without replicate data, these trends may just
be artifacts of the natural variability in the analyses. However, considering the low
concentrations (ng L-1 range) in environmental matrices (Ying et al., 2002), 100 μL of
dansyl chloride was adopted for the derivatization.
34
Figure 3-5 Derivatization efficiencies for five hormones at two concentrations each with
increasing concentration of derivating agent (dansyl chloride).
3.1.3 Method Validation
Method performance was evaluated by determining recovery, sensitivity,
linearity, and repeatability on surface water samples spiked with target compounds.
Method performances for water and soil samples are given in Tables 3-1 and 3-2. The
35
quantification limits (LOQ) of the method were the concentrations calculated from the
instrument quantification limit (IQL) which was defined as the minimum amount of
analyte in standard solution with a signal to noise ratio of 10.
Table 3-1 Method performance for surface water samples
Compounds
progesterone
melengestrol-acetate
estrone
17α-estradiol
17β-estradiol
estriol
17α-ethinylestradiol
LOQ (ng L-1)
n=3, 200ml
1.65
1.80
0.19
0.04
1.09
0.14
0.06
r2
Recovery (%)
RSD (%)
0.9989
0.9997
0.9995
0.9988
0.9996
0.9995
0.9993
91
77
95
97
95
96
94
6.04
2.78
1.46
1.81
3.03
1.57
1.00
Table 3-2 Method performance for soil samples
Compounds
progesterone
melengestrol-acetate
estrone
17α-estradiol
17β-estradiol
estriol
17α-ethinylestradiol
LOQ (ng g-1)
n=3, 5g
0.42
0.45
0.09
0.02
0.56
0.08
0.03
r2
Recovery (%)
RSD (%)
0.969
0.9723
0.9979
0.996
0.9971
0.9951
0.998
119
105
77
72
71
62
73
13.63
15.47
1.46
4.45
3.61
0.84
7.76
LOQ values for surface water samples ranged from 0.04 ng L-1 for 17α-estradiol
to 1.80 ng L-1 for melengestrol-acetate, while LOQ values for soil samples ranged from
0.02 ng g-1 for 17α-estradiol to 0.45 ng g-1 for melengestrol-acetate. Units of LOQ values
for water samples are 1000 times smaller than those for soil samples, indicating that the
LOQ values for water samples are much lower than those for soil. Linearities were
represented by the correlation coefficient (r2) of the calibration curves of each compound
and were very good for both matrices, being above 0.99 for the water samples and 0.96
36
for the soil samples. The overall recoveries ranged from 77% for melengestrol-acetate to
97% for 17α-estradiol for water samples and 62% estriol to 119% for progesterone for
soil samples. Relative standard deviations (RSD %) were evaluated from three replicates
and were below 7% and 16% for water and soil samples respectively. The sensitivities of
the developed methods for water and soil samples are comparable to or exceed other
methods reported in the literature (Gabet et al., 2007).
3.2.6 Method Application
The optimized method was tested by analyzing surface water samples from
several streams and water bodies in northwest Ohio impacted by agricultural and urban
runoff. The information about the sample sites are listed in Table 3-3 (samples collected
on 10/16/2009) and sample sites are shown in Figure 3-6. Analytical results are shown in
Figure 3-7. These sites were chosen based on their setting in drainage ditches within
agricultural fields where animal and/or biosolid wastes may have been applied or their
location near WWTP effluent discharge. In some of these sites noted with an asterisk in
Table 3-3, the occurrence of pharmaceutical and personal care products (PPCPs) were
reported (Wu et al., 2009b). Other sites, notably the Lake Erie sample at the entrance of a
drainage ditch, have been studied for their high numbers of E. coli and their possible
association with biosolids or septic discharges having been repeatedly detected.
Table 3-3 Water sample sites information (samples collected on 10/16/2009)
Site
Latitude
Longitude
NO. on map
site type
Lake Erie
41.68633
Big Ditch
41.67768
-83.37225
4
entrance of Berger Ditch
-83.40974
45
agricultural*
Heckman Ditch
41.67388
-83.42968
15
agricultural*
Sautter Ditch
41.67488
-83.35167
19
agricultural*
37
Wolf Creek
41.6749
-83.37132
28
agricultural
Maumee River
41.64583
-83.53101
115
urban
Sleepy Hollow Pond
41.65148
-83.63743
116
suburban
DS Haskins WWTP
41.48021
-83.71043
117
downstream of rural WWTP
Ottawa River DS
41.6585
-83.6172
79
suburban
Ottawa River US
41.68773
-83.7629
113
suburban
Swan Creek DS
41.63204
-83.58737
91
suburban
Swan Creek US
41.55669
-83.73776
114
suburban
Note: “DS” downstream, “US” upstream. “*”occurrence of pharmaceutical and personal
care products reported.
The five naturally occurring hormones including progesterone, estrone, 17βestradiol, 17α-estradiol, and estriol were found in many water samples, while the two
synthetic compounds melengestrol-acetate and 17α-ethinylestradiol were under their
detection limits for all samples. The total of the five compounds ranged from 42.50 ng L-1
in the Lake Erie sample to 5.86 ng L-1 in the Swan Creek upstream sample. The five
highest total hormone concentrations were found in Lake Erie (42.50 ng L-1, near the
entrance of Berger Ditch, which drains Wolf Creek into Maumee Bay), Big Ditch (40.84
ng L-1), Heckman Ditch (38.24 ng L-1), Sautter Ditch (32.82 ng L-1) and Wolf Creek
(28.77 ng L-1) all of which drain farm fields. Since these sites are in agriculture areas, the
most likely reasons for the high concentrations could be animal waste or biosolids
applications (Soto et al., 2004). Studies have shown the presence of many pharmaceutical
compounds which have been transported to these surface waters following biosolids land
application (Wu et al., 2009b). High concentrations of total hormones were also found in
the Maumee River (26.75 ng L-1), Sleepy Hollow Pond (25.55 ng L-1) and downstream of
Haskins WWPT (24.39 ng L-1), mostly in urban and suburban areas. The occurrences are
likely attributed to the use of septic systems in these areas. High concentrations found
downstream from WWPT indicates the weakness of the traditional wastewater treatment
38
to remove these compounds. Lower concentrations were found in two residential streams
(Swan Creek and Ottawa River) in areas with less likelihood of septic systems or WWTP
discharge. However, in each river, concentrations of total hormones upstream were lower
than downstream and concentrations in Swan Creek (5.86 and 10.80 ng L-1 in upstream
and downstream, respectively) were lower than in the Ottawa River (14.95 and 16.40 ng
L-1 in upstream and downstream, respectively), which may have been caused by
population variation along the river.
Figure 3-6 Water sample sites in northwest Ohio
39
Figure 3-7 Hormones found in water samples from northwest Ohio
Estrone and 17α-estradiol had the highest detection frequency among the five
detected compounds in this area. Estrone had the highest concentrations. Estrone is the
highest concentration hormone naturally excreted by women (3-20 µg person-1 day-1) and
female animals, and also by men (5 µg person-1 day-1) (Belfroid et al., 1999). This could
be a possible reason for the high concentration of estrone. 17β-estradiol was only
detected in one sample above the detection limits. With the presence of microbes and
oxygen, 17β-estradiol is mostly metabolized to estrone (Colucci et al., 2001). The small
amount of estriol at several sample sites may result from the gradual degradation of
estrone into estriol (Colucci et al., 2001). Progesterone was detected in only one sample
from Wolf Creek, suggesting the natural source (pregnant animals) for this compound in
this area. The detected hormones in this area are naturally occurring while synthetic
40
hormones were below detection limit. According to their half-lives reported in the
literature (Zuo et al., 2006), they must have entered the tested waterways no more than
1.5 days prior to testing. The concentrations of tested hormones found in this area were at
the same level as reported in other areas in the U.S. (Kolpin et al., 2002).
Overall, the developed methods for aqueous samples provide excellent data for
the northwest Ohio. Results showed the occurrence of several natural hormones in this
area.
3.3 Sorption of Hormones on Soil
Compounds detected in water samples illustrate only half of the problem
associated with hormones released into the environment. Based on their physical and
chemical properties, some hormones may be more likely to sorb onto the surfaces of soil
particles where they can remain for extended periods of time and continually be released
into waterways long after land application has ceased. Sorption kinetics are used to
calculate how fast a compound is sorbed and how long it will take for sorption to reach
equilibrium. Equilibrium time varies according to the different mechanisms of sorption
for different compounds. Usually, metals display almost instantaneous equilibrium,
which is mostly affected by their ionic charges and size. However, organic compounds,
such as pharmaceutical and personal care products (Wu et al., 2009a) and hormones often
require longer periods to reach equilibrium. Sorption kinetics of tested hormones in this
study are shown in Figures 3-8. Equilibrium is defined at the point where the remaining
aqueous concentrations of a compound change by less than 5% between sampling times.
41
Figure 3-8 Sorption kinetic results of hormones. Data are presented as concentration
remaining in solution over time.
At 3 hours, almost 85% of progesterone and melengestrol-acetate were sorbed
onto soil indicating a rapid sorption of these two compounds. Sorption of progesterone
and melengestrol-acetate reached equilibrium within less than 12 h with less than 10% of
either compound remaining in solution. Sorption of the other five compounds reached
equilibrium within 48 h. At equilibrium, about 10% of 17α-ethinylestradiol, estrone, 17αestradiol and 17β-estradiol were left in solution, while more than 15% of estriol was left.
From 24 to 72 h, the percentage of the progesterone and melengestrol-acetate left in
aqueous phase was stable thus 48 h was selected as the sampling time used in the
following batch equilibrium experiments.
Whereas the kinetic studies are used to determine equilibrium time, the batch
isotherm studies are used to describe the extent of sorption and the possible mechanisms
of sorption. Linear and Freundlich sorption isotherms constructed from the results of the
42
48-hr batch equilibrium experiments are shown in Figure 3-9 and Figure 3-10,
respectively. The linear distribution coefficients (Kd), Freundlich distribution coefficients
(Kf) and linearity constants (n) are shown in Table 3.1.
Figure 3-9 Linear sorption isotherms
Figure 3-10 Freundlich sorption isotherms
43
Table 3-4 Parameters of Freundlich and linear models
Freundlich distribution coefficient
Linear distribution coefficient
n
Kf (Ln Kg-n)
R2
Kd (L kg-1)
R2
progesterone
1.097
251.015
0.998
414.951
0.993
melengestrol-acetate
1.178
151.670
0.995
389.544
0.992
17α-ethinylestradiol
0.892
103.157
0.978
50.609
0.979
estrone
0.839
128.795
0.976
43.704
0.974
17α-estradiol
0.830
136.836
0.978
43.605
0.975
17β-estradiol
0.856
93.735
0.971
35.061
0.968
estriol
0.845
49.820
0.966
16.212
0.960
The linear sorption isotherms showed the sorption of progesterone and
melengestrol-acetate being the greatest, estriol being the least and 17α-ethinylestradiol,
estrone, 17α-estradiol and 17β-estradiol being intermediate between these extremes. Kd
values for linear sorption isotherms range from 16 to 415 L kg-1 for these hormones
attraction to the sandy loam soil. The Freundlich sorption isotherms indicate the same
relative sorption strength for the target compounds as indicated by Kd values. The Kf
values range from 49.82 Ln Kgn for estriol to 251.02 Ln Kgn for progesterone.
The experimental data were well fitted using both linear equations and Freundlich
equations. However Freundlich model fittings (R2>0.966) were slightly better than linear
sorption model fittings (R2>0.960). The n value in the Freundlich equations is close to
one for progesterone, suggesting that within the tested concentration range the sorption of
progesterone was close to linear, which indicates that in a wetland system constructed
using the sandy loam soil, the ratio of progesterone sorbed would not be affected by the
concentration introduced. Whereas for melengestrol-acetate, n is larger than 1, indicating
that the sorption strength increases as concentration increases. The ratio of melengestrol44
acetate sorbed by the sandy loam soil in the wetland system would be lower under low
introduced concentrations, while the ratio sorbed would be higher under high introduced
concentrations. For the rest of the compounds, n values are less than 1, suggesting that
the sorption strength decreases as concentration increases. Unlike melengestrol-acetate,
the ratio of these five compounds sorbed by the sandy loam soil in the wetland system
would be higher under low introduced concentrations, while the ratio sorbed would be
lower under high introduced concentrations. Sorption would be minimized when
melengestrol-acetate is introduced at low concentrations and 17α-ethinylestradiol, estrone,
17α-estradiol, 17β-estradiol and estriol are at high concentrations in the wetland system.
Even though the data from this study fit the Freundlich model and Kf better than
the linear model, much of the literature data report only Kd values. Therefore, Kd values
are used here for comparison. Values for sorption of progesterone and melengestrolacetate in soil could not be found in the literature. Sorption of estrone, 17β-estradiol, 17αethinylestradiol and estriol show a wide range of results in the literature, shown in Table
3-5.
Table 3-5 Sorption data reported in the literature
study
This study
Das, (2004)
Casey, (2005)
Hildebrand, (2006)
Kd (L kg-1)
soil type
clay
(%)
organic
(%)
estrone
17βestradiol
sandy loam
13.00
3.85
43.7
35.1
sand
6.10
0.22
3.4
3.6
-
-
silt clay loam
21.20
2.91
48.1
83.2
-
-
silt loam
26.00
9.20
93.0
84.0
-
-
sand
0.20
0.94
407.0
34.0
27.0
-
silt loam
5.81
2.66
-
-
30.0
-
clay loam
29.66
3.07
-
-
96.0
-
silt clay
48.69
3.76
-
-
121.0
-
45
17-α
ethynylestradiol
50.61
estriol
16.21
Karnjanapiboonwong,
(2010)
sand
0.00
0.10
4.7
3.8
1.0
2.2
sandy loam
16.00
1.30
67.7
115.8
176.2
8.6
silt loam
12.00
2.50
121.6
198.0
196.8
18.6
Note: “-” means none reported.
For estrone, some studies (Das et al., 2004; Karnjanapiboonwong et al., 2010)
showed increased Kd values with increasing soil organic content, suggesting sorption
capacity of estrone to soil substrate was directly related to organic carbon content. Das‟s
study also showed positive correlation between clay content and Kd value for estrone.
Casey (2005) reported high Kd value (93 L kg-1) for estrone onto a silt loam with high
organic content (9.2%), however this Kd value was lower than Karnjanapiboonwong‟s Kd
value for a silt loam soil with much lower organic content (2.5%). Hildebrand (2006)
reported that estrone sorbed rapidly and strongly (Kd=407 L kg-1) to a coarse textured soil
(sand) with low organic carbon content (0.94%), which disagrees with most of other
reported data. The tested soil has similar organic content and showed a similar Kd value
for estrone as that soil tested in Das‟s study (2004), which suggest that the sorption of
estrone onto the tested soil was most likely affected by both organic and clay contents of
the soil substrate.
For 17β-estradiol, Das (2004) and Karnjanapiboonwong (2010) reported a similar
trend as estrone, increased Kd value with increasing soil organic content. However, Das
(2004) also showed a increased Kd value with increasing clay content for 17β-estradiol.
Moreover, Casey reported a similar Kd value (84 L kg-1) on 17β-estradiol as Das‟s (2004,
Kd=83.2 L kg-1), which has similar clay content (21.2%) as Casey‟s soil (26%),
suggesting that the sorption of 17β-estradiol on soil is affected by clay content. In this
study, the tested soil has lower clay content (13%) than Das‟s (2004) silt clay loam
46
(21.2%) and Casey‟s (2005) silt loam (26%), which could explain the lower generated Kd
value (35.1 L kg-1) of 17β-estradiol on the tested soil than the other two.
Hildebrand (2006) reported increased Kd value for 17-α ethynylestradiol with
increasing of both clay and organic contents of soil substrate. However, the Kd value for
17-α ethynylestradiol for the soils in Hildebrand‟s study (27-121 L kg-1) were much
lower than the ones for soil in Karnjanapiboonwong‟s study (1-1986.8 L kg-1) with
similar clay and organic content as that in Hildebrand‟s study. In this study, the Kd value
(50.62 L kg-1) was consistent with that in Hildebrand‟s (30-96 L kg-1).
Estriol had lower reported Kd value both in the literature (2.2-18.6 L kg-1,
(Karnjanapiboonwong et al., 2010)) and in this study (16.21 L kg-1) than the other three
hormones discussed in this section. Karnjanapiboonwong‟s study suggested that estriol
was most likely only sorbed by organic compounds in soil. The Kd value generated for
estriol in this study was in the same range as in Karnjanapiboonwong‟s study, even
though this study‟s soil had slightly higher organic content.
The sorption for these four compounds generated in this study was quite
consistent with the reported values in the literature and illustrate the need to know the
texture and organic content of the substrate. The relatively strong sorption suggests that
the mobility of these four compounds in a wetland system constructed using the sandy
loam soil would be low. This study also shows that sorption of progesterone (Kd=414.95
L kg-1) and melengestrol-acetate (Kd=389.54 L kg-1) is stronger than the others, likely due
to their higher hydrophobicities. The mobility of these two in wetland systems would be
even lower than the other compounds. However, special attention should be paid to
estriol in a wetland system constructed using the sandy loam soil, since estriol would be
47
more mobile than any other tested hormones. To better understand fate and mobility,
degradation rate of these compounds also needs to be investigated.
3.4 Degradation of Hormones in Soil
Other than sorption, information on degradation is important for determining the
fate of hormones in the environment. First-order and two-compartment models are most
frequently used to describe the degradation kinetics of organic compounds. The first
order model assumes a constant relationship with time of rate of degradation and
remaining concentration. A two-compartment model assumes two compartments in the
substrate, in one of which degradation is rapid and the other degradation is slow (Kolz et
al., 2005). In this study, the degradation kinetics of progesterone and melengestrolacetate were described with both models. The degradation is shown as concentrations of
target hormones at time t (Ct) versus time in Figure 3-11. Fitting results are shown in
Figures 3-12 and the values of model-fitting parameters are shown in Tables 3-6 (a) and
(b).
48
Figure 3-11 Residuals of progesterone and melengestrol-acetate in soil with time during
the degradation experiment
Figure 3-12 Model fitting results of progesterone and melengestrol-acetate
49
Table 3-6 Model-fitting parameters
(a) First-order model
rate constant k (h-1)
hale-life t1/2(day)
r2
progesterone
0.029
1.06
0.879
melengestrol-acetate
0.012
1.94
0.945
(b) Two-compartment model
C1
k1
C2
k2
r2
progesterone
30.746
0.467
69.467
0.017
0.982
melengestrol-acetate
19.227
0.182
82.847
0.009
0.985
Both progesterone and melengestrol-acetate degraded rapidly in the first 24 hours.
Concentration of progesterone dropped from 566.91 to 317.68 µg Kg-1 (or 56%), while
melengestrol-acetate dropped from 581.01 to 425.31 µg Kg-1 (or 73%) in 24 h. Then the
degradation rate decreased from 24 h to 117 h. However at the end of the experiment
(117 hours), 13% and 31% of progesterone and melengestrol-acetate, respectively, were
left in solution.
Using first-order degradation model, which assumes a constant relationship with
time of rate of degradation and remaining concentration, the calculated half-life for
progesterone and melengestrol-acetate are 1.06 and 1.94 days, respectively. However the
two-compartment model produced a better correlation co-efficient for both compounds
(r2>0.98), suggesting the existence of the slow degradation compartment. The decrease in
degradation rate with time may correspond to sorption to soil and/or migration into
micro-pores in the soil matrix where the compounds are less accessible to microbes.
Based on the sorption experiment, strong sorption of these two compounds for the media
50
used is a reasonable cause for degradation rate decrease. If this experiment time was
extended beyond 117 hours, the hormones would be expected to slowly desorb to replace
the compounds in solution (that would continue to degrade) to maintain the equilibrium
concentration ratios described in the previous section of this study. In a remediation
wetland scenario, the sorption sink for these two compounds, and their continued input
from sorbed concentrations for an extended period of time would need to be considered
both in the substrate composition and residence time of contained waters in the system.
The fact that 31% of melengestrol-acetate remained unchanged after 117 days might
necessitate a long residence time before wetland waters can be considered safe for
discharge, with respect to this compound. The short calculated half lives, obtained from
the first order model are misleading and do not accurately represent the state of
degradation after an extended period of time.
Degradation of the other five hormones was not tested in this study. But in the
literature, half-life for 17β-estradiol in 100% non-sterilized soil at room temperature was
only 0.17 days (Xuan et al., 2008), mostly due to the effect of microbes in the soil. Even
in 20% of non-sterilized soil the half-lives for estrone, 17β-estradiol, estriol and 17αestradiol were 2.7, 0.92, 1.5 and 1.9 days, respectively (Xuan et al., 2008). Other studies
have reported low persistence in soil of these hormones with reported half-lives less than
a few days (Das et al., 2004; Lee et al., 2003). Based on the sorption data in this study,
these compounds could degrade even before they reach the sorption equilibrium in the
tested soil, which would further slow the transportations of these compounds and reduce
their concentrations in wetland discharge.
Besides microbial consideration, abiotic factors, such as photodegradation and
51
redox can affect degradation of hormones. The half-life of estrone under aerobic
conditions was much shorter than under anaerobic conditions (Ying and Kookana, 2005).
These researchers also suggested that 17-α ethynylestradiol, 17β-estradiol, estrone,
estriol, which may be present in reclaimed wastewater, would not persist in well-aerated
soil.
According to these results and literature review, in designing a successful wetland
microcosm for remediating these seven hormones, the following hypotheses were made:
(1) the five hormones (17-α ethynylestradiol, 17α-estradiol, 17β-estradiol, estrone and
estriol) with lower Kd and Kf values are expected to degrade rapidly and be effectively
removed from the system; (2) progesterone and melengestrol-acetate are also expected to
be effectively removed, however strong sorption of these two hormones would affect the
degradation and required residence time within the system.
To ensure the best
environment for degradation, the microcosm will be kept shallow, to prevent the redox
environment from becoming anaerobic.
3.5 Removal of Hormones through Constructed Wetland
Microcosm
3.5.1 Hydraulic Conditions
Soil substrates in the microcosms achieved a specific bulk density of 1.37 g cm-3.
Assuming a particle density of 2.65 g cm-1 (i.e. the density of quartz is 2.65 g cm-3), the
calculated porosity was approximately 50%. The cross sectional area was calculated as
52
138 cm2. Average velocities in the microcosm were calculated as 3.6×10-5 cm s-1 and
1.2×10-4 cm s-1 from volumetric flow rates of 0.3 and 1 mL min-1, respectively. Then
hydraulic retention time (HRT) in each cell was calculated as 7.2 and 2.2 days from
volumetric flow rates of 0.3 and 1 mL min-1, respectively. The calculated HRT in both
microcosms was longer than the sorption equilibrium time for the target compounds (up
to 2 days), which is sufficient to ensure that sorption should occur inside the microcosm.
Breakthrough times for compounds that are not affected by sorption onto soil
mineral or organic matter are used to test the packing consistency of soil substrate in
microcosms. In this experiment, bromide was used. The bromide breakthrough curves in
two different cells under the same volumetric flow rate (1 mL min-1) are shown in Figure
3-13.
Figure 3-13 Bromide breakthrough curves from two different microcosms under the same
flow rate 1 mL min-1
HRT was determined from these curves as 1.3 and 1.5 days in cell#3 and cell#2
respectively. The two curves agree well, suggesting good consistency in the packing
53
between the microcosms. The calculated HRT (2.2 days) from the velocity is greater than
the measured HRT determined by the bromide breakthrough curves, suggesting either
heterogeneity in the distribution of the porosity in the system that resulted in preferential
pathways, or a miscalculation of the bulk density. The porosity in the microcosm may be
lower at the bottom than at the top. This may cause the approximated porosity used to
calculate the velocity to be higher than the real porosity, which will ultimately cause a
longer calculated HRT. Alternatively, the negative ionic charge of the bromide ion may
cause the travel time to be lowered as repulsion forces it through the system. Whatever
the cause of the discrepancy, the similarity between the two curves in Figure 3-13 and
their smooth appearance indicate that the microcosms were prepared well and that the
sorption and degradation data will not be skewed drastically by inconsistencies in the
design.
The difference in the travel time of sorbed contaminant to the travel time for
water is defined as the retardation factor for that contaminant under the defined
environmental conditions (Domenico and Schwartz, 1997). When a contaminant displays
a linear sorption, the retardation factor can be calculated by the linear distribution
coefficient. The data in Section 3-3 above indicate that all of the tested hormones show
sorption that is close to linear, thus validating the use of this technique for this study. The
calculated
retardation
factors
for
progesterone,
melengestrol-acetate,
17α-
ethinylestradiol, estrone, 17α-estradiol, 17-estradiol, and estriol are 1137, 1068, 139, 120,
120, 97 and 45, respectively, suggesting that their sorption should highly retard the
transportation of these compounds through the tested media, especially progesterone and
melengestrol-acetate.
54
3.5.2 Fate and Transport of Hormones in Wetland Microcosm
Effluents from each microcosm were collected and analyzed for tested hormones
to investigate the removal efficiencies of the microcosm under two different flow rates.
The concentration for each tested compound introduced into both microcosms was 1 µg
L-1, which made the introduced total mass for each compound under high and low flow
rate 46.08 and 13.82 µg , respectively. At the end of the study, hormones in six soil
columns from each microcosm were analyzed for residue of progesterone and
melengestrol-acetate. The concentrations of tested hormones in effluent samples from
each cell are listed in Tables 5.1 (a) and (b). Note that, during the first day of
experiments, the water table at the outflow end of the microcosm under high flow rate
was set higher than 9 cm above the bottom, which brought the water table at the inlet end
too close to the surface. The water table was adjusted to 9 cm above the bottom
immediately after this was noticed. However, about 1.44 L flowed through the system
during this “accident”.
Table 3-7 Concentration (ng L-1) of tested hormones in effluents from microcosms
(a) Concentrations (ng L-1) in effluent from the microcosm under flow rate 0.3 ml L-1
Time(days)
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30
32
progesterone
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
melengestrol-acetate
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
17α-ethinylestradiol
-
-
6.89
2.75
1.46
0.61
0.73
0.32
-
-
-
-
0.22
0.24
-
-
estrone
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
17α-estradiol
-
-
0.31
-
0.28
0.29
0.30
0.20
-
-
-
-
0.28
0.29
-
0.37
17β-estradiol
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
estriol
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
55
(b) Concentrations (ng L-1) in effluent from the microcosm under flow rate 1 ml L-1
Time(day)
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30
32
progesterone
0
-
-
-
-
0.56
-
-
-
-
-
-
-
-
-
-
melengestrol-acetate
3.79
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
17α-ethinylestradiol
5.67
-
6.96
0.70
0.91
0.78
0.79
0.36
-
-
0.69
0.53
0.14
0.19
-
-
estrone
1.72
-
-
-
-
0.07
-
-
-
-
-
-
-
-
-
-
17α-estradiol
3.41
0.37
-
-
0.29
0.29
0.30
0.22
0.42
0.31
-
-
0.47
0.26
0.30
0.80
17β-estradiol
26.59
-
0.21
-
-
-
-
-
-
-
-
-
-
-
-
-
estriol
64.07
-
0.48
-
-
-
-
-
-
-
-
-
-
-
-
-
Note: “-” below detection limit.
Throughout the experimental time in the microcosm with low flow rate,
progesterone, melengestrol-acetate, estrone, 17β-estradiol and estriol in effluent were
below the detection limit. Only 17α-ethinylestradiol and 17α-estradiol were detected after
four days at concentrations about 150 and 3000 times lower than the input concentration
of 1 µg L-1. The concentration of 17α-estradiol remains constant for the remaining time
of the experiment, whereas 17α-ethinylestradiol concentrations steadily decline. This
much reduced concentration indicates that sorption and/or degradation is affecting all of
these compounds.
The accident mentioned above that occurred within the high flow rate microcosm
resulted in the contaminated waters filling the microcosm as the water table rose to the
surface. Inadvertently, this simulated the scenario where a body of contaminated water is
emptied rapidly into receiving waters and the subsequent faster flow rates as the waters
run through the system. The data at the first sampling time (day 2) shows the conditions
directly after this flood event. Estriol had the highest concentration 64.07 ng L-1 at day
two, and also the lowest Kd value (Kd=16.21 L Kg-1) among all target hormones. On the
other hand, progesterone had the highest Kd value (Kd=414.95 L Kg-1) and was under the
detection limit in the day 2 sample. The concentration of the rest of the compounds also
corresponded to their Kd values, suggesting that sorption plays a significant role in the
56
retention of contaminants. Once the high flow rate system was brought under control the
data paralleled the data described above for the low flow system, with 17αethinylestradiol and 17α-estradiol showing the same concentrations and trends with time.
Even after flooding the system the other five hormones are not detected in the effluent.
Soil samples in the six cores from each microcosm were analyzed for tested
hormone residues. Residuals of progesterone and melengestrol-acetate from the head to
the end of the microcosm under high flow rate are listed in Table 3-8. Assuming the
volume of the “overflow” at day one is 1.44L, mass balances of progesterone and
melengestrol-acetate in the microcosm are shown in Table 3-9.
Table 3-8 Residues (ng Kg-1) of progesterone and melengestrol-acetate in microcosm
substrate under flow rate 1 ml L-1
1 (head)
2
3
4
5
6 (end)
progesterone
92.6
115.0
158.7
41.7
158.8
242.2
melengestrol-acetate
1110.9
-
-
-
-
-
Note: “-” means under detection limit.
Table 3-9 Mass (ng) balance of the progesterone and melengestrol-acetate in microcosms
total
residues
%
in effluent
%
degradation
%
progesterone
46080
1348.6
3%
1.6
0%
44731
97%
melengestrol-acetate
46080
1851.6
4%
5.46
0%
44228
96%
Progesterone residues generally increased in concentration from the head (92.6 ng
Kg-1) to the end (242.2 ng Kg-1) of the microcosm, while melengestrol-acetate residue
was only found at the head of the microcosm in high concentration 1110.9 ng Kg-1. Based
on the sorption kinetics, although progesterone and melengestrol-acetate are both sorbed
quickly by the soil substrate (85% and 86% sorbed at 3 hours for progesterone and
57
melengestrol-acetate, respectively, in the sorption kinetic experiments), melengestrolacetate may be preferentially sorbed. Thus, progesterone was transported down gradient.
This situation suggests that the overload of one quickly and strongly sorbed contaminant
may cause the failure of sorption of other contaminants in constructed wetland systems.
The sorption or transport of the other five hormones could also be affected by the
sorption of melengestrol-acetate.
The mass balance in the microcosm under high flow rate shows almost 97%
(44731 ng) of progesterone degraded and 3% (1348.6 ng) were sorbed during the
experiment period, while almost 96% (44228 ng) melengestrol-acetate degraded and 4 %
(1851.6 ng) were sorbed. Only 1.6 ng progesterone and 5.46 ng melengestrol-acetate
were detected in the effluent under high flow rate. Sorption and degradation both
contributed to the removal of progesterone and melengestrol-acetate; however
degradation dominated in the microcosm. Although progesterone (Kd=414.95 L Kg-1) has
stronger sorption to the soil substrate than melengestrol-acetate (Kd=389.54 L Kg-1),
faster degradation of progesterone (half-life=1.06 days) than melengestrol-acetate (halflife=1.94 days) could explain the lower total mass of residual of progesterone in the
microcosm.
In the microcosm under low flow rate, residuals of progesterone and
melengestrol-acetate were under detection limit, thus data are not shown here. The lower
total mass loading may have enhanced the degradation of these compounds in the
microcosm.
Although the residues of the other five compounds were not tested in this study,
the shorter reported half-lives and lower Kd values could predict low residues of these
58
compounds. Therefore the reasons for the concentrations of tested hormones in effluent
from microcosms under either low or high flow rate could be addressed as follows:
The highest detected concentration of 17α-ethinylestradiol in the microcosm
under low flow rate was 6.89 ng L-1 at day 6. This peak should be the result of dispersion
which caused by the heterogeneity of the soil substrate in the microcosm. But the low
detected concentrations of 17-α ethynylestradiol suggest that most of 17-α
ethynylestradiol was sorbed by soil substrate and eventually mostly degraded in the
microcosm. At high flow rate, 17-α ethynylestradiol in effluent had a high detected
concentration 6.96 ng L-1 after the “accident”, which is similar to that in the low flow rate
microcosm. This suggests that flow rates tested in this study have limited effect on the
dispersion of 17-α ethynylestradiol. The declining trend of 17α-ethinylestradiol in
effluent under either flow rate may indicate acclimation of microbes for this food source.
17α-estradiol is the optical isomer of 17β-estradiol. As they are structurally similar, the
environmental behavior should be very similar. The sorption experiments in this study
showed that 17α-estradiol (Kd=43 L Kg-1) had strong sorption to soil substrate in the
microcosm. Degradation of 17α-estradiol was not tested in this study, data could not be
found in the literature either. However, the possible reason for the near constant
concentrations of 17α-estradiol in the effluent under both high and low flow rate could be
the sorption and the degradation of 17α-estradiol reached equilibrium in the microcosm.
17-α ethynylestradiol and 17α-estradiol are less sorbed than progesterone and
melengestrol-acetate to the soil substrate in the microcosms, which leaves more available
for degradation. However, both of them were detected in effluent under either flow rates,
while progesterone and melengestrol-acetate were under the detection limit. This
59
suggests that even though contaminants have slower degradation, strong sorption would
hold contaminants in the microcosm long enough for degradation. When constructing a
wetland system, long HRT is highly recommended especially for those compounds
slowly degraded or weakly sorbed to the soil substrate.
For progesterone, melengestrol-acetate and estrone in microcosm under either low
or high flow rates, high retardation caused by sorption and fast degradation have resulted
in concentrations under detection limit in the effluent. For 17β-estradiol and estriol,
which have smaller retardation factors (less than 17α-ethinylestradiol and 17α-estradiol),
rapid degradation is likely.
Including data at day two, the overall removal efficiencies for all tested
compounds were above 94% and 99% under the high and low flow rates respectively.
Shappell et al. (2007) reported a decrease of 83-93% in estrogen activities in lagoon
constructed wetlands (Shappell et al., 2007). Gray and Sedlak (2005) reported a removal
efficiency of 36% and 41% for 17β-estradiol and 17-α ethynylestradiol , respectively, in a
full-scale surface flow constructed wetland (Gray and Sedlak, 2005). One of the possible
reasons for the high removal efficiency in this study compared to those reported in the
literature could be the absence of the matrix from wastewater. Complex matrices may
introduce competition for sorption of hormones onto the soil substrate and the
degradation by the microbes in the system. Furthermore, dissolved oxygen in wastewater
is generally lower than in de-ionized water, which would decrease degradation of the
hormones (Czajka and Londry, 2006).
Overall, although no differences in the removal efficiencies in two microcosms
were identified, the total removed mass (including residues in the microcosms, data
60
shown in Table 3-10) of the tested hormones from the microcosm under high flow rate
(322.36 µg) were three times that of the one under low flow rate (96.76 µg). Thus, the
flow rate 1 ml min-1 (high flow rate) was favored due to its good removal efficiency and
the higher performance.
Table 3-10 Total removed mass (µg) of hormones in microcosms
under high and low flow rate
Low
flow
rate
High
flow
rate
progesterone
melengestrolacetate
estrone
17αestradiol
17βestradiol
estriol
17αethinylestradiol
total
13.82
13.82
13.82
13.82
13.82
13.82
13.81
96.76
46.08
46.07
46.08
46.06
46.04
45.99
46.04
322.36
3.5.3 Correlation between Removal and Physico-chemical Parameters
The high removal efficiencies of the microcosms achieved in this study agree with
those predicted in the previous chapters. Strong sorption and acute degradation play
significant roles in the removal of tested hormones in the microcosm. Similar results of
removal caused by sorption and degradation were reported for estrogens (Song et al.,
2009) and selected pharmaceuticals (Matamoros et al., 2008) in constructed wetlands.
The pH values of the effluents from each microcosm were recorded and ranged from 7.42
to 8.09. Near-neutral pH values have been reported to be favored for removal of
pharmaceuticals and personal care products (Hijosa-Valsero et al., 2010). The shallow
depth could be another contribution to the good performance of the microcosm. García et
at. (2005) reported that shallower depths (< 0.27 m) have always proven to be more
effective than deeper depths (> 0.5 m) for the removal of COD, BOD5 and ammonia. One
study discovered that highest microbial biomass was in the top 10 cm of vertical
61
constructed wetlands, attributable to the good oxygen supply (Tietza et al., 2007). Redox
conditions significantly affect degradation of estrogens (Jürgens et al., 2002; Ying et al.,
2003). It is likely that water depth influences the biochemical reaction responsible for the
degradation of tested hormones in this study.
62
Chapter 4
Conclusions and Future Research
To achieve the main objective of this thesis, which was to determine the viability
of using constructed wetlands to remediate steroidal hormones under environmental
conditions, sorption and degradation experiments were carried out and finally data were
used to interpret the results of the microcosm experiments.
Relatively strong sorption (Kd) suggests that the mobility of estrone, 17βestradiol, 17α-ethinylestradiol and estriol in a wetland system constructed using a sandy
loam soil would be low. This study also shows that sorption of progesterone and
melengestrol-acetate is stronger than the other five hormones. The mobility of these two
in a wetland system would be even lower. However, special attention should be paid to
estriol in a wetland system constructed using the sandy loam soil, since estriol would be
more mobile than any other tested hormones due to the low Kd and Kf values.
The degradation study showed the strong sorption of progesterone and
melengestrol-acetate inhibited rapid degradation of these two compounds. An extended
period of time would need to be considered both in the substrate composition and
residence time of contained waters in the system. The five hormones (17-α
63
ethynylestradiol, 17α-estradiol, 17β-estradiol, estrone and estriol) with lower Kd and Kf
values are expected to degrade rapidly and be effectively removed from the system.
The microcosm study showed throughout the experimental time, progesterone,
melengestrol-acetate, estrone, 17β-estradiol and estriol in effluent were below the
detection limit in both the high and low flow rate microcosms. Only 17α-ethinylestradiol
and 17α-estradiol were detected in much lower concentrations than the input
concentrations. An “accident” in this study suggests that sorption is significantly
important to the removal of contaminants in a constructed wetland where a body of
contaminated water is emptied rapidly into receiving waters and the subsequent faster
flow rates as the waters run through the system. Residuals of progesterone and
melengestrol-acetate in soil columns from the microcosm showed competitive sorption
between the tested hormones and that degradation dominated in the microcosm. As
predicted in sorption and degradation studies, strong sorption of progesterone and
melengestrol-acetate retard the transport and benefits the degradation of these two
compounds. Fast degradation has resulted in good removal efficiency of estrone, 17βestradiol and estriol; even though estriol would be more mobile than any other tested
hormones. Nevertheless, 17α-ethinylestradiol and 17α-estradiol were less effectively
removed from the system than previously predicted. A longer HRT is highly
recommended for the compounds which are weakly sorbed to the soil substrate or slowly
degraded. Over all, high flow rate tested in this study was favored due to its good
removal efficiency and higher performance.
To improve performance some parameters such as implementing a buffer zone for
sudden inputs will result in a longer HRT, in which additional degradation can occur in a
64
constructed wetland. Shallow depths are needed to maintain aerobic conditions. 17αethinylestradiol and 17α-estradiol need to be monitored in the effluent, while
progesterone and melengestrol-acetate need to be monitored in the soil substrate in the
wetland system. Estriol needs to be monitored when there is a sudden input.
Referring back to Figure 3-7 which shows data from surface waters in northwest
Ohio, estrone is the hormone in highest abundance. This study indicates that estrone
should be easily eliminated from these waters if it was passed through a wetland system.
Estrone is produced in very high concentrations since it‟s highly excreted by both women
and men. Therefore, without degradation, the concentration of this compound would have
been much higher. Passing through a remediation wetland could reduce these
concentrations. 17α-estradiol is also frequently detected in these surface waters, which is
analogous to this microcosm study. This compound, although with significantly reduced
concentrations, seems to survive for an extended period of time. 17α-ethinylestradiol is
not noticed in the streams sampled in this study. In the microcosm the concentration of
17α-ethinylestradiol decreased with time. This might indicate that given enough time this
compound can be effectively eliminated from surface waters. The absence of
progesterone and melengestrol-acetate might indicate its lack of presence in the samples,
or its effective elimination over time.
For further research, target hormones in environmental matrices through the
optimized microcosm design should be tested to see if introduced competition from the
matrices would affect the removal efficiencies. Selected native wetland species would be
incorporated into the microcosms and tested for their influence on hormone removal.
Addition of plants can improve the treatment performance by increasing microbial
65
activities or incorporation into plant tissues (Dordio et al., 2010). To well assess the
mechanism of the removal of hormones under these conditions, degradation and residual
effects of estrone, 17α-ethinylestradiol, 17α-estradiol, 17β-estradiol and estriol have to be
tested.
66
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