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Quantitative Analysis and Spatial Variation of
Endocrine Disrupting Chemicals
Ross McLeod
University of West Florida
Kugelman Honors Program
Advised by Dr. Matthew Schwartz
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Table of Contents
Abstract …………………………………………………………………………………………...3
Introduction …………………………………………………………………………………….4-6
Methods ………………………………………………………………………………………...6-8
Data ……………………………………………………………………………………………8-11
Discussion/Analysis ………………………………………………………………………….12-17
Conclusion …………………………………………………………………………………...17-18
References …………………………………………………………………………………...19-21
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Abstract
The increasing problems associated with endocrine disrupting chemicals (EDCs) in the
environment such as increased intersex characteristics in wildlife, increased breast cancer,
decreased sperm counts, and decreased fertility in males has created a call for increased
efficiency in the removal process. Endocrine disrupting chemicals are primarily introduced to the
environment via treated wastewater effluent. Treating wastewater to remove endocrine
disrupting chemicals is an imperfect and evolving science. This study aims to determine if point
source pollution from treated wastewater effluent is the primary mechanism for the introduction
of endocrine disrupting chemicals into the environment. Bisphenol A (BPA) is found to have an
increased concentration in the Pearl River, Guangzhou, China, and it is a point source pollutant.
Removal efficiency strategies are necessary to prevent the reintroduction of BPA to the
environment to prevent bio-accumulation and adverse effects. In addition, 4-nonylphenol is
found to have a high concentration, although it has not been determined to be a point source
pollutant, calling for the need of better removal methods in addition to the determination of the
source of its pollution.
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Introduction
Rivers and streams are places on Earth’s surface that are made up of flowing water. They
are important parts of the water cycle that provide surficial transport of water across Earth’s
continents. Streams also serve as a suitable method for discharging of human wastewater, which
introduces foreign compounds into Earth’s waterways and disrupts established natural
ecosystems.
Wastewater is treated through sewage treatment plants to remove harmful concentrations
of contaminants before the water is re-introduced to the environment. However, this purification
and removal process is not perfect, and many chemicals still remain in micro-concentrations after
the dilution process. Treated wastewater effluent, or the liquid waste discharged into the river,
contains micro-concentrations of compounds missed in the treatment process (Baronti et al.
2000). One such group of compounds that is difficult to completely rid of in the sewage
treatment process is endocrine disrupting chemicals (EDCs).
Endocrine disrupting chemicals are non-volatile, small molecular chemicals that have the
potential to cause infertility, feminization, and other adverse effects in wildlife and humans
(Colborn et al. 1993). Endocrine disrupting chemicals originate from both urban and residential
sources. There are natural estrogenic compounds, such as 17 α-ethynylestradiol (EE2), estrone
(E1), and 17 β-estradiol (E2), which originate from mainly residential sources. There are also
phenolic estrogenic compounds, such as bisphenol A (BPA) and nonylphenol (NP) that originate
from industrial sources. High concentrations of these chemicals have been found to disrupt
sexuality in fish by interrupting vitellogenin synthesis in male fish (Routledge et al. 1998).
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Vitellogenin synthesis and gene expression is a useful marker in male fish because it is a process
mainly found in female fish, which starts the egg forming process in nearly all oviparous species.
Concentrations of endocrine disrupting chemicals are not currently high enough to induce
vitellogenin responses, but the bio-accumulation of EDCs such as BPA and nonylphenol that do
not easily degrade in the environment may cause further ecological impacts in the future.
Increased anthropogenic activity is linked to increased contamination in riverine runoff,
an important contributor of contaminants to coastal zones (Wang et al. 2007). The Pearl River
Delta, which is a highly urbanized section of China undergoing population growth with over 41
million residents, receives 64% industrial sewage and 74% domestic sewage from the
Guangdong Province (Ma et al. 2005). China serves as a powerful indicator of heightened values
of pollution, therefore by analyzing the impacts that rapid urbanization is having on the
environment, a helpful precursor of knowledge can be obtained to prevent the bio-accumulation
of endocrine disrupting chemicals through better wastewater treatment methods and through use
of chemicals that degrade more easily in the environment.
The main questions to be answered by a quantitative analysis of endocrine disrupting
chemicals in the Pearl River Delta are as follows: Will endocrine disrupting chemicals be found
in riverine waterways near sewage treatment plant effluent? If found, will the endocrine
disrupting chemicals produce a spatial correlation based on the comparison between the weight
of the endocrine disrupting chemical concentrations in the riverine water based on interpolation
of the weights as the chemicals move down river, and thus away from the sewage treatment
plant? The source of sewage effluent is expected to be higher in concentration because it is a
point source of pollution in the environment. However, increased runoff in the system and other
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factors may influence the concentrations of endocrine disrupting chemicals in the river down the
gradient.
Methods
Data for concentrations of endocrine disrupting chemicals comes from the Pearl River in
Guangzhou, China. Six points of water samples were collected in the Pearl River according to
the methods laid out in Xu et al. 2014. The water samples were one hour composites collected by
a peristaltic pump at a rate of 100 mL/min from both the surface of the river and the bottom of
the river. They were then filtered using 0.5mm glass fiber filters and acidified to a pH of 3.0
using 18N sulfuric acid for preservation.
Liquid chromatography mass spectrometry was used to quantify the amount of endocrine
disrupting chemicals in the river water samples according to Xu et al. 2014. The concentrations
determined by Xu et al. 2014 were used in all geospatial analysis.
Quantification of endocrine disrupting chemicals is a difficult and evolving science. The
molecules are small, non-volatile, and have a low concentration in environmental samples due to
the dilution in the environment. Because of this, extraction procedures are necessary to increase
the relative concentration of the samples whereby a factorial can be used to determine the
original values in the sample, which are almost always <500 ng/L and sometimes <1 ng/L in
concentration.
Using gas chromatography mass spectrometry is a fast and accurate method for
determining the concentrations of endocrine disrupting chemicals, but derivitization must take
place in order to volatize the sample for gas chromatography analysis. Endocrine disrupting
chemicals are not inherently volatile, therefore volatile chemicals must be used to prepare
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samples for analysis, a method which is both time consuming and requires many materials.
Liquid chromatography is another preferred method for measuring endocrine disrupting
chemicals, but the columns may be difficult to find to produce a low enough limit of
quantification to receive a measurement.
Extraction procedures are necessary for both liquid chromatography mass spectrometry
and gas chromatography mass spectrometry. Solid phase extraction by Oasis HLB cartridges
previously conditioned using dichloromethane, methyl tert-buryl ether, methanol, and Milli-Q
water was used in this circumstance. In order to further concentrate the samples, HLB cartridges
were dried under nitrogen gas for 1h, and analytes were eluted by 5mL of methanol and further
concentrated to 1mL using a stream of nitrogen.
The final 1mL samples were used in liquid chromatography mass spectrometry to
determine the concentration of six unique endocrine disrupting chemicals: estrone (E1), 17β-
estradiol (E2), estriol (E3), 17α-ethnylestradiol (EE2), bisphenol A (BPA), and 4-nonylphenol
(4-NP). These six were chosen as they are a mixture between natural endocrine disrupting
chemicals such as E1 and E2 which naturally degrade in the environment, and synthetic
endocrine disrupting chemicals such as BPA and 4-NP which do not easily degrade in the
environment and are typically found in high amounts in industrial zones.
Spatial analysis of these chemicals was performed by using ESRI’s ArcGIS 10.2
software. Water samples were collected in the middle of the river at nearly equidistance points,
therefore the creation of an isoline across the river from bank to bank for each point collected
was created using two point values of equal weight at each bank of the river at the same latitude.
The spatial analyst license’s Nearest Neighbor Tool was used to interpolate the weights of the
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data in order to provide a visual representation of how the concentration of each endocrine
disrupting chemical changed down the river. This was repeated for each endocrine disrupting
chemical, providing six concentration gradient maps.
Data
Table 1 (Xu et al. 2015)
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Discussion/Analysis
Estrone (E1)
Estrone is primarily found in wastewater effluent, as it is reintroduced to the environment
through human excrement and waste (Lee et al. 2014). Estrone excretion is found to be
significantly higher in women than in men (Liu et al. 2015). Estrone is a natural estrogen, and
when significant amounts of estrone are introduced via the environment, medical conditions such
as breast tenderness, nausea, headache, hypertension, and leg cramps can be found in human
females (OSHA 2006). In addition, men can experience anorexia, nausea, vomiting, and erectile
dysfunction when estrone concentrations are found to be too high. Estrone is also a known
human carcinogen (OSHA 2006).
The sewage water effluent input occurs at the westernmost point in the map. The
concentration of estrone at the western edge of the study area is the lowest. Removal of estrone
through wastewater treatment is the lowest, with an 86% removal rate. However, much greater
concentrations of estrone are found further away from the point source of wastewater effluent.
Point source pollution of estrone is therefore likely to not be a significant issue in urban areas.
Runoff pollution into the Pearl River is likely a greater indicator of the introduction of estrone
into the environment, as it is found in much higher concentrations further down the river.
China’s expanding population significantly contributes to the introduction of estrone into the
environment. Treatment methods for wastewater effluent are therefore suitable; the main issue
with estrone in China would be to prevent overpopulation and population growth in the area.
Guangzhou is also a densely populated region, further contributing to higher concentrations of
estrone in the area due to increased human activity.
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17 β-estradiol (E2)/ 17 α-ethynylestradiol (EE2)
17 β-estradiol, known more commonly as estradiol, is a natural estrogen found in most
vertebrates, insects, fish, and other animals. In human females, it is necessary to maintain
oocytes in the ovaries, and acts as a growth hormone in the female reproductive system,
maintaining its organs. In addition, it plays a small role in preventing apoptosis, or the cellular
death, of sperm cells in human men (Pentikainen et al. 2000). Waterborne exposure of 17 β-
estradiol can also cause sexually mature fathead minnows to experience decreased gonad size,
causing a prominent change in male secondary sex characteristics (Miles-Richardson et al.
1999). The effects experienced in these fish is not permanent, meaning that point source
pollution is not a significant issue for overall ecological health as the secondary sex
characteristics of these fish are able to recover to suitable levels necessary for reproduction.
Estradiol is used in the medical field as a hormonal contraception, and is commonly found as a
derivative compound of many types of birth control (Glasier 2010).
17 α-ethynylestradiol (EE2) is a derivative of 17 β-estradiol, and is a common marker of
high estrogenic medication in the area, as it is an orally bioactive estrogen in oral contraceptive
pills. By measuring 17 α-ethynylestradiol in the environment alongside 17 β-estradiol,
comparisons can be made between the relative removal of each substance and its availability in
the environment.
Estradiol is primarily introduced to the environment mainly through human excretion by
areas of high oral contraceptive use. The compound 17 β-estradiol is found in higher
concentrations in the environment because it is the component that the human body breaks
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estradiol down into and it is a useful chemical marker for estrogen in the environment. The
removal rate of treated sewage water for estradiol is very high, with between 94-96% of estradiol
removed. Based on the interpolation of estradiol, it appears that the point source nearest the input
points of treated wastewater effluent are the highest in concentration, though the concentration of
estradiol quickly lowers in value as the river progresses. This is apparent in the analysis of both
the maps of 17 α-ethynylestradiol and 17 β-estradiol. This is notable, as estradiol is a natural
compound that breaks down naturally in the environment, meaning that it is available in trace
amounts <2 ng/L. Estradiol is currently not a common endocrine disrupting chemical in even
highly urban environments due to the successful sewage water treatment methods and the
chemical’s ability to break down in the environment.
Estriol (E3)
Estriol is the third and final main estrogen produced in the human body. It is mainly
produced in pregnant women to protect fetal processes. Elevated levels of estriol, when
combined with elevated levels of estradiol, typically produce adverse effects in humans. Estriol
has initially been linked as a chemical that plays a part in the development of breast cancer
(Lappano et al. 2010).
The removal of estriol in wastewater effluent is nearly 100%. It is found in high
concentrations in wastewater, near 137 ng/L, and environmental values of the treated wastewater
effluent are found to be <1 ng/L. Estriol is also a natural chemical that typically is not found in
high concentrations in environmental sources, but increased usage of estriol in medical fields as
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hormone therapy and in pregnant women with low levels of estriol may increase its prevalence in
the environment.
Like estradiol, estriol easily degrades in the environment and concentrations are very low.
The concentration of estriol is not high at the point source, as nearly all estriol is removed from
sewage water via treatment processes. Therefore, estriol in river water mainly comes from
bioaccumulation and runoff in the environment.
Bisphenol A (BPA)
BPA is a synthetic estrogenic compound that is not soluble in water and it is used to
make plastics for consumer goods. BPA mimics estrogen in the body, with a structure and
function that closely mimics estradiol. BPA is a major problem in the environment as it does not
easily break down, and it accumulates rapidly due to increasing use of plastics. It has been noted
to decrease litter size in rats, induce developmental effects on the brain in humans, decrease male
reproductive ability, and reduce sperm count (Beronius 2010).
Biodegradation and the treatment of BPA by wastewater treatment plants has been able to
reduce the amount of BPA by 92%. However, BPA is still found in higher concentrations in
wastewater effluent due to the widespread use of the chemical in plastics, making the removal
process far from perfect. BPA is therefore found to be highest in concentration at the point
source of the sewage treatment plant. It is notable that the concentration of BPA in river water
samples was found to exceed >500 ng/L, making it one of the most notable endocrine disruptors
in the environment. A contamination plume of BPA is found to decrease from >500 ng/L at the
point source of contamination down to <250 ng/L at the furthest point. Therefore, the primary
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issue moving forward is to improve the efficiency of BPA removal in sewage water to prevent its
bioaccumulation.
4-nonylphenol (4-NP)
4-nonylphenol is an organic compound that acts as an endocrine disruptor due to its
ability to act with estrogen-like activity in organisms (Mergel 2011). This ability to act as a
estrogenic compound makes 4-NP a xenoestrogen like bisphenol A. 4-NP acts like estradiol in
the human body and has the potential to cause cell death in placental cells, decreasing the
likelihood of fetal survival when introduced in elevated amounts (Bechi et al. 2009). In addition,
excess amount of nonylphenol exposure have been associated with breast cancer by increasing
the proliferation of breast cancer cells and acting as an antagonist to the protective 17 α-
ethynylestradiol (Soares 2008).
4-nonylphenol is found in high quantities in the water samples collected because it is
difficult to remove from sewage water. 89% of 4-NP is removed in treated wastewater effluent,
but because 4-NP is found in high amounts in sewage water, high concentrations between 200-
400 ng/L are found in the environment. One such method for improving the removal of 4-NP
from wastewater effluent is by using a nitrifying membrane sequence bath reactor to biodegrade
4-NP, effectively removing 62% as a single step in removal of 4-NP found in sewage water
(Buitrón et al 2015). Concentrations of 4-NP are comparatively lower than that of BPA, but the
concentration of 4-NP is not found to correlate with point source pollution for wastewater
effluent. As a result of increased 4-NP at non-point sources, it is possible that 4-NP is a bigger
problem throughout urban areas as it is high throughout samples.
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4-NP poses a significant threat because of its xenoestrogenic qualities as a endocrine
disrupting chemical and preventing bioaccumulation of 4-NP is difficult as concentrations are
found to be high even as spatial distance from point source pollution increases. Nonylphenols are
additives used as emulsifiers and solubilizers (Soares et al. 2008). Because nonylphenols are
primarily used in manufactured goods, they are also found to be in higher concentrations where
humans are present, further pronouncing the need to prevent contamination of waterways and
introduction of these chemicals via wastewater.
Conclusion
The primary endocrine disrupting chemicals found in environmental river water that
poses a significant threat to humans are bisphenol A and 4-nonylphenol. Both of these chemicals
are xenoestrogens that do not easily degrade in the environment. Bioaccumulation of
xenoestrogens in the environment is becoming a growing issue, as xenoestrogens do not easily
break down in the environment and wastewater treatment methods are not currently viable
enough to prevent the reintroduction of these chemicals into the environment.
Four of the six endocrine disrupting chemicals analyzed are likely to be point source
pollutants: BPA, both compounds of estradiol, and estriol. 4-nonylphenol was found to have
elevated levels near point source treated wastewater effluent, but concentrations were found to be
high when not near a wastewater treatment plant. Further work needs to be done to determine the
sources of 4-nonylphenol in the environment to prevent its bio-accumulation.
Close monitoring of xenoestrogens and their effects on wildlife are necessary to protect
ecosystems and prevent population loss of small fish species through development of intersex
characteristics and infertility. In addition, if the concentrations of xenoestrogens are allowed to
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increase, adverse effects could be expressed in humans, increasing the prevalence of breast
cancer and decreasing sperm counts in adult men.
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