7.1. BIOLOGICAL MATERIALS

Endosulfan, in its pure form, is a crystalline substance consisting of α- and β-isomers in the ratio of approximately 7:3. It is an organochlorine pesticide, and analysis of biological and environmental samples for endosulfan commonly results in the detection of other organochlorine pesticides and polychlorinated biphenyls. These can interfere with the determination of endosulfan unless adequate cleaning and separation techniques are used. Detection of low levels of endosulfan typically involves extraction of samples with organic solvents, a clean-up step to remove lipids and other materials that may interfere with analysis, high-resolution gas chromatography (HRGC) to separate endosulfan from other compounds in the extract, and confirmation of endosulfan by electron capture detector (ECD) or mass spectroscopy (MS). Method blanks and control samples should be used to verify method performance and ensure that the reagents and glassware are not introducing contaminants that might interfere with the determination of endosulfan isomers or endosulfan sulfate.

The method of choice for the determination of α- and β-endosulfan in blood, urine, brain, and adipose tissue is gas chromatography (GC) equipped with an electron capture detector (ECD) (Cerrillo et al. 2005; Fernandez et al. 2007; Guardino et al. 1996). This is because GC/ECD is relatively inexpensive, is simple to operate, and offers a high sensitivity for halogens (Griffith and Blanke 1974). Fernandez et al. (2007) used a GC/MS isotope dilution method for detection of a variety organochlorine pesticides in human milk samples. Detection limits ranged from 0.1 to 3 ng/mL.

A rapid headspace solid-phase microextraction (SPME) based method for the detection of organic pollutants in human serum using GC coupled with electron impact ionization mass spectrometry was described (Flores-Ramirez et al. 2014). SPME is a simple, solvent-free method of extraction for sample preparation. Detection limits for α- and β-endosulfan and endosulfan sulfate were in the ppb range.

Vidal et al. (1998) discuss a GC-tandem mass spectrometry (GC-MS-MS) method using solid-phase extraction (SPE) for the analysis of α- and β-endosulfan in urine.

Cappiello et al. (2014) describe a method using GC coupled to a quadrupole mass spectrometric (qMS) detector using liquid-solid extraction followed by SPE for the analysis of α- and β-endosulfan and endosulfan sulfate in human fetal and newborn tissues. Detection limits ranged from 1.2 to 2.0 ng/g.

Mariani et al. (1995) have used GC in conjunction with negative ion chemical ionization mass spectrometry to determine α- and β-endosulfan in plasma and brain samples with limits of detection reported to be 5 ppb in each matrix. Details of commonly used analytical methods for several types of biological media are presented in Table 7-1.

Lozowicka (2013) describe a method based on matrix solid phase dispersion (MSPD) extraction followed by GC/nitrogen-phosphorus detection (NPD)-ECD for multipesticide residues in honeybees. The use of MSPD and cleanup was reported to be an efficient extraction technique with detection limits for α- and β-endosulfan and endosulfan sulfate of 0.005, 0.006, and 0.005 μg/kg, respectively.

7.2. ENVIRONMENTAL SAMPLES

Reliable analysis of endosulfan residue concentrations in environmental samples usually involves detection of the α- and β-isomers plus endosulfan sulfate (a degradation product of endosulfan). GC/ECD has been the most widely used analytical technique for determining low-ppb to parts-per-trillion (ppt) levels of α- and β-endosulfan and endosulfan sulfate in air, water, waste water, sediment, soil, fish, and various foods (EPA 1992, 1994, 1996a, 1996b, 1996c, 1997d, 1997e, 1997f, 2007; FDA 1994, 1999a, 1999b; Gale et al. 2009; Halsall et al. 1997; Hung et al. 2002; Wania et al. 2003). Both GC and high performance liquid chromatography (HPLC) have been used to separate endosulfan and its major metabolites endosulfan ether, endosulfan sulfate, endosulfan lactone, and endosulfan diol (Kaur et al. 1997).

Table 7-1

Analytical Methods for Determining Endosulfan in Biological Samples.

The most common methods of sampling and measuring endosulfan in the atmosphere involve high-volume air samplers, where air is forced through a collection device. The collection medium is either glass fiber filters (GFFs) or polyurethane foam plugs (PUFs). The samples are then analyzed with GC/MS. This technique can measure endosulfan levels in air at the picogram level (Halsall et al. 1997; Su et al. 2007). The use of passive air samplers with XAD-2 resin filters is also common for measuring endosulfan concentrations in air. These samples are extracted with dichloromethane and methanol and analyzed with GC/ECD. Wania et al. (2003) reported detection limits of 0.15 pg/μL for α-endosulfan and 0.08 pg/μL for β-endosulfan. Gale et al. (2009) used semipermeable membrane devices (SPMDs) with low-density polyethylene tubing filled with triolein to detect endosulfans in indoor air. The samples were analyzed with GC/MS and GC/ECD and reported mean endosulfan concentrations as ng per SMPD.

GC/ECD or a halogen-specific detector (HSD) (Method 8080) is the technique recommended by EPA’s Office of Solid Waste and Emergency Response for determining α- and β-endosulfan and endosulfan sulfate in water and waste water at low-ppb levels (EPA 1994). At these low concentrations, identification of endosulfan residues can be hampered by the presence of a variety of other pesticides. Consequently, sample clean-up on a Florisil^® column is usually required prior to analysis (EPA 1994).

Methods 508, 508.1, and 525.2 (EPA 1997d, 1997e, 1997f) are applicable to drinking water and groundwater and can determine α- and β-endosulfan and endosulfan sulphate at concentrations as low as 7 ppt using liquid solid extraction (LSE) and GC/ECD.

GC/ECD and GC/MS (EPA Method 608) are the methods recommended for determining α-endosulfan, β-endosulfan, and endosulfan sulfate in municipal and industrial discharges (EPA 1996c). Sample clean-up on Florisil^® column and an elemental sulfur removal procedure are used to reduce or eliminate interferences. Sensitivity is in the sub-ppb range. Recoveries and precision are good.

Chary et al. (2012) describe a method for detection of α- and β-endosulfan and endosulfan sulfate in river water and waste water at parts-per-trillion levels using stir-bar-sorptive extraction followed by liquid desorption and GC coupled with triple-quadrupole mass spectrometry (GC/QqQ-MS-MS).

Multiresidue methods for fatty and non-fatty foods (fruits, vegetables, seeds, dairy, eggs, meats) published by FDA (FDA 1994, 1999a, 1999b). Alamgir Zaman Chowdhury et al. (2013) and Andrascikova et al. (2013) also described a multiresidue method for vegetables and oranges, respectively, using GC/MS. Limits of detection are generally in the sub-ppm to ppb range.

Dreher and Podratzki (1988) developed an enzyme immunoassay technique for detecting endosulfan and its degradation products (i.e., endosulfan diol, endosulfan sulfate, endosulfan ether, and endosulfan lactone) in aqueous media. The enzyme immunoassay technique is based on detecting antibodies raised against the diol of endosulfan by immunizing rabbits with an endosulfan-hemocyanin conjugate. Minor problems were encountered with coupling of the detecting enzyme (peroxidase) to the conjugate and with cross-reactivity with the pesticide endrin. Although the enzyme immunoassay technique does not require sample extraction, and it is rapid and inexpensive, it is not yet in common use in environmental residue analysis. A detection limit of 3 μg/endosulfan/L of sample was achieved (Dreher and Podratzki 1988; Frevert et al. 1988). Immunoassays have also been reported for endosulfan (both isomers), endosulfan sulfate, and endosulfan diol in water and soil (Lee et al. 1997a, 1997b) with limits of detection reported to be 0.2 μg/L for water and 20 μg/kg in soil. Details of commonly used analytical methods for various environmental media are presented in Table 7-2.

7.3. ADEQUACY OF THE DATABASE

Section 104(i)(5) of CERCLA, as amended, directs the Administrator of ATSDR (in consultation with the Administrator of EPA and agencies and programs of the Public Health Service) to assess whether adequate information on the health effects of endosulfan is available. Where adequate information is not available, ATSDR, in conjunction with NTP, is required to assure the initiation of a program of research designed to determine the health effects (and techniques for developing methods to determine such health effects) of endosulfan.

The following categories of possible data needs have been identified by a joint team of scientists from ATSDR, NTP, and EPA. They are defined as substance-specific informational needs that if met would reduce the uncertainties of human health assessment. This definition should not be interpreted to mean that all data needs discussed in this section must be filled. In the future, the identified data needs will be evaluated and prioritized, and a substance-specific research agenda will be proposed.

Table 7-2

Analytical Methods for Determining Endosulfan in Environmental Samples.