Assessment of QC and Uncertainty in Multiresidue Pesticides analysis in water with GC- ECD/NPD

Mukund Nagarnaik¹, Arun Sarjoshi²,Girish Pandya²*

¹Managing Director, Research and Development Division, Qualichem Laboratories Pvt. Ltd., 4,

North Bazar Road, Gokulpeth Market, Nagpur 440010

²QC Technical Manger, Research and Development Division, Qualichem Laboratories Pvt. Ltd., 4,

North Bazar Road, Gokulpeth Market, Nagpur 440010

³Sr.Scientist, Research and Development Division, Qualichem Laboratories Pvt. Ltd., 4,

North Bazar Road, Gokulpeth Market, Nagpur 440010

*Corresponding Author E-mail: pandyagh@rediffmail.com

ABSTRACT:

An assessment was carried out for simultaneous determination of 25 Organochloro and Organophos pesticides residues in drinking water by gas chromatography coupled to ECD and NPD detectors. The recovery data were obtained by spiking blank samples of drinking water with pesticides at concentration levels of 30.0 μg/L, yielding recoveries in the range 90–119%. Precision values expressed as relative standard deviation (RSD) were in the range of 6.40 – 17.49 %. Linearity was studied in the range 10–200 ug/L and the coefficient of correlation was higher than 0.98% for all compounds. Method Detection Limits (MDLs) and limits of quantification (LOQs) were established. The overall uncertainty of the method was estimated. The estimation of the uncertainty associated to analytical methods is necessary in order to establish the comparability of results. Multiresidue analytical methods lack very often information about uncertainty of results with likely implications when results are compared with levels established by regulations. An adequate identification and estimation of each uncertainty source allows to laboratories to establish the accuracy of results and to balance with time-consuming and costs. According to the validation data and performance characteristics as well as the high sample throughput, the proposed method is suitable for routine application.

KEYWORDS: Pesticides, bottled water, GC-ECD/NPD, Uncertainty.

INTRODUCTION:

Organic contaminants present in the environment are a result of different sources of pollution from anthropogenic activities .The pesticides, generated by the intensification of agriculture, are regarded as some of the most dangerous contaminants of the environment, despite their numerous merits. Not only are they toxic; they are also mobile and capable of bioaccumulation¹. On top of this, they can take part in various physical, chemical and biological processes. Many of these pesticides are characterized by a strong persistence which explains their wide presence in the different compartments of the environment^2-4. Due to these physicochemical characteristics and their extensive use, many of these pesticides end-up in surface and ground water.

They are found now a days in all surface waters and in a growing number of aquifers. Their presence in water is considered as a potential risk not only for drinking water quality and human health, but also for ecosystems. In this context, strict regulations for the control of pesticide residues concentration levels in environment have been established. Apart from water shortages at times, real and perceived needs to safeguard health has also contributed to an escalating trade in package drinking water at the national and international level. Considering the consumer’s health and safety it has become imperative to ensure that the package water offer for sale is safe and free from harmful organisms. One of the requirement under Indian Standard for Packaged Drinking water (IS 14543: 2004) ⁵is that the pesticide residue limits considered individually should not be more than 0.0001 mg/L. The total pesticide residue should not be more than 0.0005 mg/L. Such stringent limits require development and quality control of analytical method for accreditation by internationally established organization such as NABL under ISO/IEC 17025. Consideration has also been given in the Prevention of Food Adulteration Act, 1954 and the Rules framed there under regarding the presence of pesticide in samples. Many pesticides remain as residues in foodstuffs after their application, and they can be widespread in the environment (soils, surface and underground waters).The paper reports the study carried out for development of quality control in the analysis of package drinking water for organochloro and organo phosphorus pesticides.

For instrumental analysis, gas chromatography (GC) with ECD and NPD detectors are the most commonly used techniques for the quantification of pesticides in water. Consumption of packaged drinking water for drinking purposes has been increasing considerably in the country. Methods to determine pesticide residues include the extraction of the analytes from the matrix, appropriate cleanup of the raw extracts and subsequent determination by gas chromatography (GC) .Globalization of commodities market and concerns for the consumer has put pressure on regulatory agencies to increase pesticide monitoring programs in terms of specificity of analysis and number of samples analyzed.

These demands have caused the development of methods to reliably and rapidly detect as many pesticides as possible from a single extraction. Multiresidue methods with specific detectors suchas ECD and NPD installed in the same Gas chromatograph becomes advantageous and come close to meeting the needs of regulatory agencies since they allow for the determination of a broad spectrum of pesticides and metabolites in a variety of samples.. Specificity is provided by the combination of chemical structure information and retention time on an analytical GC column. These techniques are also sensitive, precise and sufficiently accurate to be useful for regulatory purposes, while being cost effective and rapid

MATERIALS AND METHODS:

Chemicals and Standards:

Pesticide reference standards were obtained from Dr. Ehrenstorfer GmbH, Augsburg, Germany. Pesticide quality chromatography grade solvents (hexane, methylene chloride, diethyl ether) were purchased from Merck, India respectively. Special grade anhydrous sodium sulfate was heated to 700ºC for 8 hours, cooled and then used for analysis. Purified RO grade water with a conductivity of 0.5 μSi/cm and Florisil: (60/100 mesh) heated at 130ºC for 8 hours then cooled slowly in a desiccators was utilized during cleanup.

Apparatus:

A Thermo Trace GC Ultra gas chromatograph (Thermo Fisher Scientific Instruments, San Jose, CA95134, USA) with electronic flow control (EFC) was used. A Thermo Autosampler TRH was attached to the gas chromatograph. The GC was equipped with ECD and NPD Detectors.

Samples were injected with a 1 ul syringe, into a split/splitless septum-equipped injector .A fused-silica analytical capillary column DB- 5( 30m x 0.25mm) was used . Helium (99.999%) at a flow rate of 1.2 mL/min was used as carrier gas.

The instrumental conditions during organochloro pesticide analysis with ECD were as follows.

Column temperature: 90°(5 min)- 20°C/min -180°C(0 min), 4°C/min-250°C(0min), -15 °/min-300°C(2 min). The injector temperature was 300°C with splitless injection. ECD was maintained at 320°C

The instrumental conditions during organophos pesticide analysis with NPD were as follows.

Column temperature: 60°(0 min)- 12°C/min -180°C(0 min), 3°C/min-230°C(1min), 15C°/min-290°C(5 min). Injector temperature: 290°C with splitless injection. NPD was maintained at 300°C

Rotary Vacuum Evaporator Model IKA RV-10 from IKA Instruments, Germany was used for concentration of the samples.

Sample Preparation:

The bottled drinking water samples were obtained from the local markets. 50 g of sodium chloride was added to 1L of water sample. 60 ml of 15% Diethyl ether in n-Hexane was added to the sample bottle and shaken for 10 minutes. The hexane layer was separated. The extraction was repeated twice, combining the hexane layers, dehydrating with anhydrous sodium sulfate, filtering and concentrating (reducing) the hexane solution to 5ml with rotary vacuum evaporator. The extract was cleaned using Florisil column and further concentrating this solution to 1ml by blowing nitrogen gas across the surface of the solution. 1μl of the extract was used for injection into GC for analysis. A blank sample was prepared as an analytical control by using the same procedure as described above. Stock standard solution was(1000 mg/l) prepared from pure certified standard reference material by accurately weighing pure reference material on a 5 decimal place analytical balance. The material was dissolved in n-Hexane and volume made up to 10 ml in certified volumetric flask. The standards were stored at low temperature in a freezer.

Figure 1. Organochloro and Organophos Pesticide Analysis

The calibration standard were at five concentration levels for each compound by adding appropriate volume of one or more stock standards to a volumetric flask and diluting to volume with n- Hexane. While preparing working standards, a record was kept of the identity and amount of all solutions and solvents employed. The standards were labeled indelibly, allocated an expiry date, and stored at low temperature in the dark in containers that prevent any loss of solvent and entry of water. The sample preparation steps are illustrated in Figure 1.

In order to carry out the quantitative analysis of the samples with GC, ECD mode was used first to separate organochloro pesticides. Identification and confirmation of the compounds was based on the use of retention time of the chromatographic peak of the analyte. The RTs were established for all the pesticides under study. About 15 organochloro pesticides were thus separated and identified. Confirmation of the remaining 10 organophos pesticides were then carried out by switching the instrument to NPD mode.

RESULTS AND DISCUSSION:

In order to carry out multiresidue pesticide analysis, it was necessary to develop an in-house quality control program for ongoing analysis of spiked samples. Ongoing data quality checks were compared with established performance criteria to meet the performance characteristics of the method. The multi-residue analysis of pesticides in water samples require validation of all procedures (steps) that were undertaken in the method. This required assessment of linearity, recovery (as a measure of trueness or bias) and precision. Linearity was studied in the range 10–200 ug/L with five calibration points by matrix-matched standard calibration.

Calibration curves for all the 25 pesticides were developed in the 10–200 ug/L range. Figure 2.illustrates the calibration for a compound like Aldrin. The resulting chromatogram is summarized in Figure 3. Linear calibration graphs were constructed by least-squares regression of concentration versus relative peak area of the calibration standards. Linearity values, calculated as determination of correlation coefficient (r²), were in the range 0.9814– 0.9999. The deviation of the individual points from the calibration curve was lower than 20%.

Figure 2. Calibration curve for Aldrin.

Figure 3. Chromatographic identification of Aldrin

Table 1. Quality Control and Uncertainty in Pesticide analysis

Name of Pesticide	RT	Mean N=7	SD	RSD	Recovery %	MDL ng/L	LOQ ng/L	Uncertainty
Alpha-HCH	14.52	29.73	4.98	16.75	99	15.63	49.8	±0.26
Beta-HCH	15.21	26.87	3.23	12.02	89	10.14	32.3	±.0.09
Gamma-HCH	15.38	33.72	4.68	13.82	112	14.69	46.8	±0.12
Delta- HCH	16.07	35.00	6.15	17.57	116	19.31	61.5	±0.12
Aldrin	18.78	28.71	4.13	14.38	96	12.96	41.3	±0.100.
2,4’-DDE	21.23	31.08	4.38	14.09	103	13.75	43.8	±0.11
Alpha-Endosulfan	21.60	28.70	4.73	7.24	95	14.85	47.3	±0.062
4,4’- DDE	22.44	30.21	2.19	7.24	100	6.87	21.9	±0.062
Dieldrin	22.59	31.21	2.00	6.40	104	6.28	20.0	±0.054
2,4’- DDD	22.81	29.14	2.17	7.44	97	6.81	21.7	±0.062
Βeta-Endosulfan	23.85	29.52	2.06	6.98	98	6.46	20.6	±0.060
4,4’-DDD	24.13	32.65	2.88	8.82	108	9.04	28.8	±0.022
2,4’- DDT	24.26	31.11	2.43	7.81	103	7.63	24.3	±0.055
Endosulfan sulfate	25.56	33.89	2.20	6.49	112	6.90	22.0	±0.056
4,4’-DDT	27.47	29.33	2.90	7.88	97	9.10	29.0	±0.065
Phorate	15.46	26.74	2.84	10.62	89.1	8.9	28.4	±0.077
Phorate sulfoxide	17.80	28.01	5.19	18.53	93.3	16.2	51.9	±0.147
Monocrotophos	18.05	31.63	5.08	16.06	105.4	15.9	50.8	±0.124
Malaxon	20.20	35.67	2.83	7.93	118.9	8.8	28.3	±0.066
Parathion ethyl	22.11	27.80	289	10.39	83.4	9.0	28.9	±0.083
Phorate sulphone	22.45	26.34	1.97	7.47	87.8	6.1	19.7	±0.063
Chlorpyriphos	22.63	28.46	4.98	17.49	94.8	15.6	49.8	±0.135
Malathion	22.71	29.35	5.10	17.37	97.8	16.0	51.0	±0.133
Methyl paraxon	30.04	31.96	4.44	13.89	106.5	13.9	44.4	±0.108
Ethion	33.32	26.77	2.42	9.03	89.2	7.6	24.2	±0.074

Accuracy was evaluated in terms of recovery by spiking blank samples of drinking water with the corresponding volume of the multi compound pesticide working standard solution. Total of seven samples, one on each day, were spiked with a concentration of 30.0 µg/L. The samples were than processed for analysis by GC. The results of day to day analyses are summarized in Table 1.

Recoveries between 90 to 119% were found in water samples. The intraday precision was expressed as percent relative standard deviation for each pesticide analysed. The minimum RSD was 6.6) and the maximum (20.8). Therefore, these results meet the requirement criteria of trueness or mean recovery for quality control. Method detection limits (MDL) were also determined for all the pesticides under this study. It provides a useful mechanism for illustrating the capability of the analytical method. MDLs were calculated for the pesticides as follows:

The sample standard deviation is multiplied by the correct Student's t-value from the statistical Tables.

In the present study seven replicates were taken, hence six degrees of freedom was considered. t is found to be 3.143. The MDL was calculated for a compound like Aldrin as follows:

MDL= (s)(t-value)= 4.13 x 3.143= 12.96 ng/L.

Rounding to the correct number of significant figures, the calculated MDL becomes 12.96 ng/L.

Similarly, LOQs were subsequently established as 10 times the Standard Deviation of the recovered pesticide. The limit of quantitation was also calculated as:

LOQ= 10 x (s)= 10 x 4.13 = 41.3 ng/L

The MDL and LOQ were thus calculated for all the pesticide under study and are summarized in Table 1.

Attempt was also made to estimate the uncertainty associated with the multiresidue analytical method in water matrix by applying a bottom-up approach. All data appearing in this study complies with NABL 17025 requirements. It was implemented in our laboratory as a pesticide residue analysis routine method and our laboratory was accredited. The uncertainty of each step was estimated identifying which of them are relevant in the global uncertainty analysis are illustrated by a cause and effect diagram as shown in Figure 4. The parameters of the measure are represented by the main branches in the diagram. Further factors are added to the diagram, considering each step in analytical procedure.

Figure 4. Cause and effect diagram for pesticide analysis

Figure 5. Uncertainty Estimation Process for Pesticides

The uncertainty estimation procedure is summarized in Figure 5.The standard uncertainties associated with each step are quantified by estimating analyte concentration from the calibration curve, calculating recovery of the sample extract. After obtaining the standard uncertainty (u(x)), expressed as a standard deviation, and combined standard uncertainty were determined. In some cases, it is feasible to use relative uncertainties which represent the value of the uncertainty normalized. It is obtained as the quotient between the standard uncertainty u(x) and the value of x:

Urel(x) = or urel(x) =

The uncertainty estimation was carried as per the procedure summarized in Figure 5.and summarized below by the following steps:

(1) Specifying the measurand. This involved making a clear statement of what is being measured, including the relationship between the measurand and the input quantities (measured quantities, constants and calibration standard values.

(2) Identifying uncertainty sources i.e listing the possible sources of uncertainty, usually specified in the above step.

(3) Quantifying uncertainty components i.e. estimating the uncertainty component associated with each potential source of uncertainty identified. The different contributions to the overall uncertainty is expressed as standard deviation which is calculated depending on the data available from a standard deviation value ( this value is directly used); from a coefficient of variation; from the standard deviation of experimental data sets; from a declared purity and uncertainty value(which is given in a certificate of calibration for reference materials) and from a

correlation coefficient of calibration curves etc.

(4) Calculate combined uncertainty by combining different contributions to the overall uncertainty according to the appropriate rules.

The combined standard uncertainty u(f) is calculated as

u(f ) = [c²(x)u²(x) + c²(y)u²(y)+· · ] ^½

Where c is a sensitivity coefficient associated to each one of variables, given by the partial derivative of the function: c(x) = ∂f/∂x.

(5)Expanded uncertainty by applying the appropriate coverage factor.

The combined uncertainty and expanded uncertainty were calculate for all the 30 pesticides under study.

The values and uncertainties for each pesticide is summarized in Table 1.The different aspects explained above for estimating the combined uncertainties have been applied to the multiresidue of 25 pesticides in water. Table 3.summarises the relevant information for calculating uncertainties associated with the preparation of primary standard solutions, volumetric materials, and analytical balance.

The expanded uncertainty was subsequently determined to develop an interval within which the value of the measurand may lie. A factor of 2 was thus used for obtaining a confidence level of 95%.

The developed method was validated in order to ensure the feasibility of the method for its application in routine pesticide analysis of drinking water. Parameters such as specificity, linearity, quantitation limits, precision, accuracy and robustness were determined. In the specificity analysis representative chromatogram of individual compounds and also in a mixture of interfering analytes was studied. Linearity and working range were demonstrated by analysis of standards three times for different concentrations. The study was made every time with serial dilutions. Linearity demonstrated by plotting graph response against concentration and the curve fitted without forcing to zero. Slope and correlation coefficient were calculated. For detection limit seven replicates of fortified samples were run and their determinations was performed. Estimation of limit of quantification and limit of detection was done by the guideline of estimation of analytical detection limit. For Precision, seven replicates of fortified samples were run for the matrix and their determinations performed. Accuracy in analysis was based on seven replicates i. e. repeatability studies. Intermediate precision obtained on different days (Reproducibility) and relative standard deviation (RSD) is determined. Robustness of analytical method was established by changing the experimental conditions such as temperature.

The method was applied to samples of packaged drinking water with several internal quality controls to ensure that the measurement process is under statistical control. Each batch of samples was processed together with a reagent blank, composed of only solvent. The reagent blank was obtained by performing the whole process without a sample. The majority of recoveries were in the range 90–119 %.

REFERENCES:

1. Pandya GH., Koel Kumar, Saravana Devi, S.Kondawar VK., Chakraborti TC., Evaluation of HCH,DDT and Endosulfan levels in soil by Gas Chromatography/ Tandem Mass Spectrometry, Soil and Sediment Contamination, 15:, 2006: 529-541

2. UNEP: Global report on regionally based assessment of persistent toxic substances, ENEP Chemicals, Geneva, Switzerland (2002).

3. Oxynos, K.,J. Schmitzer and A. Kettrup: Guidelines for environmental specimen banking in the Federal Republic of Germany, Federal Environmental Agency, Berlin(1989).

4. WHO: Public Health Impact of pesticide used in agriculture, WHO in collaboration with UNEP, Geneva(1990).

5. IS 14543:2004, Indian Standard “Packaged Drinking Water (Other than Packaged Natural Mineral Water), Bureau of Indian Standards, New Delhi, 2004

Received on 02.01.2014 Modified on 28.01.2014

Asian J. Research Chem. 7(3): March 2014; Page 304-309