Journal of Water Resource and Protection, 2012, 4, 851-858
http://dx.doi.org/10.4236/jwarp.2012.410099 Published Online October 2012 (http://www.SciRP.org/journal/jwarp)
High Performance Liquid Chromatographic Identification
and Estimation of Phthalates in Sewer Waste and a
Receiving River in Ibadan City, Southwestern Nigeria
Gregory O. Adewuyi
Department of Chemistry, University of Ibadan, Ibadan, Nigeria
Received August 2, 2012; revised September 1, 2012; accepted October 2, 2012
Medical wastes have been implicated in river pollution in developing countries and most often people depend on water
from such rivers for sources of livelihood. Phthalates (endocrine disruptors) are major components in medical wastes
and are commonly found contaminants in aquatic environment. Most sewage treatment facilities handling medical
waste are inefficient due to overus e and poor maintenance and discharg e directly into rivers. This study ai med to inves-
tigate the identity and estimates the concentration of phthalates in supposed treated medical wastes from a hospital
sewer and water from a receiving river. Samples were randomly collected before and after treatment by the sewer plants,
while samples were randomly collected along the course of the river starting from point of discharge. Control samples
were taken from upstream about 500 m along the river course. The samples were extracted by liquid-liquid chroma-
tographic process using dichloromethane, after which they were cleaned up in a column of silica gel using hexane as the
mobile solvent. The cleaned extracts were analyzed by HPLC. The concentrations (µg/L) of dimethyl-, diethyl-, di-
phenyl-, dibutyl- and di-(2-ethyl)hexyl phthalates ranged from 62.81 ± 18.53; 4.74 ± 3.57; 2.05 ± 1.80; 11.40 ± 5.58 to
141.92 ± 35.8 respectively in the sewer waste. The receiving river had a concentration (µg/L) of 9.17 ± 14.02; 0.18 ±
0.31; 0.48 ± 0.84; 2.84 ± 1.21; 61.72 ± 38.35 respectively for dimethyl-, diethyl-, diphenyl-, dibutyl- and di-(2-ethyl)
hexyl phthalates. These concentrations were higher than control and far exceeded the USEPA limits of 3 µg/l recom-
mended for phthalates in water. Contaminants of aquatic environment by untreated wastes from hospitals has serious
implications on public health and environment as human risks for phthalate esters downstream are high and this calls
for urgent need to develop strategy to build incentives for compliance in treatment and discharge of wastes into river
Keywords: Sewage Treatment; Phthalates; Liquid-Liquid Extraction; Clean-Up; Effluent; Toxicity
The impact of some synthetic industrial chemicals on
environment has attracted a lot of attention worldwide
due to their overwhelming environmental significance. In
the last five decades, there has been a growing interest
and concern on the study of the impacts of some of these
chemicals especially phthalate esters on wild-life, humans
and the environment. Phthalate esters are dialkyl- or
alkyl/aryl esters of 1,2-benzenedicarboxylic acid (phthalic
acid); they have a myriad of commercial uses and are
considered ubiquitous environmental contaminants. Glo-
bally over 8 billion tones of phthalate esters are used
each year primarily as additives to poly (vinyl) chloride
pla stics, as industrial solvents and as components of many
consumer products . Phthalates have been implicated
as possible carcinogenic or tetratogenic agents for hu-
mans [2-4]. Some other well documented human health
problems in which phthalate esters are implicated include
early puberty in girls , genital defects and reduced
testosterone production [6,7], testicular cancer impaired
sperm quality and sperm damage in men [6,8,9], pre-
matu re d el iv ery [ 8,1 0 ] r es p ira to ry health problems like air
obstruction, lung malfuncti on  and asthma [12,13].
However, it is the possible action of phthalates as
endocrine disrupters in humans that has caused the most
serious concern [5,14-16]. In most of the compounds that
utilizes phthalates, they are not chemically bound to the
matrix, and hence they can easily diffuse or leach out
into the environment overtime [17,18]. The general
population is exposed to phthalates through consumer
products as well as through diet and medical treatments
. One of the main routes of exposure is via water;
also, these chemicals find their ways into rivers through
effluent discharges, leaching from waste dumps and
opyright © 2012 SciRes. JWARP
G. O. ADEWUYI
through diffuse sources . The potential health risk of
exposure to phthalates is higher in the developing coun-
tries considering the fact that waters for domestic activi-
ti es are sourced directly from streams with little or no treat-
ment. Considering the widespread use of polyvinylchlo-
ride medical devices in the healthcare delivery services,
hospital sewage is therefore highly suscep tible to contain
high level of phthalate esters . Most hospitals hardly
treat their effluents before discharge into surrounding
water bodies. Though liquid waste from University Col-
lege Hospital in Ibadan are channelled into a sewage
plant before the effluents are discharged directly into a
receiving river that serves as the major source of water to
some local populations who use the water for domestic,
irrigational and recreational activities. Therefore, for the
purpose of establishing the effectiveness of the sewage
treatment plant, this paper aims at determining the levels
of phthalate esters in the supposedly treated hospital
effluents and in the waters of the receiving river using
high perfo rmance liqui d chromat ography.
2. Materials and Methods
2.1. Reagents and Standards
All chemicals used in this study were of analytical
reagent grade. Acetonitrile, n-hexane, and ethyl acetate
were of HPLC grade. All solvents were further purified
by distillation. Phthalate esters and n-butyl benzoate were
purchased from Merck and Aldrich Chemical Company.
Organic free water was sourced from International Insti-
tute for Tropical Agriculture, Ibadan. Sodium chloride,
sodium carbonate, anhydrous sodium sulphate and alumi-
nium oxide; were purified by heating in a muffle furnace
at 450˚C for 4 hours.
2.2. Description of the Study Area
The sampling areas were the sewage treated plant of the
University College Hospital, Ibadan where the effluent
samples were collected and Olojuor o river in Ibadan city
which receives the effluents discharges. The sewage
system, as indicated on the map (Figure 1), receives
waste waters and sewage from the school of nursing staff
residential quarters, hall of residence, the hospital com-
plex and the farm. These are collected together in the
collecting box, the grits and other settleable solids settle
under gravitational action to form the sludge in the sedi-
ment ation tank. The sewage receives no further treat ment,
except that it is dozed with chlorine before it is finally
discharged into Olojuoro river.
The effluents from the sewage plant were sampled in
three different d esignated sampling points S 1-S3 of about
10 m apart, as shown in Figure 1. The receiving stream
was similarly sampled, but with more sampling points
(S4-S7) from the point of discharge of the effluent and
downstream, this method has been adjudged as the most
effective way of monitoring point source pollution in
Amber bottles were used for sampling to prevent
bacteria growth. All bottles an d other glassware used for
samplin g w ere thoroug h ly wash ed an d so aked in ch ro mic
acid as described by , the bottles were rinsed seve-
Figure 1. Description of the study areas.
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G. O. ADEWUYI 853
rally with organic free water. Bottles were dried in oven
for 2 hrs at 105˚C and finally rinsed with dichlorome-
thane (DCM) before employed for sampling. Sampling
locations were as indicated on the map (Figure 1). Sam-
ples were collected once every week for six weeks (from
the third week of the month of January to the end of the
month of February 2009). The samples S1-S3 are sewage
samples. Sample SR is the reference sample collected
from the receiving stream, at a point, about 20 m up-
stream from the point of effluent discharge into the
stream. Samples S4-S7 are samples from the stream from
few metres away from the point of effluent discharge into
the stream until the stream enters the Oyo State fishpond.
These samples were collected at about 20 m apart. The
sample bottles were first rinsed with samples before final
collection. The pH of the samples was immediately ad-
justed to 2 by the addition of concentrated hydrochloric
acid after collection; this was to reduce the activity of
microorganisms. Samples were collected to the brim of
the bottles and covered with metal caps. Thereafter sam-
ples were stored in ice bucket and transported to the la-
boratory for analysis.
2.4. Extraction Procedures
Cold liquid extraction procedure was used for this experi-
ment. 500 ml of the sample was measured into a sepa-
ratory funnel. The sample was saturated with about 10 g
of sodium chloride to prevent the formation of persistent
emulsions from the solvent . It was then extracted
with three portions of 25 ml dichloromethane. The three
portions of extracts from each sample were added toge-
ther in another separatory funnel. Free-fatty acid inter-
ferences from the organic constituents were removed by
further extraction with 5 × 10 ml 0.1 M sodium carbonate
. The extracts were then dried over anhydrous sod-
ium sulphate in a glass fibre filter [25,26]. The solvent
was evaporated using rotary evaporator, after which the
residue was re-dissolved in 2 ml of dichloromethane for
About 12.5 g of activated alumina prepared in a slurry
form with n-hexane was packed in a column of about 10
ml. The extract was chromatographed through the packed
column. Hydrocarbons and phthalate esters were eluted
successively from the column with 20 ml of n-hexane
and 30 ml ethyl acetate. The ethyl acetate eluate was
concentrated to 1 ml by purging with nitrogen gas. 1 ml
of acetonitrile was added to the residue for HPLC
analysis [24-26]. The extracts were kept in refrigerator
below 4% until instrumental analysis was conduc ted.
2.5. Instrumental Analysis
High performance liquid chromatographic analysis was
carried out with Hellanco series HPLC system available
at the Department of Chemistry, Laboratory of Analytical
Chemistry, University of Athens, Athens Greece. The
instrument was equipped with a degasser, a quaternary
pump, an autosampler, ultra violet and fluorescence
detector, a thermostated column compartment, variable
wavelength detector and a computer system. Chromato-
graphic separation was carried out using a 150 mm × 4.6
mm i.d. Zorbax Eclipse XDB C18 analytical column with
particle size of 5 µm. Detection of phthalate ester was
done at 226 nm wavelength. Chromatographic separation
was performed under gradient elution condition using
acetonitrile and water (80:20 v/v) as mobile phase. Under
these condition separation lasted for about 18 minutes
with flow rate of 0.3 m1/min. 20 µl was used as the
injection volume and the column temperature was set at
40˚C. Identification of phthalate ester was based on its
retention time and quantification was by combination of
internal standardization and response factor. Individual
phthalate esters in the standard mixture and samples were
identified according to their retention times.
2.6. Preparation of Stock Standard Solution and
Determination of Response Factors
A stock solution (100 mg/L) of the mixture of the pure
phthalate ester analyte standards and n-butyl benzoate
internal standards was prepared by weighing out 1.0 mg
each of the phthalate ester standards and internal stand-
ards into the same container, and making up to 10 mL
mark with acetonitrile. This solution was run on high
performance liquid chromatography for the determination
of response factor for the respective phthalate esters us-
ing the expression shown below .
Response Factor = Peak Area of Phthalate/Peak area
of Internal Standard.
2.7. Recovery Studies and Quality Assurance
Preparation of sampling materials, sampling procedures,
reagents and materials, extraction and analytical proced-
ures were carefully quality controlled. Quality assurance
study was carried out in terms of recoveries of phthalate
in order to ascertain the efficiency of the extraction and
the analytical procedures since no certified reference
material was available. The recovery study was carried
out to assess the efficiency of the methodology. The
recovery study was carried out by blank water samples
spiked with known concentration of mixture of dimethyl-,
diethyl-, diphenyl-, dibutyl- and diethylhexyl phthalates.
3. Results and Discussion
The efficiency and suitability of the analytical procedure
was assessed in terms of recovery and sensitivity. Using
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G. O. ADEWUYI
high performance liquid chromatography with gradient
elution, the phthalates were eluted from the column in
the order of dimethyl-, diethyl-, diphenyl-, dibutyl, and
di-(2-ethyl hexyl) phthalates. The Rf values are as indi-
cated in Table 1. This elution pattern was similar to the
observations of  and . The recovery study was
used to establish the efficiency of the procedure adopted
in this work. The percentage recoveries ranged from
57.48 to 96.95 for the ph thalates (Table 1). The range of
the result is comparable to percentage recoveries obtained
in some previous works on phthalates which were 46.36%
- 71 .5 % [2 4 ], 23.78% - 85.5%  and 50% - 10 5 % [ 2 7] .
These comparable results further establish the validity of
the recovery study and confirmed the efficiency of the
analytical procedure adopted for this work.
The values for response factor of the detector as deter-
mined using the mixture of phthalates standards and the
limits of detection were recorded in Table 1. The limits
of detection were calculated as twice the standard deviat-
ion of peak areas of respective phthalates for ten runs on
the high performance liquid chromatography . The
very low values obtained for limits of detection confirm
the high sensitivity of the analytical procedure adopted
for the study. In this study, phthalates were not detected
in the uncontaminated water (organic free water) blank.
This equally confirmed that problems relating to back-
ground contamination that have been identified as major
flaws in the liquid phase analysis of phthalates  has
been adequately solved. A representative chromatogram
of phthalates standards and the internal standard is shown
in Figure 2. The peaks are well resolved which makes it
easy for identification of the phthalates in the sample.
The results of quantitative analysis of phthalates in
designated sampling points which included sewage efflu-
ents, receiving stream and reference samples for a period
of six weeks (third week of the month January to the end
of the month of February) are presented in Table 2. The
levels established in this study showed wide occurrence
of phthalates in both the sewage effluents and the receiv-
ing stream samples.
A total of five phthalate esters were identified in the
sewage effluents and the receiving stream. The promin-
ent phthalate esters detected during the period of this
study in order of prominence were diethylhexyl-, dime-
thyl-, dibutyl phthalates. Diphenyl-, diethyl phthalates
levels are extremely low in most samples especially the
receiving stream (Figure 3).
A cursory look at the concentrations of phthalate esters
in the reference samples, SR, (Table 2) shows the ub iqui-
ty of phthalate esters in the environment. The mean
concentrations of the phthalate esters ranged from not
detected to 2.69 µg/L. The background presence of the
detected phthalates in the reference samples may be
attributed to background contamination of the stream by
Table 1. Values for response factor, retention time and re-
covery of phthalate esters from spiked samples.
times (min) % Recovery Limit of detection
DMP 0.62 7.23 89.02 0.87
DEP 0.56 8.26 77.93 0.98
DPhP 0.6 10.09 57.48 1.23
DBP 0.48 13.2 83.5 1.02
DEHP 0.53 16.45 96.95 0.60
Table 2. Mean levels of phthalates in the sampled site s.
SP*DMP DEP DPhP DBP DEHP
SRND ND 0.97 ± 1.27 1.99 ± 1 .69 2.69 ± 1.14
S148.69 ± 8.169.78 ± 3.134.57 ± 3.47 12.95 ± 4.90 93.09 ± 24.29
S250.74 ± 8.022.34 ± 1.97ND 3.92 ± 3.38 154.52 ± 35.06
S388.99 ± 16.962.11 ± 1.861.57 ± 1.27 17.32 ± 4.62 178.17 ± 50.70
S433.40 ± 13.22ND 1.93 ± 1.33 4.37 ± 1.34 12 7.47 ± 42.35
S5ND ND ND 3.63 ± 0.59 35.82 ± 9.35
S60.72 ± 0.912.44 ± 1.291.94 ± 0.77 ND 48.68 ± 8.24
S70.83 ± 0.98ND ND 1.41 ± 1.57 34.92 ± 9.84
Concentrations are mean values ± standard deviation for n = 6; ND = not
detected; *Sampling points.
Figure 2. Representative chromatograph for phthalate ester
standards and the internal standard.
DMP DEP DPhP DBP DEHP
SRS1 S2 S3 S4 S5 S6 S7
Figure 3. Multiple bar chart representing variation levels of
phthalate esters in the study area with the sampling points.
phthalate ester plasticizers that may have leached from
the plastic products containers, which the local resident
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G. O. ADEWUYI 855
commonly employed for domestic purposes upstream.
Table 2 showed the mean concentrations of phthalate
esters in the sewage stream (S1-S3) the receiving stream
(S4-S7) and the reference samples for the entire period of
six weeks. For the sewage samples, the values ranged
from 48.69 µg/L to 88.99 µg/L for dimethyl phthalate,
2.11 µg/L to 9.78 µg/L for diethyl phthalate, Not De-
tected to 4.57 µg/L for diphenyl phthalate, 3.92 µg/L to
17.32 µg/L for dibutyl phthalate and 93.09 µg/L to
178.17 µg/L for diethylhexyl phthalate. The expected
trend for the concentration of the phthalate esters is that
the level of phthalates in the sewage effluents stream will
be decreasing downstream . However, a cursory
survey of the dominant phthalate esters levels in the
sewage effluent stream shows the reverse of the expected
trend as shown in Figure 2. This could likely be attrib-
uted to complexation of phthalate esters with various
drugs and other chemical substances present as solute in
the sewage system. These solutes could mask the phth-
alate esters and thus reduce the detectable level of these
phthalates. This observation is substantiated by the fact
that it has been discovered that complexes formed be-
tween solutes and phthalates ester solvents are likely to
be of Van der Waal type perhaps augmented by dipole-
type interaction and hydrogen bond acid solutes .
The complexation of phthalate esters with various chemi-
cal solvents has been investigated, for example, the effect
-cyclodexterin on complexation of phthalate ester
had been traced to the formation of inclusion complex
between the two compounds . Formation of complex
product insoluble in common organic solvent formed by
the reaction of some phthalates and polyvinyl pyrolidone
(PVP) in ethanol and aqueous medium  is another
fact that lays more credence to the observed trend.
However, the interaction of phthalate esters is weak
and of Van der Waal type which contributes to the grad-
ual decomplexation of phthalate ester observed. This de-
complexation is suspected to be enhanced by aqueous
dilution, and this occurs downstream as sewage effluents
from the halls of residence join that from the hospital
wards laden with drugs and other chemicals at the collec-
tion tank and gradually continues to mix progressively
downstream. The contribution of all these factors is be-
lieved to have strongly contributed to the observed trend
(Figure 3) in contrast to the expected trend in the sewage
For the samples from the receiving stream, the values
ranged from not detected (ND) to 33.40 µg/L for dime-
thyl phthalate, ND to 0.72 µg/L for diethyl phthalate, ND
to 1.93 µg/L for diphenyl phthalate, 1.41 µg/L to 4.37
µg/L for DBP and 34.92 µg/L to 127.47 µg/L for dieth yl-
hexyl phthalate (Table 2). At sampling point 4 (S4),
considering the level of dilution (about 100 fold dilution )
that occurs when the sewage effluent stream joins, Oluju-
oro stream (the Receiving stream) was expected to be
very much lower than that of sewage samples. However,
the determined concentrations of phthalate esters were
higher than expected. This observation can no doubt be
equally attributed to decomplexation of phthalate-solute
complexes as earlier explained.
At point S5, the observed levels of phthalate esters
were lower than that of S4. This is in agreement with the
result obtained by . The reasons could be attributed
to adsorption of phthalates on the sediment  and
biodegradation of the phthalates by some micro-organ-
ism [18,33]. At point S6 however, higher concentrations
of phthalate esters were detected. This increasing trend
corresponds to the farming and recreational activities
occurring within the vicin ity of th is point. Tourists patro-
nizing Agodi zoological garden, often pass empty plastic
containers of drinks and other household waste within
the vicinity of point S6. This practice could have con-
tributed to the unexpected increase in the level of phtha-
late esters at this point. Concentrations of ph thalate esters
were still noticeable at S7, where the stream entered Oyo
State fish pond.
Table 3 shows the group mean levels of phthalates in
the sewage, receiving stream and reference samples.
Paired t-test for test of significance was used in compar-
ing the values for sewage and receiving stream with the
reference values. The t-score values were shown in Ta-
ble 4. The concentrations of phthalate esters in the sew-
age effluent stream for all the phthalate esters studied
were significantly different from the respective values for
the reference samples. For the receiving stream, the con-
centrations of dimethyl-, diethyl-, diphenyl- and dibutyl
phthalates were not significantly different from the re-
spective values for the reference samples, whereas the
concentration of diethylhexyl phthalate was significantly
different from that of the reference sample.
However, a test of significance be tween the total phth-
alate in the receiving stream and that of the reference
sample confirmed that there is a significant difference
Table 3. Group mean levels of phthalates in the sewage,
receiving stream and reference samples.
Reference SRSewage (S1-S3) Receiving stream (S4-S7)
DMP (µg/L)ND 62.81 ± 18. 53 9.17 ± 14.02
DEP (µg/L)ND 4.74 ± 3.57 0.18 ± 0.31
DPhP (µg/L)ND 2.05 ± 1.80 0.48 ± 0.84
DBP (µg/L)1.99 ± 1.6911.40 ± 5.58 2.84 ± 1.21
DEHP (µg/L)2.69 ± 2.0441.92 ± 35.85 1.72 ± 38.35
Total (µg/L)4.68 ± 1.69220.87 ± 40.94 74.39 ± 40.86
Concentrations are mean values ± standard deviation for n = 6; ND = not
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G. O. ADEWUYI
Table 4. t-score values for test of significance.
Sewage (S1-S3) Receiving stream (S4-S7)
DMP (µg/L) 8.305 1.603
DEP (µg/L) 3.258 1.417
DPhP (µg/L) 2.996 1.409
DBP (µg/L) 3.956 1.002
DEHP (µg/L) 9.615 3.769
between the aggregate level of phthalate esters in the re-
ceiving stream and that of the reference sample. It is
therefore obvious that the bulk of phthalate esters pollu-
tion load on the receiving stream comes from di-(2-ethyl
hexyl) phthalate. This is not unexpected as the main pla-
sticizer used for most PVC medical devices is diethilhe-
xyl phthalate .
The results of this study show that there is a possible
negative impact of the sewage effluents on the receiving
stream and possibility of deleterious effects of the ph tha-
lates on aquatic biota and people that depend on the
receiving stream for fishing and recreational purposes.
This observation is noteworthy as some of phthalate
esters are endocrine disruptors and affect the develop-
ment of the male reproductive system and production of
normal sperm in young animals , while majority are
carcinogenic, teratogenic and mutagenic organic pollut-
The pollution data obtained for the receiving stream in
this study are much higher than those reported for rivers
in the developed countries polluted with industrial sewage
and chemicals [18,36,37]. Furthermore, values obtained
in this study from the receiving stream are at least 103
much higher than the water criteria of 3 g/L recom-
mended by the US Environmental Protection Agency for
the protection of fish and other aquatic organisms in
water . The values are also higher than Suggested
No-Adverse Effect Level of 7.5 - 38.5 g/L for drinking
water . This is environmentally significant as the
stream flows directly into Oyo State fish pond (Figure 1)
where commercial fish farming is practiced.
4. Conclusion and Recommendations
The evaluation of the efficiency of the sewage treatment
plant in removing or reducing the high concentration of
phthalate esters in the hospital effluents is low. This was
indicated in this study where high levels of the phthalate
esters were found in the treatment effluent samples from
th e s e w a ge plant an d the sa mples fro m the receiv ing r i v e r .
The situation is further compounded by the fact that re-
ceiving river empties into Oyo State fish pond where
commercial fish farming is at present being practiced.
This poses health risk to consumer of the fish sourced
from the pond. Therefore, there is an urgent need for the
authorities in charge of the hospital to upgrade the sew-
age treatment facility so as to improve its efficiency,
thereby reducing the pollution load of the effluent dis-
charged into the surrounding river. Also, the state envi-
ronmental agency needs to develop a routine monitoring
programme in order to ensure that the levels of this pol-
lutants discharged into the aquatic ecosystem are in
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