Levels and Potential Health Hazards of Chlorinated Pesticides in Surface Water Samples of Charsadda Area of Pakistan Using SPME-GC-ECD Technique

Round 1

Reviewer 1 Report

The manuscript water-1328943 entitled “Levels and potential health hazards of chlorinated pesticides in surface water samples of Charsadda area of Pakistan using SPME-GC-ECD technique” is focused on the extraction and determination of 20 organochlorine pesticides in surface water samples. The subject of contamination of water sources is of topical interest, and therefore this paper is timely. On the other hand, many aspects of the manuscript need to be extensively improved. In my opinion, the authors should consider the following suggestions:

1. The English is readable, but some sentences are not finished and in some cases, grammar and spelling errors occur. The help of a native English speakers would help to overcome this issue.
2. The weakest part of the manuscript is “Results and Discussion”. The author should decide what will be part of the paper or provide sufficient discussion to all topics mentioned in this part. For example, there is a complete lack of information about optimization of SPME (what type of fiber was selected and why, why 45 min of extraction time, etc.). What types of fibers have you compared? What was the main selection criteria? The discussion should be supported by relevant figures etc.
3. The quantification of the detected pesticides is not clear. Did you use matrix-matched calibration curves?
4. Any explanation for the calculation of the recoveries at the highest concentration levels is need. I suggest adding recovery studies also on lower concentration levels, which are near to the lowest calibration levels.

After these checks, the work can be considered for publication on Water.

Author Response

Reviewer 1

The manuscript water-1328943 entitled “Levels and potential health hazards of chlorinated pesticides in surface water samples of Charsadda area of Pakistan using SPME-GC-ECD technique” is focused on the extraction and determination of 20 organochlorine pesticides in surface water samples. The subject of contamination of water sources is of topical interest, and therefore this paper is timely. On the other hand, many aspects of the manuscript need to be extensively improved. In my opinion, the authors should consider the following suggestions:

Q1.  The English is readable, but some sentences are not finished and in some cases, grammar and   spelling errors occur. The help of a native English speakers would help to overcome this issue.

 

• Worthy reviewer, we try our best to remove grammar and spelling errors in revised manuscript.

 

Q2. The weakest part of the manuscript is “Results and Discussion”. The author should decide what will be part of the paper or provide sufficient discussion to all topics mentioned in this part. For example, there is a complete lack of information about optimization of SPME (what type of fiber was selected and why, why 45 min of extraction time, etc.). What types of fibers have you compared? What was the main selection criteria? The discussion should be supported by relevant figures etc.

 

 

• Worthy reviewer, amendments as per following details have been made.
• Worthy reviewer, solid-phase micro-extraction (SPME) fiber used was divinylbenzene polydimethylsiloxane (DVB-PDMS) (Supelco, USA). The fiber was conditioned before the first application by using hot injector of GC at 250 °C and detector at 300 °C for 1h. The column and detector were cleaned for 30 min at 280 and 310 °C, respectively (Given in the manuscript). The optimization of SPME for the adsorption and desorption of pesticides were determined. Two types of fibers i.e. (DVB-PDMS) and polydimethylsiloxane (PDMS) were compared. Bothe the fiber provides good extraction results for the selected pesticides, however, the efficiency of DVB-PDMS fiber is somewhat better as compared to the (PDMS) in the establishing of equilibrium for all the selected compounds. As SPME is equilibrium dependent process which involving partitioning of the analytes from the liquid sample into the stationary phase and concentration of analyte adsorbed depends on the mass and extraction time.  Therefore, to achieve the equilibrium between the pesticides aqueous solution and fiber, extraction of pesticides by SPME fiber were performed at various extraction times (5–55 minutes). It is clear from the figure that the fiber DVB-PDMS, extraction efficiency of analytes is gradually increased with the increase of extraction time for fiber. The maximum extraction time (45 min) was selected for the fiber DVB-PDMS for establishing equilibrium and no major increase in the extraction efficiency of fiber was achieved at a longer time.

 

Fig. Effect of extraction times on behavior of the organochlorine compounds through SPME fiber at different time intervals.

 

 

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Q3.  The quantification of the detected pesticides is not clear. Did you use matrix-matched calibration curves?

 

• Worthy reviewer, for quantification of detected pesticides calibration plots and straight line equation (Y = mx + C, where Y is peak area, m is slop, x is concentration and c is intercept) is used. For calibration plots, fresh standard working solutions of various concentrations i.e. 0.01, 0.05, 0.1, 0.3, 0.5, 1 and 2 μg L^-1 were prepared from stock solution in ultra-pure water containing all the 20 pesticides. From Calibration plots of standards, slope and intercept is obtained. Then for quantification of detected pesticides in unknown sample, straight line equation (Y = mx + C) is used. Where Y is peak area of detected pesticide in sample, x is the concentration of detected pesticide. However, the numerical values of slop and intercept is obtained from Calibration plots of standards to calculate the concentration of pesticide in water samples. By putting the values in equation, we can easily determine the concentration of detectable pesticides in the water sample.

 

Q4. Any explanation for the calculation of the recoveries at the highest concentration levels is need. I suggest adding recovery studies also on lower concentration levels, which are near to the lowest calibration levels.

 

• Worthy reviewer, calibration plots were prepared in the range of (0.01, 0.05, 0.1, 0.3, 0.5, 1 and 2 μg L^-1) Therefore, recovery studies at 1 and 2 μg L^-1 levels is within the calibration range. We have also studied calibrations for somewhat higher concentration i.e. 3, 4 and 5 μg L^-1, which also given good linear response. It is also cleared that in micro-extraction as the concentration decreases, the result become more good. Therefore, 1 and 2 μg L^-1 level is good for recovery studies.

Reviewer 2 Report

This manuscript provides data on the content of some previously used agricultural pesticides in the natural waters of northern Pakistan. The authors use well-known analytical methods and describe their adaptation to their analytical capabilities.

These data can be used by environmental chemists to compare with the results of other researchers working in different parts of the world, and therefore this manuscript can be published after careful revision in the form of a short correspondence. The authors must answer all the questions I have.

1.Introduction - very long - most of the information is widely known even from popular literature. A detailed consideration (nicely written in paragraphs 3-5) should be given to the production and use of pesticides in Pakistan.

It should be indicated why exactly these 20 pesticides were determined (I understand that the authors studied only those compounds that they had in a standard solution, but this is not enough for comprehensive monitoring)

2. Materials and Methods

2.1.-2.4 -OK

2.5.-Samples Collection. – Washed and clean sampling bottles – how were they cleaned? For trace analyzes, there are special procedures for thorough cleaning of the samplers, including the mandatory baking up of the samplers at high temperatures. How did authors deal with this using plastic bottles? It is imperative to provide data of blanks for the field, laboratory, and instrumental tests.

What was the volume of water samples for analysis? What was the final sample volume before injection into the chromatograph? How long were samples stored between collection and instrumental analysis?

2.6. – Analytical instrumentation – Authors must provide information about internal standards and surrogate standards. Figure 2 is not necessary – it is just a chromatogram of purchased calibration standards. Instead, authors must present a chromatogram including IS and SS with marked RT.

3.2.- Method validation. At present, chlorinated hydrocarbons are routinely analyzed by GC/MS. If the authors have only GC-ECD at their disposal, the samples should be fractionated, since many chemical compounds have similar retention times (PCBs, toxaphenes, PBDEs, and many others). I am very surprised by Figure 3, where only the beforementioned pesticides are present in the water samples - apparently, something is missing in the text. Misidentification possibly explains the high concentration of aldrin, which has not been used for over 45 years and has long been degraded in the environment. It is also desirable to compare the ratio of HCH isomers and DDT metabolites to each other to confirm the presence of these compounds in water and trace their fate in the ecosystem. Also, apparently, the authors cited an instrumental MDL 3 s./n. It is preferable to use the 7-point US EPA method. Finally, the authors did not explain their performance check (Did they run RSM?)

3/4-4 – OK

References – OK, but I would prefer links to more recent information about similar studies in Asian countries

                                                                          

 

Comments for author File: Comments.pdf

Author Response

Reviewer 2

           

This manuscript provides data on the content of some previously used agricultural pesticides in the natural waters of northern Pakistan. The authors use well-known analytical methods and describe their adaptation to their analytical capabilities.

These data can be used by environmental chemists to compare with the results of other researchers working in different parts of the world, and therefore this manuscript can be published after careful revision in the form of a short correspondence. The authors must answer all the questions I have.

1.Introduction - very long - most of the information is widely known even from popular literature. A detailed consideration (nicely written in paragraphs 3-5) should be given to the production and use of pesticides in Pakistan.

It should be indicated why exactly these 20 pesticides were determined (I understand that the authors studied only those compounds that they had in a standard solution, but this is not enough for comprehensive monitoring)

• Worthy reviewer, the selected 20 organochlorine pesticides are very stable and they resist to biodegradation in the environment. They are mostly persistant organic polutants. The study of these OCPs is important, because of their highly toxic effects to the aquatic life and human health and their persistence in the environment for a very long time. We agree with valuable suggestion of Reviewer to study more pesticides for comprehensive monitoring. In this connection, we have already started work to analyze more number of organochlorine pesticides and organophosphorus pesticides in various environmental samples, water, soil, fruits and vegetables samples.

Materials and Methods

• Worthy reviewer, refined  1.-2.4 –in materials and method.

2.5.-Samples Collection. – Washed and clean sampling bottles – how were they cleaned? For trace analyzes, there are special procedures for thorough cleaning of the samplers, including the mandatory baking up of the samplers at high temperatures. How did authors deal with this using plastic bottles? It is imperative to provide data of blanks for the field, laboratory, and instrumental tests.

• Worthy reviewer, we taken 100 ml plastic bottles (new and disposable) only used to collect water samples from various locations during sampling. In the Lab, for GC analysis of the samples, COMBI PAL CTC autosampler was used for extraction of pesticides from water samples. In COMBI PAL CTC autosampler, three types of tray for different vials were available i.e for 2 ml vials, 10 ml vials and 20 ml vials. We have used 20 ml glass vials made by Agilent technologies, USA. The vials are specially designed for CTC autosampler and containing magnetic caps.

What was the volume of water samples for analysis? What was the final sample volume before injection into the chromatograph? How long were samples stored between collection and instrumental analysis?

• Worthy reviewer, we have used 20 ml water samples in 20 ml vials (made by Agilent technologies, USA). A Solid-phase micro-extraction (SPME) fiber was used for pesticide extraction from water samples. The SPME fiber used was DVB-PDMS (Supelco, USA). When using CTC autosampler, then SPME fiber is fixed in the holder assembly of autosampler. The autosampler immersed the SPME fiber in the 20 ml vials for 45 minutes with agitation. The SPME fiber extract the organics from water samples. After 45 minutes, SPME fiber come out from vial and directly injected to the GC injector, where hot flow of Nitrogen gas at 250 ^◦C desorb the organics (pesticides) from SPME fiber to injector and then to column of the GC for separation. Samples are stored 7-10 days for processing time.

Note: SPME fiber is used to save the organic solvents and prevent the environmental pollution.

When using SPME fiber no organic solvent is injected to GC but only fiber is injected. However, when sample is prepared in organic solvent, then 1-2 microliter is injected to GC.

• – Analytical instrumentation – Authors must provide information about internal standards and surrogate standards. Figure 2 is not necessary – it is just a chromatogram of purchased calibration standards. Instead, authors must present a chromatogram including IS and SS with marked RT.

 

• Worthy reviewer, the SPME fiber used was DVB-PDMS for extraction of pesticide from water samples. Using SPME fiber, the percent recoveries studies at 1 µg L^-1 and at 2 µg L^-1 concentrations of pesticides ranged from 87.5 % to 106.0 % and 88.5 % to 109.2 %, respectively. It showed good results. When using SPME fiber, mostly IS is not needed, because sample valume injected to GC mostly effect the results, which is in very much minor amount i.e. 1 microlitre. In Fig. 2 chromatogram for 20 pesticides is based on peak area versus retention time. However, peaks are marked with no. for easiness purpose.

3.2.- Method validation. At present, chlorinated hydrocarbons are routinely analyzed by GC/MS. If the authors have only GC-ECD at their disposal, the samples should be fractionated, since many chemical compounds have similar retention times (PCBs, toxaphenes, PBDEs, and many others). I am very surprised by Figure 3, where only the beforementioned pesticides are present in the water samples - apparently, something is missing in the text. Misidentification possibly explains the high concentration of aldrin, which has not been used for over 45 years and has long been degraded in the environment. It is also desirable to compare the ratio of HCH isomers and DDT metabolites to each other to confirm the presence of these compounds in water and trace their fate in the ecosystem. Also, apparently, the authors cited an instrumental MDL 3 s./n. It is preferable to use the 7-point US EPA method. Finally, the authors did not explain their performance check (Did they run RSM?)

• Worthy reviewer, GC with MS detector, ECD and FID detector is a modern and sensitive instrument for trace analysis. GC/MS is not available with us, as it is expensive and its expenditure is also more because it only use helium gas instead of Nitrogen etc. We have GC-ECD and FID. The GC-ECD is best because Electron Captured Detector (ECD) is very much sensitive for chlorinated compounds. Because in environmental samples pesticides are present in very small amount, so we can easily detect it by GC-ECD when standards of the compounds are available. The retention time of the standards and retention time of the analyte in the samples is matched. The retention time of compound depends on molecular weight, size, shape, dipole moment, polarity, molecular structure etc. therefore, the retention time of the compounds cannot be the same at same condition of temperature and pressure. The isomers of the same compounds also have the different retention time on same conditions. For examples the isomers of BHC (α-BHC;  β-BHC;  γ-BHC; and δ-BHC) all have different retention time. Aldrin is detected in water samples but in very small amount. The maximum concentration of Aldrin detected was  014 µg L^-1, whis is very small as compared to WHO guide line value for Aldrin  (0.03 µg L^-1).The reason  of presence of Aldrin is that it was smugled to Khyber pakhtunkhwa, Pakistan through Afghanistan from black market up to 10-12 earlier. However, it is stoped from last 10 years. The 4, 4'-DDT and its metabolites 4, 4'-DDE and 4, 4'-DDD were also analyzed.  However, only 4, 4'-DDD was detected in only 5 samples in very small amount. MDL or Lower limits of detection (LOD) (the lowest concentration of an analyte in a sample that can be detected by analytical instrument, but not necessarily quantified, under the stated conditions of the test) were obtained on the basis of signal-to-noise ratio (S/N) of 3:1.

 

• Worthy reviewer, 3/4-4 – OK
• Worthy reviewer, references – OK, but I would prefer links to more recent information about similar studies in Asian countries
• Worthy reviewer, asian countries already have been added in the References. For example, References from China, India and Pakistan already added.

Hu, Y.; Qi, S.H.; Zhang, J.P.; Tan, LZ.; Zhang, J. Q.; Wang YH, et al. Assessment of organochlorine pesticides contamination in underground rivers in Chongqing, Southwest China. J Geochem Explor. 2011, 111, 47–55.

Sankararamakrishnan, N.; Kumar Sharma, A.; Sanghi, R. Organochlorine and organophosphorous pesticide residues in ground water and surface waters of Kanpur, Uttar Pradesh, India. Environ Int. 2005, 31(1), 113-120

Teng, Y., Li J., Wu J., Lu, S., Wang, Y., &  Chen, H., 2015. Environmental distribution and associated human health risk due to trace elements and organic compounds in soil in Jiangxi province, China. Ecotox Environ Safe. 122, 406–416.

Yang, Y., Yun, X., Liu, M., Jiang, Y., Li, Q.X., & Wang, J., 2014. Concentrations, distributions, sources, and risk assessment of organochlorine pesticides in surface water of the East Lake, China. Environ Sci Pollut Res. 21(4), 3041-3050.

Younas, A., Hilber, I., &  Rehman. S., 2013. Former DDT factory in Pakistan revisited for remediation : severe DDT concentrations in soils and plants from within the area. Environ Sci Pollut Res. 20, 1966–1976.

Zhou, Q., Wang, J., Meng, B., Cheng, J., Lin, G., Chen, J., Zheng, D., &  Yu, Y., 2013. Distribution and sources of organochlorine pesticides in agricultural soils from central China. Ecotox  Environ Safe. 93, 163–170.

Liu, W.X., He, W., Qin, N., Kong, X., He, Q., Ouyang, H., &  Xu, F., 2013. The residues, distribution and partition of organochlorine pesticides in the water, suspended solids, and sediments from a large Chinese lake ( Lake Chaohu ) during the high water level period. Environ Sci Pollut Res. 20, 2033-2045.

Round 2

Reviewer 1 Report

without comments

Author Response

Thank you worthy reviewer for accepting our paper

Reviewer 2 Report

I am not 100% satisfied with the authors' response to my comments.

Firstly, the authors did not answer several critical questions regarding QA/QC.

Data on blanks is still not provided. This is a very important indicator when examining trace concentrations of contaminants, especially considering that the authors do not carry out special cleaning of laboratory glassware. Data on surrogate and performance tests are still not provided, as well as drift test information.

I disagree with the statement that comparing the retention times of the standard and the sample completely identifies the chemical compound. PCBs, PBDEs, Toxaphenes, and many other organohalogenated compounds under the same chromatographic conditions have very similar retention times.

The authors point out that it will take 7 days from the moment of sampling to instrumental analysis - how far is the laboratory from the collection point?

Explain the discrepancy in Table 4 and Figure 3. The authors provide a chromatogram of the sample with identified alpha-CD and Endosulfan 2, although the table indicates that these pesticides were not identified in any sample.

Second, the authors did not include the answers to my questions in the revised text.

After answering ALL of my questions and including the answers in the final test, the article could be recommended for publication.

Author Response

-

Author Response File: Author Response.docx

Round 3

Reviewer 2 Report

OK, I see that this time the authors have tried to answer most of my questions. I will note that they have put a lot of effort into carrying out this project. Most of my questions are cleared. And although I have some remarks, I give the green light to the publication of this manuscript.