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 Products are often sold even though as tests continue, and are withdrawn only when adverse reactions occur (The Star, 2001).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	nd	nd
2	ISR02	1.46	0.78	nd	0.10
3	ISR03	6.78	2.24	nd	nd
4	ISR04	6.14	1.41	nd	nd
5	ISR05	3.87	1.16	nd	nd
6	ISR06	6.43	1.80	nd	nd
7	ISR07	4.43	1.30	nd	nd
8	ISR08	8.24	1.76	nd	nd
9	ISR09	2.44	0.80	nd	0.07
10	IT01	nd	0.24	nd	nd
11	IB01	nd	0.49	nd	nd
12	IB02	8.58	0.69	nd	nd
13	IB03	0.25	1.04	nd	nd
14	IB04	0.26	0.97	nd	nd
15	IB05	0.13	0.86	nd	nd
16	IK01	nd	0.81	nd	nd
17	IK02	nd	0.48	nd	nd
18	IK03	nd	0.29	nd	nd
19	IK04	nd	0.39	nd	nd
20	IK05	nd	0.17	nd	nd
21	IK06	0.38	0.30	nd	0.12
22	IK07	0.25	1.02	nd	nd
23	IK08	0.14	0.49	nd	0.06
24	IK09	0.37	1.09	nd	0.26
25	IK10	nd	0.61	nd	0.10
26	IK11	nd	0.36	nd	nd
27	IK12	0.22	0.38	nd	nd
28	IK13	0.42	0.34	nd	nd
29	IK14	1.08	0.76	nd	nd
30	IK15	nd	0.16	nd	nd
31	IK16	nd	0.37	nd	0.25
32	IK17	6.47	2.71	nd	nd
33	IK18	0.30	0.59	nd	nd
34	IK19	3.32	1.40	nd	nd
35	IK20	0.81	0.58	nd	0.23
36	IK21	5.16	2.12	nd	nd
37	IK22	4.00	1.70	nd	nd
38	IK23	0.33	1.00	nd	0.27
39	IK24	5.06	1.75	nd	nd
40	IS01	5.89	1.62	nd	nd
41	IS02	7.85	2.19	nd	nd
41	IS03	5.10	1.312	nd	nd
43	IL04	0.14	0.29	nd	nd
44	IL17	0.48	0.20	nd	nd

Table 8. Residue levels of phthalates in various locations for the month of Feb 2002

No	Station No	Concentration, ng/ml
		DEP	DBP	DOA	DEHP
1	ISR01	nd	nd	nd	nd
2	ISR02	nd	nd	nd	nd
3	ISR03	nd	nd	nd	nd
4	ISR04	nd	0.13	nd	nd
5	ISR05	nd	0.08	nd	nd
6	ISR06	nd	nd	nd	nd
7	ISR07	nd	nd	nd	nd
8	ISR08	nd	nd	nd	nd
9	ISR09	nd	nd	nd	nd
10	IT01	nd	nd	nd	nd
11	IB01	nd	0.52	nd	0.24
12	IB02	0.16	1.34	nd	nd
13	IB03	0.28	0.95	nd	0.96
14	IB04	0.27	1.42	nd	0.93
15	IB05	0.17	0.42	nd	nd
16	IK01	nd	0.59	nd	nd
17	IK02	0.20	1.02	nd	nd
18	IK03	0.22	2.27	nd	nd
19	IK04	0.34	1.27	nd	nd
20	IK05	0.36	3.52	nd	nd
21	IK06	0.25	0.64	nd	nd
22	IK07	0.48	1.26	nd	nd
23	IK08	0.62	0.62	nd	nd
24	IK09	0.85	0.59	nd	nd
25	IK10	0.30	0.69	nd	nd
26	IK11	0.50	2.68	nd	nd
27	IK12	0.89	5.78	nd	0.21
28	IK13	0.18	0.15	nd	nd
29	IK14	0.88	0.94	nd	nd
30	IK15	0.20	2.42	nd	nd
31	IK16	1.21	0.62	nd	nd
32	IK17	0.90	1.04	nd	0.05
33	IK18	0.22	0.61	nd	nd
34	IK19	0.58	0.52	nd	nd
35	IK20	0.49	0.59	nd	0.13
36	IK21	1.68	1.26	nd	0.21
37	IK22	0.37	0.71	nd	0.08
38	IK23	1.68	0.41	nd	nd
39	IK24	0.33	1.85	nd	nd
40	IS01	nd	0.52	nd	nd
41	IS02	nd	0.66	nd	nd
41	IS03	0.15	0.57	nd	nd
43	IL04	nd	nd	nd	nd
44	IL17	nd	0.17	nd	nd

For the month of December 2001, the range of the detectable residue levels of phthalates in the samples were as follows (nanogram per liter): DEP (nd - 8.58); DBP (nd – 2.71); DOA (nd) and DEHP (nd – 0.27).

For the month of February 2002, the range of the detectable residue levels of phthalates in the samples were as follows (nanogram per liter): DEP (nd – 1.68); DBP (nd – 5.78), DOA (nd) and DEHP (nd – 0.96).

From the results above, it was obvious that the concentrations of DBP and DEHP were higher during the dry season as compared to the rainy season. This could be due to evaporation of phthalates from the rivers during the hot season whereas the season of heavy rainfall usually falls between the month of October and December. Heavy rainfall may also cause the river to swell drastically and hence causes the residue of phthalates in the river to be diluted to an undetectable amount. However the concentration of DEP was lower at the dry season as compared to the rainy season. Generally, no discernible trend was observed throughout the study.

Table 9. A comparison between results from the present study and water quality criteria

	Concentration, ng/ml
	DEP	DBP	DOA	DEHP
Present study	nd – 8.58	nd – 5.78	nd	nd – 0.96
Water quality criteria @	na	34 000	na	6
Water quality criteria #	4910	36.5	na	100

@ Water quality criteria in drinking water; set by the U. S. EPA

# Water quality criteria in fresh water; set at Oklahama

nd = Not detected

na = Not available

It was also observed that many sampling stations in the present study which contained phthalate residues had levels which were much lower than the water quality criteria in drinking water and fresh water (Table 9).

4.0 CONCLUSIONS

Phthalate residues continue to be detected in the water columns of Malaysia. The present study reflected the contamination level by phthalates in Malaysian water. The observed levels did not exceed the water quality criteria in drinking water and fresh water. However, the detectable residue levels of DEHP, although in trace amount is a cause of concern because it has been reported that DEHP causes animal toxicity in many organ systems, including the liver, male and female reproductive organs, heart, lungs, kidneys and developing embryo and fetus. In addition, the toxicities which appear to be of most concern for humans, based on the potential for effects from low doses of exposure, are hepatic and testicular (Health Effects Reviews, 2000).

APPENDICES

Sample Descriptions

Results December

Results February

5.0 REFERENCES

1. The United Nations University (2001) Endocrine disruptor compounds (EDCs) in water: Manual for sample collection and analysis, pp 1-71.

2. Castillo, M. and Barcelo, D. (1997) Analysis of industrial effluents to risk and hazard assessment on health and environmental effects of endocrine-disrupting chemicals. Trends in Analytical Chemistry, 16: 574-583.

3. Colborn, T., Dumanoski, D. and Myers, J. P. (1996) Our Stolen Future, Penguin Books, USA

4. Health Effects Review (2000), Volume 3: Issue 4, International Joint Commission.

5. Bhattacharjee, A. (2001), Living with Toxic Chemicals. The Star, Section 2, pp 3.