Determination of some Endocrine Disruptors in
a Sewage Treatment Oxidation Pond and a
Receiving Stream by High Performance Liquid
Chromatography and Inductively Coupled
Plasma-Mass Spectrometry
N. Torto
Department of Chemistry, Rhodes University,
Grahamstown 6140, South Africa
A.O. Ogunfowokan, E.K. Okoh, and
A.A.Adenuga
Department of Chemistry, Obafemi Awolowo
University, Ile-Ife, 220005 Nigeria
What are Endocrine disruptors?
Endocrine disruptors (sometimes referred to as
hormonally active agents) are exogenous substances that
act like hormones in the endocrine system and disrupt the
physiologic function of endogenous hormones
Substances that stop the production or block the
transmission of hormones in the body and often interfere
with development
Endocrine disrupting compounds encompass a variety of
chemical classes, including pesticides, compounds used in
the plastics industry and in consumer products, and other
industrial by-products and pollutants
A large number of environmental pollutants including
alkylphenolic compounds, polychlorinated biphenyls, and
heavy metals including lead, mercury, cadmium and arsenic
have been shown to disrupt endocrine functions in animals
(Susan and John, 2001; Wu et al., 2003; Lee et al., 2003).
Any Adverse Health Effects?
Endocrine disrupting chemicals have been proposed as a potential
cause of a numerous human health problems such as:
Birth defects; alterations in sexual and functional development
(Thomas and Colborn 1992);
neurologic disorders, diabetes mellitus, immunologic disorder
(Smoger et al,1993)
early puberty in young girls (Colon et al., 2000;Zacharids et al..,
1970),
Breast cancer (Wolff et al., 1993, Steingraber et al.,1997,
contribution to subfertility (Newbold, 1995),
Reduced physical stamina (Guillette,et al., 1998)
Reduced sperm counts and un-descended testes (Toppari et
al.,1996) and
Enlargement/reduction of prostate (vom Saal et al., 1997).
Inter-sex in fish e.g. fish from River Tames in London
Occurrence of Endocrine
Disruptors (Phthalates)
Phthalates are components of many consumables, including:
Personal- care
Paints
Industrial plastics, and
Certain medical devices and pharmaceuticals (ATSDR, 1993, David
et al., 1999).
Surface waters of south western Nigeria (Ogunfowokan and Fatoki
1993a&b; Ogunfowokan et al. 2006; Torto et al. 2007)
Phthalates are moderately persistent and, as a consequence of their
wide use, are the most abundant man-made chemicals in the
environment (Jobling et al., 1995).
They are not chemically bound to the polymer matrices of the
products but are present as a mobile component, significant
migration of them into the environment is inevitable.
Occurrence of Endocrine
Disruptors (Lead, Cadmium and
Arsenic)
Sources of Lead include: paint, Inks and dyes, Vehicular
emission, Plastics and chemicals, Dust in the roof void
etc (http://www.lead.org.au/fs/fst2.html)
Cadmium is released into the environment from mining
and metal processing operations, burning fuels, making
and using phosphate fertilizers, and disposing of metal
products.
Arsenic is found in the natural environment in some
abundance in the Earth’s crust and in small quantities in
rock, soil, water and air. It is present in many different
minerals. Industrial processes such as mining, smelting
and coal-fired power plants are other sources
What are Phthalate Esters?
Phthalates are dialkyl or alkyl
aryl esters of phthalic acid with
the general structure shown
below where R1 and R2 can
be various combinations of
straight and branched alkyl
chain or aryl group.
Are persistent organic
pollutants (POPs) and liable to
undergo significant
biomagnification
Chemicals that have high
environmental toxicity to
humans and other organisms
Health Effects of Pb, Cd and As
Lead
For pregnant women, elevated Pb concentrations increase the risk
of hypertension and birth defects (Rabinowitz, 1988); reduction in
the IQ of Children (Ogunfowokan et al. 2000).
Cadmium
Health effects of Cd are: Ittai-Ittai diseases, it also has mutagenic,
carcinogenic and teratogenic effects (Fischer, 1987; Friberg, et al.,
1986; Heinrich, 1988).
Arsenic
Health effects of Arsenic are: decreased production of red and
white blood cells, skin changes and lung irritation, infertility and
miscarriages with women, and it can cause skin disturbances,
heart disruptions and brain damage with both men and women,
inorganic arsenic can damage DNA
(http://www.lenntech.com/periodic-chart-elements/asen.htm#ixzz0QXN5ykWO)
Humans may be exposed to Arsenic, Lead and Cadmium through
food, water and air.
Generally, endocrine disruptors have the
potential to mimic, or in some cases
block, the effects of the endogenous
hormone. They are therefore, described
as endocrine disrupting chemicals,
hormone disruptors or estrogen
mimickers (Roberts, 1999; Colon et al.,
2000).
They are not under normal control and
cause unregulated activities, hence in
some cases there may be hyper-function
(excessive function) or hypo-function
(under function) The main target of the
endocrine disruptors is the endocrine
system
ENDOCRINE SYSTEM
The endocrine system is instrumental in regulating metabolism, growth, development,
puberty, tissue function and also plays a part in determining mood
Endocrine system
Major endocrine glands: 1. Pineal gland 2. Pituitary gland 3. Thyroid gland 4. Thymus 5.
Adrenal gland 6. Pancreas 7. Ovary 8. Testes. Source:
http://en.wikipedia.org/wiki/Endocrine_system
Our Early efforts on Endocrine disruptors
Development of methods for the quantitative determination of
Phthalate Esters in Surface waters, sewage treatment oxidation
pond and Tap Water (Fatoki and Ogunfowokan 1993a &b,
Ogunfowokan et al., 2006)
Other work from the study area includes the determination of some
physicochemical parameters in the sewage treatment oxidation
pond (Ogunfowokan et al. 2005 & 2008)
Fatoki, O.S. and Ogunfowokan, A.O. (1993b): Environment International 19, 619-623
Fatoki, O.S. and Ogunfowokan, A.O. (1993a): International Journal of Environmental
Studies: 44, 237-243.
A.O. Ogunfowokan, , N.Torto , A.A.Adenuga and E.K.Okoh (2006):
Environmental Monitoring and Assessment 118 , 457-480 The Netherland)
N. Torto, Lesego C. Mmualefe, J.F. Mwatseteza, B. Nkoane, L. Chimuka, M.M. Nindi and
A.O. Ogunfowokan (2007): Journal of Chromatography A, 1153: 1–13
Ogunfowokan A.O., Adenuga A.A., Torto N., E.K.Okoh (2008) Environmental Monitoring
and Assessment. The Netherland 143: 25-41
Ogunfowokan A.O., E.K. Okoh, A.A. Adenuga and O.I. Asubiojo (2005):Journal of
Applied Sciences 5 (1): 36-43.
Why this study?
Endocrine disruptors are of concern because of their
subtle toxicity effects since they affect the normal
function of the endocrine system
Small but critical changes in the chemical makeup of an
environment are enough to trigger outcomes that could
lead to population decline and loss of bio-diversity
This study therefore, focused on the identification and
quantification of some phthalate esters and some heavy
metals that have been implicated as endocrine
disruptors in a sewage treatment oxidation pond; as a
source and the impact on a receiving stream.
Study area and sampling points
Figure 1 is the map of the study area showing the sampling sites. Measurement points
from the sampling sites have been designated S1 to S8. Sample SR represents the
reference point upstream before discharge of the effluent into the stream and serves
as control.
The Obafemi Awolowo University campus has two Oxidation ponds A and B lying side
by side. (Figure1). Each pond measures 150m by 32m, and is about 1.2m deep. A 5m
wide dyke separates the ponds. The wastes are conveyed to the ponds through a
network of concrete pipes of different diameters. Only one pond receives influents
and waste water at a time. The sewage is retained in the pond for about 2 weeks
during which algae, bacteria and other microorganisms act on them.
Each pond has a discharge channel through which effluents are discharged into the
receiving stream. Site S1was located 30m away from the inlet to the pond, before the
influent enters the pond. S2 was located at the inlet to the pond and S3 was located
right on the pond about 50m away while site S4 represents the point of exit of
effluents from the oxidation pond. S5 was a point located at the place where the
pond's effluent enters the receiving river while sites S6 and S7 were located down
stream, at approximately 15m and 100m respectively away from S5. S8 was located
about 350m away from the confluence of Opa River and the receiving stream, which is
about 800m away from S5. Site SR was the reference point and was located about
20m up stream from the point of discharge of the effluents into the receiving stream
(S5).
S4
S1
S3
S5
B
S2
S6
S7
S8
Abattoir
Experimental
Chemicals and Reagents
All reagents used in this study were of analytical
grade and were further purified before use.
Ultra pure water was from a Milli-Q system supplied
by Millipore (Bedford, MA, USA). N-hexane and
ethyl acetate were from Ultrafine Limited,
(Finchely,London). Acetonitrile was from Riedelde Haën (Sigma-Aldrich, Laborchemikalien
GmbH, Germany). Other reagents used were:
sodium chloride from Associated Chemical
Enterprises, (Glenvista, RSA), sodium
carbonate from SAARCHEM, (Muldersdrift,
RSA), anhydrous sodium sulphate and
dichloromethane were obtained from Rochelle
chemicals (Johannesburg, RSA) while
aluminum oxide was from Fluka Chemica
(Switzerland). HNO3 and HClO4 were obtained
from BDH Chemicals (Poole, England).
Sample Handling
Samples for phthalate esters analysis : were
collected in 2.5liter amber Winchester glass
bottles, which had been thoroughly washed and
rinsed with triply distilled water, oven dried and
finally rinsed with triply distilled
dichloromethane. The bottles were rinsed
thoroughly with the samples before they were
finally filled. The mouths of bottles were
covered with glass stopper after filling them
with samples to avoid contamination from
plastic covers. Alteration of the organics due to
microbial activities was prevented by
acidification of the samples to pH 2 with
concentrated hydrochloric acid immediately
after collection and samples were stored in
refrigerator at about 40C prior to analysis.
Samples for metals analysis : were collected using
plastic bottles that have been previously
cleaned by washing in detergent, rinsed with
tap water, and later soaked in 10% HNO3 for 72
hours and finally rinsed with deionised water
prior to usage. During sampling, sample bottles
were rinsed with sampled water several times
and then filled to the brim. The samples were
transported to the laboratory immediately and
stored in the refrigerator at about 4ºC prior to
analysis.
Trace Organics analysis (Phthalates)
Extractions
Ultra pure water was used as the blank
sample. 1000mL aliquot of ultra
pure water was measured into a 2 L
beaker and acidified with
concentrated HCl to pH 2, then
saturated with about 20g of NaCl.
This was extracted three times with
15ml of CH2Cl2 each time. The
CH2Cl2 extracts were combined and
they contained phthalate esters and
other organic contaminants. The
free fatty acids (FFA) interferences
were removed by further extraction
with 3 x 10mL 0.1M Na2CO3. The
organic extracts after alkali
washing, were then dried over
anhydrous Na2SO4. The
dichloromethane was subsequently
evaporated by purging with
nitrogen gas. The same procedure
was employed for real samples.
Sample clean-up
A 10 mL column was packed with
about 12.5g of activated alumina
prepared in a slurry form in nhexane. The residue extracts were
redissolved in 2ml CH2Cl2 and
chromatographed through the
packed column.
Hydrocarbons and phthalate esters
were eluted successively from the
column with 20mL of n-hexane and
30mL ethyl acetate. The ethyl
acetate eluate was concentrated to
1ml by purging with nitrogen gas.
This was diluted with 1mL
acetonitrile for LC/MS analysis.
Quality assurance study
Quality assurance was carried out by recovery
experiments in order to ascertain the precision and
efficiency of the analytical procedure. This was
done by extracting a sample spiked with 10 mL of a
mixture of five authentic phthalate esters at a
concentration of 1 mg/mL. Using a spiked sample
has the limitation that some analytes strongly
retained on the particles may not be extracted.
However, since no certified reference material was
available to us when this study was conducted, this
method was used for method validate.
Analyses of Metals
Digestion of water samples
The samples were digested using mixture of acids. The method
adopted was described by Carrondo et al. (1979). 10mL of water
sample was put in a pretreated Teflon beaker and 30ml of concentrated
HN03 was added and the mixture was evaporated to dryness on a hot
plate in a fume cupboard. This was allowed to cool and 5mL HNO3,
2mL 60% HClO4 and 6mL 40% HF acids were added. The resulting
mixture was evaporated to dryness, and then 2mLHNO3 and HClO4
acids were added and evaporated to dryness to ensure that silicon and
fluoride were removed. The final residue was re-dissolved in 2.5mL of
2M HNO3 and brought up to the mark of 25mL standard flask with
distilled water.
A blank digestion was as well carried out for background correction.
The worked-up samples above were analyzed using ICP-MS.
Instrumental
Phthalate Ester
Determination of phthalate esters was
achieved after separation using a HP1100
series HPLC system, Agilent
Technologies (Waldbronn, Germany),
equipped with a diode array detector and
thermostated column compartment.
Chromatographic separation was carried
out using a 250mm x 4.6mm i.d. Kromasil
100 C18 column with particle size of
10μm, from SUPELCO (Bellefonte, USA).
Separation was performed under
gradient elution conditions using
acetonitrile and water as mobile phase,
with an injection volume of 25 µl and flow
rate of 1 ml/min, with the column
temperature was set at 40 C. The elution
gradient started with 50% acetonitrile,
which was increased linearly to 75% over
four minutes. Then over four minutes, it
was changed to 100%, and the condition
was maintained for twelve minutes
before returning to initial percentage in
four minutes
Metals
The metals As, Cd, and Pb were analyzed
after acid digestion using a Finnigan MAT
Element 2 High Resolution Inductively
Coupled Plasma - Mass
Spectrophotometer Finnigan (Bremen,
Germany). For ICP-MS analysis, the
isotopes of the elements determined
were; 111Cd, 75As and 208Pb.
The RF power was 1.158kW, nebulizer gas
flow rate was 1.0L/min, cooling gas flow
rate was 14.89L/min, with a detector
voltage 2398V.
RESULTS AND DISCUSSION
% Recovery of Phthalate Esters from Spiked Water
Samples
Phthalate esters
Percentage recovery %
Standard
deviation
Coefficient of
variation(Cv)%
Dimethyl phthalate (DMP)
68.43
2.56
3.74
Diethyl phthalate (DEP)
72.35
2.40
3.32
Diphenyl phthalate (DPhP)
46.36
1.81
3.91
Dibutyl phthalate (DBP),
85.60
2.06
2.42
Bis(2-ethylhexyl) phthalate (DEHP)
106.34
4.11
3.87
1750
1500
9.145 DPhP
10.326 DBP
2000
6.889 DEP
5.177 DMP
2250
9.541 BUTYL BENZOATE
*DAD1 A, Sig=254,16 Ref=850,40 (190504\STD-L4OC.D) - FLD1 A, Ex=235, Em=307 (190504\STD-L4OC.D)
mAU
1000
18.167 DINP
750
16.755 DOP
I5.863 DEHP
1250
500
250
0
0
5
10
15
20
Figure 2: Representative Chromatogram of mixture of Phthalate Esters
obtained on HPLC
min
Levels of phthalate Esters in the Oxidation
pond and the receiving Stream
120
100
concentration (mg/L)
80
DMP
DEP
60
DPhP
DBP
DEHP
DOP
40
DINP
20
0
S1
S2
S3
S4
SR
S5
sampling site
S6
S7
S8
USEPA criteria of 3μg/L phthalates recommended for the protection of fish and other aquatic life
in water and the SNAEL of 7.5–38.5μg/L for drinking water. This should give cause for great
environmental concern. Peoples’ health downstream is at stake and so is the ‘health’ of the
ecosystem.
Interpretation of infrared spectrum of phthalate esters in samples from
Sewage treatment oxidation pond
Wave number cm-1
Assigned functional group
2,907 – 2,880 (s)
C-H stretching vibration of CH3, CH3, and CH.
1720 (m)
C =O stretching vibration of ester
1600-1400 (m)
C=C aromatic ring
1140 – 1110 (m)
C-O stretching vibration of ester
1250 1280 (m)
C-O stretching vibration of benzoates
690 – 720 (w)
di-substituted aromatic compound
S – strong absorption, m – medium absorption, w –
weak absorption
AbeyDBP1 #16-137 RT: 0.11-0.98 AV: 122 NL: 4.15E8
T: + c m s [ 125.00-325.00]
278.8
100
95
90
85
80
75
70
65
60
55
50
45
40
35
30
25
20
279.8
15
204.7
10
5
0
149.1
135.9 148.3 150.1 163.0 182.8 189.6
140
160
180
205.8
200
221.8
220
m /z
236.7 252.9 259.9
240
278.1
260
Mass Chromatogram of Dibutyl Phthalate Ester
280.8
280
295.4
300
317.8
320
Mean values for metals in the sewage treatment oxidation
pond
Month
Metals
Cd µg/L
As µg/ L
Pb µg/ L
July
17.59
±
3.02
46.67
±
12.25
21.69
±
10.88
August
15.61
±
4.13
62.03
±
11.00
42.18
±
9.13
September
4.70
±
0.52
92.30
±
20.13
51.56
±
10.25
October
19.05
±
2.71
94.22
±
23.69
43.84
±
4.99
November
11.13
±
1.43
104.67
±
1.76
64.57
±
7.00
December
11.17
±
1.51
96.27
±
2.92
65.55
±
13.85
Overall
mean
13.26
±
5.27
82.69
±
22.88
48.24
±
16.36
Average values for metals in the receiving stream
Month
Metal
Cd µg/L
As µg/L
Pb µg/L
July
16.03
±
10.62
21.50
±
9.81
8.29
±
3.98
August
5.52
±
2.40
45.45
±
14.46
31.28
±
4.85
September
5.98
±
3.07
98.88
±
12.49
24.18
±
6.27
October
19.57
±
2.55
74.74
±
22.50
42.78
±
4.97
November
5.05
±
3.18
74.88
±
11.07
39.93
±
6.80
December
10.98
±
2.78
40.06
±
14.95
51.75
±
8.94
Overall
mean
10.53
±
6.12
59.42
±
28.53
33.12
±
15.23
Average values for metals in the reference samples
Metal
Month
Cd µg/L
As µg/L
Pb µg/L
July
5.78
±
1.16
<0.5
<0.50
August
3.54
±
0.16
3.02
±
1.01
3.35
±
0.02
September
1.75
±
0.08
4.88
±
0.85
2.83
±
0.06
October
4.14
±
0.72
5.30
±
2.01
5.34
±
0.18
November
2.32
±
0.07
1.10
±
0.76
6.81
±
0.78
December
2.27
±
0.03
5.62
±
2.26
7.20
±
1.20
Overall
mean
3.30
±
1.51
3.98
±
1.90
5.11
±
1.98
oxidation pond
receiving stream
reference samples
Overall
mean
Cd µg/L
As µg/ L
Pb µg/ L
13.26
±
5.27
82.69
±
22.88
48.24
±
16.36
Overall
mean
10.53
±
6.12
59.42
±
28.53
33.12
±
15.23
Overall
mean
3.30
±
1.51
3.98
±
1.90
5.11
±
1.98
Maximum allowable threshold of metals in water intended for human consumption
and that will not constitute a threat to health: As = 10 μg/L (DWAF 1996); Pb = 100
μg/L (FEPA); Cd = 3 μg/L (WHO 1993)
Conclusion
•
The levels of phthalate ester plasticizers and metals were studied in a
sewage lagoon effluents and its receiving stream in this study. Liquid–
liquid extraction and high performance liquid chromatography and
Inductively Coupled Mass Spectrometry have been used for
quantitative analysis of phthalate esters and metals.
•
The results obtained showed high levels of phthalate esters, As, Cd
and Pb in the sewage lagoon and the receiving stream with the former
being higher in phthalates and metals than the latter.
•
The discharge of the effluent from the sewage lagoon into the
receiving stream led to increase in the concentrations of the analytes
downstream and has therefore impacted the receiving stream.
Conclusion/Recommendation
This is unfortunate because lives of fish and aquatic biota in the
receiving stream are at risk and the rural dwellers that depend on the water
from the receiving stream for various domestic purposes downstream
untreated are at great risk of serious health effects due to phthalate esters
and metals pollution.
The sewage lagoon serves as point source pollution into the receiving
stream. There is therefore a need for urgent action by the authority in
charge of the sewage lagoon to upgrade it to improve its treatment
performance.
Thorough analysis and study of water from the stream before being used for
domestic applications is therefore recommended to minimize the health
risk. Future work should be carried out on fish and sediments of the
receiving stream to ascertain biomagnifications of these analytes in these
matrices.
Food for thought !!!!
Therefore, my beloved brethren, be ye
steadfast, unmoveable, always abounding in the
work of the LORD, for as much as ye know that
your labour is not in vain in the Lord (I
Corinthians 15 verse 58) KJV

Acknowledgement
I would like to appreciate the Local organizing committee of the Humboldt
International conference and Stiftung /Foundation for the full sponsorship
granted to me
Thank You all for your attention !!!!!!!!