Environmental Monitoring and Governance in the East Asian Hydrosphere
Monitoring of POPs in the East Asian Region

Persistent Organic Pollutants (POPs) in Water

Manual for Sample Collection and Analysis

January 2003

I. Introduction

Persistent Organic Pollutants (POPs) are chemical substances that persist in the environment, bioaccumulate through the food web, and pose a risk of causing adverse effects to human population and the environment. There has been a realization that these pollutants, upon exposure of human population, can cause serious health effects ranging from increased incidence of cancers to disruption of hormonal system. These effects have also been observed and recorded for various animal species. Developing countries are particularly vulnerable due to often indiscriminate use and disposal of POPs.

There is also significant evidence that there is long-range transport of these substances to regions where they have never been used or produced. The obvious ramification is that POPs pose threats to the environment of the whole globe, and therefore, the international community has focused on global actions to reduce and eliminate releases of these chemicals. It is now internationally acknowledged that Arctic ecosystems and indigenous communities are particularly at risk because of the biomagnification of POPs in the food chain and contamination of traditional foods in the Tundra region is a public health issue.

An equally important issue is to identify future actions needed to minimize and prevent entry of POPs into the environment. This includes re-thinking production processes and utilization of chemicals in various manufacturing activities. It is also critical to develop adequate methodologies for disposal of wastes that contain POPs. Implementing any such remediation approaches in developing countries would likely entail considerable capacity development and technology transfer.

There has been a significant level of debate on these issues at the international level, particularly during the 1990’s. This has culminated in the development of the Stockholm Convention On Persistent Organic Pollutants on 22 May 2001; this was a major milestone in the efforts to curb the impacts of POPs. At this time, there are 151 signatories and 11 parties to this convention.

The East Asian region has also been actively involved in the POPs debate. However, there is still a need for developing extensive database of the level of POPs in various compartments of the environment. In its previous activities since 1996, UNU has targeted the monitoring of many POPs compounds. This project builds on the previous work and compiled database of POPs, to launch a comprehensive set of monitoring activities linked closely to thematic discussions on environmental quality. This can lead to concrete mechanisms for identifying existing and impending threats from POPs.

II. Overview of UNU’s Previous Activities

2.1 1996-1999 “Environmental Monitoring and Analysis in the East Asian Region”

UNU led the project: ‘Environmental Monitoring and Analysis in the East Asian Region’ since 1996 under a three-year programme. This project was primarily sponsored by Shimadzu Corporation. The countries/territories involved were: China, Indonesia, Japan, Korea, Malaysia, Singapore, Taiwan, Thailand, and Viet Nam. Individual research laboratories were selected in each country and a National Project Coordinator (NPC) was designated. The NPC was responsible for managing the project within its laboratory, interfacing with the local government and communicating with UNU. An extremely useful aspect of this project was the development of a network of environmental researchers within the East Asian region and internationally.

During the first year of the project, the primary emphasis was on evaluating pesticide contamination in foods. Rice was selected as the representative crop due to its widespread use as staple of the diet in the East Asian region. Additionally, soil samples were also analyzed to establish a correlation between soil and rice contamination. During the second year of the project, the monitoring emphasis was on water contamination. For this purpose, tap water samples were collected to evaluate the quality of drinking water in East Asia. A few seawater samples were also analyzed at some locations. During the third and final year of the project, the emphasis was placed on monitoring air pollution. The contaminants particularly targeted were aldehydes and volatile organic compounds (VOCs).

2.2 1999-2002 “Environmental Monitoring and Governance in the East Asian Coastal Hydrosphere”

This project contained three major components: (1) Environmental Monitoring and Governance - EDC Pollution in the East Asian Coastal Hydrosphere; (2) Cooperative International Research Project on Marine and Coastal Environment; and (3) Asia-Pacific Cooperation On Research And Conservation Of Mangroves. The three components were closely linked together and implemented with collaboration of the UNU network on coastal issues.

The first component: EDC Pollution in the East Asian Coastal Hydrosphere was managed with close cooperation and major funding from Shimadzu Corporation. The project collected data regarding presence of endocrine disruptor compounds (EDCs) in the coastal hydrosphere. This information was used to develop consistent and rational guidelines for coastal management programmes in East Asia (Adeel and King, 2002). The following countries were involved: China, Indonesia, Japan, Korea, Malaysia, the Philippines, Singapore, Thailand, and Viet Nam. In continuation of the previous (1996-99) project, the individual research laboratories were maintained as partners and implementation units within each country

During the first year of the project, DDT and its breakdown components were monitored by all the participating organizations. In addition, several other pesticides were also reported. During the second year of the project additional pollutants were included in the list of those monitored; these were primarily alkylphenols and bisphenol-A. In the third year, a new group comprising various phthalates was added.

2.3 Outcomes of the Previous Project Activities

The previous project activities have resulted in generating reliable environmental data in the East Asian region as well as capacity building in the participant laboratories. The data generated as part of this project have gone through a quality assurance and quality control (QA/QC) process jointly overseen by Shimadzu and UNU. This has led to confidence in the information provided by these data.

The benefits of the project can be briefly summarized as:

Development of a network of professionals
Building and maintaining the capacity for environmental monitoring

§ Providing technical support for environmental monitoring

§ Training of young professionals in environmental monitoring techniques

Dissemination of environmental-friendly information
Awareness-raising in participating countries through meetings
Generation of reliable environmental quality data

III. Project Description

3.1 Focus & Objective

The main focus of the project is on monitoring of Persistent Organic Pollutants (POPs) in rivers and fresh water bodies close to the coastal areas. The objective is to develop an early-warning system to counter and minimize environmental pollution; this will be achieved through periodic and systematic monitoring.

3.2 Participating Institutions

China – China-Japan Friendship Center for Environmental Protection
Indonesia – Environmental Management Center - SARPEDAL
Japan – National Institute for Environmental Studies (NIES)
Korea – Korean Ocean Research and Development Institute (KORDI)
Malaysia – University of Malaya
The Philippines – National Science Research Institute, University of the Philippines
Singapore – Department of Chemistry, National University of Singapore
Thailand – Environmental Research and Training Center (ERTC)
Viet Nam – Viet Nam National University (VNU)

3.3 Project Components:

A. Capacity Building Element

Cross-laboratory calibration exercise: This will require a systematic development of calibration methodology including statistical design, sample preparation and distribution among the participating laboratories. This exercise will be closely linked to the training in analytical methods. A group of experts may be consulted if there are problems encountered for some of the laboratories.
Training in laboratory analysis of pollutants: This will be conducted in the form of short training workshops in close collaboration with Shimadzu Corp.
Development of training manuals (to be published by UNU as a book by 2005)

B. Environmental Monitoring

Monitoring of organic pollutants – POPs – in freshwater and sediments in the vicinity of coastal areas. A number of the POPs will be targeted, including aldrin, chlordane, DDT, dieldrin, endrin and heptachlor. Other POPs may also be considered for the monitoring programme.
Maintaining LandBase for data coordination and dissemination
Guidelines for Environmental Quality will be developed to evaluate the threats to ecosystems and human population.

C. Information Dissemination

Annual International Symposia will be held in the participating countries; the international symposium in 2005 will be held in Tokyo to mark the completion of the project.
Internet-based quarterly newsletter. This will be targeted to the general public with the objective of widely disseminating the project findings and increasing the visibility of the project globally, particularly in the East Asian region.
Publication of project findings as symposium proceedings and through electronic media such as the UNU website and CD-ROMs.

3.4 Project Output

The benefits of the project can be briefly summarized as:

a. Continued collaboration of the existing network of professionals

b. Capacity building in participating developing countries for environmental monitoring

c. Development of an early-warning capacity in the region against environmental pollution to water bodies

d. Generation of reliable data on water quality and pollution sources in East Asia

e. Training of young professionals in environmental monitoring techniques

f. Awareness-raising in participating countries through meetings and publications

g. Development of environmental management approaches to control pollution sources

IV. Sample Collection and Preparation for Analysis

1. QA/QC

QA/QC is very important, and necessary for the analysis.

Fig. 1 shows the Flow Chart of the analysis.

Flow chart 1 : from set up GC/MS to end

Flow chart 2 : the case of calibration curve has made yet

1. Set up GC/MS

2. Check GC/MS using software “System check” and Auto tuning

3. Analyze standard sample (1ppm).

Check Mass Spectra, Retention Time, Sensitivity (Peak height), Profile of Peak, Decomposion of Pesticides

4. Analyze blank sample (n-Hexane)

5. Make the calibration curve of pesticides

Check the calibration curve

6. Analyze standard sample (10ppb) at several times (minimum 5 times)

Check the detection limit

7. Analyze the monitoring sample

After the analysis of 20 samples

8. Analyze standard sample (10ppb)

Check Retention Time, Sensitivity (Peak height), Profile of Peak

9. End of analysis

Allowance

Retention time less 0.1min

Peak height less 20%

Profile of peak tailing factor

Repeatability CV(%) less 10%

Blank less 1ppb

Fig.1 QA/QC Flow Chart

2. Sample for Analysis

2.1 Water Sample

The water samples will be collected from rivers or coastal areas. The sample collection and analysis methods are the same for both freshwater and seawater samples.

The water sample will be collected as close to the surface as possible. In the case of river water samples, the sample should be collected in the middle of the stream.

The water sample will be collected in a glass container and the volume to be collected is 10 liters. It is preferable to store the sample in ice and away from sunlight during shipment to the laboratory. In the laboratory, the sample should be stored in a refrigerator at all times.

The form for recording sample collection is provided in Appendix 1.

2.2 QA/QC Sample

The samples for QA/QC are diluted by 1000times with the mineral water. Then they are taken the same preparation, and analysis as the water samples.

3. Target compounds (Pesticides for POPs)

The target compounds are :

1. Aldrin

2. Dieldrin

3. Endrin

4. Chlordane

5. Heptachlor

6. Hexachlorobenzene

7. p,p’-DDT

4. Internal Standard compound (IS) and Surrogate compound

The following compounds will be used as internal standard and surrogate compounds for the GC/MS operation.

1. Pyrene - d10 (Internal Standard compound)

2. p,p’-DDT-¹³C12 (Surrogate compound)

5. Reagents, Materials and Apparatus

The following reagents are required for the analysis:

1. NaCl

2. N-Hexane

3. Na₂SO₄ (anhyd)

4. Acetone

5. Standard stock solutions (100ppm: 100mg/L)

Dissolve 10mg of each Pesticides with 100ml of Acetone + n-Hexane

Stock at a refrigerator

6. Surrogate stock solutions (100ppm: 100mg/L)

Dissolve 10mg of Surrogate Compound with 100ml of n-Hexane

Stock at a refrigerator

7. Internal standard stock solutions (100ppm: 100mg/L)

Dissolve 10mg of Internal Standard Compound with 100ml of Acetone

Stock at a refrigerator

The following equipments are required for sample preparation and analysis:

1. Separating funnel

2. Rotary evaporator

3. Florisil Cartridge (Bond Elut FL 500mg)

6. Sample for Calibration Curve

Standard solution Ⅰ (1ppm: 1mg/L)

100μl of Standard stock solution (100ppm) and Surrogate stock solution (100ppm) are injected into 10ml of n-Hexane.

Standard solution Ⅱ (100ppb: 100ng/ml)

10μl of Standard stock solution (100ppm) and Surrogate stock solution (100ppm) are injected into 10ml of n-Hexane.

Standard solution Ⅲ (50ppb: 50ng/ml)

5ml of 100ppb solution is injected into 10ml of n-Hexane.

Standard solution Ⅳ (10ppb: 10ng/ml)

1ml of 100ppb solution is injected into 10ml of n-Hexane.

Standard solution Ⅴ (5ppb: 5ng/ml)

1ml of 50ppb solution is injected into 10ml of n-Hexane.

Standard solution Ⅵ (1ppb: 1ng/ml)

1ml of 10ppb solution is injected into 10ml of n-Hexane.

Internal standard solution Ⅰ (10ppm: 10mg/mL)

100μl of Internal standard stock solution (100ppm) is injected into 1ml of

n-Hexane.

Surrogate standard solution Ⅰ (2ppm: 2mg/ml)

2μl of Surrogate standard stock solution (100ppm) is injected into 1ml of

n-Hexane.

Inject 5μl of Internal standard solution Ⅰ (10ppm) into 1ml of each solutionⅡ,Ⅲ,Ⅳ,Ⅴ,

Make the calibration curves of each Pesticides and Surrogate compound using the internal standard method.

7. Summary of Method for Preparation (Flow chart)

The following flow chart provides an overview of the method required for sample preparation prior to GC/MS analysis.

Water (1L)
↓ ↓	← ←	NaCl 30g Surrogate Compound 100μl 2ppm
↓	←	n-Hexane 50ml
Extraction by shaking
↓	←	Na₂SO₄ (anhyd) about 3g
Dehydration
↓
Concentration	1ml by rotary evaporator and N₂ gas
↓
Clean up	Florisil chromatography
↓
Concentration	1ml by N₂ gas
↓	←	IS 5μｌ 10ppm
Mess up	n-Hexane to 1ml
↓
Analysis by GCMS	2μｌ injection

V. Analytical Methods

1. Preparation for Analysis

The following steps should be followed in preparing the sample for analysis:

1. Add 1L of river water into 2L of separating funnel

2. Add 30g of NaCl into separating funnel

3. Add 100μl 2ppm of Surrogate compound into separating funnel (Refer to step 1)

4. Add 50ml of n-Hexane into separating funnel

5. Shaking it for 10 min. for extraction

6. Transfer n- Hexane from separating funnel

7. Add 50ml of n-Hexane into water layer

8. Shaking it for 10 min. for extraction

9. Transfer n- Hexane from separating funnel into flask

10. Add about 3g of Na₂SO₄ (anhyd) into n-Hexane layer for dehydration

11. Stay for 20 min.

12. Condense to less 1ml by Rotary Evaporator and N₂ gas

13. Transfer n-Hexane layer into tube

14. Flow n-Hexane layer into Florisil column (Refer 2)

15. Flow 7ml of 2% Acetone/n-Hexane

16. Condense to less 1ml by N₂ gas

17. Add 5μl of 10ppm IS (Refer 3)

18. Measure 1ml by n-Hexane

19. Inject 2μl to GCMS

Refer 1 Surrogate standard solution

2ppm of p,p’-DDT-¹³C12

Refer 2 Method of Column

Wash Florisil Column by 5ml of Acetone and 15ml of n-Hexane

Refer 3 Internal Standard solution

10ppm of Pyrene-d10

Notice

It is necessary to check an insert at GC injection port before analysis of pesticides.

If the insert is dirty, p,p'-DDT is decomposed.(Show Fig. 6.)

When peaks appear, you must change a new insert.

2. Analysis Condition

--- GC ---

Column DB-1 30m 0.32mm ID 0.25μm df

Column Temp. 70℃ (1min.)－20℃/min.－130℃－5℃/min.－210℃－

15℃/min.－300℃ (4min.)

He Liner Velocity

Pressure 49.9kPa

Total Flow 44.9ml/min

Column Flow 2.32ml/min

Liner Velocity 55cm/sec

Purge Flow 10.0ml/min

Split Ratio 14.0

Injection port Temp. 250℃

Injection Method Spritless （1min.）

Injection Volume 2μl

--- MS ---

Ion Source Temp. 230℃

Interface Temp. 270℃

Scanning Range m/z 35 ～ 500

Scanning Interval 0.5sec.

SIM Sampling Rate 0.2sec.

	Compounds	M/Z
1	Hexachlorobenzene	283.9	285.9	248.9
2	Heptachlor	100.0	271.9	336.9
3	Aldrin	262.9	264.9	292.9
4	trans-Chlordane	374.8	372.8	407.85
5	Cis-Chlordane	374.8	372.8	407.85
6	Dieldrin	262.9	278.9	379.85	242.9
7	Endrin	262.9	278.9	242.9	316.9
8	p,p'- DDT	235.0	237.0	165.0
IS	Pyrene-d10	212.1	106.0
Surrogate	p,p'- DDT-13C12	247.0	249.0	224.0

3. Analysis of Recovery Test (100ng/L)

Add 100μl of Standard Sample Ⅱ(1ppm) into 1L of pure water (mineral water)

Take same preparation and analyze

	Compounds	M/Z	1	2	3	4	5	Average	Deviation	CV(%)
1	Hexachlorobenzene	283.9
2	Heptachlor	271.9
3	Aldrin	262.9
4	trans-Chlordane	374.8
5	cis-Chlordane	374.8
6	Dieldrin	262.9
7	Endrin	262.9
8	p,p'- DDT	235.0
IS	Pyrene-d10	248.0	-	-	-	-	-	-	-	-
Surr	p,p'- DDT-13C12	262.9

4. Repeatability Test (10pg)

Analyze 2μl of Standard Sample Ⅴ(5ng/ml)

	Compounds	M/Z	1	2	3	4	5	Average	Deviation	CV(%)
1	Hexachlorobenzene	283.9
2	Heptachlor	271.9
3	Aldrin	262.9
4	trans-Chlordane	374.8
5	cis-Chlordane	374.8
6	Dieldrin	262.9
7	Endrin	262.9
8	p,p'- DDT	235.0
IS	Pyrene-d10	248.0
Surr	p,p'- DDT-13C12	262.9

5. Data

Fig. 1 QA/QC Flow Chart

Fig. 2-1 TIC of Pesticides (1ppm)

Fig. 2-2 TIC of Pesticides (1ppm)

Fig. 3-1 SIM of Pesticides (100ppb)

Fig. 3-2 SIM of Pesticides (70ppb)

Fig. 3-3 SIM of Pesticides (50ppb)

Fig. 3-4 SIM of Pesticides (25ppb)

Fig. 3-5 SIM of Pesticides (10ppb)

Fig. 3-6 SIM of Pesticides (5ppb)

Fig. 3-7 SIM of Pesticides (0ppb)

Fig. 4-1 Calibration Curve of Pesticides (0～100ppb)

Fig. 4-2 Calibration Curve of Pesticides (0～500ppb)

Fig. 5 SIM of River Water Sample

Fig. 6-1 TIC of p,p'-DDT by good system

Fig. 6-2 TIC of p,p'-DDT by bad system

Table 1 Recovery of Pesticides (100ppb)

Table 2 Repeatability of Pesticides (5ppb)

Table 3 Results of River Water Sample

Fig.1-1 TIC and Mass Spectra of Pesticides

Fig.1-2 TIC and Mass Spectra of Pesticides

Fig. 3-1 SIM of Pesticides (100ng/ml)

Fig. 3-2 SIM of Pesticides (70ng/ml)

Fig. 3-3 SIM of Pesticides (50ng/ml)

Fig. 3-4 SIM of Pesticides (25ppb)

Fig. 3-5 SIM of Pesticides (10ng/ml)

Fig. 3-6 SIM of Pesticides (5ng/ml)

Fig. 3-7 SIM of Pesticides (0ng/ml)

Fig. 4-1 Calibration Curve of Pesticides (0～100ng/ml)

Fig. 4-2 Calibration Curve of Pesticides (0～500ng/ml)

Table 1 Recovery of Pesticides (100ppb)

Table 2 Repeatability of Pesticides (5ppb)

Fig. 5 SIM of River Water Sample

Table 3 Results of River Water Sample

River Water 1L -> 1ml
	Value for GCMS (ng/ml)	River conc. (μg/L)

Hexachlorobenzene	No Peak	-
Heptachlor	No Peak	-
Aldrin	No Peak	-
trans-Chlordane	No Peak	-
cis-Chlordane	No Peak	-
Dieldrin	No Peak	-
Endrin	No Peak	-
p,p'-DDT	No Peak	-
p,p'-DDT-13C12	Recovery　89.458%

River Water 1L (Add 50ng) -> 1ml
	Value for GCMS (ng/ml)	River conc. (μg/L)

Hexachlorobenzene	43.447	0.043
Heptachlor	44.481	0.044
Aldrin	43.969	0.044
trans-Chlordane	50.051	0.050
cis-Chlordane	49.113	0.049
Dieldrin	44.698	0.045
Endrin	46.195	0.046
p,p'-DDT	48.398	0.048
p,p'-DDT-13C12	Recovery　92.629%

Fig. 6-1 TIC of p,p’-DDT by good system

Fig. 6-2 TIC of p,p’-DDT by bad system

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Table of Contents

I. Introduction

II. Overview of UNU’s Previous Activities

III. Project Description

IV. Sample Collection and Preparation for Analysis

1. QA/QC

2. Sample for Analysis

3. Target compounds (Pesticides for POPs)

4. Internal Standard compound (IS) and Surrogate compound

5. Reagents, Materials and Apparatus

6. Sample for Calibration Curve

7. Summary of Method for Preparation (Flow chart)

V. Analytical Methods

1. Preparation for Analysis

2. Analysis Condition

3. Analysis of Recovery Test (100ng/L)

4. Repeatability Test (10pg)

5. Data