High Performance Liquid Chromatographic Identification and Estimation of Phthalates in Sewer Waste and a Receiving River in Ibadan City, Southwestern Nigeria

doi:10.4236/jwarp.2012.410099

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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

Email: [email protected]

Received August 2, 2012; revised September 1, 2012; accepted October 2, 2012

ABSTRACT

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

waters.

Keywords: Sewage Treatment; Phthalates; Liquid-Liquid Extraction; Clean-Up; Effluent; Toxicity

1. Introduction

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 [1]. 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 [5], 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 [11] 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

[19]. 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

G. O. ADEWUYI

852

through diffuse sources [20]. 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 [21]. 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.

2.3. Sampling

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

water bodies.

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 [22], the bottles were rinsed seve-

Figure 1. Description of the study areas.

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 [23]. 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

[24]. 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

clean-up process.

Clean-Up Procedure

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 [20].

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

G. O. ADEWUYI

854

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 [20] and [24]. 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% [25] 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 [28]. 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 [20] 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.

Phthalate

esters Responses

factors Retention

times (min) % Recovery Limit of detection

(µg/L)

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

100

120

140

160

180

200

SRS1 S2 S3 S4 S5 S6 S7

Sampling points

Concentration (g/L)

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

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 [26]. 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 [29].

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 [30]. 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 [31] 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

stream (S1-S3).

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 [26]. The reasons could be attributed

to adsorption of phthalates on the sediment [32] 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.

Sampling sites

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

detected.

G. O. ADEWUYI

856

Table 4. t-score values for test of significance.

Sampling sites

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 [18].

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 [34], while majority are

carcinogenic, teratogenic and mutagenic organic pollut-

ants [25,35].

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 [38]. The values are also higher than Suggested

No-Adverse Effect Level of 7.5 - 38.5 g/L for drinking

water [26]. 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

agreement with the internation al standards.

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