Dissipation Pattern of Quinalphos in Cauliflower, Tomato and Bean Samples

J Consum Prot Food Saf (2018) 13:63–67 Journal of Consumer Protection and Food Safety https://doi.org/10.1007/s00003-017-1143-8 Journal fu¨ r Verbraucherschutz und Lebensmittelsicherheit

RESEARCH ARTICLE

Dissipation pattern of quinalphos in cauliﬂower, tomato and bean samples

1 1 1 Zerin Sultana Munia • Mohammad Shoeb • M. I. R. Mamun • Nilufar Nahar1

Received: 17 June 2017 / Accepted: 13 November 2017 / Published online: 24 November 2017 Ó Bundesamt fu¨r Verbraucherschutz und Lebensmittelsicherheit (BVL) 2017

Abstract Quinalphos, an organothiophosphate 0.50 lgg-1 in cauliflower, tomato and bean) value chemical, is chiefly used as an insecticide. A field within 6, 4 and 7 days after application, respectively. experiment was conducted at Bangladesh Agricul- tural Research Institute (BARI), to study the Keywords Bean Á Cauliflower Á Dissipation Á dissipation of quinalphos in tomato, cauliflower and Quinalphos Á GC-ECD Á Tomato bean samples when sprayed at its recommended dose and were collected from 0 (2 h after application) 1 Introduction to 15 successive days. To determine the residual value of quinalphos, the samples were extracted in quick, Vegetables are healthy foods that are low in fat, high easy, cheap, effective, rugged, and safe (QuEChERS) in dietary fibres and containing various minerals, method and run through a gas chromatography- vitamin C etc. Today, vegetables are available in electron capture detector (GC-ECD). Higher, middle Bangladesh throughout the year, but especially are and lower concentration calibration curves were plentiful during winter and spring. However, as obtained with linear correlation coefficient (r2)of farmers try to increase yields they frequently use 0.998, 0.995 and 0.993, respectively. Limit of detec- pesticides during vegetable cultivation, sometimes tion (LOD) and limit of quantification (LOQ) were more than the recommended pesticide use in veg- found to be 0.009 and 0.027 lgmL-1, respectively. etables, which is becoming a consumer food safety The percentages of the recovery of quinalphos resi- concern (Nahar et al. 2012, 2016). In Bangladesh, dues in spiked vegetable samples at three different 50–70% of vegetables on the market are contami- levels (0.625, 2.5 and 5.0 lgmL-1) were in the range nated by residues of toxic pesticides (Karanth 2000), of 74–86%. The amount of quinalphos residue in mainly due to harvesting the crops before the safe tomato, bean and cauliflower were in the range of waiting period. -1 0.05–6.3, 0.05–3.1 and 0.06–6.5 lgg and dissipated Quinalphos, C12H15N2O3PS, (O,O-diethyl-O-(quinox- below maximum residue limit (MRL; 0.20, 0.20 and alin-2yl), is an organothiophosphate pesticide, which is widely used to control pests in vegetables (Suvard- han et al. 2005). It is solid at low temperature (melting point 31.5 °C) and is very toxic to mammals -1 Electronic supplementary material The online version of (LD50 = 71 mg kg ) (Anonymous 2011). It is mainly this article (https://doi.org/10.1007/s00003-017-1143-8) contains used for the management of Diamond-Back Moth supplementary material, which is available to authorized users. (DBM) (Plutella xylostella) on vegetables and Tobacco Caterpillar (Manduca sexta) on tobacco. The recom- & Mohammad Shoeb mended dose is 500 g a.i. ha-1 on cabbage [email protected] (Anonymous 2009) and could control 100% DBM on 1 Department of Chemistry, University of Dhaka, Dhaka cauliflower (Ramzan and Singh 1980). It is also a good 1000, Bangladesh 123 64 Z. S. Munia et al. controller of eggplant fruit and shoot borer (Leucin- The extraction process followed the quick, easy, odes orbonalis) (Gupta and Kauntey 2007). However, cheap, effective, rugged, and safe (QuEChERS) using excess of quinalphos can pose human health method of Anastassiades et al. (2003). To the hazards both to farmers and consumers (Banerjee homogenized sample (10 g) in a Teflon test tube ethyl et al. 2006). Due to its high toxicity, it has been acetate (20 mL) was added. The tube was vortexed for banned in many countries. 1 min, after which 6 g MgSO4 and 1.5 g NaCl were Quinalphos is not an approved pesticide in Ban- added and then again vortexed for 1 min. The mix- gladesh. Despite of not being registered, it is ture was then centrifuged for 5 min at 5.000 rpm frequently found in Bangladeshi produce. Therefore, maintaining temperature 20 °C (SIGMA 2-16 Bench- it is necessary to determine the fate of quinalphos in top, 10,000 rpm). Of the supernatant, 10 mL was Bangladeshi crops under the climatic and soil prop- transferred into a round bottom flask and the solvent erties of Bangladesh. Such information is important was reduced to dryness using rotary evaporator for decisions regarding legislation on the use of this (Bu¨chi R-210, Switzerland). n-Hexane (5 mL) was pesticide in Bangladesh, whether it should be regis- added to the dried mass and from there 2 mL solu- tered for use or if stricter actions should be taken to tion was transferred into a screw cap test tube. remove it from the market. The objective of this study For clean-up, 150 mg primary secondary amine was the dissipation pattern of quinalphos on tomato, (PSA) and 750 mg MgSO4 were added and the test bean and cauliflower under the condition of Ban- tube vortexed for about 1 min. It was then cen- gladeshi agriculture, ultimately to collect trifuged for another 5 min. and then the extract was information on the possible safe harvesting period passed through pipette with cotton plugged inside before produce can be admitted onto the local which was washed successively with methanol, ace- market. tone and finally with n-hexane. Quinalphos residues were identified and quantified using a Shimadzu 17A gas chromatograph (GC) 2 Materials and methods equipped with an electron capture detector (ECD). Separation was on an ultra-performance quartz cap- The standards of quinalphos (99% pure) were pur- illary column (HP 5MS, 30 m, 250 lm internal chased from Dr. Ehrenstorfer, Germany. Primary diameter, 0.25 lm film thickness). The injector and secondary amine (PSA) from Supelco USA, was used in detector temperatures were set to 280 and 300 °C, clean-up steps. Analytical grade magnesium sul- respectively, and the column temperature was pro- phate, sodium chloride (anhydrous), ethyl acetate grammed from 165 °C as initial temperature, held for and ultra-pure GC-grade n-hexane were obtained 2 min, raised at 15 °C min-1 to 255 °C, which was from Merck KGaA, Germany. held for 4.0 min. Nitrogen gas was used as carrier Seeds of bean (Phaseolus vulgaris), tomato (Lycop- and make up gas, and the flow rate was 1 mL min-1. ersicon lycopersicum) and cauliflower (Brassica Samples were injected manually in the splitless oleracea var. botrytis) were sown in a nursery bed at (1 min)/split mode. the research fields of the Entomology Division, The recovery experiments of quinalphos in spiked Bangladesh Agricultural Research Institute (BARI), control vegetable samples were carried out in three Gazipur, Bangladesh. Quinalphos, 0.03 percent different concentrations levels, 0.625, 2.5 and aqueous emulsion prepared from Convoy 25 EC was 5.0 lgmL-1 and were extracted and cleaned up fol- sprayed at a recommended dose (1 mL L-1 of water lowing the same procedure as described above. ha-1). Three replicate samples from each bed of three vegetables were collected from day 0 (2 h after spraying) and gradually each day up to 15 days 3 Results and discussion after spraying. Before spraying, control samples (untreated) were harvested. All the samples were The residual level of quinalphos in tomato, bean and immediately brought to the laboratory and kept in a cauliflower was analysed by GC-ECD using external freezer below -20 °C. The collected samples of each standard calibration method. The recovery percent- day were cut into small pieces and then homoge- age of quinalphos at 0.625, 2.5 and 5.0 lgmL-1 nized by kitchen blender; the required amount of fortification levels was 85, 74 and 86%, with relative homogenized samples was transferred into Teflon standard deviation (RSD) of 6.2, 4.2 and 5.5%, tube and then preserved in the freezer until respectively, (Table 1) which is within accept- analysis. able range (Codex 2010). The calibration curves 123 Dissipation pattern of quinalphos in cauliflower, tomato and bean samples 65

Table 1 Linearity range, correlation coefﬁcients (r2), LOD, LOQ, Percent recovery, ± SD and percent RSD of quinalphos Calibration Linearity range Correlation coefﬁcient LOD (lg LOQ Spiking level % ± SD % curve (lgmL-1) (r2) mL1) (lgmL1) (lgmL1) Recovery RSD

Higher conc. 2.5–6.5 0.998 0.009 0.027 0.625 85 5.3 6.2 Middle conc. 1.0–2.5 0.995 2.5 74 3.1 4.2 Lower conc. 0.06–0.13 0.993 5.0 86 4.7 5.5 showed excellent linear coefficients (r2) of 0.998, were below the detection limit after 10, 11, 11 days 0.995 and 0.993 for higher, medium and lower con- cauliflower, tomato and bean, respectively. The centration calibration curves, respectively, and the maximum residue limit (MRL) of quinalphos in linearity ranges were 2.5–6.5, 1.0–2.5 and tomato, bean and cauliflower are 0.20, 0.50, 0.20 lg/ 0.06–0.13 lgmL-1, respectively (Fig. S1). LOD and LOQ g, respectively (EPA 2011). In our study the quinalphos of quinalphos in tomato, bean and cauliflower were residue dissipated to below the MRL within 4, 7 and 0.009 and 0.027 lgmL-1, respectively. The unwanted 6 days after application on/in cauliflower, tomato and interfering compounds with quinalphos were and bean, respectively. examined by comparing the chromatograms of the Among the vegetables grown in Bangladesh, standard, blank sample and fortified sample. There tomato, cauliflower and bean are most popular and were no interference peaks at the retention time of highly valued. It has been published that these veg- quinalphos. etable are attacked by pests and farmers use The quinalphos residues were 6.3 ± 0.35, pesticides quite often, and even every day (Sanghi 3.1 ± 0.18, 0.05 ± 0.07 and 6.5 ± 1.06 to and Tewari 2001). In the current experiment, quinal- 0.06 ± 0.03 lgg-1 in tomato, bean and cauliflower, phos residue was found to be the highest in respectively on day zero samples (2 h after pesticide cauliflower, then in tomato and bean. The dissipation application). Quinalphos dissipated with time and percentage of pesticide residues in/on vegetables de- went to 0.05 ± 0.009, 0.12 ± 0.18, 0.11 ± 0.23 in pends on the climatic conditions, type of application, tomato, bean and cauliflower, respectively on day plant species, dosage, the interval between applica- nine which indicates that the percentage of dissipation and harvest. In case of cauliflower, the pesticide tion in these vegetable samples were 99, 96 and 98% reaches to the target point properly. Cauliflower (Table 2). In the present study, residual quinalphos grows in upper direction and its surface is large, as

Table 2 Amount of quinalphos (Av. ± SD, lgg-1) in tomato, bean and cauliﬂower Day Tomato Bean Cauliﬂower Amount (lgg-1) % of dissipation Amount (lgg-1) % of dissipation Amount (lgg-1) % of dissipation

0 6.3 ± 0.15 3.1 ± 0.18 6.5 ± 1.1 1 4.5 ± 0.19 29 2.0 ± 0.16 35 4.9 ± 0.26 25 2 2.7 ± 0.25 57 1.1 ± 0.03 63 4.8 ± 0.18 27 3 2.0 ± 0.25 67 0.88 ± 0.08 71 3.4 ± 0.11 47 4 1.5 ± 0.20 76 0.59 ± 0.05 80 2.7 ± 0.16 58 5 1.1 ± 0.17 82 0.42 ± 0.02 86 1.8 ± 0.03 72 6 0.19 ± 0.18 94 0.28 ± 0.02 90 1.1 ± 0.03 83 7 0.10 ± 0.06 98 0.21 ± 0.02 93 0.8 ± 0.19 87 9 0.05 ± 0.09 99 0.12 ± 0.18 96 0.11 ± 0.23 98 10 bdl – 0.05 ± 0.07 98 0.06 ± 0.03 99 11 bdl – bdl – bdl – 12 bdl – bdl – bdl – 13 bdl – bdl – bdl – 14 bdl – bdl – bdl – 15 bdl – bdl – bdl – bdl below detection limit 123 66 Z. S. Munia et al.

y = 9.621e-0.58x y = 2.603e-0.36x y = 12.08e-0.47x cauliflower. He reported that for cauliflower, half-life R² = 0.914 R² = 0.993 R² = 0.930 was 2.2 days and the safe waiting period was 8 days t = 1.4 t = 2 1/2 1/2 t1/2 = 1.9 8 which correlates with our finding also. 7 Quinalphos in 6 tomato 5 Quinalphos in 4 Conclusions 4 bean 3 Quinalphos in Quinalphos gradually dissipated from cauliflower, 2 cauliflower tomato and bean following first order kinetics. The 1

Concentration (µg g-1) 0 residual quinalphos were below the MRL within 7, 6 0510and 4 days after ﬁnal application on/in cauliﬂower, Days tomato and bean, respectively.

Fig. 1 Dissipation curves of quinalphos in tomato, bean and Acknowledgements ZSM thanks Dr. Md. Sultan Ahmed, cauliflower Entomoly Division, Bangladesh Agricultural Research Institute (BARI), Gazipur, Bangladesh for experimental fields and Bangabandhu Fellowship on Science and ICT Project, Bangla- well as non-homogeneous. So, the pesticide has desh for fellowship. Authors are grateful to International Science Program (ISP), Uppsala, Sweden and Higher Education much space to accommodate easily. Tomato grows Quality Enhancement Project (HEQEP) for financial supports. hanging down and its skin is smoother, compared to the outer surface of cauliflower. This may be the Compliance with ethical standards reason that the residue was lower in tomato than Conflict of interest There is no conflict of interest associated cauliflower. Due to the structural alignment of bean with this work. in the plant and comparatively harder surface, less accumulation of the pesticide was also observed there. The surface area of cauliflower is larger than References those of tomato and bean, and contains nooks and crevices that might reduce photochemical degrada- Anastassiades M, Lehotay SJ, Stajnbaher D, Schenck FJ (2003) tion of the quinalphos. This may be accounted for Fast and easy multiresidue method employing acetonitrile quinalphos to dissipate to below the detection limit extraction/partitioning and dispersive solid-phase extraction for the determination of pesticide residues in in longer days (11 days) in cauliflower than in tomato produce. J AOAC Int 86:412–431 or bean. Anonymous (2009) In: package of practices for vegeta- The degradation of a component is described by bles crops. Punjab Agricultural University, Ludhiana -kt Anonymous (2011) http://sitem.herts.ac.uk/aeru/footprint/en/ the first order function (Ct = C0xe ). The half-lives Reports/576.htm of the pesticides were obtained by the equation Banerjee K, Upadhyay AK, Adsule P, Pandurang G, Patil SH, -1 t = ln2/k, where Ct is the concentration (lgL )at Oulkar DP (2006) Food Add Contam 23:994 time t (days) after application, C0 is the initial con- Chawla RP, Dhaliwal GS (1979) Dissipation of quinalphos centration (lgL-1) and k is the first order rate residues on cauliflower (Brassica oleracea L. var.capitata). -1 Phytoparasitica 7:23–29 constant (day ) (Zhang et al. 2012). Dissipation Codex Alimentarius Commission (2010) Pesticide residues in curves of quinalphos for tomato, bean and cauli- food. FAO/WHO, Rome flower were made. The half-life of quinalphos for EPA (2011) Environmental Protection Agency. Washington, DC tomato, bean and cauliflower were found to be 1.4, Gupta YC, Kauntey RPS (2007) Efficacy of some insecticides against brinjal shoot and fruit borer, Leucinodes orbonalis 2.0 and 1.9 days, respectively (Fig. 1). Gurminder S. Guenee. Proc Zool Soc India 6(2):31–38 analyzed degradation dynamics of quinalphos on Gurminder S, Singh G, Bhardwaj U, Takkar R, Battu RS, Singh B cabbage under subtropical conditions of Ludhiana, (2011) Degradation dynamics of quinalphos on cabbage India (Gurminder et al. 2011). The initial amount went under subtropical conditions of Ludhiana, Punjab, India. Orbital Electr J Chem 3(2):104 below detection limit after 7th days. A. R Pathan Karanth NGK (2000) Abstract in proceedings of the interna- reported the degradation pattern of quinalphos 25 tional workshop, CIRAD-FAO EC in/on aubergine, locally called brinjal (Solanum Nahar N, Shoeb M, Mamun MIR, Ahmed S, Hasan MM, Kabir A melongena var. esculentum) and soil (Pathan et al. (2012) Studies of dissipation pattern of cypermethrin in tomato. J Bangladesh Chem Soc 25(2):200–203 2012). The half-life was 2.0 days and safe waiting Nahar N, Hossain MM, Uddin-Al-Mahmud MN, Shoeb M, Latifa period was 6 days. Chawla and Dhaliwal (1979) ana- GA, Kabir KH (2016) Dissipation of cypermethrin in bean lyzed the dissipation percentage of quinalphos in and cauliflower. Dhaka Univ J Sci 64(1):89–90

123 Dissipation pattern of quinalphos in cauliﬂower, tomato and bean samples 67

Pathan A, Parihar N, Sharma B (2012) Dissipation study of Suvardhan K, Kumar SK, Chiranjeevi P (2005) Extractive quinalphos (25 EC) in/on brinjal and soil. Environ Contam spectrofluorometric determination of quinalphos using Toxicol 88(6):894 fluorescein in environmental samples. Environ Monit Ramzan M, Singh D (1980) Chemical control of the tobacco Asses 108:217–227 caterpillar, Spodoptera litura Fabr. on cauliflower. J Res Zhang X, Shen Y, Yu XY, Liu XJ (2012) Dissipation of chlorpyrifos Punjab Agric Univ India 17(2):236–239 and residue analysis in rice, soil and water under paddy Sanghi R, Tewari V (2001) Monitoring of pesticide residues in field conditions. Ecotox Environ Safe 78:276–280 fruits and vegetables from Kanpur, India. Bull Environ Contam Toxicol 67(4):587–593

123 Environ Monit Assess (2015) 187:743 DOI 10.1007/s10661-015-4965-9

Analysis of DDT and its metabolites in soil and water samples obtained in the vicinity of a closed-down factory in Bangladesh using various extraction methods

M. N. U. Al Mahmud & Farzana Khalil & Md. Musfiqur Rahman & M. I. R. Mamun & Mohammad Shoeb & A. M. Abd El-Aty & Jong-Hyouk Park & Ho-Chul Shin & Nilufar Nahar & Jae-Han Shim

Received: 13 August 2015 /Accepted: 2 November 2015 # Springer International Publishing Switzerland 2015

Abstract This study was conducted to monitor the same laboratory, at three fortified concentrations (n=4). spread of dichlorodiphenyltrichloroethane (DDT) and DDTs extracted from water samples by liquid-liquid its metabolites (dichlorodiphenyldichloroethylene partitioning and all samples were analyzed by gas chro- (DDE), dichlorodiphenyldichloroethane (DDD)) in soil matography (GC)-electron capture detector (ECD) and and water to regions surrounding a closed DDT factory confirmed by GC/mass spectrometry (GC/MS). Linear- in Bangladesh. This fulfillment was accomplished using ities expressed as determination coefficients (R2)were inter-method and inter-laboratory validation studies. ≥0.995 for matrix-matched calibrations. The recovery DDTs (DDTand its metabolites) from soil samples were rate was in the range of 72–120 and 83–110 %, with extracted using microwave-assisted extraction (MAE), <15 % RSD in soil and water, respectively. The limit of supercritical fluid extraction (SFE), and solvent extrac- quantification (LOQ) was 0.0165 mg kg−1 in soil and tion (SE). Inter-laboratory calibration was assessed by 0.132 μgL−1 in water. Greater quantities of DDTs were SE, and all methods were validated by intra- and inter- extracted from soil using the MAE and SE techniques day accuracy (expressed as recovery %) and precision than with the SFE method. Higher amounts of DDTs (expressed as relative standard deviation (RSD)) in the were discovered in the southern (2.2–936×102 mg kg−1)

M. N. U. Al Mahmud and Farzana Khalil contributed equally to this work.

M. N. U. Al Mahmud : M. M. Rahman : J.

M. N. U. Al Mahmud : F. Khalil : M. I. R. Mamun : M. Shoeb : N. Nahar (*) Department of Chemistry, University of Dhaka, Dhaka 1000, Bangladesh e-mail: [email protected] J.

Samples MAE was performed according to a modified version of the EPA (US Environmental Protection Agency) meth- Samples were collected from regions surrounding the od 3546 (EPA method 3546). A simple microwave oven CCC area in the southern, southwestern, and eastern (700 W), generally utilized in conventional households, directions. These regions are situated in the vicinity of was applied for the extraction process. An open-system the Barabkundu Bazar in the Chittagong district, south- MAE was employed, where the maximum temperature eastern Bangladesh (22.584000° N, 91.685005° E) was determined based on the boiling point of the solvent (Fig. 1). Soil samples were collected at a depth of 0– at atmospheric pressure. Two grams each of the homog- 20 cm by spade from 21 different locations. Three enized soil samples was placed in 1-L round-bottomed samples per location at the point of marked 1-ft triangu- flasks, and 2 mL of 0.2 M NH4Cl solution was added to lar area were mixed together and kept in one plastic zip- these, in order to open up the soil structure (Akerblom

Table 1 Retention times (RT), determination coefficients (R2), limit of detection (LOD), and limit of quantification (LOQ) for DDTs in soil and water samples and a DDT recovery experiment in water

Pesticide Linear range RT Linearity LOD LOQ Accuracy (% recovery)±RSD% (water) (ppm) (min) (R2) Spiking level 4.0 Spiking level 20.0 μgL−1 μgL−1 p,p′-DDE 0.005–0.5 11.062 0.998 0.005 mg kg−1 (soil) 0.0165 mg kg−1 (soil) 83±2.55 98±6.15 μ −1 μ −1 p,p′-DDD 11.584 0.9990.04 gL (water) 0.132 gL (water) 89±2.71 105±6.14 o,p′-DDT 11.634 0.998 86±2.29 102±6.3 p,p′-DDT 12.040 0.995 87±3.68 110±9.31

RSD relative standard deviation) 743 Page 4 of 12 Environ Monit Assess (2015) 187:743

Fig. 1 Sample collection sites a Bangladesh and b CCC, Chittagong. c Map showing sampling points

1995). The flask was agitated by hand for 1 min. A (10 mL), and only 3 mL was purified from all interfering

30-mL mixture of acetone/n-hexane (1:1) was added to substances with 98 % concentrated H2SO4 (3 mL), this mixture, and the sample was refluxed in the micro- vortex-mixed for 1 min, and centrifuged at 4000 rpm wave for 4 min at medium power. The sample was for 5 min. At last, the samples were gradually diluted cooled for a few minutes and filtered through a (dilution factor 2500–1,500,000) and analyzed by GC- Whatman-41 filter paper-lined separation funnel. Dis- ECD. tilled water (100 mL) and saturated NaCl (20 mL) solution were added to the filtrate and agitated by hand for Supercritical fluid extraction (SFE) 1 min. The organic layer was separated and evaporated in a rotary evaporator until 0.5 mL was remained. The The homogenized soil samples (2 g) were placed in concentrated extract was reconstituted in n-hexane high-pressure vessels (10 mL capacity; Jasco, Tokyo, Environ Monit Assess (2015) 187:743 Page 5 of 12 743

Japan). Carbon dioxide with 25 % methanol (modifier) evaporation, re-dissolved in n-hexane (2 mL), purified was used as an extracting solvent. CO2 was passed with H2SO4, and analyzed by GC-ECD. through a CO-965 column placed in an oven, using a PU-980 pump (Jasco). The back pressure was regulated Gas chromatography-electron capture detector using a back-pressure regulator (880-81; Jasco). The (GC-ECD) analysis experimental variables used were as follows: oven temperature, 80 °C; pressure, 280 kg cm−2; dynamic extrac- A gas chromatograph equipped with an electron capture tion time, 30 min; and flow rate, 3.0 mL min−1,as detector (7890A; Agilent Technologies, Palo Alto, CA, adapted from our previous studies (Shim et al. 2003; USA) and an auto injector (7683B; Agilent Technolo- Choi et al. 2006;Kangetal.2006; Kim et al. 2006;Jeon gies) was used for the analysis of all cleaned extracts. et al. 2007). The total extract was transferred to a round- The analysis was conducted in a HP-5MS fused silica bottomed conical flask, and the rest of the methodology capillary column (30 m×250-μm inner diameter and was as above mentioned in MAE with slight variation in 0.25-μm film thickness). Nitrogen was used as the car- dilution factor (1600–1,200,000). rier (flow rate, 2 mL min−1) and make-up gas (flow rate, 60 mL min−1). Oven temperature was programmed to Solvent extraction (SE) 100 °C (1-min holding time) and increased up to 280 °C, at intervals of 15 °C min−1 (3-min holding time). The This extraction process was conducted as per a previ- injector and detector temperatures were set at 270 and ously described method, with minor modifications 290 °C, respectively. All samples (1-μL volume) were (Akerblom 1995). The homogenized soil samples (2 g) injected in a split mode with a 10:1 split ratio. A were dissolved in NH4Cl solution (2 mL, 0.2 M) in Shimadzu 17A gas chromatograph (Shimadzu Corp., 250-mL round-bottomed conical flasks and agitated by Tokyo, Japan) adjusted to the same conditions was used hand for 1 min. A mixture of n-hexane/acetone (1:1, for the analysis of pesticide residues in the base labora- 60 mL) was added and the mixture shaken at 200 rpm tory in Bangladesh. for 1 h. The mixtures were filtered through a Whatman- 41 filter paper-lined separation funnel. Water (100 mL) Confirmation of DDT and its metabolites by gas and saturated NaCl (20 mL) were added to the filtrates, chromatography/mass spectrometry (GC/MS) and the mixtures were manually agitated for 1 min. The organic layers were collected in round-bottomed conical The presence of DDTs in the samples was investigated flasks and preceded as above mentioned in MAE. The using an Agilent 6890 gas chromatograph equipped dilution factor was ranged from 1600–1,200,000 times. with a 7683B auto-sampler and 5973 N mass selective detector (Agilent Technologies). An HP-5MS capillary Water sample preparation by liquid-liquid column (30 m×250-μm inner diameter, 0.25-μmfilm extraction (LLE) thickness) was used for the separation of analytes. Helium (1.0 mL min−1) was used as the carrier gas. Liquid-liquid extraction was conducted as per the stan- The oven temperature was programmed as follows: dard procedure provided by the US EPA (EPA Methods initial temperature of 100 °C for 1 min; increase up to 500 Series). Water samples (250 mL) were placed in 280 °C, at intervals of 15 °C min−1; maintained at 500-mL separation funnels, to which saturated NaCl 280 °C for 3 min; and a post-run of 2 min. The injector (50 mL) solution and dichloromethane (30 mL) were and detector temperatures were set to 270 and 290 °C, added. The funnels were vigorously agitated in a me- respectively. Injection volume used was 2 μL, in chanical shaker for 3 min and allowed to stand until the splitless mode. The samples were ionized using the complete separation of the two phases. The organic electron ionization mode (70 eV) and analyzed using phase was collected in a round-bottomed conical flask quadrupole mass analyzer. The detection was carried out by filtration using a cotton filter coated with 20 g anhy- at the selected ion monitoring (SIM) mode. In this mode, drous sodium sulfate. The aqueous phase was further three characteristic ions (mass to charge ratio (m/z)) separated using dichloromethane (30 mL). The organic including 176.1, 246, and 318 (for p,p′-DDE); m/z= phase collected from this secondary separation was 165.1, 199, and 235 (for p,p′-DDD); and m/z=165.1, collected in the same flask. The extract was dried by 199, and 235 (for DDT) were monitored. 743 Page 6 of 12 Environ Monit Assess (2015) 187:743

Fig. 2 Gas chromatography electron capture detection of a blank 100 ft (30.48 m) south of the factory storage area (sequence of soil sample, b DDT (matrix matched) standards at 0.5 mg L−1, c peak: DDE, 11.062; DDD, 11.584; o,p-DDT, 11.634; and p,p- fortified samples at 0.25 mg kg−1,andd field soil samples obtained DDT, 12.040) Environ Monit Assess (2015) 187:743 Page 7 of 12 743

Table 2 Intra-day and inter-day accuracy and precision in the analysis of DDTs in soil samples by microwave-assisted extraction (MAE), supercritical fluid extraction (SFE), and solvent extraction (SE)

Accuracy Pesticide Spiking conc. MAE SFE SE (mg kg−1) Accuracy Precision Accuracy Precision Accuracy Precision (recovery %) (RSD %) (recovery %) (% RSD) (recovery %) (RSD %)

Intra-day (n=4) p,p′-DDE 0.025 109 4.06 105 4.05 88 1.95 0.05 74 4.07 96 1.98 95 4.04 0.25 92 3.26 75 4.18 94 4.41 p,p′-DDD 0.025 120 1.60 102 9.14 76 10.23 0.05 73 1.78 101 2.82 119 2.79 0.25 116 2.67 101 5.49 116 4.79 p,p′-DDT 0.025 83 6.54 102 4.2 73 3.98 0.05 86 6.38 100 10.27 73 2.71 0.25 114 4.24 119 14.54 109 6.50 Inter-day (n=12, p,p′-DDE 0.025 110 4.15 107 10.60 89 4.11 3 day × four 0.05 73 2.99 96 10.25 95 4.50 replicates) 0.25 79 4.59 79 8.07 96 3.16 p,p′-DDD 0.025 120 2.59 102 10.54 79 8.97 0.05 76 5.08 102 4.55 119 3.01 0.25 102 4.32 96 10.79 119 4.08 p,p′-DDT 0.025 72 3.14 105 7.51 72 3.49 0.05 92 6.77 88 7.71 72 3.31 0.25 116 6.86 120 7.34 112 9.98

Method performance curves were constructed at seven concentrations (0.5, 0.25, 0.125, 0.05, 0.025, 0.01, and 0.005 ppm). A control soil sample, collected from non-cultivated A solvent blank or a soil/water blank (control sam- land located at a hilly area in the vicinity of the Chitta- ples; triplicates) was injected into the system prior to the gong district, was used for the fortification experiments. injection of the extracts, in order to get a smooth base- The three methods described above were conducted, line to ensure the absence of any residual DDT peaks. evaluated, validated, and compared on the basis of re- To determine the LOD, working standard solutions covery experiments. For each method, the intra-day were serially diluted with blank extract to get desired accuracy (expressed as recovery) of p,p′-DDT, p,p′- concentrations. The diluted standard solutions were DDD, and p,p′-DDE was carried out independently at injected one by one, and the limits of detection three fortification levels: 0.025, 0.050, and (LODs) of the tested analytes were determined using 0.250 mg kg−1 (n=4). To evaluate the inter-day accura- S/N of 3 with reference to the background noise obtain- cy, the same fortification levels were tested for three ed for the blank sample, whereas the limits of quantifi- consecutive days (n=4). The recovery for water samples cation (LOQs) were determined with S/N of 10. was carried out in triplicates at two fortification levels (4 and 20 μgL−1) using LC-grade water. The spiked samples (soil and water) were incubated for 4 h and subse- Results and discussion quently extracted and analyzed using the procedures described above. Method validation The DDT residue levels in fortified and incurred samples were quantitatively determined by matrix- Endogenous compounds interfering with the analytes matched calibration curves. Linear DDT calibration were investigated by comparing the chromatograms of 743 ae8o 2EvrnMntAssess Monit Environ 12 of 8 Page

Table 3 Comparison of DDTs obtained from soil samples using different extraction methods, with an associated analysis of inter-method, inter-laboratory, and inter-person calibration

Sample MAE-Korea SFE-Korea SE-Korea Inter-method calibration (Korea) SE-Dhaka Inter-laboratory Inter-person calibration (n=3) calibration (% RSD (% RSD between D1 DDTs DDTs DDTs %RSDbetween % RSD between %RSDbetween DDTs DDTs between K and D1) and D2) (n=6) − − − − − (mg kg 1) (mg kg 1) (mg kg 1) MAE and SFE SFE and SE MAE and SE (mg kg 1) (mg kg 1) (n=6) (×103) (×103) (×103)(K) (n=6) (n=6) (n=6) (×103)(D1) (×103)(D2) First person First person First person First person Second person

20 ft S 65.5 20.5 77.9 58.23 64.95 13.75 66.4 74.8 10.38 7.30 60 ft S 6.6 7.9 10.0 10.71 14.83 23.82 8.7 9.5 7.97 5.83 100 ft S 8.6 1.6 6.9 75.42 68.43 14.10 6.1 6.7 8.87 6.48 200 ft S 0.4 0.2 0.4 47.08 44.35 12.83 0.5 0.6 7.44 10.99 330 ft S 0.4 0.2 0.4 45.22 41.06 9.23 0.5 0.5 8.46 11.16 20 ft Et 2.1 1.8 1.3 14.86 21.90 27.65 1.7 1.8 14.85 8.27 30 ft E 1.4 0.5 2.1 54.60 68.49 20.42 2.1 1.9 1.04 11.08 40 ft E 2.6 0.7 3.2 64.91 73.42 15.97 3.6 4.2 10.56 10.10 50 ft E 217.3 34.7 257.5 79.55 85.08 13.88 239.4 235.7 9.51 4.64 70 ft E 6.1 2.5 4.6 49.03 36.90 14.85 4.2 4.2 6.54 6.30 ft feet, S south, E east, DDTs (p,p′-DDE + p,p′-DDD + p,p′-DDT), K Korea, D Dhaka (2015)187:743 Environ Monit Assess (2015) 187:743 Page 9 of 12 743

Table 4 DDTs extracted from soil/sediment samples by solvent extraction (SE), in Dhaka

Sample p,p′-DDE p,p′-DDD o,p′-DDT p,p′-DDT DDTs (DDE + % Moisture (n=3) (mg kg−1) (mg kg−1) (mg kg−1) (mg kg−1) (mg kg−1) DDD)/ (×102) (×102) (×102) (×102) (×102) p,p′-DDT

20 ft S 66.9±1.9 6.1±0.9 272±5.9 591±10.2 936 0.12 25.24 30 ft S 15.0±2.1 71.0±10.4 29.7±2.3 195±17.5 311 0.44 25.09 50 ft S 10.3±1.9 60.8±4.9 24.9±3.1 191±4.1 287 0.37 24.55 60 ft S 6.2±0.2 0.2±0.01 32.8±1.6 80.2±3.1 119 0.08 24.74 70 ft S 5.0±0.6 21.0±3.8 21.1±1.5 118±17.4 165 0.22 23.98 100 ft S 3.7±0.4 1.2±0.2 15.7±0.5 56.1±1.9 76.6 0.09 24.67 150 ft S 1.2±0.08 3.9±0.5 1.8±0.03 5.8±0.2 12.8 0.75 23.54 200 ft S 0.2±0.02 0.1±0.01 1.4±0.09 4.4±0.1 6.1 0.08 22.97 250 ft S 0.2±0.03 0.4±0.07 0.1±0.004 1.4±0.2 2.1 0.47 23.78 330 ft S 0.2±0.02 0.03±0.001 0.9±0.07 4.5±0.3 5.6 0.06 19.27 20 ft S-W 9.0±1.1 585±16.9 5.4±0.3 1468±92.8 2067 0.40 21.58 30 ft S-W 61.5±4.7 459±31.3 30.4±3.0 563±26.2 1114 0.92 20.14 50 ft S-W 29.0±2.4 23.3±2.1 3.3±0.1 30.7±5.1 86.3 1.70 20.62 20 ft E 0.05±0.001 0.05±0.002 0.7±0.06 16.7±0.1 17.6 0.01 23.60 30 ft E 0.04±0.001 0.04±0.001 1.7±0.1 20.9±0.2 22.7 0.004 19.51 40 ft E 0.2±0.01 2.1±0.08 4.8±0.2 33.8±1.3 41.0 0.07 20.42 50 ft E 81.3±6.5 5.3±0.3 510±39.9 2308±162 2905 0.04 21.64 70 ft E 0.8±0.04 0.6±0.03 6.5±0.2 40.7±1.5 48.7 0.03 20.57 200 ft E 0.04±0.006 0.4±0.06 0.04±0.002 0.7±0.01 1.2 0.63 22.07 250 ft E 0.07±0.001 1.1±0.1 0.2±0.005 3.5±0.2 4.8 0.33 21.79 300 ft E 0.06±0.005 0.2±0.04 0.001±0.0002 0.7±0.08 1.0 0.39 22.22 70 ft S-W (sediment) 5.0±1.1 4.4±0.4 0.3±0.02 6.4±1.1 16.2 1.48 28.94 300 ft S (sediment) 0.9±0.1 0.8±0.1 0.1±0.003 5.06±0.9 6.9 0.33 29.96

DDTs (p,p′-DDE + p,p′-DDD + o,p′-DDT + p,p′-DDT), S south, W west, E east the matrix-matched standard, blank soil/water control field samples (soil, sediment) were also determined sample, and the fortified samples (Fig. 2). No interfering using the matrix-matched calibration curves (Tables 1, peaks were detected during the time of DDT retention. 2, 3, and 4). Matrix-matched calibration curves were prepared for The LODs and LOQs were found to be 0.005 and p,p′-DDE, p,p′-DDD, o,p′-DDT, and p,p′-DDT, by plot- 0.0165 mg kg−1 for p,p′-DDE, p,p′-DDD, o,p′-DDT, and ting the peak area against the concentration of each of p,p′-DDT for all tested methodologies in soil/sediment the standards. The matrix-matched calibration curves samples. On the other hand, the values were 0.04 and were linear to the determination coefficients (R2)≥ 0.132 μgL−1, respectively, in water (Table 1). At vari- 0.995 (Table 1). The residual DDTs in the spiked and ance to our results, Rissato et al. (2006) reported a lower

Table 5 Amount of DDTs in water samples

Sample (n=7) p,p′-DDE p,p′-DDD o,p′-DDT p,p′-DDT DDTs (p,p′-DDE + p,p′-DDD)/ (μgL−1) (μgL−1) (μgL−1) (μgL−1) (μgL−1) p,p′-DDT

70 ft S-W 0.53±0.04 0.43±0.10 BQL 0.98±0.10 1.94 0.98 300 ft S 0.65±0.08 0.83±0.11 BQL 1.54±0.12 3.01 0.96 350 ft S (pond) 0.20±0.07 0.21±0.02 BQL 0.19±0.04 0.59 2.15

DDTs (p,p′-DDE + p,p′-DDD + o,p′-DDT + p,p′-DDT) 743 Page 10 of 12 Environ Monit Assess (2015) 187:743

LOQ (0.00001 mg kg−1) and Kihampa and Mato (2009) in the estimated analysis results of the highly contam- found a lower LOD (0.0002 mg kg−1) for p,p′-DDE, p,p inated soil samples with DDTs. In the same way, inter- ′-DDD, o,p′-DDT, and p,p′-DDT in soil, the findings, person reproducibility (RSD) was established in home which are might be due to the variations in the used laboratory and it was between 4.64 and 11.16 % methods and/or machine. (Table 3). The inter-method calibrations were also Accuracy and precision (for soil recovery) were eval- calculated between MAE and SFE, SFE and SE, and uated by intra- and inter-day analyses. In soil, the accu- MAE and SE (Table 3). Clearly, from the inter-method racy (intra and inter-day), expressed as a percentage of calibration, MAE and SE gave more or less similar recovery, was within the range, 70–120 % (Table 2; result. Codex Alimentarius 1993). Precision (intra-day and inter-day repeatability) was expressed as the relative standard deviation (RSD). The analytical precision of DDTs concentrations within the factory site DDTs extracted from spiked soil matrices, using the MAE, SFE, and SE methods, is presented in Table 2. The soil/sediment surrounding the factory was discov- The lowest RSDs were observed in samples extracted ered to be highly contaminated with DDT and its me- by MAE (1.6–6.86 %), followed by SE (1.95–10.23 %) tabolites (Tables 3 and 4). The DDT concentrations were and SFE (1.98–14.54 %). The recovery percentages decreased with increasing distance from the factory in (accuracy) in water were within the range 83–110 %, the southern direction. However, this trend was not with precision (RSD) <9.5 (Table 1). observed in the eastern direction. DDTs (p,p′-DDE + p,p′-DDD + p,p′-DDT) in CCC soil were quantified as Confirmation of presence of DDTs by gas 0.4×103–217.3×103 mg kg−1 by MAE, 0.2×103– chromatography/mass spectrometry 34.7×103 mg kg−1 by SFE, and 0.4×103–257.5× 103 mg kg−1 by SE in Korea and 0.5×103–239.4× The samples subjected to GC may contain interfering 103 mg kg−1 by SE in Dhaka (Table 3). The amounts compounds that could result in false-positive readings. of individual DDTs quantified by Dhaka laboratory In this respect, mass spectrometry (MS) is a valuable using SE method were 0.04× 102–81.3×102 mg kg-1 technique for the identification and confirmation of the (for p,p′-DDE), 0.03×102 –585×102 mg kg-1 (for p,p′- presence of an analyte in a given sample. p,p′-DDT and DDD), 0.001×102–510×102 mg kg-1 (for o,p′-DDT), o,p′-DDT expressed identical molecular ion peaks and and 0.7×102–2308×102 mg kg-1 (for p,p′-DDT). The fragmented ions were confirmed from their retention moisture content of the soil samples was 19.27–29.96 % times. (Table 4). In water samples, DDTs were found to be 0.59–3.01 μgL−1 (Table 5). Inter-method, inter-laboratory, and inter-person The concentrations of p,p′-DDT were found to be calibration greater than those of o,p′-DDT in soil, sediment, and water (Tables 4 and 5). These clearly indicate that the Soil samples were extracted (n=3) in Chonnam Nation- contamination was carried out by technical-grade DDT, al University, Gwangju, Republic of Korea, using the which primarily consists of 65–80 % p,p′-DDT and 15– three above-mentioned techniques. Inter-laboratory cal- 21 % o,p′-DDT (Tomlin 2000). The highest contamina- ibrations were established only for the SE method, as the tion of soil by DDTs was found up to 290 g kg−1 (29 %) facilities for SFE and MAE methods were not available (Table 4) in 50-ft east samples. In line, Younas et al. in Dhaka laboratory. The inter-laboratory calibration (2013) reported more concentrations of DDTs up to was established via six-replicate analysis of the samples 65 % in surface soil samples at a former DDT- between Korean (three replicates) and Dhaka laborato- producing factory in Amman Garh near Nowshera, ries (three replicates) by single analyst. The reproduc- Khyber Pakhtunkhwa, Pakistan. They also found more ibility referred as Bbetween-laboratory precision^ p,p′-DDT than those of o,p′-DDT and their metabolites (RSD) was found to be 1.04–14.85 % (Table 3)(FDA (p,p′-DDE and p,p′-DDD). In addition, Elfvendahl et al. 2012). The majority of the quantitative results between (2004) reported similar concentrations up to 28 % total the Korean and Dhaka’s lab were satisfactory match DDTs in surface soil samples at a former pesticide stock each others, except 20 ft south, 20 ft east, and 40 ft east site in Tanzania. Environ Monit Assess (2015) 187:743 Page 11 of 12 743

Conclusions De Miguel, E., Llamas, J. F., Chacon, E., Berg, T., Larssen, S., Royset, O., & Vadset, M. (1997). Origin and patterns of distribution of trace elements in street dust: unleaded petrol The three extraction methods produced good validation and urban lead. Atmospheric Environment, 31,2733–2740. results (accuracy and precision) based on fortification Elfvendahl, S., Mihale, M., Kishimba, M. A., & Kylin, H. (2004). with a contact time of 4 h in control soil samples. The Pesticide pollution remains severe after cleanup of stockpile – MAE method was deduced to be the best, based on a of obsolete pesticides at Vikuge, Tanzania. Ambio, 33,504 509. comparison of accuracy and precision. The MAE pro- EPA method 3546, Microwave assisted extraction (2007), http:// cess offers many advantages over SE, such as shorter www.epa.gov/osw/hazard/testmethods/sw846/pdfs/3546. extraction time and lower consumption of solvents. On pdf. the other hand, good inter-laboratory reproducibility EPA Methods 500 Series (http://www.accustandard.com/asi/epa_ was established using the SE technique. However, the downloads.php3#500_series). FDA foods program guidelines for chemical methods, Version 1.0 major drawbacks of the SE process include low sample 2/28/2012, http://www.fda.gov/downloads/ScienceResearch/ concentration due to the use of manual concentration FieldScience/UCM298730.pdf. steps and the large volumes of organic solvents required. Goncalves, C., & Alpendurada, M. F. (2005). Assessment of SFE minimizes the solvent and glassware expenses; pesticide contamination in soil samples from an intensive horticulture area, using ultrasonic extraction and gas chroma- however, the extraction time for this process is very tography–mass spectrometry. Talanta, 65(5), 1179–1189. long. A major drawback of the SFE process is the high Jeon, H. R., Abd El-Aty, A. M., Cho, S. K., Choi, J. H., Kim, K. initial cost of the equipment and the need for careful Y., Park, R. D., & Shim, J. H. (2007). Multiresidue analysis manipulation, in order to achieve good results. of four pesticide residues in water dropwort (Oenanthe We discovered that the surrounding soil or sediment javanica) via pressurized liquid extraction, supercritical fluid extraction, and liquid–liquid extraction and gas chromato- was extremely contaminated with DDTs. These results graphic determination. Journal of Separation Science, 30, will be directed for the attention of the Bangladesh 1953–1963. Chemical Industrial Corporation and Department of Kang, C. A., Kim, M. R., Shen, J. Y., Cho, I. K., Park, B. J., Kim, I. Environment, The Government of Bangladesh. We wish S., & Shim, J. H. (2006). 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Distribution Akerblom, M. (1995). Environmental monitoring of pesticide of heavy metal contents of urban soils in parks of Seville. – residues—guidelines for the SADC region. Uppsala: Chemosphere, 49, 1301 1308. Department of Environmental Assessment, Swedish Mielke, H. W., Gonzales, C. R., Smith, M. K., & Mielke, P. W. University of Agricultural Sciences. (1999). The urban environment and children’s health: soils as Chen, T. B., Zheng, Y.M., Lei, M., Huang, Z. C., Wu, H. T., Chen, an integrator of lead, zinc and cadmium in New Orleans, H., Fan, K. K., Yu, K., Wu, X., & Tian, Q. Z. (2005). Louisiana, USA. Environmental Research, 81,117–129. Assessment of heavy metal pollution in surface soils of urban Nahar, N. (2006). Survey and research on DDT and PCBs in food parks in Beijing, China. Chemosphere, 60,542–551. items and environmental samples, Department of Chen, L., Song, D., Tian, Y., Ding, L., Yu,A., & Zhang, H. (2008). 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