UNU Project on EDCs Pollution in the East Asian Coastal Hydrosphere

The monitoring of phthalates in various rivers in Peninsular Malaysia from December 2001 to February 2002

Prepared by Melissa Chan Pui Ling¹, Dr. Mustafa Ali Mohd¹ and Dr. Abdul Rani Abdullah²

¹Department of Pharmacology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, ²Alam Sekitar Malaysia (ASMA) Sdn. Bhd.,

MALAYSIA

CONTENTS

1.0	Introduction		3
2.0	Materials and method		4
	2.1	Gas chromatograph-Mass spectrometer (GC-MS) parameters	4
3.0	Results and discussion		7
4.0	Conclusions		12
5.0	References		12
Appendix 1: In situ measurements and activities for the months of December 2001 and February 2002			13

1.0 INTRODUCTION

Environmental pollutants which have endocrine-disrupting properties currently number more than 50 and include industrial chemicals, pesticides and byproducts of manufacturing processes as well as products of incineration of industrial and household wastes. Known EDCs include PCBs, organochlorine pesticides and other types of pesticides, dioxins, alkylphenols. polyethoxylates, pentachlorophenols, bisphenol A, styrenes and phthalate esters (Colborn et al., 1996; Castillo and Barcelo, 1997). Because of the widespread use of many of these chemicals, exposure of biota, including man, to EDCs is generally considered significant. Of the estimated 70 000 chemicals that enter the market as consumer products, only 4 000 to 5 000 have been investigated by manufacturers and researchers. In addition, there are hazardous byproducts created when compounds react with the environment and hence, can cause adverse health effects to mankind. Products are often sold even though as tests continue, and are withdrawn only when adverse reactions occur (The Star, 2001).

The results of numerous environmental surveys on organic pollutants with reported endocrine-disrupting characteristics including pesticides, PCBs and phthalate esters involving both biotic and abiotic components in the global environment have indicated that the contamination of the general environment by such chemicals may be significant. In addition to the physicochemical nature of the chemical, tropical climatic conditions and agricultural and industrial practices and policies play important roles in determining the fate and distribution of EDCs in the global environment.

Phthalates are a ubiquitous class of compounds used most commonly as a softener for products made with polyvinyl chloride (PVC). Between 1985 and 1990, 300 million pounds a year of phthalates were manufactured annually. Phthalates are a widespread environmental micropollutant in Europe and North America including in the Great Lakes Basin (Health Effects Review, 2000).

The present work is aimed at assessing the state of the water quality of the various rivers in Peninsular Malaysia with respect to the levels of phthalate residues.

2.0 MATERIALS AND METHOD

Sampling was carried out in December 2001 (rainy season) and February 2002 (dry season). There were a total of 44 sampling stations. The sampling locations were selected based on the variety of activities at each location. The activities at each sampling station are indicated in Appendix 1. Water samples from each station were collected from a depth of 1 meter using a van Darn water sampler. The water samples were extracted within 24 hours of sampling. The extraction and analysis of phthalates were conducted in accordance to the UNU protocols (Figure 1) (UNU, 2001).

Distilled water was spiked with 20ng of phthalates to validate the accuracy and precision of the analysis.

2.1 Gas chromatograph-Mass spectrometer (GC-MS) parameters

A Shimadzu QP5000 gas chromatograph coupled with mass spectrometer was used for the quantitative analysis of residue of endosulfan and metabolites in plasma and tissues. The J&W DB1-MS fused silica capillary column (30m x 0.32mm i.d.) was used. The operating conditions for the Gas Chromatograph are as follows:

Oven temperature: Initial temperature 70°C

Ramp 20°C/min to 120°C, then 5°C/min to 245°C

followed by 20°C/min to 320°C and hold for 3 minutes

Injector temperature: 320°C

Interface temperature: 300°C

Carrier gas: Helium (Highly purified)

Carrier gas pressure: 50.0 kPa

Carrier flow rate: 54.2 ml/min

Column flow rate: 2.3 ml/min

Injection mode: Splitless

Injection volume: 2ml

Mass spectrometer:

The detection was done using quadrupole detector with electron ionization

mode detection.

Data acquisition mode: Selected Ion Monitoring (SIM)

Ion monitored: DEP (149, 177); DBP (149, 223.1, 205.1); DOA (129, 147, 241.1) and DEHP (149, 167, 279.1)

Phthalates were quantified using selected ion monitoring (Table 1). Calibration was done using external standards.

Figure 1. Method for sample preparation

Table 1. Selected ion monitoring (SIM)

No	Compound	M/Z			Time (min)
		1	2	3
1	DEP	149	177		10.315-12.315
2	DBP	149	223.1	205.1	17.613-19.613
3	DOA	129	147	241.1	25.882-27.882
4	DEHP	149	167	279.1	28.020-30.020

Figure 2. Total ion chromatogram of DEP (1), DBP (2), DOA (3) and DEHP (4) in scan mode

Figure 3. Total ion chromatogram of DEP in SIM mode

Figure 4. Total ion chromatogram of DBP in SIM mode

DEHP

DOA

Figure 5. Total ion chromatogram of DOA and DEHP in SIM mode

3.0 RESULTS AND DISCUSSION

Detection limit for the chemicals were as follows: 2ng/ml (DEP); 0.2ng/ml (DBP); 20ng/ml (DOA) and 0.2ng/ml (DEHP).

In determining error factor contributed by the GC, three different concentrations of the standards solution mixture of DEP, DBP, DOA and DEHP were repeatedly injected into the GC. The results obtained by repeated injections are tabulated in Table 2-5.

Table 2. Precision of the GC analysis for DEP

Concentration, ng/ml	Area recovered					C. V.
	1	2	3	4	5
20	26499	24479	25997	25735	26755	3.4
50	84584	81356	86801	84869	83394	2.4
100	225589	263749	234272	223645	258514	7.8

Table 3. Precision of the GC analysis for DBP

Concentration, ng/ml	Area recovered					C. V.
	1	2	3	4	5
20	56012	52374	56012	57434	53674	3.7
50	155240	151660	152953	164459	153698	3.3
100	382901	475079	404621	379582	399144	9.5

Table 4. Precision of the GC analysis for DOA

Concentration, ng/ml	Area recovered					C. V.
	1	2	3	4	5
20	20893	21866	21764	22611	21390	2.9
50	49404	47661	46820	52878	52234	5.4
100	93348	115883	102218	97944	103713	8.2

Table 5. Precision of the GC analysis for DEHP

Concentration, ng/ml	Area recovered					C. V.
	1	2	3	4	5
20	106737	106928	108221	112309	108938	2.1
50	217267	216869	207759	226150	218945	3.0
100	378475	467868	410284	387290	416395	8.5

The coefficient variations of repeated GC injections range from 2.4 to 7.8% (DEP), 3.3 to 9.5% (DBP), 2.9 to 8.2% (DOA) and 2.1 to 8.5% (DEHP). These do not appear to be a correlation between coefficient variation and the concentrations of the injected samples.

Distilled water fortified with 20ng of phthalates gave recoveries ranging from 93.9 to 118.4% with coefficient variations ranging from 1.2 to 20.2% (Table 6)

Table 6. Percentage recovery of DEP, DBP, DOA and DEHP

Compound	Replicate					Average	S. D.	C. V.
	1	2	3	4	5
DEP	97.6	117.8	109.4	114.6	112.8	110.4	7.8	7.1
DBP	101.6	82.2	83.7	116.6	85.2	93.9	14.9	15.9
DOA	111.1	107.6	110.2	109.8	108.9	109.5	1.3	1.2
DEHP	137.9	128.1	83.2	138.0	104.6	118.4	23.9	20.2

The results of the analysis reported are shown in Table 7 (December 2001) and 8 (February 2002).

Table 7. Residue levels of phthalates in various locations for the month of Dec 2001

No	Station No	Concentration, ng/ml
		DEP	DBP	DOA	DEHP
1	ISR01	1.71	0.95