Estrogenic Endocrine
Disrupting Compounds
Review and Analytical Procedures for
GC-MS and ELISA Analyses
January
TR 2010/005
Auckland Regional Council
Technical Report No.005 January 2010
ISSN 1179-0504 (Print)
ISSN 1179-0512 (Online)
ISBN 978-1-877540-52-3
Technical Report – first edition
Reviewed by:
Approved for ARC Publication by:
Name:
Judy-Ann Ansen
Name:
Paul Metcalf
Position:
Team Leader
Land and Water Team
Position:
Group Manager
Environmental Programmes
Organisation: Auckland Regionl Council
Organisation: Auckland Regional Council
Date:
Date:
24 March 2010
12 May 2010
Recommended Citation:
SINGHAL, N.; SONG, Y.; Johnson, A.; SWIFT, S. (2009). Estrogenic Endocrine
Disrupting Compounds. Prepared by UniServices for Auckland Regional Council.
Auckland Regional Council Technical Report Number TR 2010/005
© 2008 Auckland Regional Council
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Estrogenic Endocrine Disrupting Compounds
Dr Naresh Singhal
Dr Yantao Song
Anthea Johnson
Dr Simon Swift
Prepared for
Auckland Regional Council
Environmental Research
Uniservices Client Report
October 2009
Uniservices Project Number: 13454.00
Auckland Uniservices Ltd.
Department of Civil and Environmental Engineering and
Molecular Medicine & Pathology, School of Medical Sciences
University of Auckland
Private Bag 92019
Auckland
New Zealand
Contents
1
Executive Summary
2
2
Introduction
4
2.1
The Endocrine System
4
2.2
Endocrine Disrupting Compounds (EDCs)
5
2.2.1
Chemicals Displaying EDC Behaviour
5
2.2.2
Impacts
6
2.2.3
Mechanism of Action
9
2.2.4
Scope of Study
11
3
EDC Sources, Fate and Transport
12
3.1
Sources
12
3.2
Fate and Transport
12
3.2.1
e-EDC Degradation in Wastewater Treatment Plants
17
4
Concentration of EDCs in the Environment
19
4.1
Pesticides
19
4.2
Polychlorinated Biphenyls
21
4.3
Dioxins
21
4.4
Phenols
21
4.5
Phthalates
22
4.6
Estrogens
22
5
Methods for Analysing EDCs
24
5.1
Approach
24
5.1.1
Chromatographic Techniques
24
5.1.2
Non-Cellular Assays
24
5.1.3
Biologically Based Assays
25
5.2
Comparison of Analytical Techniques
27
5.3
Selection of Techniques for e-EDC Analysis
30
6
Determination of Steroid Estrogens in Seawater and Sediment using ELISA and
GC-MS
32
6.1
Materials and Methods
32
6.1.1
Chemicals and Materials
32
6.1.2
Preparation of Standard Solutions
32
6.1.3
Sediment and Artificial Seawater Sample Preparation
34
6.1.4
Extraction Protocol
34
6.1.5
GC-MS and ELISA Analysis
37
1)
Derivatisation-GC-MS analysis
37
2)
ELISA analysis
37
6.1.6
Method Validation
40
6.2
Results and Discussion
41
6.2.1
Optimisation of the Derivative Conditions prior to GC-MS
41
6.2.2
Standard Calibration and Detection Limits
42
1)
Derivatisation and GC-MS analysis
42
2)
ELISA analysis
45
6.2.3
C18 Cartridge Loading Capacity and Breakthrough
45
6.2.4
Recovery Efficiencies
47
1)
Spike Recovery Efficiencies Obtained For GC-MS Analysis
47
2)
Spike Recoveries Obtained For ELISA Analysis
49
6.3
Summary
50
References
51
Appendices
63
A)
Summary of Preliminary ELISA Analysis Results
63
B)
Typical Calibration Curves for E1, E2, and E3 ELISA Analysis
65
List of Tables
Table 1 EDC Effects
8
Table 2 Estrogenic Compounds
10
Table 3 EDC Pathways to Waterways
13
Table 4 Properties of e-EDCs
15
Table 5 Relative Potency of Environmental Pollutant e-EDCs
19
Table 6 Detected e-EDC Levels
20
Table 7 Estrogen concentrations in wastewater treatment plant effluent
23
Table 8 Non-Cellular Assays
25
Table 9 Organisms Used to Indicate EDC Effects
26
Table 10 In Vitro Bioassays
27
Table 11 E2 concentrations in wastewater
29
Table 12 Comparison of analysis methods
29
Table 13 Method Detection Limits for e-EDCs in Wastewater Reported in Literature
30
Table 14 Properties of E2 Commercial ELISA Assay Kits
31
Table 15 Properties of E3 Commercial ELISA Assay Kits
31
Table 16 Chemical and material used for e-EDC GC-MS and ELISA analysis
33
Table 17 Composition of artificial seawater
34
Table 18 Linearity and detection limits for e-EDC analysis with derivatisation-GC-MS
43
Table 19 Quantitation range and detection limits for e-EDC analysis with ELISA
45
Table 20 Loss of EDCs (pg/L) from SPE C18 cartridges during the loading capacity tests
47
Table 21 Recovery efficiency (R%
GC-MS
Table 22 Recovery (R%
RSD) of e-EDCs from seawater samples analysed using
48
RSD) of e-EDCs from sediment samples analysed using GC-MS 48
Table 23 Recovery R% ( RSD) of e-EDCs analysed with ELISA
49
Table 24 Testing Cayman E2 standards with Tokiwa E2 ELISA kit
49
List of Figures
Figure 1 Major endocrine glands
4
Figure 2 Estrogen Excretion
12
Figure 3 EDC Transport Pathways
14
Figure 4 Nonylphenol ethoxylate degradation
16
Figure 5 Estrogen Degradation
17
Figure 6 Wastewater treatment
18
Figure 7 NP in sewage sludge
22
Figure 8 Comparison of data from HPLC and ELISA analyses
28
Figure 9 Procedure for e-EDC extraction and analysis
35
Figure 10 Procedure of extracting target compounds using SPE
36
Figure 11 Procedure for E1 analysis with Tokiwa Chemical ELISA kit
38
Figure 12 Procedure for E3 analysis with Cayman Chemical ELISA kit
39
Figure 13 Sequence used for testing EDC breakthrough in SPE C18 cartridge
41
Figure 14 Effects of derivatisation time and temperature on GC-MS TIC peak area of e-EDCs
42
Figure 15 GC-MS chromatogram of TMCS derivatives of E1, E2 and E3 in SIM mode (2µL of
2000ng/L mixture standard)
43
Figure 16 Calibration curves of E1(a), E2 (b) and E3 (c) from GC-MS analysis.
44
Figure 17 Standard curves of E1(a), E2 (b) and E3 (c) obtained from ELISA analysis. Bcorrected standard binding absorbance, B0-corrected maximum binding absorbance.46
Figure 18 Mass (a) and fraction (b) of non-retained e-EDCs in breakthrough for increasing load
application.
47
List of Abbreviations
2,4-D
2,4-Dichlorophenoxyacetic acid
2,4,5-T
2,4,5-Trichlorophenoxyacetic acid
AChE
Acetylcholinesterase
APEs
Alkylphenol ethoxylates
BBAs
Biologically based bioassays
BPA
Bisphenol-A
BSTFA
Bis(trimethylsilyl)trifluoroacetamide
CMC
Critical micelle concentration
CPRG
Chlorophenol red β-D-galactopyranoside)
DBP
Di-n-butyl-phthalate
DDE
Dichlorodiphenyldichloroethylene
DDT
Dichlorodiphenyltrichloroethane
E1
Estrone
E2
17β-Estradiol
E3
Estriol
EDCs
Endocrine disrupting chemicals
e-EDCs
Estrogenic endocrine disrupting chemicals
EE2
17α-Ethinylestradiol
EEF
Estradiol equivalency factor
ELISA
Enzyme linked immunosorbent assay
ELRA
Enzyme linked receptor assay
ER
Estrogen receptors
ER-CALUX
Estrogen responsive chemically activated luciferase expression.
ERE
Estrogen responsive elements
FIA
Fluorescence immunoassay
GC-MS
Gas chromatography-mass spectrometry
GC-MS/MS
Gas chromatography-mass spectrometry (tandem)
hER
Human estrogen receptor
HGELN
HeLa Gal-ER-Luc-Neo transgenic human cell line
HPLC
High-performance liquid chromatography
HPLC/ESI-MS/MS High-performance liquid chromatography with positive electrospray
ionisation and tandem mass spectrometry.
IDL
Instrument detection limit
LC-MS/MS
Liquid chromatography-mass spectrometry (tandem)
LOQ
Limit of quantitation
MVLN
MCF-7-p-Vit-tk-Luc-Neo transgenic human cell line
NP
Nonylphenol
OP
Octylphenol
PCBs
Polychlorinated biphenyls
RIANA
River analyser
SIM
Single ion monitoring
S/N
Signal to noise ratio
SPE
Solid phase extraction
SPME–HPLC
Solid-phase microextraction high performance liquid chromatography.
TMCS
Trimethylchlorosilane
TMB
Tetramethylbenzidine
WHO
World Health Organisation
YES
Yeast Estrogen Screen
Reviewed by:
Approved for release by:
Formatting checked
………………………
1
1
Executive Summary
Endocrine disrupting chemicals (EDCs) are chemicals that can have an effect on the
endocrine system of animals including humans. Several EDCs have been detected in
water, air, and soil environments. Estrogenic EDCs (e-EDCs) are able to induce an
estrogen-like response in organisms and are of particular importance due to the
sensitivity of organisms to these compounds and their suspected links to particular
hormone-dependent cancers. This document presents an overview of e-EDC sources,
impacts on organisms, characteristics relating to transport and degradation in the
environment and wastewater treatment systems, and analytical methods that can be
used to quantify them.
The e-EDCs enter the environment from a range of point and non-point sources;
however, wastewater treatment plants are generally acknowledged as the primary
sources of e-EDCs in the environment. The most common e-EDCs listed in literature
are Estrone (E1), 17β-Estradiol (E2), Ethinylestradiol (EE2), Estriol (E3), Bisphenol A
(BPA), Nonylphenol (NP), Nonylphenol ethoxylates (NPnEO), and Octylphenol. Impacts
of EDCs have been reported for wildlife and in controlled laboratory studies. Because
of this, it is suspected that exposure to EDCs is the cause for a number of disorders in
humans. However, it has not been possible establish a direct link between EDC
exposure and a human disorder. Some countries have adopted a cautious approach
and placed bans on, or severely curtailed, the use of certain EDCs.
Most of the e-EDCs have low water solubility and are associated with solids. The
natural estrogens E1, E2 and E3 and the synthetic estrogen EE2 show variable
degradability in wastewater treatment systems. All of these estrogens are degradable
under aerobic conditions, with the rate of degradation being E3>E2>E1>EE2. Little
degradation of these estrogens is expected under anaerobic conditions. The long chain
NPnEO degrade under aerobic conditions to carboxylated NPnEO and short chain
NPnEO, which further degrade under anaerobic condition to NP. Degradation of these
compounds leads to a lowering of solubility and an increase in estrogenicity. The
highest concentrations of EDCs have been detected in sediments and wastewater;
however, the small amounts present in air and drinking water can also be
estrogenically active. There is little data available on the concentrations of EDCs
present in the local environment and therefore monitoring of EDCs in effluent
discharges and in receiving environments is necessary in order to assess the risk that
these compounds pose to New Zealand’s environment and communities.
Two approaches used to detect e-EDCs are the determination of specific chemical
concentrations and the quantification of overall estrogenicity. Specific chemical
concentrations can be determined using a variety of chromatographic techniques (e.g.
GC-MS/MS) and non-cellular assays. The estrogenic response can be assessed using
in vivo or in vitro bioassays. An assessment shows that all of the above methods have
2
advantages (such as high sensitivity) and disadvantages (such as requiring high level of
expertise to operate). Enzyme linked immunosorbent assay (ELISA) is a widely used
non-cellular assay that is available for specific EDCs. The use of ELISA kits can offer a
rapid and cost-effective test for quantifying e-EDCs. However, different kits can give
different responses and the ELISA assay can suffer from cross-reactivity between the
different estrogens present in the sample. GC-MS of derivatised samples can
overcome these problems while giving similar sensitivity to GC-MS/MS analyses.
Analytical methods for derivatisation-GC-MS and ELISA were developed for E1, E2,
and E3 using synthetically contaminated seawater and sediment samples. Procedures
for sample cleanup using solid phase extraction (SPE) cartridges were developed and
their loading capacity determined. Various solvents for tested to determine the solvent
giving the highest efficiency of estrogen extraction from the SPE cartridges. The
calibration curves for GC-MS showed excellent linearity, while those for the ELISA kits
were in close agreement with the results provided by the kit manufacturers. The limit
of quantitation (LOQ) for ELISA was estimated to be slightly higher than that of the
GC-MS.
3
2
2.1
Introduction
The Endocrine System
The endocrine system is a combination of glands and the hormones that they produce,
which affect biological reproduction, growth, development, and behaviour of animals,
including humans. The major glands of the endocrine system (Figure 1) are the
hypothalamus, pituitary, thyroid, parathyroids, adrenals, pancreas, pineal body, and the
reproductive organs (ovaries and testes). Hormones are chemical messengers created
by the body to transfer information from one set of cells to another in order to
coordinate the functions of different parts of the body. They are secreted by ductless
endocrine glands into the bloodstream and transported to receptors where they trigger
responses.
Figure 1 Major endocrine glands
Major endocrine glands. (Male left, female on the right) 1. Pineal gland 2. Pituitary gland
Thyroid gland
4. Thymus
5. Adrenal gland 6. Pancreas
3.
7. Ovary 8. Testes. (Figure obtained
from SEER Training Modules, 2009).
Hormones are grouped into three classes – amines, peptides, and steroids, with most
hormones being peptides. Amines are derived from the amino acid tyrosine and are
secreted from the thyroid and the adrenal medulla (Purves et al., 1995). Peptides are
short chains of amino acids, molecules containing both amine and carboxyl functional
groups and are secreted by the pituitary, parathyroid, heart, stomach, liver, and
kidneys. Steroids are lipids derived from cholesterol and are secreted by the gonads,
adrenal cortex, and placenta. These are the main hormones controlling sexual
Estrogenic Endocrine Disrupting Compounds - Review
4
determination, differentiation and development, and can be further distinguished as
estrogens and androgens. In mammals, the main estrogens are estrone, estriol and
17 -estradiol (responsible for many female sex characteristics), while the main
androgens include testosterone (the male sex hormone) and 5 -dihydrotestosterone.
In fish, the main androgen is 11-ketotestosterone. The estrogens and androgens are of
particular importance because of their central role in reproductive function.
An important objective of the endocrine system is to maintain some form of
homeostasis, avoiding wild swings in hormone levels or responses that might
otherwise have detrimental metabolic effects (Norman and Litwack, 1998).
Homeostasis is achieved using cycles and feedback to regulate physiological functions
and the secretion of hormones, a process that can be thought of as the ‚seesaw‛
principle. Increased secretion of hormone A, which regulates production of hormone B,
causes increased secretion of hormone B, which in turn exerts negative feedback to
regulate the secretion of hormone A (WHO, 2002). In this way, homeostasis (i.e., the
correct levels of A and B) is maintained. As per this simplified representation of the
hormonal regulatory process, the target cells send feedback signals (usually negative
feedback) to the regulating cells, with the result that secretion of the target cell–
stimulating hormone is altered (usually reduced) by one or more of the products of the
target cells (Darlington and Dallman, 1995).
2.2
2.2.1
Endocrine Disrupting Compounds (EDCs)
Chemicals Displaying EDC Behaviour
EDCs are ubiquitous in our environment and can be found in all media (air, water, soil).
They are detected in food products (soybeans, legumes, flax, yams), plants
(phytoestrogens present in fruits, vegetables, beans, grasses), household products
(degradation products of detergents and associated surfactants, including nonylphenol
and octylphenol), pesticides (DDT, endosulphan, atrazine, nitrofen), plastics (bisphenol
A, phthalates), pharmaceuticals (drug estrogens - birth control pills, cimetidine),
industrial chemicals (PCBs, dioxin and benzo(a)pyrene), by-products of incineration,
paper production, and fuel combustion, and metals (cadmium, lead, mercury) (PUBH
5103, 2003a).
EDCs may be natural or synthetic. For example, plants such as soybeans and garlic
produce EDCs as a defence mechanism. However, most of the known EDCs are
synthetic chemicals, such as pharmaceuticals, phthalates (used as plasticizers),
alkylphenols (industrial detergents), and bisphenol A (food packaging). In addition, a
large number of chemicals act as potential EDCs, i.e., these exogenous substances or
mixtures possess properties that might be expected to lead to endocrine disruption in
an intact organism, or its progeny, or (sub)populations (EC, 1996). Several
comprehensive lists of known and potential EDCs have been compiled – for example,
Estrogenic Endocrine Disrupting Compounds - Review
5
a comprehensive list of 966 proven and potential EDCs has been compiled by IEH
(2005) while another list of 95 known EDCs is available from www.ourstolenfuture.org
(Our Stolen Future, ND).
2.2.2
Impacts
Over the past two decades concerns have been expressed over the potential adverse
effects caused by exposure to chemicals that can alter the normal functioning of the
endocrine system (WHO 2002). It is well understood that EDCs can block, mimic,
stimulate or inhibit production of natural hormones, and disrupt homeostasis. As such,
endocrine disruption is not a toxicological endpoint, but a functional change leading to
adverse effects (McGovern and McDonald, 2003; WHO, 2002).
EDC exposure can result in a diverse range of physiological and behavioural effects in
both wildlife and humans. These include abnormal sex determination (e.g., uneven sex
ratios in offspring), sexual differentiation (e.g., imposex - the presence of male
reproductive organs in females; and intersex – the presence of both male and female
reproductive organs) and development (delay in sexual maturity, reproductive disorders
and failure). The adverse effects of EDCs gained attention in the late 1940s and early
1950s when significant population decline of the bald eagle, the national symbol of the
United States, was first observed in Florida and subsequently in other states (Broley,
1952). This has since been attributed to eggshell thinning as well as aberrant
behavioural changes and physical deformities resulting in lower offspring survival from
exposure to EDCs from pesticides and polychlorinated biphenyls (Colborn et al., 1996).
EDC-induced physiological changes can also affect reproduction, as was discovered
during the 1970s when population decline in the dog-whelk (Nucella lapillus) along the
south coast of England was found to be caused by the presence of male sex
characteristics in females (Bryan et al., 1986). In this case, breeding was prevented
due to the development of a penis in females which blocked the oviduct, preventing
egg release and ultimately resulting in female death. Cases of ‘imposex’ such as this
have been linked to exposure to the anti-fouling agent tributyl tin (TBT) (Bryan et al.,
1987). Imposex in dog-whelk has been found to occur at TBT concentrations of only
1ng/L, while complete reproductive failure, female sterility and local extinctions have
been observed where environmental concentrations of TBT have been in the 6-8ng/L
range (Bryan et al., 1986). There is a strong correlation between imposex in the whelk
Buccinum undatum and the intensity of shipping traffic (Tenhallers-tjabbes et al.,
1994) and no whelk populations in Europe have been found to be unaffected by TBT
(Oehlmann et al., 1996). While TBT is a masculinising rather than estrogenic EDC, this
example highlights the significant impact on the survival of marine populations that
may result from exposure to even low concentrations of EDCs.
Estrogenic Endocrine Disrupting Compounds - Review
6
Larger animal species are no more immune: the 1980s saw a population decline of the
alligator in Lake Apopka, Florida, due to reduction in male penis size and low plasma
testosterone levels (Guillette et al., 1996), presumably the result of a spill of the
organochlorine dicofol in the lake. In the mid 1980s a female yolk protein vitellogenin
was discovered in the blood of freshwater male fish ( Rutilus rutilus) in a British river
(Purdom et al., 1994). This was thought to be due to exposure to one or more of the
many chemicals released into the river from sewage discharges.
Additionally, the timing of EDC exposure can influence sex determination, sexual
differentiation and sexual development. In reptiles and some fish species, the gonadal
and genetic sex is determined by the environmental temperature during a critical
period of embryonic development. However, exposure to EDCs during the incubation
period while the sex is undefined may alter the sexual determination of the embryo.
Further stages of differentiation and development may therefore also be influenced by
EDCs. In mammals and birds, sexual determination is genetically controlled, thus
exposure to EDCs will not affect the sex of offspring. However, embryo exposure to
EDCs can influence sexual development and behaviour (Vom Saal, 1981). In fish, birds
and some amphibians, sexual differentiation is also changeable, and thus may be
influenced by e-EDC exposure at abnormal concentrations or times. A summary of
representative environmental health effects from exposure to potential and known
estrogens is presented in Table 1.
Human exposure to EDCs has led to speculation on linkages to a range of disorders
including cancers of the reproductive organs and decreased sperm count in males.
Whilst high levels of exposure to some EDCs could theoretically increase the risk of
such disorders, no direct evidence is available at present. Correlations have been found
in some cases, while other studies have not confirmed the trends. In the Netherlands,
lower sperm counts were found to correlate with higher levels of PCBs in blood serum
(Dallinga et al., 2002). Trends in the incidence of some of these disorders are difficult
to discern and, when they are found, are difficult to interpret because of
inconsistencies in method. EDCs are but one of a variety of potential risk factors, both
environmental and genetic. On the basis of limited animal data, identified
environmental EDCs appear to pose minimal risk to humans on their own, but the risk
from mixtures of compounds is unknown (The Royal Society, 2000).
The US has already banned the use of some known EDCs – PCBs, DDT, and chlordane
– because of their carcinogenic effects rather than their estrogenic effects. Many
putative EDCs appear in EPA’s National Toxics Rule and in state regulations governing
discharges of toxic substances. Among European countries, Norway has banned the
production import, distribution and most uses of nonylphenol and octylphenol
ethoxylates – these two compounds along with ethinylestradiol account for 90% of the
wastewater estrogenic potential (McGovern and McDonald, 2003).
Estrogenic Endocrine Disrupting Compounds - Review
7
Table 1 EDC Effects
Environmental effects linked to estrogens present in wastewater. (Cited from Teske and Arnold,
2008.)
EDC
SAMPLE SITE
SPECIES
EDC EFFECT
Mix of WW with
PCB, PBDE,
APEOs, pesticides
(hormones not
identified)
Potomac River,
Washington, DC
Micropterus
dolomieu/ small
mouth bass
Intersex (oocytes in testes)
WWTP effluent –
unidentified mix of
compounds
United Kingdom:
WWTP receiving
waters (rivers)
Rutilus rutilus/
roach fish
Intersex (vitellogenin, ova, and
tissue changes) characteristics in
males
Bisphenol A
Review of
several studies
Human
Prostate cancer development
Bisphenol A
Review of
several lab
studies
Human
Polycystic ovary syndrome,
uterotrophic effects, decreased
sperm, increased prolactin
release
Octylphenol
Lab study
Fisher 344 and
Donyru/2 rat
strains
Persistent estrus
Ethinylestradiol
(EE2)
Lab study
Oryzias latipes/
Medaka fish
Intersex in males: testes ova and
abnormal tissue development
Ethinylestradiol
(EE2)
Review of
several studies
Human
Prostate cancer development
Nonylphenol (NP)
Lab study
Sprague-Rawley
female rats
Irregular estrous cycles and
advanced onset of tissue
development
Nonylphenol (NP)
Lab study
Human males
Decrease in sperm production
17 -Estradiol (E2)
Review of
several studies
Rats
Delay in age of first estrus and
vaginal opening; irregular then
persistent estrus; disorders in
ovarian and mammarian
development
17 -Estradiol (E2)
Field study
Chrysemys
pictal/ female
painted turtles
Increased E2 levels needed for
vitellogenin induction of female
eggs
APEs: Alkylphenol ethoxylates (including nonylphenol ethoxylates)
PCBs: Polychlorinated biphenyls
Estrogenic Endocrine Disrupting Compounds - Review
8
2.2.3
Mechanism of Action
EDCs can alter the functioning of the endocrine system by a variety of different
mechanisms (PUBH 5103, 2003a; Soto et al., 1995):

By mimicking the sex steroid hormones (estrogens and androgens) by binding to
their natural receptors (i.e., behaving as agonists, and binding to receptors to
produce a similar response).

By antagonizing the effects of hormones (i.e., blocking hormones or preventing the
binding of hormones to receptors).

By altering the pattern of synthesis and metabolism of natural hormones.

By modifying the production and functioning of hormone receptors by altering
hormone receptor levels.
Much of the environmental research has focussed on EDCs that are able to induce
estrogen-like responses in organisms, referred to as estrogenic EDCs (e-EDCs),
because of the central role of estrogen in reproductive function. This category of
chemicals includes both natural and synthetic estrogens (e.g., xenoestrogens and
pseudoestrogens). Specific examples of e-EDCs include natural hormones and
pharmaceutical estrogens (e.g., E2, EE2 and phytoestrogens including isoflavonoides
and coumestrol), surfactants (e.g., alkyphenol-ethoxalates), pesticides (e.g., atrazine,
dieldrin, and toxaphene), industrial chemicals (e.g., bisphenol A), and heavy metals
(e.g., cadmium, nickel, lead). These e-EDCs are environmentally significant as many of
these have the potential to cause an estrogenic response at very low concentrations
(parts per billion to parts per trillion) (Campbell et al., 2006).
In order to bind to steroid receptors, e-EDC compounds must have structural
similarities to the steroid hormones. Compounds that actively mimic estrogens are
based on polycyclic hydrocarbons and are able to exist in a planar form (Waller et al.,
1996). Due to differences in the chemical structures of natural hormones and e-EDCs,
it is not possible to use the chemical structure as the sole means of determining
whether a chemical is an endocrine disruptor or not. Also, because the structures of
endocrine disruptors are so variable and unpredictable, they are sometimes
synthesized unintentionally as by products in chemical manufacture (PUBH 5103,
2003a). Examples include polychlorinated biphenyls (PCBs) and the pesticides DDT and
lindane – all of which have estrogenic activity and were originally synthesised for a
completely unrelated purpose. Table 2 presents the structural diversity among
chemicals in the environment reported to be estrogenic.
Estrogenic Endocrine Disrupting Compounds - Review
9
Table 2 Estrogenic Compounds
Examples of estrogenic chemicals found in the environment and their sources. (Adapted from
McLachlan, 2001).
STEROIDS
Estrone (E1)
17β-Estradiol (E2)
PHARMACEUTICAL
17α-Ethinylestradiol (EE2)
Estriol (E3)
PESTICIDES
Dichlorodiphenyl
trichloroethane (DDT)
2,4-Dichlorophenoxyacetic
acid (2,4-D)
ENVIRONMENTAL POLLUTANTS
Bisphenol A (BPA)
Polychlorinated Biphenyls
(PCBs)
Nonylphenol
PHYTOESTROGENS
Flavones
Estrogenic Endocrine Disrupting Compounds - Review
Coumestrol
Coumarin
10
2.2.4
Scope of Study
While EDCs have been monitored in studies based overseas, there is a lack of
research specific to New Zealand. The need for such research is highlighted by the
current activity in Australia, which bears similarity to New Zealand in terms of its
location, peoples living conditions and expectations of standard of life, on monitoring
EDCs in the Australian environment. The effects of EDCs are being investigated in all
Australian state capitals, and monitoring tools are used in Brisbane, Canberra,
Melbourne and Adelaide. Research into EDC and PCPP treatment is being conducted
in Sydney and Wollongong (Kookana et al. 2007).
This document presents an overview of the sources, characteristics, degradation in
wastewater systems and natural environment, environmental impact, and methods of
analysis for EDCs with the aim of assessing the need for EDC quantification in the
Auckland Region and appropriate analytical methods. However, the literature on EDCs
is extensive and it is not possible for a single document to cover all aspects. The
emphasis of this report is on providing information relating to e-EDCs that manifest an
estrogen receptor mediated response (e.g. bisphenol A, E2 and EE2). As such, a wide
variety of EDCs are not specifically addressed in this report, including anti-estrogens
(e.g. dioxin, endosulphan), anti-androgens (e.g. vinclozolin, DDE), toxicants reducing
steroid hormone levels (e.g. fenarimol and other fungicides, endosulphan), toxicants
affecting reproduction primarily through effects on the central nervous system (e.g.
dithiocarbamate pesticides, methanol), and other toxicants that affect hormonal status
(e.g. cadmium, benzidine-based dyes).
Estrogenic Endocrine Disrupting Compounds - Review
11
3
3.1
EDC Sources, Fate and Transport
Sources
Wastewater treatment facilities are generally implicated as the major sources of eEDCs (Sumpter, 1995; Kolpin et al., 2002; Legler et al., 2002), the actual sources are
upstream discharges to these facilities. Potential upstream sources include natural
hormones and pharmaceutical estrogens flushed down home toilets, household
cleaners containing nonylphenol (NP), industrial processes using cleaners containing
NP and plastics containing Bisphenol A (BPA), or agrochemicals containing alkylphenol
and nonylphenol ethoxylate surfactants (Staples et al., 1998; Ying et al., 2002; Snyder
et al., 2003). The measured daily amounts of estrogens excreted by humans are
presented in Figure 2. Other potential sources of e-EDCs include agricultural practices
such as use of wastewaters from dairying and aquaculture (Kolodziej et al., 2004),
agricultural runoff containing estrogenic surfactants (e.g. nonylphenol ethoxylates) in
pesticide and fertilizer formulations (Staples et al., 1998; Ying et al., 2002), and
excretion of hormones in manure and urine from livestock feed lots (Hanselman et al.,
2003; Tashiro et al., 2003; Soto et al., 2004). Spawning fish may locally increase the
estrogen concentrations in river water (Kolodziej et al., 2004).
Figure 2 Estrogen Excretion
Daily amounts of estrogens excreted by humans (Cited from Sayles and Marsh, 2001).
3.2
Fate and Transport
EDCs persist in the environment and can bioaccumulate. Some EDCs (e.g., personal
care products) degrade more quickly but are present in the environment due to their
Estrogenic Endocrine Disrupting Compounds - Review
12
wide usage and continuous introduction. Other EDCs (e.g., antibiotics, birth control
pills, livestock drugs) are designed to be persistent in order to be effective. It is
estimated that 50-90% of typical drug dosage can be excreted unchanged to the
environment where it can exist for years. Environmental fate and transport refers to
the outcome of a contaminant in the environment as a result of its potential to be
transported, transformed (physically, chemically, or biologically), or accumulated in one
or more media. These processes are controlled by the compound’s physical and
chemical properties and the nature of the media through which the compound is
migrating. The common categories of EDCs and the primary pathways by which these
enter waterways are summarised in Table 3 and EDC distribution in the environment is
schematically illustrated in Figure 3.
Table 3 EDC Pathways to Waterways
Categories of known and suspected EDCs and primary pathways to waterways (cited from WMI,
2003).
CATEGORY
EXAMPLES
PRIMARY PATHWAY TO WATERWAYS
Prescription
and nonprescription
drugs
Birth control pills,
steroid-based
medications,
chemotherapy
medications
Drugs partially metabolized in the body. Remaining drug
and metabolites excreted in urine and faeces.
Wastewater treatment facilities may partially remove or
break down these chemicals. Remainder of drugs and
metabolites discharged to surface water.
Improper disposal of leftover medication.
Household
products
Detergents,
surfactants, and
personal care
products
Commonly rinsed down sinks and flushed down toilets.
Wastewater treatment facilities may partially remove or
break down these chemicals. Remainder of compounds
and break down products discharged to surface water.
Industrial
chemicals
and metals
Polybrominated
biphenyl ethers,
bisphenol-A, PCBs,
phthalates,
styrenes, mercury,
lead, dioxins and
furans
Discharged to sewers from households, and industrial
and commercial facilities. Wastewater treatment
facilities may partially remove and/or break down these
chemicals. Remainder of compounds and break down
products discharged to surface water.
Fungicides
Hexachlorobenzene,
maneb, tributyltin
Outdoor uses lead to runoff into storm drains which
discharge directly to surface waters.
Indoor use or cleaning of contaminated equipment and
clothing leads to discharge to wastewater treatment
plants where partial removal or break down may occur.
Remainder discharged to surface water.
Herbicides
2,4-D, 2,4,5-T,
atrazine
Outdoor uses lead to runoff into storm drains which
discharge directly to surface waters.
Indoor use or cleaning of contaminated equipment and
clothing leads to discharge to wastewater treatment
plants where partial removal or break down may occur.
Insecticides
Carbaryl, chlordane,
dieldrin, lindane,
parathion
Outdoor uses lead to runoff into storm drains which
discharge directly to surface waters.
Indoor uses or the cleaning of contaminated equipment
and clothing leads to discharge to the wastewater
treatment plant where partial removal or break down
may occur. Remainder discharged to surface water.
Animal
husbandry
products
Steroid-based
supplements to
increase production
Drugs are partially metabolized in animal's body.
Remaining drugs and metabolites are excreted in urine
and faeces where they run off to surface waters.
Estrogenic Endocrine Disrupting Compounds - Review
13
Figure 3 EDC Transport Pathways
Schematic illustration of the EDC transport pathways through different environmental media.
Specific physical and chemical parameters of interest include the partitioning
coefficient of e-EDCs between the aqueous and solid phases (estimated via several
different parameters including the organic carbon partition coefficient, K oc, and the
distribution coefficient, Kd), sorption to biotic tissue (octanol-water partition coefficient,
Kow), maximum dissolved concentration of the chemical in water at a specified
temperature (solubility), relative estrogenic activity (Estradiol equivalency factor, EEF),
enhancement in e-EDC solubility due to micelle formation (critical micelle
concentration (CMC) values), increased solubility at elevated pH (indicated by the
chemicals acidity constant, pKa), and the first-order decay rate. These values are
presented in Table 4.
Estrogenic Endocrine Disrupting Compounds - Review
14
Table 4 Properties of e-EDCs
Properties of selected e-EDCs from the literature. (Adapted from Campbell et al., 2006; Suárez et al., 2008; Teske and Arnold, 2008.)
MOL.
WT.
(g/mol)
EDC
Estradiol
C
LOG Koc
(L/kg)
LOG
Kow
(L/kg)
D
LOG Kd (L/kg)
FOR SLUDGE
BIOLOGICAL
DIGESTED
SOLUBILITY
(mg/L)
EEF
CMC
(mg/L)
pKa
DECAY
RATE
(L/gSSday)
B
A
272.4
2.55–4.01
NA
NA
NA
13.0–32.0
1.0a
NA
10.5–10.71
NA
17β-Estradiol (E2)
272.4
3.10–4.01
3.9-4.0
2.4-2.8
2.3-2.5
13.0
1.0b
NA
10.71
300-800
Estrone (E1)
270.4
2.45–3.34
3.1-3.4
2.4-2.9
2.4-2.6
6.0–13.0
0.1–1.0a,
0.01–0.1b
NA
10.3–10.8
200-300
Ethinylestradiol (EE2)
296.4
2.91–3.04
2.8-4.2
2.5-2.8
2.3-2.6
4.8
0.8–1.9b
NA
NA
7-9
Estriol (E3)
288.4
2.13–2.62
NA
NA
NA
32
0.01–0.08b
NA
10.4
NA
NA
9.6–11.3
NA
5–13
10.28
NA
NA
NA
NA
NA
Bisphenol A (BPA)
228.0
2.50–6.60
NA
NA
NA
120–300
-4
-5
-5
-4
5.0x10 – 6.0x10 b
Nonylphenol (NP)
220.2
3.56–5.67 3.8->4.75
NA
NA
4.9–7.0
2.3x10 –9.0x10 a
-7
-2
7.2x10 –1.9x10 b
Nonylphenol
ethoxylates (NP1EONPnEO; n ≤ 20)
264.01101.6
3.91–5.64
NA
NA
NA
3.02–31.9
2.0x10 –1.3x10 b
Octylphenol
206.3
3.54–5.18
NA
NA
NA
12.6
1.0x10 –4.9x10 b
-7
-5
-5
-4
4.25x10
-5
150 (Triton
X-100)
Notes:
a) Estrogen equivalent factor effect relative to estradiol (a) and relative to 17β-estradiol (b) – ranges include various difference bioassays and estrogen receptors including ERCALUX, YES, E-Screen transgenic zebrafish, and sheepshead minnows, as well as, both hEH- α and hEH- β receptors.
b) Critical micelle concentration.
c) Estradiol here is presented separate from 17β-estradiol as it may include a larger class of compounds including 17α-estradiol and 17β-Estradiol, and the specific compound used
was not clarified in all sources.
d) Not available or not found in the literature.
Estrogenic Endocrine Disrupting Compounds - Review
15
Many e-EDCs have moderate to high log Koc values and low solubility, so the mass that
does not remain soluble often ends up in organic complexes in, or sorbed to,
sediments or suspended organic material. This tendency to partition to the organic
fraction is reflected in the log Kd values for these compounds, which remain elevated
even in digested sludge. In the sediments there is the potential for biological uptake,
degradation and transformation to less mobile or more mobile forms. In some cases eEDCs have been detected in groundwater and drinking water, possibly due to the
presence of more soluble precursors in media, colloid facilitated transport, and
enhancement in solubility due to elevated pH or micelle formation (Campbell et al.,
2006). Bisphenol A (BPA) sorbs to solids and appears to be aerobically biodegraded;
however, its degradation products are more estrogenic than BPA and nonylphenol
(NP). The degradation of nonylphenol ethoxylates (Figure 4) results in shortening of the
ethoxylate chain, less foaming, and formation of break down products that have lower
solubility, greater partitioning onto solids, more persistence and greater estrogenic
activity.
Figure 4 Nonylphenol ethoxylate degradation
Biodegradation of nonylphenol ethoxylate under aerobic and anaerobic conditions (redrawn after
Porter and Hayden, ND.)
The natural (E1, E2, E3) and synthetic estrogen (EE2) appear to be biodegradable at
varying levels and the observed biodegradation order (highest to lowest) is estriol (E3)
> 17β-estradiol (E2) > estrone (E1) > 17α-ethinylestradiol (EE2). Under aerobic
conditions E2 is rapidly oxidized to E1. Due to the increase in estrogenicity under
anaerobic conditions, suspicions of E1 reducing to E2 have been raised, but not
confirmed. The by-products of EE2, E1, E3 degradation are currently unknown. These
transformations are shown in Figure 5. Compared to freshwater systems it has been
suggested that E2 degradation in estuarine waters is considerably longer – 6–10 days
vs. 14 up to 49 days, respectively (Jürgens et al., 1999). In addition, increased water
temperature can be expected to result in more rapid microbial degradation of E2.
Estrogenic Endocrine Disrupting Compounds - Review
16
Figure 5 Estrogen Degradation
Biodegradation of estrogens E1, E2, E3 and EE2
Estrone (E1)
Estradiol (E2)
Aerobic
Unknown
byproducts
Ethinyl estradiol (EE2)
Estriol (E3)
3.2.1
e-EDC Degradation in Wastewater Treatment Plants
Conventional wastewater treatment processes (Figure 6) were not developed to
remove the many trace organic chemicals present in municipal wastewater. Such
compounds frequently elude treatment practices that are designed primarily for waste
stabilization, clarification, disinfection and nutrient removal. Several authors (e.g., Joss
et al. 2004; Clara et al. 2004; Andersen et al. 2003; Johnson et al. 2005) have
examined the amount and mechanisms of estrogen removal in wastewater treatment
plants (WWTPs). Preliminary treatment (screening, grit removal, comminution, etc.)
does not significantly contribute to estrogen removal but sludge removal in primary
and secondary clarifier can be significant as sorption is an important process in the
removal of estrogens from the liquid phase (Holbrook et al. 2004). High pH leads to
desorption of E2 and EE2 from sludge (Clara et al., 2004).
The reported removal efficiencies for estrogens vary widely. For example, the
observed removal efficiency for E2 ranges from 30% (Adler et al., 2001) to 98%
(Andersen et al., 2003). Increasing the hydraulic retention time (HRT) and sludge
retention time (SRT) increases the amount of E1 removal, and enhances the removal
of other biodegradable estrogens as well (Joss et al. 2004; Johnson et al. 2005). For
EE2 the observed removal efficiencies varying from 34% (Cargouet et al. 2003) to
almost 100% (Vethaak et al. 2002); however, unlike E1, no relationship between SRT
and EE2 removal has been established (Clara et al., 2004). Greatest biodegradation of
estrogens is seen in aerobic systems. In particular, nitrifying activated sludge systems
Estrogenic Endocrine Disrupting Compounds - Review
17
are able to degrade all estrogens, while activated sludge processes without nitrification
do not degrade EE2. Under denitrifying conditions, only E1 and E2 are degraded; EE2
degrades only under aerobic conditions. E2 and E1 in sludges are not degraded during
methanogenic digestion (Andersen et al., 2003). Of concern though is the observation
that both aerobic and anaerobic sludge digestion processes increase the overall
estrogenic activity (Holbrook et al., 2004). As the wastewater treatment systems were
not designed for removal of e-EDCs, this observation highlights one of the limitations
of the conventional treatment systems in dealing with this new category of pollutants.
Improving the performance of wastewater treatment systems to overcome this
limitation in currently an area of major research activity (for example, see Pauwels et
al., 2008).
Several microorganisms capable of E1, E2, E3 and EE2 transformation have been
isolated from activated sludge processes. E1, E2 and E3 were degraded by
Nitrosomonas europaea (Vader et al. 2000; Shi et al. 2004), Ralstonia sp.,
Achromobacter xylosoxidans (Weber et al. 2005), Rhodococcus zopfii and
Rhodococcus equi isolates (Yoshimoto et al. 2004). During E2 degradation, most
strains formed E1 and subsequently biodegraded it. EE2 was only degraded by
Nitrosomonas europaea, Rhodococcus zopfii and Rhodococcus equi,
Sphingobacterium sp. JCR5 (Haiyan et al. 2007) and the fungus Fusarium proliferatum
(Shi et al. 2002).
Figure 6 Wastewater treatment
Schematic layout of a conventional Wastewater Treatment Plant (adapted from Gomes et al.,
2003).
Influent
Preliminary
treatment
Primary
sedimentation
Biological
treatment
Screenings
Grit
Primary
sludge
Secondary
sludge
Waste
disposal
Anaerobic
digestion
(sludge
treatment)
Effluent
Estrogenic Endocrine Disrupting Compounds - Review
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4
Concentration of EDCs in the Environment
Measurable concentrations of EDCs have been found in wastewater, surface waters,
sediments, groundwater, and drinking water (Benfenati et al., 2003; Petrovic et al.,
2003; Snyder et al., 2003; Petrovic et al., 2004). While the highest concentrations have
been observed in sediments and wastewaters, the smaller quantities present in air and
drinking water may still be estrogenically active (Campbell et al., 2006). The effect of
concentrations of various estrogenic compounds cannot be directly compared without
reference to their relative affinity for the estradiol receptor (selected values shown in
Table 5). A summary of concentrations reported for selected e-EDCs in different
environmental media is presented in Table 6.
Table 5 Relative Potency of Environmental Pollutant e-EDCs
Affinities of pollutant e-EDCs for the estradiol receptor. Data collated from the comprehensive
review by Tyler et al. (1998).
COMPOUNDS
AFFINITY FOR ESTRADIOL RECEPTOR
(Estradiol Equivalency Factor)
PCBs (ortho-substituted)
2x10 - 0.02
Methoxychlor
1x10 - 1x10
Methoxychlor metabolites
3x10 - 0.01
-3
-5
-4
-3
-5
APEs
1x10 - 5x10
BPA
5x10
-4
-4
APEs: Alkylphenol ethoxylates (including nonylphenol ethoxylates)
BPA: Bisphenol-A
PCBs: Polychlorinated biphenyls
4.1
Pesticides
Organochlorine pesticides with estrogenic properties include DDT, methoxychlor,
lindane and kepone. Although the use of DDT as a pesticide is widely restricted, it is
still commonly used as a pesticide in some developing countries, where it has been
found in water sources at concentrations up to 10µg/L (Begum et al., 1992). Generally
the concentrations are not sufficiently high to cause adverse affects, but it persists in
many areas due to its recalcitrance and tendency to bioaccumulate and sorb to solid
phase, with a half-life of over 50 years (Cooke and Stringer, 1982). Methoxychlor has
been found in surface waters at concentrations up to 2.8µg/L (Castillo et al., 1997; Dua
et al., 1996) while lindane concentrations of up to 10µg/L have been detected (Begum
et al., 1992).
Estrogenic Endocrine Disrupting Compounds - Review
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Table 6 Detected e-EDC Levels
Global concentrations of e-EDCs detected in various environmental media. (Modified from Campbell et al., 2006.)
EDC
SURFACE
WATER
(ng/L)
SEDIMENTS
(µg/g)
GROUNDWATER
(CONTAMINATED)
(ng/L)
DRINKING
WATER
(ng/L)
WASTEWATER
EFFLUENT
(ng/L)
SEWAGE
SLUDGE
(µg/g)
AIR
3
(ng/m )
E2
<0.1-6.0
0.9-2,480
13-80
0.20-2.1
<0.1-650
0.00057
NA
E1
<0.1-4.1
<0.04-2,520
NA
0.20-0.60
<0.1-19
0.00143
NA
EE2
0.1-5.1
<50-500
NA
0.15-0.50
<0.4-8.9
0.00061
NA
E3
1.0-2.5
0.5-1.5
NA
NA
5.0-7.3
NA
NA
BPA
0.5-250
NA
3-1,410
0.50-44
4.8-258
NA
NA
NP
6.7-15,000
<0.003-154
200-760
2.50-2,700
18-770
3-8,000
<0.001-81
NP1EO-NPnEO
<20-97,600
<0.003-30
<10-38,000
100-300
320-1,570
<0.5-254
<0.001-14
OP and OPEO
0.8-13,000
<0.005-8.8
NA
0.20-4.9
2.2-358
<0.5-12.6
0.01-2.5
Notes:
NA: Not available or not found in the literature
Estrogenic Endocrine Disrupting Compounds - Review
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4.2
Polychlorinated Biphenyls
Certain polychlorinated biphenyls (PCBs) and their hydroxylated metabolites have been
found to have estrogenic properties (Korach et al., 1988), although the concentrations
required to elicit physiological effects in laboratory studies are far greater than those
found in the environment. PCBs are found at concentrations generally less than DDTcompounds (up to 1µg/L in aquatic systems), however, these levels may increase due
to the current widespread use of PCBs, particularly in developing countries.
Furthermore, PCBs are known to readily bioconcentrate in tissues, with the result that
organisms may be exposed to concentrations very much higher than those in the
surrounding media (Guiney et al., 1979; Nimi, 1983).
4.3
Dioxins
Polychlorinated dibenzo-p-dioxins are generally found in very low concentrations in the
water phase of the aquatic environment (up to 1.4µg/L) due to their hydrophobic nature
and tendency to partition into organic matter. They are, however, extremely estrogenic
at even very low concentrations. It is thought that the highest concentrations of
dioxins released into soils and the aquatic environment are from landfills, and in
bleached kraft mill effluents, where concentrations up to 40µg/L have been found
(Merriman et al., 1991).
4.4
Phenols
Alkylphenol ethoxylates, including their degradation products the alkylphenols
octylphenol (OP) and nonylphenol (NP), have long been known to be estrogenic (Dodds
and Lawson, 1938). A survey of rivers in the U.K. found that NP concentrations were
generally less than 10µg/L, concentrations of 1.9µg/L were detected in the Kurose
River in Japan (Shoko et al., 2003), while most of the rivers surveyed in the U.S.A. had
concentrations less than 0.1µg/L (Blackburn and Waldock, 1995; Harries et al., 1997).
The findings from a review of NP detected in sewage in different countries are
presented in Figure 7, which suggests that concentrations of these compounds in
waste streams globally can vary greatly, Significant quantities of alkylphenols
compounds are released into the aquatic environment from municipal (µg/L range) and
industrial (mg/L range) effluents. In aquatic environments that receive effluent inputs,
the concentrations of alkylphenolic compounds may be sufficiently high to adversely
affect the organisms present.
Bisphenol-A (BPA) is an estrogen mimic that is used in the manufacture of plastics,
lacquers, and packaging materials. It has been found at concentrations of more than
1µg/L in water pipes where it has been applied as a liner and is thought to leach out of
plastic and lacquer-coated food packaging (Brotons et al., 1995). BPA has been found
at concentrations up to 0.75µg/L in the Kurose River in Japan (Shoko et al., 2003).
Estrogenic Endocrine Disrupting Compounds - Review
21
Figure 7 NP in sewage sludge
Concentrations of NP in sewage sludge in different countries. (Redrawn after Petrovic and
Barceló, 2004).
USA
Canada
UK
Switzerland
Spain
Germany
Finland
Denmark
Austria
1
10
100
1000
10000
NP C oncentration (mg/kg)
4.5
Phthalates
Phthalates are abundant in the environment and at least some have been found to be
weakly estrogenic at high concentrations. Di-n-butyl-phthalate (DBP) is the most
commonly detected phthalate in the environment. While environmental concentrations
of DBP are high - up to 30µg/L in developed countries, and reportedly up to 1472mg/L
in the River Owena, Nigeria (Fatoki and Ogunfowokan, 1993), the response dose in
many organisms is often higher than for other types of EDCs.
4.6
Estrogens
Synthetic estrogens such as EE2 can be even more potent than naturally-occurring
estrogenic compounds. Used in pharmaceuticals such as the contraceptive pill, the
main source of synthetic estrogens in the aquatic environment is sewage discharge
(Stumpf et al., 1996). They have been detected at concentrations up to 7µg/L in
sewage effluent in the U.K. (Routledge et al., 1998). The concentrations of E1, E2, and
EE2 detected in the discharge from wastewater treatment plants in different countries
summarised in Table 7 suggest that these are generally small, typically <10 ng/L. The
potency of EE2 as an estrogenic chemical greatly increases the risk of adverse effects
in the environment, despite the very low concentrations that may be present.
Estrogenic Endocrine Disrupting Compounds - Review
22
Table 7 Estrogen concentrations in wastewater treatment plant effluent
Median concentrations (ng/L) of the three most biologically active estrogens discharged from
domestic activated sludge sewage treatment plants into environmental waters of various
countries. (Cited from Baronti et al., 2000).
COUNTRY
NO. OF SAMPLES
E2
EE2
E1
Italy
30
1
0.45
9.3
Netherlands
6
0.9
<1.8
4.5
Germany
16
<1
1
9
Canada
10
6
9
3
Brazil
6
<0.2
1
7
England
6
5
<1
3
Estrogenic Endocrine Disrupting Compounds - Review
23
5
5.1
Methods for Analysing EDCs
Approach
Two analytical approaches are used to detect EDCs: (i) determine concentration of
compound and (ii) screen for estrogenic activity. The methods used to achieve this can
be broadly separated into chromatographic techniques, non-cellular assays and
biologically based assays. These methods (described further below) have differing
strengths and limitations with respect to compound specificity, sensitivity, sample
preparation, time requirement and cost-effectiveness and are compared in section 5.2.
5.1.1
Chromatographic Techniques
The chemical concentration of specific e-EDCs can be determined using
chromatographic techniques such as HPLC-FLD (Fan et al. 2005), GC/MS (Hernando et
al. 2004), GC-MS/MS (Huang and Sedlak, 2001), LC-MS (Petrovic and Barceló, 2000;
Petrovic et al., 2002) and LC-MS/MS (Benjits et al. 2004). These chromatographic
techniques provide excellent sensitivity and precision for quantifying the mass of
EDCs. All of these techniques involve separation of individual chemicals using a
chromatographic column followed by chemical quantification by a detector. Examples
of the methodologies used for analysing EDCs by these techniques are available in
Huang and Sedlak (2001), Zhang et al. (2004), and Fan et al. (2005).
5.1.2
Non-Cellular Assays
Non-cellular assays (see Table 8) offer an alternative to chromatographic techniques for
quantifying e-EDCs in the environment. The enzyme-linked immunosorbent assay
(ELISA) is based on the specific binding of an antibody (a protein) to an EDC and the
production of fluorescence by binding of enzymes to free residual antibodies (Huang
and Sedlak, 2001; Sun et al., 2001). The enzyme-linked receptor assay (ELRA) is similar
to ELISA with the difference that it involves the use of a physiologically relevant
receptor instead of an antibody as a linking protein (Seifert, 2004). The RIver ANAlyser
(RIANA) is a multi-analyte immunosensor that uses total internal reflection
fluorescence to determine the levels of specific organic analytes in waters. By
changing the binding ligand utilised, RIANA can be adapted for the detection of various
compounds, and has been successfully used for E1, E2 and EE2 (Rodriguez-Mozaz et
al., 2004, Le Blanc et al., 2009). Quantifying concentrations is useful for assessing eEDC fate and transport in the environment. However, concentrations do not provide
information on estrogenic effects or synergistic or anti-estrogenic influences from
multiple estrogenic compounds (Campbell et al., 2006).
Estrogenic Endocrine Disrupting Compounds - Review
24
Table 8 Non-Cellular Assays
Commonly used non-cellular assays for detection of e-EDCs. (Adapted from Campbell et al.,
2006.)
5.1.3
ASSAY NAME
e-EDC RESPONSE
QUANTIFICATION
Enzyme-linked immunosorbent
Assays (ELISA)
Colorimetric
Spectrophotometer
Enzyme-linked receptor assay
(ELRA)
Luminescent,
colorimetric
Luminometer,
spectrophotometer
River analyser (RIANA)
Fluorescence
Fluorometer
Biologically Based Assays
Biologically based assays (BBAs) can be used to detect and monitor the estrogenic
activity of EDCs and offer an alternative approach in assessing the impacts of EDCs. In
BBAs, living organisms are exposed to EDCs, either in the laboratory ( in vitro) or in the
environment (in vivo), and EDC effects are measured. BBAs can be used to monitor
EDC activity in natural environments, or to assess compounds and effluent
contamination in laboratory experiments. The effect of EDCs upon multicellular
organisms can be assessed through measurements ranging from characteristic effects
upon anatomy (e.g. gonad abnormalities), to atypical levels of protein expression (e.g.
production of vitellogenin in male fish). BBAs in marker species are especially useful in
monitoring EDC effects in the natural environment, but may also be applied to
investigate EDC effects under controlled laboratory conditions. In vivo life-cycle studies
are especially pertinent when determining the endocrine-disrupting effect on biota of
for example, wastewater entering surface waters or the presence of contaminated
sediment (Jobling et al., 1998). The effect of EDCs on a wide range of whole
organisms has been studied in the natural environment and under controlled laboratory
conditions – some of the whole organisms that have been used in these assessments
are listed in Table 9.
In vitro assays are, by comparison to in vivo assays, rapid, cost effective tools requiring
smaller concentration factors and can achieve lower detection limits than chemical
analysis because of their specificity. Detection in an in vitro BBA may occur by a
number of mechanisms, including cell proliferation, vitellogenin induction, or induction
of reporter genes in recombinant organisms (Campbell et al., 2006). The E-SCREEN
provides an example of how cell proliferation can be used to estimate the estrogenicity
of samples by quantification of the mitogenic effect of samples upon the esterogenresponsive human breast cell line MCF-7 (Soto et al., 1995). Vitellogenin is a yolk
protein normally found in female fish that is produced in response to estrogens. It can
be quantified following extraction from plasma of male fishes, providing an indication
of endocrine disruption (Jimenez, 1997). The induction of a reporter gene is illustrated
by both the ER assay and the YES assay. The ER assay involves the binding of
estrogens to receptors (ER) which, when activated, bind to estrogen response
elements (ERE), triggering gene expression. The recombinant luciferase gene is placed
downstream of the ERE resulting in luciferase expression upon exposure to EDCs.
Luciferase can be quantified by its ability to catalyse the oxidation of the luciferin
substrate, measured by luminescence (Legler et al., 2002). The ERE-luciferase
Estrogenic Endocrine Disrupting Compounds - Review
25
construct has been used to transfect cultured human cells and also to develop
transgenic zebrafish that are effective sensors of (xeno)estrogens (Legler et al., 2002).
The Yeast Estrogen Screen (YES) is a promising technique for quantifying compounds
capable of binding to the human estrogen receptor using a recombinant yeast
construct that induces an assayable enzyme activity in response to exposure to
estrogens. The yeast expresses the recombinant ER and activates an ERE-linked
reporter gene in the presence of estrogens. The method is not commercially available,
but strains are freely available and have previously been imported into New Zealand by
ESR. Some examples of bioassays for in-vitro studies are presented in Table 10.
Table 9 Organisms Used to Indicate EDC Effects
Examples of organisms used in studies as indicators of estrogenic endocrine disruption in the
environment or in whole organism in vivo studies under controlled laboratory conditions.
(Adapted from Campbell et al., 2006; Schmitt et al., 2007)
SPECIES
COMMON NAME
EDC EFFECT
Rana pipiens
Leopard frogs
Gonadal abnormalities
Chrysemys picta
Painted turtle
Vitellogenin induction
Rainbow trout
Reproductive deficiencies, egg and
offspring development, and
vitellogenin induction
Fathead minnow
Gonad development; reproductive
deficiencies, development;
vitellogenin induction
Oncorhynchus mykiss
Pimephales promelas
Cyprinodon variegates
Sheephead minnow
Vitellogenin induction
Zebrafish
Gonad development; physiological
development; vitellogenin induction,
transgenic ER-dependent luciferase
expression
Medaka fish
Gonadal development; reproductive
success; transgenic estrogen
dependent GFP expression from the
medaka choriogenin (ChgH)
promoter region. ChgH is an egg
envelope protein synthesized in liver
with the stimulation of estrogen
(Kurauchi et al., 2008)
Flounder
Vitellogenin induction; gonad
development; physiological
development;
Atlantic Salmon
Zona radiata protein and vitellogenin
induction
Halineetus leucocephalus
Bald eagle
Reproductive and teratogenic effects
Coturnix coturnix japonica
Colinus virginianus
Japanese quail and
bobwhite quail
Sexual behaviour; embryo
development; egg shell thickness
Domestic chicken
Embryo development; egg shell
thickness
Water flea
Physiological and biochemical
disruption
Tisbe battagliai
Marine copepod
Fecundity, longevity, and rate of
development
Potamopyrgus antipodarum
New Zealand mudsnail
Increased reproduction
Danio rerio Brachydanio reri
Oryzias latipes
Platichthys flesus
Salmo salar
Gallus domesticus
Daphnia magna
Estrogenic Endocrine Disrupting Compounds - Review
26
Table 10 In Vitro Bioassays
Examples of in vitro bioassays for detection of e-EDCs detailed in the literature. (Adapted from
Campbell et al., 2006.)
5.2
COMMON NAME
CELL TYPE
e-EDC EFFECT
E-SCREEN
MCF-7 breast cancer cell
Cell proliferation response
Yeast Estrogen Screen (YES) –
including LYES and BLYES variations
Various (Saccharomyces
spp., Cryptococcus spp.,
and Candida spp.)
Colorimetric and
luminescent response
ER-luciferase assay with HEK 293
cells
Human embryonic kidney
(HEK)
Luminescent response
Estrogen responsive chemically
activated luciferase expression (ERCALUX® - commercially available kit)
T47D human breast
adenocarcinoma cell
Luminescent response
Comparison of Analytical Techniques
Chromatographic techniques are valuable for the identification and quantification of
individual compounds. However, these methods do not provide information regarding
the potency, and efficacy of a compound in modulating endocrine function. Method
development for chromatographic techniques is often difficult and time-consuming,
particularly when samples contain mixtures of compounds. In addition, the methods
require large sample volumes and extensive clean-up procedures to achieve sample
purification (Maurício et al., 2006).
While in vivo methods are crucial in identifying the connection between exposure and
biological effects, they are expensive, cannot accommodate high throughput
screening, and cannot characterize individual compounds. In addition, in vivo studies
are subject to inter-individual, seasonal, and temporal variability in conditions and
responses, which may make interpretation difficult. In most cases, the significant
limitation for in vivo studies is the greater time required to obtain results.
In comparison, in vitro bioassays are relatively inexpensive, can be conducted with
large throughput, and are performed quickly. As in vitro methods are simplified
biological systems, the responses are highly sensitive and allow for analysis of
individual compounds (PUBH 5103, 2003b), or can be used to provide an integrated
measure of the synergistic effects of all compounds in a given sample. Some
bioassays also provide superior sensitivity and allow for detection of compounds that
exert effects at levels below analytical detection limits. Additionally, many in vitro
bioassays are capable of detecting compounds for which there are no analytical
methods available (PUBH 5103, 2003b). Although useful, in vitro bioassays have
limitations. For one, the relevance of the measured response for estimating in vivo
activity depends on the mechanism the assay was based on. Consequently,
knowledge about the mechanism of action of a compound must be attained prior to
analysis of the compound. Also, in vitro bioassays are mechanism specific and do not
account for many factors that may affect the mechanism in vivo such as cross-talk
between biological pathways and environmental influence.
Estrogenic Endocrine Disrupting Compounds - Review
27
Non-cellular assays, such as ELISA, are relatively fast, simple and cost-effective
methods for quantitative analysis of EDCs (Goda et al., 2000). Castillo et al. (2000)
determined surfactants in wastewater using liquid chromatography (LC) coupled with
mass spectrometric (MS) or fluorescent (FL) detection and a test-tube ELISA kit and
reported that while the ELISA (only used for nonylphenolpolyglycol ether
determination) values were higher than those obtained from LC-MS due to
interference of cross-reacting substances, the ELISA technique gave acceptable
results. Another example illustrating the appropriateness of using ELISA in the form of
comparison of the ELISA and HPLC analyses is presented in Figure 8. Due to unknown
cross-reactivity in new applications, ELISA results should be validated against a
chromatographic technique such as LC-MS,
Figure 8 Comparison of data from HPLC and ELISA analyses
Comparison of ELISA (X axis) and HPLC (Y axis) results for 23 river water samples (100mL for
ELISA and 500mL for HPLC). Solid circles represent data when alkylphenol polyethoxylates and
nonylphenoxycarboxylic acids are both present while hollow circles represent data when only
alkylphenol polyethoxylates are present in solution (Goda et al. 2004). Reprinted with permission
from Elsevier.
However, use of ELISA kits from different manufacturers requires some caution. Table
11 presents E2 measurements in the primary effluent, aeration tank and secondary
effluent of a wastewater treatment plant measured using LC-MS/MS and ELISA kits
manufactured by Japan EnviroChemicals, Assay Designs, Cayman Chemical, Neogen,
and R-Biopharm. The values obtained using the ELISA kits were up to 65 times higher
than those obtained using LC-MS/MS (Hirobe et al. 2004).
Estrogenic Endocrine Disrupting Compounds - Review
28
Table 11 E2 concentrations in wastewater
Average E2 concentrations in a wastewater treatment plant as measured using LC-MS/MS and
ELISA kits from different manufacturers (Hirobe et al. 2004).
AVERAGE E2 CONCENTRATION (ng/L)
SAMPLE
a
N
LCMS/MS
Japan
EnviroChemicals
Assay
Designs
Cayman
Chemical
Neogen
RBiopharm
8.4
12.5
21.1
81.4
97.6
202.2
Primary effluent 10
a
Aeration Tank
13
1.9
2.1
4.4
9.4
7.8
31.6
Secondary
effluent
5
1.2
2.6
5.3
15.1
18.8
75.9
N = number of samples
From the above studies it can be concluded that while the chromatographic methods
are more accurate and give more reliable results, the simplicity and lower cost of
ELISA makes it an attractive technique for analysing EDCs, although with somewhat
lower accuracy, which varies with the specific ELISA kit used. Furthermore, GC-MS
with derivatisation may offer an alternative method of analysing EDCs, especially when
there are concerns for interference due the likely occurrence of a combination of EDCs
in the sampled media. Some studies have reported that GC/MS with derivatisation can
give similar sensitivity to GC-MS/MS without derivatisation (Hernando et al. 2004).
Table 12 compares the main advantages and limitations of the analytical techniques
discussed. The typical detection limits for various analytical techniques are presented
in Table 13. For many analytical techniques, the method detection limit will be close to
or even higher than the EDC levels that may be expected to be detected in the
environment (compare to levels presented in Table 6 and Table 7). Concentrations of eEDCs in samples of drinking water, surface water and even wastewater effluent will
often be too low for detection by most techniques other than chromatography. In
these cases, environmental samples will require a pre-concentration step during
routine sample preparation that will be necessary for all methods, thus the detection
limit does not apply directly to the raw sample.
Table 12 Comparison of analysis methods
Advantages and disadvantages of analytical techniques. (Adapted from Conroy, 2006.)
METHOD
PRIMARY ADVANTAGES
PRIMARY DISADVANTAGES
Chromatographic Techniques
Rapid, Compound-specific
May not detect non-target
estrogen mimics
Provide no information on
estrogenic potency
Non-Cellular Assays
(e.g. ELISA, RIANA, ELRA)
Rapid, Cost-effective
Higher detection limit
In Vivo Organism Studies
Physiological response
closer to humans, Relevant
to aquatic organisms
Time-consuming
In Vitro Bioassays
(e.g. YES)
Indicates (anti-) estrogenic
response, Rapid
Use simple model organisms
and cells
Estrogenic Endocrine Disrupting Compounds - Review
29
Table 13 Method Detection Limits for e-EDCs in Wastewater Reported in Literature
Typical limits of detection reported for the different methods used to detect e-EDCs in
wastewater.
ANALYTICAL
TECHNIQUE
METHOD
DETECTION
LIMIT (ng/L)
REFERENCES
Chromatography
GC–MS
0.008-17.0
Mouatassim-Souali et al., 2003,
Hernando et al., 2004
GC–MS/MS
0.1 - 27.5
Huang and Sedlak, 2001, Hernando
et al., 2004, Belfroid et al., 1999
LC–MS/MS
0.4 – 7.0
Salvador et al., 2007, Lagana et al.,
2004
ER-CALUX
0.14
Murk et al., 2002
ER Binding
272.4
Murk et al., 2002
YES
0.1 – 16
Heisterkamp et al., 2004, Murk et al.,
2002
In-vitro bioassay
Non-cellular assay
ELISA
0.1
Huang and Sedlak, 2001
ELISA: Enzyme-linked immunosorbent assay.
ER-CALUX: Estrogen responsive chemically activated luciferase expression.
ER Binding: Estrogen receptor competitive ligand binding assay
GC–MS: Gas chromatography mass spectrometer.
GC–MS/MS: Gas chromatography tandem mass spectrometer.
LC–MS/MS: Liquid chromatography tandem mass spectrometer.
YES: Yeast estrogen screen.
5.3
Selection of Techniques for e-EDC Analysis
At the present time there is little understanding of e-EDC concentrations in the local
environment and the selection of the analytical technique is driven by the need to
quickly develop a method that can be used to quantify e-EDC concentrations in
environmental media.
Non-cellular assays, such as ELISA, offer a number of important advantages in that
they are rapid to conduct and are cost effective methods for screening large numbers
of samples. ELISA kits are available for several estrogen compounds, surfactants,
pesticides, antibiotics, and personal care products (for example, from Neogen Corp
Lexington, KY; ALPCO Bio-Quant Inc., San Diego, CA; Cayman Chemical Company,
Ann Arbor, MI; Immuno-Biological Laboratories, Inc., Minneapolis, MN). The
sensitivity and working range of commercially available ELISA kits varies by
manufacturer, with specific kits available for E1, E2, EE2, E3 and total estrogens. A
certain degree of cross-reactivity exists between the various compounds for most kits,
with this data specified by the manufacturer. In general, most E2 kits are capable of
detecting E2 concentrations above 10pg/mL, with the detection limits of E3 kits
approximately an order of magnitude greater. Table 14 and Table 15 show the
detection limits and range of commercially available kits for E2 and E3 respectively.
Although the detection limits are higher than for other methods, a preconcentration
step during sample preparation can overcome this potential limitation. Following
sample preparation (a step required for all analytical methods), the time required to
obtain results from ELISA assay kits ranges from one to several hours. This makes the
technique reasonably rapid, and many samples can be determined simultaneously.
Estrogenic Endocrine Disrupting Compounds - Review
30
The ELISA assay can suffer from interference in complex matrices, and when multiple
estrogens are present. Derivatisation of estrogens can overcome this potential
problem by making the target compounds more volatile and thermally stable for
enhanced detection of the individual compounds by GC-MS and LC-MS (Gomes et al.,
2003). The sensitivity of LC-MS analysis is increased by derivatisation of target
estrogens (Gomes et al., 2003). Additionally, GC-MS analysis of derivatised samples
can give similar sensitivity as analysis using GC-MS/MS without derivatisation
(Hernando et al, 2004). Thus, GC-MS with derivatisation may serve as a cost-effective
technique for quantifying individual EDCs.
Table 14 Properties of E2 Commercial ELISA Assay Kits
SUPPLIER
SENSITIVITY (pg/mL)
ASSAY RANGE (pg/mL)
Alpco Diagnostics
10
20 – 3200
(Ultra-sensitive)
3
3 – 200
ARP American Research Products, Inc.
10
25 – 200
Alpco Diagnostics
Bio-Quant Inc.
1
Not available
Calbiotech, Inc
10
Not available
Cayman Chemicals
8
Not available
Diagnostic Systems Laboratories, Inc.
7
20 – 6000
GenWay Biotech, Inc.
10
Not available
Immuno-Biological Laboratories, Inc.
9.7
0 – 2000
Neogen Corporation
Not available
100 – 2000
Table 15 Properties of E3 Commercial ELISA Assay Kits
SUPPLIER
SENSITIVITY (pg/mL)
ASSAY RANGE (pg/mL)
Alpco Diagnostics
3
5 – 40,000
ARP American Research Products, Inc.
20
300 – 40,000
Assay Designs
59.6
122 – 500,000
Bio-Quant Inc.
140
Not available
Calbiotech, Inc
20
Not available
Cayman Chemicals
20
8-43
Diagnostic Systems Laboratories, Inc.
40
100 – 30,000
Immuno-Biological Laboratories, Inc.
75
0 – 40,000
Neogen Corporation
80
40 – 4000
Estrogenic Endocrine Disrupting Compounds - Review
31
6
Determination of Steroid Estrogens in
Seawater and Sediment using ELISA and
GC-MS
This chapter describes the development and validation of ELISA and GC-MS
methodologies for determining estrone (1,3,5(10)-estratrien-3-ol-7-one), 17ß-estradiol
(1,3,5(10)-estratriene-3,17ß-diol), and estriol (1,3,5(10)-estratriene-3,16á,17ß-triol) in
seawater and sediment.
6.1
Materials and Methods
6.1.1
Chemicals and Materials
The chemical and materials used in this work are listed in Table 16. Chemicals used in
this work are analytical grade or higher.
6.1.2
Preparation of Standard Solutions
Standards of estrone, 17ß-estradiol, and estriol with concentrations in ethanol of 200,
400, and 200 µg/L, respectively were purchased from Cayman Chemical for GC-MS
analysis. These were stored in the dark at -20 oC until use. The calibration curves were
prepared by serial dilution of the stock standards after derivatisation (discussed in
section 6.1.5).
ELISA kits for E1, E2 and E3 purchased from Cayman Chemical (Ann Arbor, MI, USA)
were used in an earlier series of tests. The analytical results from these kits were not
consistent. The problem was attributed to possible contamination during the four
months storage time prior to experimentation (Appendix A). To avoid potential
problems from reoccurring in the current series of tests, kits for E1 and E2
manufactured by Tokiwa Chemical Industries Co. Ltd, Japan were used to analyze eEDCs in environmental samples following other studies (Maurício et al. 2006). The E3
ELISA kit was not available from this company and was purchased from Cayman
Chemical. The estrone and 17ß-estradiol standard serial dilutions were supplied by
Tokiwa Chemical. However for E3 ELISA analysis, standard serial solutions for
calibration were prepared by diluting the stock E3 standard according to the
instructions for using the Estriol EIA Kit supplied by Cayman Chemical.
Estrogenic Endocrine Disrupting Compounds - Review
32
Table 16 Chemical and material used for e-EDC GC-MS and ELISA analysis
CHEMICAL/MATERIAL
Estrone (E1)
a
a
17ß-Estradiol (E2)
Estriol (E3)
Estrone
a
SOURCE
Standard
Cayman Chemical (Ann Arbor, MI, USA)
Standard
Cayman Chemical (Ann Arbor, MI, USA)
Standard
Cayman Chemical (Ann Arbor, MI, USA)
b
17ß-Estradiol
Estriol
GRADE OR
PURITY
Cayman Chemical (Ann Arbor, MI, USA)
b
Cayman Chemical (Ann Arbor, MI, USA)
b
Cayman Chemical (Ann Arbor, MI, USA)
BSTFA+1% TMCS
Derivatisation
Supelco
Ethyl acetate
Analytical
Romil Pure Chemistry
n-hexane
Analytical
Romil Pure Chemistry
Acetone
Analytical
Romil Pure Chemistry
Methanol
Analytical
Romil Pure Chemistry
Ethanol
Analytical
Merck
Sodium chloride
Analytical
BDH
Sodium sulfate
Analytical
BDH
Potassium chloride
Analytical
BDH
Sodium bicarbonate
Analytical
ECP
Potassium bromide
Analytical
BDH
Magnesium chloride
Analytical
BDH
Calcium chloride
Analytical
BDH
Water
c
MilliQ 18 M
Water
d
ChromAR
HPLC
cm
Environmental Engineering
University of Auckland
Laboratory,
Mallinckrodt Chemicals
Estriol EIA kit
Cayman Chemical (Ann Arbor, MI, USA)
Estrone ELISA kit
Tokiwa Chemical Industries Co. Ltd, Japan
17ß-Estradiol ELISA kit
Tokiwa Chemical Industries Co. Ltd, Japan
Solid
phase
extraction
cartridges C18 (6mL column
with 500 mg sorbent)
Cayman Chemical
a
Estrone, 17ß-estradiol, and estriol stock standards were used to obtain the calibration curves for
GC-MS.
b
Other EDC stock solutions were prepared by dissolving particle EDCs in ethanol. Their
concentrations were calibrated against the standards with GC-MS.
c
MilliQ water was used to wash labware, prepare artificial seawater and precondition SPE
cartridges
d
HPLC grade water was used in ELISA analysis.
Estrogenic Endocrine Disrupting Compounds - Review
33
6.1.3
Sediment and Artificial Seawater Sample Preparation
Sediment collected from Mission Bay, Auckland was air dried and cleaned using a
sequence of solvents (hexane, ethyl acetate, methanol, and MilliQ water). In this
procedure the sediment was completely immersed under the solvent during ten
minutes of ultrasonication, after which the supernatant was drained and replaced with
fresh solvent. The cleaned sediment was then dried at 105oC for 48 hours. This
procedure is expected to remove organic matter from the sediment (although the total
organic carbon of the solvent-washed sediments was not measured). Pre-weighed
sediment samples were spiked with individual e-EDCs or a mixture of E1, E2 and E3,
to obtain a concentration of 1 µg/kg for each e-EDC in the sediment samples.
Artificial sea water was prepared using the chemicals presented in Table 17 following
the method of Kester et al (1967). The artificial seawater was spiked with individual eEDCs or a mixture of E1, E2 and E3 to obtain a concentration of 10 ng/L for each EDC.
Table 17 Composition of artificial seawater
CHEMICAL
CONCENTRATION
WEIGHT (g/mol)
(g/kg)
Sodium chloride (NaCl)
58.44
23.926
Sodium sulfate (Na2SO4)
142.04
4.008
Potassium chloride (KCl)
74.56
0.667
Sodium bicarbonate (NaHCO3)
84.00
0.196
Potassium bromide (KBr)
119.01
0.098
Sodium fluoride (NaF)
41.99
0.003
61.83
0.026
a
Boric acid (H3BO3)
Magnesium chloride hexahydrate (MgCl2.6H2O) 203.30
10.831
Calcium chloride dihydrate (CaCl2.2H2O)
147.01
1.519
266.64
0.024
a
Strontium chloride hexahydrate (SrCl2.6H2O)
a
6.1.4
MOLECULAR
Boric acid and strontium chloride hexahydrate were not included in this work.
Extraction Protocol
Sample preparation was performed to extract and concentrate the target compounds.
The e-EDCs in water samples were extracted using solid phase extraction (SPE) while
those in sediments were extracted with organic solvents under ultrasonication. The
procedure used in this work for e-EDC extraction and analysis is depicted in Figure 9.
The SPE system has been widely and successfully used to extract and concentrate
target e-EDCs from aqueous samples prior to analysis. The principles of SPE involve a
partitioning of compounds between two phases: The analytes to be extracted are
partitioned between a solid and a liquid phase. Ideally target analytes should have a
greater affinity for the solid phase than for the matrix. Compounds retained on the
solid phase can be eluted with a solvent which has a higher affinity for the analytes.
Estrogenic Endocrine Disrupting Compounds - Review
34
Figure 9 Procedure for e-EDC extraction and analysis
A general SPE procedure is given in Figure 10, where the sorbent bed of the cartridge
retains the target compounds along with some impurities. The SPE cartridge is first
pre-conditioned by flushing with a solvent followed by water. The sample is then
introduced during the loading stage. A specific solvent is used to wash away some of
the impurities, leaving the compound of interest on the column for subsequent elution
in an appropriate solvent or solvent mixture. The type of SPE cartridge sorbent and the
eluting reagent are two important factors governing extraction efficiency. Oasis HLB
and C18 cartridges have been used in extracting various organic contaminants from
environmental samples (Gabet et al. 2007; Petrovic et al. 2002). The Supelco C18 SPE
cartridges were used to extract target EDCs from water samples in this work due to
low interference with complex matrices (López De Alda and Barceló 2001; RodgersGray et al. 2000). Three types of commonly used eluting solvents - ethyl acetate,
acetone/hexane (65/35, v/v), methanol/acetone (50/50,v/v), were tested, and their
elution and extraction efficiencies were determined.
The SPE cartridges generally require preconditioning with various solvents
(Mouatassim-Souali et al. 2003; Peck 2006; Sarmah et al. 2006; Zhou et al. 2009). In
this work the 6 mL SPE C18 cartridges (500 mg sorbent) were conditioned with 5.0 mL
methanol followed by 5.0 mL water prior to loading water samples containing the
target e-EDCs. The spiked artificial seawater (50mL) was applied to a conditioned
cartridge at a flow rate of 10.0 mL/min as higher (e.g. 25 mL/min) flow rates have been
reported to decrease the extraction efficiency (Peck 2006). The cartridges were then
dried under a gentle stream of nitrogen, and then eluted with 5 mL of organic solvent
at a flow rate of 4mL/min (Mouatassim-Souali et al. 2003). Three types of organic
solvents (ethyl acetate, acetone/hexane [65/35, v/v] and methanol/acetone [50/50 v/v])
were tested and compared as e-EDC eluting reagents. The eluate was collected and
evaporated to dryness in a vacuum drying oven at room temperature.
Estrogenic Endocrine Disrupting Compounds - Review
35
Figure 10 Procedure of extracting target compounds using SPE
Ultrasonically-assisted organic solvent extraction is generally used to recover e-EDCs
from solid matrices (Hájková et al. 2007). The e-EDCs were extracted from the spiked
sediment in an ultrasonic bath using the same organic solvents as those used to elute
the SPE C18 cartridges for artificial seawater samples. About 2mL of the organic
solvent (i.e. ethyl acetate, acetone/hexane (65/35, v/v) or methanol/acetone (50/50,v/v))
was added to 1.5 g of spiked sediment so that the sediment was fully immersed. The
suspension was ultrasonically agitated for 15 minutes, the supernatant was collected
and fresh organic solvent was added to the sediment. This procedure was repeated
three times for each sediment sample. The supernatant from each extraction step was
combined. Possible sediment particles in the supernatant were removed by passing it
through an SPE C18 cartridge. The flow rate was 4 mL/min (Mouatassim-Souali et al.
2003). The SPE C18 cartridge used in this step had been preconditioned with 5 mL
methanol, 5mL MilliQ water, 5mL methanol followed by 5 mL of the organic solvent
used for extraction. The resulting eluate was collected and evaporated to dryness in a
vacuum drying oven at room temperature.
Estrogenic Endocrine Disrupting Compounds - Review
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6.1.5
GC-MS and ELISA Analysis
Dry e-EDC residues obtained from the procedures described in section 6.1.4 were
either derivatised for GC-MS analysis or dissolved in the solvent specified by the ELISA
kit manufacturer for ELISA analysis.
1) Derivatisation-GC-MS analysis
For GC-MS analysis the dry e-EDC residues were derivatised by adding 100 µL
derivatisation reagent, a combination of N,O - bis(trimethylsilyl)trifluoroacetamide and
trimethylchlorosilane (BSTFA+ 1%TMCS). The capped vials were stirred by vortex at
20,000 rpm for one minute and then allowed to react in an incubator at 40oC for 100
minutes. Derivatised samples were cooled at ambient temperature and the solution
was evaporated to dryness under a gentle stream of nitrogen, followed by the
dissolution of the residue in anhydrous hexane.
Aliquots of the reaction mixtures were vortex-stirred and analysed using a Shimadzu
GC 2010 gas chromatograph equipped with a non-polar SLB-5MS 30m × 0.25mm x
0.25 µm capillary column (Supelco, U.S.A.). The temperature of the injector (with a
split/splitless insert) was set at 250oC , and the oven was programmed to provide a
temperature of 175oC for 0.5 minutes, ramp at 15oC/min to 295oC, and then maintain
at this temperature for 10 minutes. The carrier gas was helium with a constant linear
velocity of 1mL/min. Qualitative analysis of EDCs was performed on the basis of
retention time and mass spectrum in a full scan mode, while the quantitative
determination was made in a selected ion monitoring (SIM) mode, i.e. the intensities
of several specific ion beams are recorded rather than the entire mass spectrum.
2) ELISA analysis
As described in Section 6.1.2, E1, E2 and E3 were analyzed using ELISA kits supplied
by different manufacturers. The E1 and E2 kits were provided by Tokiwa-Chemical
while the E3 ELISA kit was purchased from Cayman Chemical. The principles and the
procedures of the ELISA kits are similar and schematically illustrated in Figure 11 and
Figure 12.
a)
E1/E2 ELISA analysis (Tokiwa-Chemical 2005a; b)
The analysis of E1 (or E2) with ELISA kits from Tokiwa Chemical was based on the
specific reaction between E1 (or E2) antibody with E1 (or E2). The test protocol
can divided into six steps: sample pre-treatment, mixing sample/standard with
antigen-enzyme conjugate solution, competitive reaction under incubation,
washing unbound material, colour development and absorbance measurement
(Figure 11).
Estrogenic Endocrine Disrupting Compounds - Review
37
Figure 11 Procedure for E1 analysis with Tokiwa Chemical ELISA kit
E1 (or E2) in 10% methanol in H2O and an E1 (or E2)-enzyme conjugate were
mixed and added to specific antibody coated microplate wells, where competition
occurred for the limited number of binding sites of the immobilised specific
antibodies coating the surface of the microplate wells. This competitive reaction
was normally carried out at 18-25oC for 60 minutes. Unbound estrogen and excess
estrogen-enzyme conjugated were then washed out using the specific washing
buffer provided with the kit. A chromogenic substrate was then added, which was
catalysed by the antibody bound enzyme-labelled E1 (or E2) to a coloured product.
This colour development was generally carried out at 18-25oC for 30 minutes
before the reaction was stopped by the addition of an acid. The absorbance of the
solution in each well was then measured at 450 nm with a microplate reader,
within 15 minutes of the colour development reaction being stopped. Higher E1 (or
E2) concentrations will compete with the E1 (or E2)-enzyme conjugate for antibody
binding sites and therefore result in less antibody-bound enzyme-labelled E1 (or
E2), generating a lighter colour and lower absorbance.
The standard calibration curve for E1 (or E2) ELISA analysis was obtained from a
series of known concentrations of E1 (or E2) standards prepared in 10% methanol.
Estrogenic Endocrine Disrupting Compounds - Review
38
The E1 (or E2) concentrations in samples were then calculated using the standard
calibration curve.
b) E3 ELISA analysis (Cayman-Chemical 2008)
The analysis of E3 with ELISA kits from Cayman Chemical was based also on the
competition between E3 and E3-acetylcholinesterase (AChE) conjugate for a
limited number of E3-specific rabbit antiserum binding sites coating the surface of
the microplate wells. The procedure includes six steps: sample pre-treatment,
mixing the sample/standard with antigen-enzyme conjugate solution, competitive
reaction under incubation, washing unbound material, colour development and
absorbance measurement.
Figure 12 Procedure for E3 analysis with Cayman Chemical ELISA kit
Unlike the E1/E2 ELISA kits from Tokiwa Chemical where 10% methanol in H 2O was
used to dissolve target estrogens, the E3 ELISA kit from Cayman Chemical used EIA
buffer supplied with the kit to dissolve the samples. Compared to the Tokiwa Chemical
ELISA kits, Cayman Chemical ELISA kits require a longer competition reaction time;
the former requires 60 minutes at 18-25oC without shaking, while the latter requires
either 120 minutes at room temperature on an orbital shaker or overnight at 4 oC.
Moreover, Cayman Chemical ELISA kits require a longer colour development time (60Estrogenic Endocrine Disrupting Compounds - Review
39
120 minutes with orbital shaking), while Tokiwa Chemical recommends 30 minutes for
colour development without shaking. The Tokiwa Chemical kits required an additional
step of adding dilute acid to stop the colour development reaction prior to the
absorbance measurement at 450 nm, whereas the Cayman Chemical E3 ELISA kit
involves measuring the absorbance at 405-420 nm directly after colour development
(i.e. without an additional step to stop the colour development reaction).
Samples containing E3 in this work were analysed with the Cayman Chemical E3
ELISA kit following the manufacturer’s guidelines. The competition reaction was
carried out overnight and 90-120 minutes of colour development followed the plate
washing step. The samples were then measured with a microplate reader at 405 nm.
6.1.6
Method Validation
Validation of the methodology involved determining the recovery efficiency of e-EDCs
from SPE cartridges for each of the three solvent mixtures, developing the standard
calibration curves, and estimating the detection limits. The recovery efficiencies and
the method reproducibility were determined for spiked sediment and artificial sea
water samples. The instrument detection limit (IDL) and the limit of quantitation (LOQ)
were estimated (Bliesner 2006; Raman et al. 2008). The LOQ is ‚the minimum
concentration of analyzed substance that can be determined at an acceptable degree
of reproducibility and accuracy under rated conditions of analysis by a given method‛
(Épshtein N. A., 2002)
The IDL was determined for GC-MS analysis by injecting derivatised e-EDCs standards
(5000 ng/L in hexane) into the GC-MS system. Values were derived by subsequently
injecting lower concentrations so that an S/N (signal-to-noise) ratio of 3:1 was
produced. The LOQ was determined by analysing spiked and unspiked sediment or
artificial seawater samples. The LOQ values for e-EDCs were obtained from the
concentration at an S/N ratio of 10 taking into consideration the sample quantity and
dilution factors in the extraction, elution and derivatisation procedures.
For ELISA analysis the quantitation ranges for E1, E2 and E3 were provided by the kit
supplier. The LOQ in this work was based on 10-fold multiplication of the absorbance
of unspiked sediment or artificial seawater sample. This value was used to determine
the concentration using the standard curves. The LOQ for ELISA analysis of sediment
and seawater were then calculated after taking sample quantity and dilution factors
into account.
In addition, the target e-EDC loss from SPE cartridges was determined using GC-MS.
This was estimated by step-wise loading of an SPE cartridge with 50mL, 100mL,
200mL, and 400mL for a total of 750mL solution of the artificial seawater spiked with
50 ng/L of E1, E2 and E3 as a mixture. Eluates from the 50mL, 100mL, 200mL, and
400mL loading steps were separately collected and loaded to individual fresh SPE
cartridges (referred to as cartridges 2, 3, 4, and 5, respectively) as illustrated in Figure
13. The e-EDCs retained by these cartridges were eluted and the e-EDC breakthrough
from the originally loaded C18 cartridge (referred to as cartridge 1) was analysed using
GC-MS.
Estrogenic Endocrine Disrupting Compounds - Review
40
Figure 13 Sequence used for testing EDC breakthrough in SPE C18 cartridge
750mL
(applied in
steps of 50mL,
100mL, 200mL
and 400mL)
50mL
Cartridge 1
Cartridge 2
100mL
200mL
400mL
6.2
Results and Discussion
6.2.1
Optimisation of the Derivative Conditions prior to GC-MS
Cartridge 3
Cartridge 4
Cartridge 5
As the reported derivatisation time and control temperature vary widely depending on
the target compound being derivatised, the derivative reaction temperature and time
for E1, E2 and E3 were examined to obtain higher sensitivity. Hexane was used as the
solvent for BSTFA+TMCS derivatised products (Zhou et al. 2009). Testing samples
were prepared using e-EDC standards with the same concentration of E1, E2 and E3.
They were derivatised at different temperatures and with different derivatisation times.
The GC-MS total ion chromatography (TIC) peak area of E1, E2 and E3 obtained from
these samples are shown in Figure 14. In general higher peak areas were obtained for
samples derivatised at 40oC instead of 70oC with the same treatment time. Increasing
the derivatisation reaction duration did not always result in larger peak area values;
samples derivatised over the longest duration of 16h gave the lowest peak areas. The
largest peak areas for E1, E2, and E3 were obtained at 40oC with 100 minutes of
derivatisation time. These conditions were therefore selected as being optimal for
derivatising E1, E2 and E3 prior to GC-MS analysis.
Estrogenic Endocrine Disrupting Compounds - Review
41
Figure 14 Effects of derivatisation time and temperature on GC-MS TIC peak area of e-EDCs
1600
40C -60min
40C -100min
T IC peak area
1200
40C -16h
70C -60min
70C -100min
800
70C -16h
400
0
E1
6.2.2
E2
E DC s
E3
Standard Calibration and Detection Limits
1) Derivatisation and GC-MS analysis
Blank sediment sample extracts, artificial seawater extracts, and e-EDC standards
were derivatised under the optimal conditions described in Section 6.2.1 prior to GCMS analysis. A GC-MS chromatogram in the SIM mode for analyzing E1, E2 and E3
mixture is shown in Figure 15. Calibration curves were prepared for individual e-EDCs
as well as a mixture of E1, E2, and E3. Good linearity was observed, with correlation
coefficients greater than 0.99 obtained for e-EDC concentrations ranging from 50 to
5000 ng/L (Figure 16). The IDL and LOQ for E1, E2 and E3 in sediment and artificial
seawater samples were estimated and are summarised in Table 18. The LOQ values
for sediment sample GC-MS analysis were 1.5, 1.0 and 3.0 µg/kg for E1, E2 and E3,
respectively. These were lower than the values reported in a previous study
undertaken by the National Institute of Water and Atmospheric Research Ltd. (NIWA)
for Auckland Regional Council (Stewart et al., 2009), where the LOQ for GC-MS
analysis of e-EDCs in sediment samples was determined to be 5 µg/kg for E1 and E2,
and 50 µg/kg for E3 (Stewart et al. 2009). A possible reason for this difference may be
that the NIWA report analysed field samples while this report analysed pre-cleaned
sediment samples.
Estrogenic Endocrine Disrupting Compounds - Review
42
Figure 15 GC-MS chromatogram of TMCS derivatives of E1, E2 and E3 in SIM mode (2µL of 2000ng/L
mixture standard)
Table 18 Linearity and detection limits for e-EDC analysis with derivatisation-GC-MS
2
e-EDC
LINEARITY (R )
IDL(ng/L)
E1
0.992
E2
E3
LOQ
SEDIMENT (µg/kg)
SEAWATER (ng/L)
100
1.5
20
0.998
50
1.0
19
0.994
150
3.0
30
Estrogenic Endocrine Disrupting Compounds - Review
43
Figure 16 Calibration curves of E1(a), E2 (b) and E3 (c) from GC-MS analysis.
Open and solid symbols represent results obtained from individual and mixture of e-EDC
standards, respectively.
a)
4000
3500
y = 0.6842x
r2 = 0.9921
E1
3000
intensity
2500
2000
1500
1000
500
0
b)
8000
7000
E2
y = 1.4918x
r2 = 0.9976
6000
intensity
5000
4000
3000
2000
1000
0
c) 3000
E3
2500
y = 0.4698x
r2 = 0.9937
intensity
2000
1500
1000
500
0
0
Estrogenic Endocrine Disrupting Compounds - Review
1000
2000
3000
e-EDC, ng/L
4000
5000
6000
44
2) ELISA analysis
Standard calibration curves for E1, E2 and E3 analysis with ELISA were obtained by
analysing serial dilutions of e-EDC standards following the manufacturer’s guidelines
(Cayman-Chemical 2008; Tokiwa-Chemical 2005a; b). These standard curves are given
in Figure 17 and their shapes are in close agreement with the typical standard curve
examples provided by the kit manufacturers (Appendix B). These curves were used to
calculate e-EDC concentrations from sediment and artificial seawater samples. The
quantitative range for e-EDC ELISA analysis was obtained from user manuals for the
ELISA kits and is listed in Table 19. The LOQ for ELISA analysis was determined by
assessing unspiked control blank samples as described in Section 6.1.6. The LOQ
values determined for e-EDCs in sediment sample analysis are in close agreement
with that reported by Stewart et al (2009), where the LOQ values for E1 and E2 were
0.5 and 0.4 µg/kg, while the values obtained in this work are 1.0 and 0.7 µg/kg,
respectively.
Table 19 Quantitation range and detection limits for e-EDC analysis with ELISA
e-EDC
6.2.3
LOQ
SEDIMENT (µg/kg)
SEAWATER (ng/L)
E1
50~2000
1.0
8.4
E2
50~1000
0.7
7.0
0.2
2.7
E3
a
QUANTITATION RANGE
PROVIDED BY ELISA KITS (ng/L)
a
12 ~2000
Estimated from the overnight incubation format with LOD = 4 ng/L.
C18 Cartridge Loading Capacity and Breakthrough
Environmental samples with volumes of up to several litres of water have been applied
to individual SPE cartridges for EDC extraction in many studies (Gabet et al. 2007).
However little information is publically available on the loading capacity of different
SPE cartridges. The quantity of a compound that can be isolated using SPE depends
on the capacity of the sorbent bed. Low loading capacities may result in the
underestimation of compound concentrations due to the loss of target compounds
caused by poor retention efficiency. Generally, the combined quantity of the target
compound and impurities that can be retained is estimated to be approximately 5%
w/w of the total sorbent bed. The SPE C18 cartridges used in this work have 500 mg
sorbent and the loading capacity is therefore theoretically estimated to be 25 mg
organic compounds per cartridge.
Target e-EDC loss from SPE C18 cartridge was tested in this work and the results are
presented in Table 20 and Figure 18. The e-EDC loss for water samples using the C18
cartridges was in the order of E1<E2<E3. Although trace losses of E1, E2 and E3 were
observed in all cases, loss of the target e-EDC was less than 0.016% of the amount
loaded. Loading capacity has been also expressed as the absolute amount of analyte
loaded into the column in conditions in which more than 1% (w/w) of the sample is not
retained by the stationary phase (Baggiani et al 2007). The SPE C18 cartridge in this
Estrogenic Endocrine Disrupting Compounds - Review
45
work has a loading capacity for e-EDCs higher than 37.5 ng e-EDCs per cartridge,
which is sufficient for extracting e-EDCs from aqueous samples in this work.
Figure 17 Standard curves of E1(a), E2 (b) and E3 (c) obtained from ELISA analysis. B- corrected
standard binding absorbance, B0-corrected maximum binding absorbance.
100
a)
B/B0%
E1
10
10
100
1000
10000
100
b)
B/B0%
E2
10
10
100
1000
100
c)
% B/B0
E3
10
1
Estrogenic Endocrine Disrupting Compounds - Review
10
100
e-EDC, ng/L
1000
10000
46
Table 20 Loss of EDCs (pg/L) from SPE C18 cartridges during the loading capacity tests
CARTRIDGE NO.
LOADING
a
VOLUME (mL)
E1
1
750
NM
2
50
3
4
E2
E3
b
NM
NM
ND
c
ND
5.1
100
ND
2.0
7.6
200
ND
4.7
4.0
5
400
1.4
3.0
Artificial seawater was spiked with 50ng/L E1, E2 and E3
2.1
a
b
Not measured
b
Not detected
Figure 18 Mass (a) and fraction (b) of non-retained e-EDCs in breakthrough for increasing load
application.
a)
b) 0.02
6
e-EDC released, % of total
e-EDC released, pg
E1
4
2
0.012
E2
E3
0.008
0.004
0
0
50
150
350
750
Loading volume, mL
6.2.4
0.016
2.5
7.5
17.5
37.5
e-EDC loaded, ng
Recovery Efficiencies
1) Spike Recovery Efficiencies Obtained For GC-MS Analysis
The use of different organic solvents to elute target EDCs from SPE cartridges has
been reported to result in recovery efficiencies ranging from 0 to 110% (Hájková et al.
2007; Labadie et al. 2007; Quintana et al. 2004; Zhou et al. 2009). Three types of
organic solvents were tested for their ability to elute target e-EDCs from SPE C18
cartridges loaded with artificial seawater samples and extract e-EDCs from sediment
samples in conjunction with ultrasonication. The results obtained from GC-MS analysis
Estrogenic Endocrine Disrupting Compounds - Review
47
are presented in Table 21 and Table 22. Recovery efficiency (R %) was calculated as
the percentage of the total quantity of the target e-EDC extracted (Eq. 1).
R% =
Qe
×100%
Qt
Eq. 1
where Qe and Qt are the extracted and the original quantities of e-EDC
Table 21 Recovery efficiency (R%
RSD) of e-EDCs from seawater samples analysed using GC-MSa
b
E2
b
E3
b
E1
c
E2
c
E3
c
ELUTING
SOLVENT
E1
Ethyl acetate
68.1 4.1
71.2 1.6
82.4 2.8
90.5 2.4
94.9 0.4
88.5 1.9
Acetone/hexane
69.4 3.6
74.4 6.3
81.8 2.5
86.7 3.8
91.4 2.8
86.7 1.5
86.4 0.7
79.4 3.5
79.2 1.6
92.0 5.9
94.6 0.7
96.5 6.6
(65/35, v/v)
Methanol/acetone
(50/50, v/v)
a
Three types of eluting solvents were used
b
Sample contains individual e-EDC
c
Sample contains a mix of E1, E2 and E3
Table 22 Recovery (R%
RSD) of e-EDCs from sediment samples analysed using GC-MSa
b
E2
b
E3
b
E1
c
E2
c
E3
c
ELUTING
SOLVENT
E1
Ethyl acetate
60.8 4.8
56.2 0.7
86.3 0.8
78.3 6.4
81.2 3.4
88.6 2.8
Acetone/hexane
75.3 3.0
62.3 1.8
86.9 0.3
75.3 4.5
92.8 5.0
91.8 1.7
86.3 6.2
63.2 1.1
89.5 1.6
86.5 4.6
95.7 5.1
87.5 4.3
(65/35, v/v)
Methanol/acetone
(50/50, v/v)
a
Three types of eluting solvents were used
b
Sample contains individual e-EDC
c
Sample contains a mix of E1, E2 and E3
The recovery efficiencies obtained using three types of solvents ranged from 68% to
96% for aqueous samples, and from 56% to 96% for sediment samples. A
comparison of the recovery efficiencies for samples spiked with individual e-EDCs and
those for e-EDC mixtures shows that the extraction from samples with a mixture of
EDCs is more efficient, presumably due to greater co-extraction when other
compounds are present.
In general, the 50/50 (v/v) methanol/acetone mixture gave higher recovery efficiencies
than that of ethyl acetate and the 65/35 (v/v) acetone/hexane mixture for both aqueous
and sediment samples. This mixture has been considered as a suitable eluting reagent
in e-EDC sample extraction with SPE in other studies (Hájková et al. 2007; Zhou et al.
Estrogenic Endocrine Disrupting Compounds - Review
48
2009). Therefore this solvent mixture was subsequently used to elute or extract eEDCs from samples for ELISA and GC-MS analysis.
2) Spike Recoveries Obtained For ELISA Analysis
Recovery efficiencies obtained using ELISA for e-EDCs in sediment and artificial
seawater were found to be above 50% for all samples (Table 23). Similar to the results
obtained with GC-MS analysis, recovery efficiencies for samples with e-EDC mixtures
were observed to be higher than those with only one EDC present.
Table 23 Recovery R% ( RSD) of e-EDCs analysed with ELISAa
a
b
E2
b
E3
b
E1
c
E2
c
E3
c
SAMPLE
TYPE
E1
Seawater
92.9 4.7
62.4 2.3
93.3 6.9
107.2 1.1
65.4 1.7
149.9 3.1
Sediment
69.9 3.5
56.2 2.3
79.2 4.6
92.1 4.9
57.4 6.5
106.1 8.4
E1(or E2) and E3 samples were analysed using ELISA kits from Tokiwa Chemical and Cayman
Chemical, respectively
b
Sample contains individual e-EDC
c
Sample contains a mix of E1, E2 and E3
In general the recovery efficiency results for E1 analysed with ELISA are higher than
70%, which are in close agreement with those obtained using GC-MS. Recovery
efficiencies for E2 samples determined with ELISA were slightly lower (by
approximately 10%) than those obtained using GC-MS. A possible reason may be that
e-EDC standards were purchased from different sources, i.e. E2 standard for GC-MS
analysis was supplied by Cayman Chemical, while the E2 standard for ELISA analysis
was from Tokiwa Chemical. To test this, three E2 standards prepared from Cayman E2
standard stock were tested against Tokiwa E2 standards using the Tokiwa E2 ELISA kit.
Results showed that the Tokiwa E2 ELISA kit underestimated Cayman E2 standards, i.e.
the concentration of Cayman E2 standards was determined by the Tokiwa kit to be
lower than the labeled value of the standard (Table 24).
Table 24 Testing Cayman E2 standards with Tokiwa E2 ELISA kit
CAYMAN E2 STANDARDS (ng/L)
E2 CONCENTRATION MEASURED WITH
TOKIWA ELISA KIT (ng/L)
1000
631
500
345
250
157
High recovery efficiencies (>79%) for E3 samples were obtained with ELISA analysis.
However, very high recovery efficiencies were found for samples containing a mixture
of e-EDCs. Although co-extraction was presumably a reason, it is possible that E1/E2
Estrogenic Endocrine Disrupting Compounds - Review
49
might interfere with E3 ELISA analysis, although this phenomenon was not significant
for E1/E2 ELISA analysis.
6.3
Summary
Two analytical methods (derivatisation-GC-MS and ELISA) to determine trace EDCs in
seawater and sediment were developed and validated for three selected e-EDCs. This
report presents the optimal conditions for sample preparation and target e-EDC
derivatisation prior to GC-MS analysis, as well as the procedure for e-EDC analysis with
ELISA kits from two suppliers.
Target e-EDCs in aqueous samples were extracted with SPE and then eluted with an
organic solvent. Sediment bound e-EDCs were extracted with an organic solvent in
conjunction with ultrasonication. A methanol/acetone (50/50 v/v) mix gave the highest
recovery among three types of organic solvents tested. The calibration curves for GCMS showed excellent linearity with the correlation coefficient ranging from 0.992 to
0.998 for E1, E2 and E3, respectively. For ELISA analysis, e-EDC standard curves were
plotted on a log-log graph and were in close agreement with the typical results
presented in the ELISA kit manuals. Detection limits were estimated for both GC-MS
and ELISA methods. The LOQ of ELISA is estimated to be slightly higher than that of
GC-MS.
Estrogenic Endocrine Disrupting Compounds - Review
50
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Appendices
A) Summary of Preliminary ELISA Analysis Results
In February 2009 e-EDC samples were analysed using Cayman ELISA kits. Figure
A.1 presents the setup format for E1 ELISA plates, while the measured
absorbance values obtained from the microplate reader are presented in Table
A.1. Upon data analysis it was found that triplicate readings for each sample had
very high standard deviation and the blank (BLK) values were higher than 0.3.
These values represent the background absorbance caused by Ellman’s reagent
and subtracted from the absorbance readings obtained from the other wells in
the plate. Similarly the non-specific binding (NSB) value was extremely high
(>1.3), and should be less than 0.035 (according to the manufacturer).
As the BLK and NSB values were larger than the measurements for the samples
in several cases, it was not possible to obtain values for EDCs present in the
samples. For each e-EDC, ELISA kits were purchased as a set of four plates in
one package. One plate from each EDC pack had previously been used to
analyse E1, E2, and E3, with the remaining three stored in the freezer without
being properly sealed. It is possible that abnormalities in the initial tests had
arisen from either the contamination or deterioration of plates during the 4-month
storage period. Another possible reason is that the microplate reader measuring
method was not correctly set up by the previous operator.
Figure A.1 Microplate setup format for E1 ELISA analysis. BLK-plate blank, TA-total activity, NSB-nonspecific binding, B0-maximum binding, Std-standards 1-8, WB- seawater blank sample, W19-36seawater sample #19-36.
1
2
3
4
5
6
7
8
9
10
11
12
A
NSB
NSB
NSB
Std6
Std6
Std6
W4
W4
W4
W30
W30
W30
B
TA
TA
TA
Std7
Std7
Std7
W5
W5
W5
W31
W31
W31
C
B0
B0
B0
Std8
Std8
Std8
W6
W6
W6
W32
W32
W32
D
Std1
Std1
Std1
WB1
WB1
WB1
W7
W7
W7
W33
W33
W33
E
Std2
Std2
Std2
WB2
WB2
WB2
W8
W8
W8
W34
W34
W34
F
Std3
Std3
Std3
W1
W1
W1
W9
W9
W9
W35
W35
W35
G
Std4
Std4
Std4
W2
W2
W2
W28
W28
W28
W36
W36
W36
H
Std5
Std5
Std5
W3
W3
W3
W29
W29
W29
BLK
BLK
BLK
Estrogenic Endocrine Disrupting Compounds - Review
63
Table A.1 Measured absorbance values at 414 nm for E1 samples listed in Figure A.1.
SAMPLE
READING 1
READING 2
READING 3
BLK
1.0254
0.3286
2.7633
NSB
3.0197
2.3953
1.2895
TA
0.1431
0.1845
0.1568
B0
0.2706
0.1537
0.4339
Std1
0.4598
0.4169
0.7930
Std2
0.5582
0.5283
0.8215
Std3
0.3736
0.3999
0.8036
Std4
0.3403
0.2209
0.4268
Std5
2.7148
1.7955
1.1358
Std6
1.9860
1.1485
0.9301
Std7
0.3082
0.4843
0.7252
Std8
0.8893
1.0989
1.4047
WB1
0.9724
0.9210
0.6582
WB2
1.1480
1.1415
0.8914
W1
0.8658
0.7842
0.6064
W2
0.6226
0.8658
1.0156
W3
0.6657
0.8998
1.3990
W4
1.3307
1.3592
1.3562
W5
1.0521
1.1017
0.9806
W6
0.7458
0.7458
0.9148
W7
0.3224
0.3206
0.5918
W8
0.3305
0.3332
0.3274
W9
0.5001
0.4674
0.5639
W28
1.2576
1.2333
1.0263
W29
1.5395
1.3841
1.1908
W30
1.1088
1.1622
1.9748
W31
0.7504
0.5065
0.2422
W32
0.9148
0.9128
0.7930
W33
0.5918
0.8667
0.8651
W34
0.4884
0.8212
0.9193
W35
0.8513
0.9476
0.8754
W36
0.9501
0.9060
0.6634
Estrogenic Endocrine Disrupting Compounds - Review
64
B) Typical Calibration Curves for E1, E2, and E3 ELISA Analysis
The concentration of e-EDCs in individual samples were determined using
calibration curves as presented in Figure 17. Typical standard curve examples for
E1, E2 and E3 provided by the kit manufacturers are shown in Figures B.1 and
B.2.
Figure B.1 Example of typical E1 and E2 calibration curves and concentration estimation method
(Tokiwa Chemical 2005a,b).
Estrogenic Endocrine Disrupting Compounds - Review
65
Figure B.2 Example of typical E3 calibration curve (redrawn from Cayman Chemical 2008).
100
% B/B0
80
60
40
20
0
0.1
1
10
100
1000
10000
Es triol ( pg/mL)
Estrogenic Endocrine Disrupting Compounds - Review
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