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Qualification of organochlorine pesticides by Gas Chromatograph Electron Capture Detector

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Micro-extraction apparatus

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*»Ÿπ¬å«‘®—¬·≈–Ωñ°Õ∫√¡¥â“π ‘Ëß·«¥≈âÕ¡ °√¡ à߇ √‘¡§ÿ≥¿“æ ‘Ëß·«¥≈âÕ¡ ‡∑§‚π∏“π’ µ.§≈ÕßÀâ“ Õ.§≈ÕßÀ≈«ß ®.ª∑ÿ¡∏“π’ 12120 ‚∑√. 0-2577-1136 ‚∑√ “√. 0-2577-1138 Environmental Research and Training Center, Department of Environmental Quality Promotion. Technopolis. Klong 5 Klong Luang, Pathumthani 12120 e-mail: Ruchaya@deqp.go.th

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ABSTRACT Water samples have been analyzed for 22 compounds of Organochlorine pesticides by using micro-extraction technique.

The technique was easy to perform, flexible and required minimal glassware and sample handling . This approach not only fulfilled the practical requirements of this program but also gave reliable data. A method based on simple extraction theory for determining recoveries and quality control by using Oxychlordane and Pentachloronitrobenzene (PCNB) as internal standards. A step of recovery and recovery factor were considered to compensate extracting and transferring of analyte from the matrix.Recovery factor of Oxychlordane was used for correction volume of extracting solvent. HP-1 (crosslinked to methyl silicone gum ) 0.2 mm. id x 50 m x 0.5 mm film thickness was used to separate 22 compounds in 45 min. The detection limit was 0.02 - 0.10 ppb and recovery 75-90 % which depends on chemical properties of each Organochlorine pesticides.

1. Introduction Organochlorine pesticides were worldwidely used to increased agricultural yield and protect livestock.Environmental and human health hazards associated with the use of there chemical in Developing Countries. On the other hand, Organochlorine pesticides are toxic and can be hazardous to human being because of bioaccumulation, persistent in the environment (Ramesh, A et al.,1900) and food chain (Macek et al.,1970) such as HCHs, Aldrin, Dieldrin, Endrin and DDTs etc. Development of analytical method for determination 22 compounds of Organochlorine pesticides in water sample by using °-32

microextraction technique introduced to support rapidly screening in river water sample.

The main objective of this development program is to determine the Organochlorine pesticide residues within the shortest possible time, small amount of solvent or chemical and required minimal glassware.

Microextraction technique employed common technique perform by using small amount of solvent to extraction. The method based on simple extraction theory for determining recoveries with spikes in water sample. In some cases an internal standard was added to the extraction solvent to compensate for injection error and solvent evaporation (Lawrence H.Keith ,1981). Because this technique used a volatile organic solvent to extract chlorinated compound from water sample. The analysis of water samples was extracted with volatile organic solvent, in which the solvent-to-water ratio range from 1:40 to 1:1000 . This approach was developed for use in the U.S. Environmental Protection Agency (EPA) verification sampling and analysis program.

Recovery factor was considered for quality control in quantitative data for each step. The 3 recovery factors investigated matrix, spike level and optimum of extraction time .

2. Methods and Materials 2.1 Apparatus (a) Extraction system . mechanical rotation model (b) Gas chromatograph . Hewlett packard 5890 series II plus ,equipped with a Ni 63 electron capture detector. HP-1 (crosslinked methyl silicone gum) capillary column (50m x 0.2 mm.id x 0.5 µm film thickness) was used to determine the 22 target compounds. The temperature program was as follows: The initial column »Ÿπ¬å«‘®—¬·≈–Ωñ°Õ∫√¡¥â“π ‘Ëß·«¥≈âÕ¡ °√¡ à߇ √‘¡§ÿ≥¿“æ ‘Ëß·«¥≈âÕ¡

temperature was held at 70 ÌC for 1 min. after injection and increased at 20 Ì C /min to 150 ÌC hold 1min.and increase 3 Ì C /min to 185 ÌC hold 1min. and increase 1 ÌC/min to 198 Ì C hold 18 min. The injection and detector were 220 ÌC and 300 ÌC respectively. The carrier gas is hydrogen (flow rate ca 0.7 ml/ min). The nitrogen was used for making up gas (flow rate ca 40 ml/min) . (c) Vertex genic 2. model G-560 E, USA

2.2 Material (a) Standards. 22 target compounds were obtained from GL science Inc, Japan.

The purity of all compounds were greater than 98%. Stock solution of the 22 target compounds were prepared in pesticide grade hexane 200 µg/ml ; working solution were prepared by using serial solution of composite stock solution about 10 ng/ml . (b) Solvent. Hexane, Acetone (Pesticide grade from J.T.Beaker, America)

(c) Internal standard. Oxychlordane and Pentachloronitrobenzene (PCNB) were obtained from Wako Pure Chemical Industrials LTD, Japan.

2.3 Experimental design The experimental design consisted of the experiment runs as shown in Table 1.

Each time period was replicated 4 times to determine the optimum extraction time for microextraction technique. 2 matrix types were used (tap water and river water) Concentration for recovery test 3 level was used the mixed standard in river water to determine the detection limit at 12 replication in each level as shown in Table 2.

In this study recovery factor (RF) were used to correct the extraction time and detection limit by using internal standards (oxychlordane and pentachloronitrobenzene).

Table 1 . experiment design A. The matrices were checked for decision of further clean up step by analyzing tap water sample and river sample.

B. Determination of method of detection limit.

Chemical standard mixture

15

Extraction time (min) 30 60

120

C. Concentration of spiked mixed standard for determination of method detection limit.

Chemical α-HCH HCB β-HCH γ-HCH δ-HCH Heptachlor Aldrin Heptachlor epoxide oxy-Chlordane trans-Chlordane

Spike (ng/ml) 31 41 316 63 100 104 63 58 40 60

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concentration of spiking ng/l ng/l ng/l 15 20 158 32 50 52 20 50 20 30

9.2 12 95 19 30 31 19 17 12 18

6.1 8 63 12 20 21 13 12 8.0 12 °-33

Chemical o,p-DDE Endosulfan I cis-Chlordane Dieldrin p,p′-DDE o,p-DDD Endrin Endosulfan II p,p′-DDD o,p-DDT Endosulfan sulfate p,p′-DDT Methoxychlor

Spike (ng/ml) 135 417 104 90 100 150 126 158 125 158 244 274 405

concentration of spiking ng/l ng/l ng/l 67 208 52 45 50 75 63 79 63 79 122 137 202

40 125 31 27 30 45 39 47 38 47 73 82 121

27 84 21 18 19 30 25 32 25 32 49 55 81

(1) The recovery factor ( X1 ) was used to determine extraction volume by using Oxychlordane as internal standard .

Concentration of spike * Volume of spike = Final Volume * A * C B X1 where X1 = extraction volume ( ml. ) A = area of Oxychlordane in sample B = area of Oxychlordane in standard C = concentration of Oxychlordane ( ng./ml. ) (2) Determination of recovery factor X2 by using PCNB as internal standard in certain technique for error injection .

3. Results and discussion The results showed that this method can analyse the 22 target compounds that were spiked in water samples. The river water and tap water sample were tested to skip the clean up step. The chromatogram of river water samples was very clear to identify the target compounds in Figure 1, that were compared with a chromatogram of river water sample by ordinary method as show in Figure 2. and also the chromatogram of spiked mixed standard in water sample was clear in Figure 4. when compared with a chromatogram of standard in Figure 3.

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Determination of the optimum extraction time were confirmed by 4 replication of each time period (15 min, 30 min, 60 min. and 120 min) The optimum time was 60 min. and appropriate to design all the experiment because of small %RSD and high of the percentage of recoveries by using recovery factor as shown in Table 3 and ordinary calculation in Table 2. The method of using recovery factor shown that variation of a recovery and %RSD of all extraction time were less than ordinary calculation, especially %RSD of 60 min the level was <10%, for example α - HCH, HCB and β-HCH as shown in Figure 5 and 6. »Ÿπ¬å«‘®—¬·≈–Ωñ°Õ∫√¡¥â“π ‘Ëß·«¥≈âÕ¡ °√¡ à߇ √‘¡§ÿ≥¿“æ ‘Ëß·«¥≈âÕ¡

Figure 1 Chromatogram of river water sample by using microextraction technique

Figure 2 Chromatogram of river water sample by using ordinary method

Figure 3 Chromatogram of 24 mixedstandards

Figure 4 Chromatogram of spiked river water sample

In the micro extraction technique, the evaporation of solvent and the recovery factor X1 and X2 were used to determine

extraction volume and error of injection into the GC

Table 2.

Experiment design for optimization of extraction time by using general calculation of percent recovery

Chemical

Spike (ng/L)

% R.(±%RSD) 15 mins

% R.(±%RSD) 30 mins

% R.(±%RSD) 60 mins

% R.(±%RSD) 120 mins

α-HCH HCB β-HCH γ-HCH δ-HCH Heptachlor Aldrin Heptachlor epoxide Oxy-Chlordane Trans-Chlordane o,p-DDE

15 20 158 31 50 52 20 50 20 30 67

75 ( ±20 ) 48 ( ±25) 87 ( ±22 ) 75 ( ±22 ) 55 ( ±44 ) 86 ( ±44 ) 84 ( ±38) 83 ( ±28 ) ** 83 ( ±45 ) 83 ( ±29 )

100 ( ±14 ) 52 ( ±14 ) 130 ( ±16 ) 99 ( ±15 ) 63 ( ±33 ) 130 ( ±34 ) 120 ( ±17 ) 120 ( ±17 ) ** 130 ( ±19 ) 130 ( ±19 )

94 ( ±30 ) 53 ( ±32 ) 110 ( ±30) 94 ( ±30) 65 ( ±44 ) 110 ( ±31 ) 110 ( ±23 ) 110 ( ±19 ) ** 110 ( ±15 ) 110 ( ±17 )

89 ( ±17 ) 45 ( ±19 ) 100 ( ±23 ) 82 ( ±19 ) 55 ( ±32 ) 100 ( ±27 ) 97 ( ±21 ) 100 ( ±17 ) ** 100 ( ±18 ) 100 ( ±17 )

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Chemical

Spike (ng/L)

% R.(±%RSD) 15 mins

% R.(±%RSD) 30 mins

% R.(±%RSD) 60 mins

% R.(±%RSD) 120 mins

Endosulfan I Cis-Chlordane Dieldrin p,p′-DDE o,p-DDD Endrin Endosulfan II p,p′-DDD o,p-DDT Endosulfan sulfate p,p′-DDT Methoxychlor

208 52 45 49 75 63 79 63 79 120 140 200

82 ( ±26 ) 83 ( ±30 ) 82 ( ±23 ) 83 ( ±30 ) 82 ( ±23) 87 ( ±30) 80 ( ±23 ) 81 ( ±27 ) 85 ( ±39 ) 76 ( ±17 ) 87 ( ±41 ) 87 ( ±45 )

120 ( ±20 ) 120 ( ±20 ) 120 ( ±16 ) 130 ( ±19 ) 120 ( ±15 ) 130 ( ±28 ) 120 ( ±25 ) 120 ( ±18 ) 120 ( ±28 ) 110 ( ±32 ) 130 ( ±33 ) 130 ( ±42 )

110 ( ±18 ) 110 ( ±19 ) 110( ±15 ) 110( ±17 ) 110( ±16 ) 110( ±24 ) 100 ( ±16 ) 110 ( ±16 ) 110 ( ±21 ) 100 ( ±21 ) 110 ( ±24 ) 110 ( ±31 )

99 ( ±18) 100 ( ±17) 100 ( ±10 ) 100 ( ±20 ) 100 ( ±15 ) 110 ( ±20 ) 95 ( ±14 ) 100 ( ±16 ) 100 ( ±27 ) 91 ( ±17) 110 ( ±34 ) 110 ( ±42 )

** internal standard

Figure 5. Variation of % recovery on extraction time by using general calculation

Figure 6. Variation of % recovery on extraction time by using Recovery factor

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Table 3.

Experiment design for optimization of extraction time by using percent of recovery.

Chemical

Spike (ng/L)

% R.(±%RSD) 15 mins

% R.(±%RSD) 30 mins

% R.(±%RSD) 60 mins

% R.(±%RSD) 120 mins

α-HCH HCB β-HCH γ-HCH δ-HCH Heptachlor Aldrin Heptachlor epoxide oxy-Chlordane trans-Chlordane o,p-DDE Endosulfan I cis-Chlordane Dieldrin p,p′-DDE o,p-DDD Endrin Endosulfan II p,p′-DDD o,p-DDT Endosulfan sulfate p,p′-DDT Methoxychlor

15 20 160 32 50 52 20 50 20 30 67 210 52 45 50 75 63 79 63 79 120 140 200

34 ( ±17 ) 53 ( ±22 ) 61 ( ±19 ) 52 ( ±20 ) 38 ( ±38 ) 60 ( ±30 ) 59 ( ±27 ) 58 ( ±35 ) ** 59 ( ±28 ) 59 ( ±20 ) 58 ( ±24 ) 59 ( ±29 ) 59 ( ±26 ) 59 ( ±27 ) 58 ( ±20 ) 61 ( ±23 ) 56 ( ±23 ) 57 ( ±22 ) 60 ( ±35 ) 54 ( ±13 ) 61 ( ±33 ) 61 ( ±34 )

72 ( ±9.8 ) 36 ( ±13 ) 88 ( ±16 ) 68 ( ±12 ) 43 ( ±17 ) 87 ( ±16 ) 84 ( ±20 ) 84 ( ±30 ) ** 87 ( ±14 ) 86 ( ±13 ) 84 ( ±10 ) 85 ( ±16 ) 85 ( ±9.5 ) 86 ( ±16 ) 85 ( ±18 ) 87 ( ±12 ) 81 ( ±12 ) 83 ( ±11 ) 85 ( ±15 ) 74 ( ±13 ) 86 ( ±20 ) 86 ( ±20 )

82 ( ±7.8 ) 46 ( ±9.8 ) 99 ( ±8.2 ) 82 ( ±7.8 ) 56 ( ±10.0 ) 93 ( ±10.6 ) 95 ( ±6.9 ) 97 ( ±6.1 ) ** 96( ±12) 96 ( ±6.7 ) 96 ( ±6.0 ) 95 ( ±6.5 ) 98 ( ±6.8 ) 95 ( ±7.2 ) 97 ( ±6.9 ) 98 ( ±9.8 ) 92 ( ±6.5 ) 97 ( ±6.4 ) 93 ( ±10 ) 87 ( ±7.8 ) 95 ( ±12 ) 92 ( ±14 )

75( ±9.2 ) 38 ( ±10 ) 88 ( ±10 ) 70( ±12 ) 46( ±12 ) 84( ±10 ) 82 ( ±8.2 ) 85 ( ±5.4 ) ** 84 ( ±12 ) 85 ( ±11 ) 84 ( ±12 ) 84 ( ±11 ) 86 ( ±9.0 ) 85 ( ±11) 85 ( ±8.8 ) 91 ( ±8.4 ) 80 ( ±11 ) 85 ( ±13 ) 88 ( ±14 ) 76 ( ±10 ) 97 ( ±9.8 ) 93 ( ±15 )

The recovery factor can be used to determine the concentration in sample in case of RSD <10 % such as δ-HCH and HCB that have %recovery in the range of 40-50 % and RSD < 10 % .

4. Conclusion The recovery factor was applied to develop the analysis of organochlorine pesticides by using micro extraction technique to compensate the volume in the extraction and to determine concentration of target compounds. The recovery factor was used to correct for determining the

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optimum of time consume and % recovery.

The detection limit of this method is at the range of 0.02-0.1 ppb as shown in Table 4.

However the application of recovery factor should depend on the chemical properties such as decomposition during extraction or injection that results in low recovery and % RSD should less than 10 %.

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Table 4.

A average percent recoveries (±%RSDs) and method detection limit (MDL) of 22 organochlorine pesticide compounds by using micro extraction technique.

Chemical

α-HCH HCB β-HCH α-HCH δ-HCH Heptachlor Aldrin Heptachlor epoxide Trans-Chlordane o,p-DDE Endosulfan I Cis-Chlordane Dieldrin p,p′-DDE o,p-DDD Endrin Endosulfan II p,p′-DDD o,p-DDT Endosulfan sulfate p,p′-DDT Methoxychlor

Spike (ng/L)

%R(±RSD) (n=12)

MDL (ng.L)

6.1 8.2 63 13 20 21 13 12 12 27 84 21 18 20 30 25 32 25 32 49 55 81

79 ( ±6.2 ) 40 ( ±6.1 ) 73 ( ±9.3 ) 73 ( ±5.9 ) 33 ( ±5.6 ) 97 ( ±10 ) 84 ( ±5.7 ) 91 ( ±5.4 ) 80 ( ±6.0 ) 85 ( ±5.9 ) 92 ( ±5.3 ) 87 ( ±5.9 ) 89 ( ±6.2 ) 92 ( ±6.9 ) 87 ( ±5.4 ) 96 ( ±2.1 ) 91 ( ±6.3 ) 90 ( ±5.5 ) 93 ( ±0.9 ) 84 ( ±9.4 ) 100 ( ±2.5 ) 98 ( ±4.8 )

1.4 1.0 21 2.7 9.3 3.1 9.3 2.0 2.8 6.7 7.6 5.2 4.9 6.2 7.0 2.0 23 6.1 1.0 18 4.8 13

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G. H. Jeffery, J. Bassett, J. Mendham, R. C. Denney. 1989. çVogelûs Textbook Of Quantitative Chemical Analysisé Fifth edition,.

Junk G.A., I. Ogawa and H. J. Svac.1981. çExtraction of Organic Compounds from Water using Small Amount of Solventé Adv. Identif. Anal.Org. Pollut.

Water (Keith ; L.H.) , Inst. Appl. Sci. Texas : pp. 281-292.

Macek . K . J & Korn , S. (1970) J. FISH. Board Canada 27,1496-1498.

M. M. Thomson and W. Bertsch. J. Chromatogr.

1983, 279, pp.383-393.

Morrison, G. H. and H. Freisher, Solvent

Extraction in Analytical Chemistry , John Wiley & Sons Inc., U.S.A.,1975.

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Ramesh, A., Tanabe. S., Murase.1991. H., Subramanian & Tatsukawa R. Environment . Poll . 74 : pp 293-307.

Rhodes J. W. and C. P. Nulton. 1981. çMicroextraction as an Approach to Analysis for Priority Pollutions in Industrial Waste wateré Adv. Identif.

Anal. Org. Pollut. Water (Keith ; L.H.) ,pp.241 Inst. Appl. Sci.Texas : pp. 241252.

Thrun., K.E., K.E. Simmons and J.E. Oberhtzer.

J. 1980. Environmental science and Health, 415, 5 : pp. 485-501.

Thielen Dr., G. Olesen., A. Davis., E. Bajor., J. 1987. Stefanovski and J. Chodkowski.

J. Chromatogr. Sci., 25: pp. 12-16.

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