A refined method for analysis of 4,4′-dicofol and 4,4′-dichlorobenzophenone
The acaricide, dicofol, is a well-known pesticide and partly a substitute for dichlorodiphenyltrichloroethane (DDT). Only few reports on environmental occurrence and concentrations have been reported calling for improvements. Hence, an analytical method was further developed for dicofol and dichlorobenzophenone (DCBP) to enable assessments of their environmental occurrence. Concentrated sulfuric acid was used to remove lipids and to separate dicofol from DCBP. On-column injection was used as an alternative to splitless injection to protect dicofol from thermal decomposition. By the method presented herein, it is possible to quantify dicofol and DCBP in the same samples. Arctic cod (Gadus morhua) were spiked at two dose levels and the recoveries were determined. The mean recovery for dicofol was 65% at the low dose (1 ng) and 77% at the high dose (10 ng). The mean recovery for DCBP was 99% at the low dose (9.2 ng) and 146% at the high dose (46 ng). The method may be further improved by use of another lipid removal method, e.g., gel permeation chromatography. The method implies a step forward in dicofol environmental assessments.
KeywordsKelthane On-column injection α-Cl-DDT Cod Analysis
A number of studies have shown that 1,1-dichloro-2,2-bis(4-chlorophenyl) ethane (4,4′-DDE), the major metabolite of 1,1′-bis(4-chlorophenyl)-2,2,2-trichloroethane (4,4′-DDT), is present at high levels in different environmental media in China (Grung et al. 2015; Yin et al. 2015; Zhang et al. 2013; Zhou et al. 2016a) even though technical DDT was prohibited for agricultural use in 1983 (Yu et al. 2005). Qiu et al. (2005) stated that the use of dicofol in China has become an important source of DDT after the prohibition. This can be explained by the manufacturing process of 4,4′-dicofol being synthesized from DDT. China was the largest consumer of dicofol globally, from 2000 to 2012, with a cumulative usage of 19,500 MT, occupying 69% of the global total amount (Li et al. 2015).
Dicofol (trade name, Kelthane) has been widely used as an organochlorine acaricide that is applied to protect citrus and cotton cultivations from mites (Thiel et al. 2011; Vonier et al. 1996). Dicofol is produced from technical DDT through a pathway including chlorination to form the intermediate 1,1-bis(4-chlorophenyl)-1,2,2,2-tetrachloroethane (α-Cl-DDT), and followed by hydrolysis thereof to form dicofol (Qiu et al. 2005). It is estimated that 93% of 4,4′-DDT is converted to 4,4′-dicofol while only 37% of 2,4′-DDT is converted to 2,4′-dicofol probably due to steric hindrance for the formation of 2,4′- α-Cl-DDT (Qiu et al. 2004). The lower reactivity of 2,4-DDT results in an increased ratio of 4,4′-dicofol/2,4′-dicofol to around 10 (Qiu et al. 2004). Hence, dicofol analyzed in the present study refers to 4,4′-dicofol.
Due to its structural similarity to DDT, dicofol is considered to be of similar concern as DDT and its metabolites DDE and dichlorodiphenyldichloroethane (DDD). These concerns relate to persistence, bioaccumulation, environmental long-range transport, and adverse effect in wildlife and humans (UNEP 2015a). In 2011, the Japanese government rejected the import of Chinese-produced eel due to high levels of dicofol residues, reflecting a severe contamination of this acaricide in China (Wang et al. 2011a).
Dicofol has been listed as a persistent toxic compound in a series of multilateral agreements, e.g., Convention on Long-range Transboundary Air Pollution Protocol on persistent organic pollutants (POPs), and banned in many developed countries (Li et al. 2015). Dicofol has been proposed (2013) as a candidate for POPs in the Stockholm Convention (UNEP 2015a). However, the inclusion of dicofol among the legacy POPs is controversial. For example, there is a lack of evidence of dicofol’s environmental stability. Dicofol is not stable under alkaline condition when it decomposes to dichlorobenzophenone (DCBP) (UNEP 2015a). Dicofol has been demonstrated to easily undergo transformation to DCBP when classical gas chromatography (GC) injection techniques, such as high-temperature split/splitless injectors, are used (Fujii et al. 2011).
The environmental research on dicofol and exposure to dicofol is limited. It is mainly restricted by analytical difficulties. Fujii et al. (2011) used GC coupled to mass spectrometry (MS) in splitless mode to compare dicofol and its related pesticides in breast milk from China, Korea, and Japan assuming dicofol decomposed to DCBP completely. However, according to our experience, dicofol cannot be expected to be quantitatively transformed to DCBP. In addition, it is known that other acaricides, e.g., chlorobenzilate and chloropropylate, may form DCBP (Knowles and Ahmad 1971). Even DDT itself is metabolically transformed to DCBP (Heberer and Dünnbier 1999). Accordingly, dicofol is not the only source of DCBP.
Wiemeyer et al. (2001) used GC with an electron capture detector (ECD) for analysis of dicofol residues in eggs and carcasses of captive American kestrels (Falco sparverius). The authors injected dicofol in a temperature-programmed mode at several concentration levels of the analyte and measured the corresponding DCBP amount being formed. They then corrected for dicofol residues in the samples. However, the decomposition ratios are suspected to vary among single injections. Consequently, the use of classical GC injection techniques is problematic for the analysis of dicofol.
Other pretreatment and instrumental techniques, e.g., reversed-phase high-performance liquid chromatography (Han et al. 2011), spectrophotometry (Pandey et al. 2015), and molecularly imprinted solid-phase extraction (Wang et al. 2011a), have been applied according to the scientific literature.
In the present study, a simple solution for improved analysis of dicofol and DCBP is proposed by using an on-column injection technique. The sample was injected directly onto the GC column at a temperature below the boiling point of the solvent. The advantage with this technique is that little or no thermal degradation occurs compared to what happens when splitless injections are done. The applicability of the method is evaluated by the recovery of dicofol and DCBP in spiked cod samples.
Material and methods
Solvents and chemicals
The solvents were all of the highest quality available on the market. All glassware were heated at 300 °C overnight before being used. Aluminum oxide (90 active basic, 0.063–0.200 mm) was supplied from Merck and heated to 130 °C overnight prior to use. Cod (Gadus morhua) muscle from the North Atlantic was bought from ICA Supermarket, Sweden, for the recovery study.
The recovery study included both dicofol and DCBP. Dicofol was spiked to cod samples at two levels (1 and 10 ng). DCBP, with two levels (9.2 and 46 ng), was spiked to another batch of cod samples. The lower level of each compound is approximately around ten times the limit of quantification whereas the higher level is still in the linear range of ECD. For each compound and concentration level, five replicates were analyzed. In addition to those 20 samples spiked with standards, three cod samples were left unspiked, as control samples. CB-200 and MSF-IS, which have been commonly used in our research group as internal standard (Hovander et al. 2006; Zhou et al. 2016b), were added to the samples as surrogate standards for dicofol and DCBP, respectively. CB-201 was added as volumetric standard prior to analysis for recovery calculation.
Extraction and clean-up
Recovery (%) of 4,4′-dicofol (Difocol), 4,4′- dichlorobenzophenone (DCBP), and their corresponding surrogate standards, 2,2′,3,3′,4,5,6,6′-octachlorobiphenyl (CB-200) and 4′-Me-5′-MeSO2-CB106 (MSF-IS), respectively, in cod samples
Extraction of DCBP
DCBP acts as a Lewis base and partitions into the conc. sulfuric acid in the lipid removal treatment step and needs to be re-extracted from the acid. The acid phase fraction was placed in an ice-bath and diluted with cold distilled water (2 mL) which was subsequently extracted with iso-hexane (3 mL) twice. The combined iso-hexane phase was concentrated to 0.5 mL by a gentle nitrogen flow. The samples were further cleaned up on a basic aluminum oxide column (0.9 g), packed in a Pasteur pipette. The column was preconditioned with n-hexane/DEE (9:1, v/v, 5 mL). The sample was applied to the column and analytes eluted with n-hexane/DEE (9:1, v/v, 12 mL), and thereafter with DEE (10 mL). The solvent volume was reduced by a gentle flow of nitrogen. Prior to instrumental analysis, CB-201 was added as the volumetric standard. The final volume was adjusted to 0.1 mL for GC-ECD analysis.
A Varian 450-GC equipped with a Varian CP-8400 autosampler and a Varian 1079 programmed temperature vaporizing (PTV) inlet was used. To be able to do an on-column injection, a 2-m non-polar methyl deactivated capillary precolumn (J&W Scientific) with an inner diameter of 0.53 mm, big enough to allow a 26-gauge needle tip to enter the bore, is press-fit connected to an injector liner that is tapered in the middle. The liner is positioned in such a way that the needle tip releases the sample inside the precolumn bore. Automated injections of 1 μL were made in splitless mode, and the inlet temperature of the injector was programmed as follows: initial temperature of 55 °C for 2 min, a 200 °C/min ramp to a final temperature of 280 °C which was maintained throughout the analysis; compressed air was used to cool the injector to the starting temperature. The analytical column (TR-5MS, 30 m × 0.25 mm × 0.1 μm, Thermo Scientific) was connected to the precolumn with a zero dead volume adapter. Helium was used as carrier gas at a constant flow of 1.2 mL/min. The temperature program for the oven started at 55 °C for 2 min, a 15 °C/min ramp up to 300 °C, and maintained there for 8 min. An ECD was used for detection and operated at 325 °C with nitrogen as make-up gas (25 mL/min).
One procedural solvent blank was used in each batch of five samples. Three unspiked cod samples (control samples) were analyzed in parallel with the spiked samples. Neither dicofol nor DCBP was detected in any procedural blank or control sample. The replicate samples were mixed and analyzed in different batches to avoid any systematic bias or contamination.
Results and discussion
Summary of dicofol residue in environmental samples reported in the scientific literature
(Singh et al. 2009)
GPC, silica gel column
5.8–64 (9.6) ng/g l.w.
(Fujii et al. 2011)
GPC, silica gel column
0.8–3.0 (1.9) ng/g l.w.
(Fujii et al. 2011)
GPC, silica gel column
<0.1–2.7 (0.32) ng/g l.w.
(Fujii et al., 2011)
Microwave-assisted steam distillation
(Ji et al. 2007)
(Salghi et al. 2012)
Human adipose tissue
2.91 ng/g fat
(Wang et al. 2011b)
(Syed and Malik 2011)
(Lv et al. 2010)
(Malik et al. 2011)
(Malik et al. 2011)
Conc. sulfuric acid is commonly used for lipid removal for POP analysis. However, this step will partition Lewis bases to the conc. sulfuric acid phase, a property first applied in environmental analysis to isolate aryl methyl sulfone compounds, as methylsulfonyl-PCBs (MeSO2-PCBs) from other neutral organohalogen compounds (Jensen and Jansson 1976). Also, dibenzophenone compounds are Lewis bases that are partitioning to the conc. sulfuric acid phase in a partitioning with e.g. hexane. Since the sulfuric acid has been commonly discarded, this can explain why DCBP is rarely detected and reported from actual environmental samples.
Summary of method for dicofol analysis reported elsewhere
Sulfuric acid basic aluminum column
0.01 ng/g l.w.
1 and 10 ng
Molecularly imprinted solid-phase extraction
(Wang et al. 2011a)
Water, milk, tomato, beans, grapes
(Pandey et al. 2015)
139 (250, 141)
(Chen et al. 2009)
(Ribeiro et al. 2000)
Dispersive solid phase extraction (d-SPE)
250, 252, 251, 254
(Zhang et al. 2010)
In the present study, a method composed of liquid extraction, sulfuric acid clean-up and separation, on-column injection, was developed for dicofol and its major decomposition product DCBP. Dicofol does not break down when on-column injection is used. It should be pointed out that the application of the method may not be limited to analysis of dicofol. On-column injection can be used for compounds that are thermo-labile.
The sulfuric acid treatment can separate chemicals containing a ketone group from neutral compounds in addition to removing lipids. However, improvements and/or alternatives are suggested, e.g., use gel permeation chromatography to remove lipids before sulfuric acid, further clean-up for DCBP phase. The method is a step forward in dicofol environmental assessment and particularly promising to be applied to those environmental samples from dicofol hot spot areas.
Sune Eriksson is acknowledged for the valuable discussion on the method development. The work was funded by the Swedish Research Council (No. 639-2013-6913).
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