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7. ANALYTICAL METHODS
The purpose of this chapter is to describe the analytical methods that are available for detecting, measuring, and/or monitoring cobalt, its metabolites, and other biomarkers of exposure and effect to cobalt. The intent is not to provide an exhaustive list of analytical methods. Rather, the intention is to identify well-established methods that are used as the standard methods of analysis. Many of the analytical methods used for environmental samples are the methods approved by federal agencies and organizations such as EPA and the National Institute for Occupational Safety and Health (NIOSH). Other methods presented in this chapter are those that are approved by groups such as the Association of Official Analytical Chemists (AOAC) and the American Public Health Association (APHA). Additionally, analytical methods are included that modify previously used methods to obtain lower detection limits and/or to improve accuracy and precision.
7.1 BIOLOGICAL MATERIALS
Entry of cobalt and its radioisotopes into the human body can be gained through ingestion, inhalation, or penetration through skin. The quantities of cobalt within the body can be assessed through the use of bioassays that are comprised of either in vivo and/or in vitro measurements. In vivo measurements can be obtained through techniques that directly quantitate internally deposited cobalt using, for example, whole body counters. These in vivo measurement techniques are commonly used to measure body burdens of cobalt radioisotopes (i.e., 60 Co), but cannot be used to assess the stable isotope of cobalt (^59 Co). Instead, in vitro measurements provide an estimate of internally deposited cobalt (both the stable and radioactive isotopes), utilizing techniques that measure cobalt in body fluids, feces, or other human samples. Examples of these analytical techniques are given in NRCP Report No. 87 (1987) and are also listed in Tables 7-1 and 7-2.
7.1.1 Internal Cobalt Measurements
In vivo measurement techniques are the most direct and widely used approach for assessing the burden of cobalt radioisotopes within the body. The in vivo measurement of these radioisotopes within the body is
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Table 7-1. Analytical Methods for Determining Stable Cobalt in Biological
Materials
Sample Analytical Sample Percent matrix Preparation method method detection limit recovery Reference Urine Direct injection
Addition of magnesium nitrate and nitric acid matrix modifiers and equal volume dilution of sample with water Sample chelated with dithiocarbamic acid derivative, solvent extracted Sample wet digested with acid and chelated with 2, butanedion dioxide and complex preconcentrated at hanging mercury drop electrode Direct injection
Whole Sample diluted with a blood homogenizer
Sample wet digested with acid and chelated with 2,3-butanedion dioxine and complex preconcentrated at hanging mercury drop electrode Sample acid digested, complexed with thiocyanate and N-phenylcinnamo hydroxamic acid and ex tracted into ethyl acetate Serum Direct injection
GF-AAS with Zeeman back ground correction GF-AAS with Zeeman back ground correction
GF-AAS with Zeeman back ground correction DPCSV
GF-AAS with Zeeman back ground correction GF-AAS with D 2 background correction DPCSV
Colorimetric
GF-AA with Zeeman back ground correction
0.3 μg/L
2.4 μg/L
0.1 μg/L
0.2 μg/L
0.1 μg/L
2 μg/L
0.8 μg/L
0.15 mg/L
0.02 μg/L
101% at Bouman et al. 40μg/L 1986 107.6% at Kimberly et al. 50 μg/L 1987
No data Alexandersson 1988; Ichikawa et al. 1985 No data Heinrick and Angerer 1984
No data Sunderman et al. 1989 No data Heinrick and Angerer 1984 No data Heinrich and Angerer 1984
No data Afeworki and Chandravanshi 1987
No data Sunderman et al. 1989
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Table 7-2. Analytical Methods for Determining Radioactive Cobalt in Biological
Samples
Sample matrix Preparation method
Analytical method
Sample detection limit a
Percent recovery Reference Urine Direct count of sample γ-spectrometry with NaI detector
No data (<MDL) No data Miltenberger et al. 1981 Soft tissue
Sample wet-ashed γ-spectrometry (NaI)
No data No data Baratta et al. 1969 Sample directly counted in detector
γ-spectrometry 5 pCi/g No data Rabon and Johnson 1973 Sample digested in acid, oxidized with HClO 4 , con centrated by precipitation with AMP, purified by resin column, precipitated with hexachloroplatinic acid
-counter 0.1 pCi/g 40–85% Nevissi 1992
Feces Direct count of sample γ-spectrometry No data No data Smith et al. 1972 Blood Red cells separated from plasma and washed
γ-spectrometry with NaI detector
No data No data Smith et al. 1972 a1 Bq=2.7x10-11 (^) Ci=27 pCi
AMP = ammonium molybdophosphate; MDL = minimum detectable level; NaI = sodium iodide
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performed with various radiation detectors and associated electronic devices that are collectively known as whole body counters. These radiation detectors commonly utilize sodium iodide (NaI), hyperpure germanium, and organic liquid scintillation detectors to measure the 1,172 and 1,332 keV gamma rays from the decay of 60 Co. Because of the relatively low attenuation of the high energy gamma rays emitted from 60 Co by most tissues, cobalt radioisotopes can easily be detected and quantified using whole body counting techniques (Lessard et al. 1984; NCRP 1987; Raghavendran et al. 1978; Smith et al. 1972; Sun et al. 1997). Many configurations of the whole body counter and scanning methods have been utilized, ranging from unshielded single-crystal field detectors to shielded, multi-detector scanning detectors (IAEA 1962, 1970, 1972, 1976, 1985; NCRP 1987). Where appropriate, shielding of the room that houses the whole body counter and/or the detector is often used to increase the detection sensitivity of the equipment by minimizing background radiation. Additionally, care must be exercised to insure that external contamination with radioactive cobalt or other gamma-emitting radioisotopes on the clothing or skin of the individual to be scanned has been removed. Also, in vitro measurements of cobalt (see Section 7.1.2) are often used in conjunction with whole body counting when monitoring individuals working with cobalt, especially in conjunction with the assessment of individuals who have experienced accidental exposures to cobalt (Bhat et al. 1973).
Calibration of whole body counters is achieved through the use of tissue-equivalent phantoms. These phantoms are constructed to mimic the shape and density of the anatomical structure using tissue equivalent materials such as water-filled canisters or masonite (Barnaby and Smith 1971; Bhat et al. 1973; Sun et al. 1997). For example, the bottle mannequin absorber (BOMAB) consists of a series of water- filled polyethylene canisters constructed into seated or reclined human forms (Sun et al. 1997). 60 Co standards are measured either as point sources along the phantom or dissolved within the water-filled canisters. Comparisons of the actual counts obtained from the phantom to the known activity of the cobalt standards are used to determine the efficiency of the counting technique and, thus, provide the basis for calibrating the technique. Even so, differences in whole body measurement techniques, calibration methods, and background radiation count calculations between different laboratories can complicate the direct comparisons of body burden measurements and clearance rates for cobalt radioisotopes and should be taken into consideration when comparing data obtained from independent laboratories.
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For the in vitro analysis of cobalt radioisotopes in human samples, the majority of the analytical methods measure the cobalt radioisotopes directly in the samples, without the requirement for an extensive sample preparation procedure, using gamma spectrometry techniques. Of the cobalt radioisotopes that have been detected in the environment (e.g., 57 Co, 58 Co, and 60 Co), 60 Co is the most common. Consequently, most of the analytical methods that will be described in this chapter are those developed for the detection and quantitation of 60 Co in biological (see Table 7-2) and environmental samples (see Table 7-4).
The radiochemical analysis of 60 Co in urine has been used in conjunction with whole body scanning methods to assess acute and long-term body burdens of this isotope. The analysis of 60 Co in urine is the same as that described for a standardized method of analysis of cesium radioisotopes in urine (Gautier 1983). A urine sample of approximately 2 L is collected (either over 24 hours or before and after bedtime) and a 1-L aliquot is transferred to a Marinelli beaker for counting in a gamma-ray spectrometer (Gautier 1983). This simple procedure offers high recoveries of cobalt (98%) and the minimum detection sensitivity (100 pCi/L [3.7 Bq/L]) that is required to evaluate individuals for exposures to radioactive cobalt (Gautier 1983). Direct counting methods are also used for the analysis of cobalt radioisotopes in tissues, feces, and blood (Smith et al. 1972, Table 7-2). However, some of these methods may require sample preparation to reduce volume or increase concentration.
Accuracy of in vivo and in vitro measurements of cobalt is determined through the use of standard, certified solutions or radioactive sources with known concentrations or activities of cobalt. Certified standards for stable cobalt can be obtained through a number of commercial sources. The primary source of certified cobalt radioisotope standards is the National Institute of Standards and Technology (NIST). Gamma ray point sources for 60 Co (SRM 4200, 60,000 Bq [1.6 μCi] and SRM 4207, 300,000 Bq [56 μCi]) and standard solutions of 60 Co (SRM 4233, 600,000 Bq/g [16 μCi/g]) are available from NIST. Also, the determination of accuracy of a method often requires standard reference materials (SRMs). Unfortunately, very few biological SRMs are available. An SRM for cobalt in animal muscle is available from the International Atomic Energy Agency (IAEA), Vienna; an SRM for bovine liver (SRM-1577) is available from NIST (formerly the National Bureau of Standards) (Adeloju et al. 1985; Smith and Carson 1981).
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7.2 ENVIRONMENTAL SAMPLES
There are two common approaches for measuring cobalt in the environment. Cobalt radioisotopes can either be measured directly in the field ( in situ ) using portable survey instruments or samples can be procured from the field and returned to the laboratory for quantitation. However, quantitation of the stable cobalt isotope 59 Co in environmental samples is generally conducted in the laboratory.
7.2.1 Field Measurements of Cobalt
In situ measurement techniques are extremely useful for the rapid characterization of radionuclide contamination in the environment, such as soils, sediments, and vegetation, or when monitoring personnel for exposure to radionuclides. The measurement of gamma-ray-emitting radionuclides, like cobalt, in the environment is conducted with portable survey instruments such as Gieger-Mueller detectors, sodium iodide scintillation detectors, and gamma-ray spectrometers. However, the use of gamma-ray spectrometers in field survey equipment is preferred for measuring cobalt in the field because of its selectivity and sensitivity. The relatively high energy and penetrability of the gamma ray that is emitted during the decay of 60 Co provides an advantage for assessing the level of cobalt both on and below the surface using portable field survey instruments such as the gamma-ray spectrometer. These gamma-ray spectrometers are equipped with a high purity germanium detector that is able to selectively and sensitively differentiate the 1,173 and 1,332 keV gamma rays emitted from 60 Co from the gamma-rays emitted from other radionuclides, for example 40 K or 137 Cs (USNRC 1997). Minimum detectable activities (MDAs) of 0.005 Bq/g (0.05 pCi/g) for 60 Co are routinely achieved using p-type germanium gamma-ray spectrometers with 10-minute counting times (USNRC 1997). However, counting errors can occur where the simultaneous detection of the 1,173 and 1,332 keV gamma rays produces a sum peak at 2,505 keV or a count in the continuum between the individual peaks and the sum peak (APHA 1998; USNRC 1997). These errors can be minimized by changing the geometry of the detector or the distance of the detector from the source of radioactivity. Computational methods have been derived to aid in determining the concentrations and distributions of 60 Co in different soil types and depths (USNRC 1997). The concentrations and distributions of 60 Co that have been derived from the computational analysis of the survey data are often verified by laboratory-based analyses of soil samples procured from the survey area.
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Table 7-3. Analytical Methods for Determining Stable Cobalt in Environmental
Samples
Sample Sample Analytical detection Percent matrix Preparation method method limit recovery Reference Air Weighed filter irradiated INAA 0.17 μg/m 3 No data Haddad and (workplace) in a reactor Zikovsky 1985 Sample filter digested Flame-AAS with 0.4 μg/m^3 98% with 12– NIOSH 1984 by wet acid ashing background 96 μg spiked correction (NIOSH filter method 7027) Sample filter digested ICP-AES (NIOSH 0.5 μg/m^3 95–100% with NIOSH 1984 by wet acid ashing method 7300) 2.5– 1,000 spiked filter Water (low Direct injection GF-AAS with <0.5 μg/L 93–115% at Fishman ionic strength) Zeeman or 8.5–30 μg/L et al. 1986 deuterium back ground correction Lake water Sample complexed with ICP-AES <0.004 μg/L No data Nojiri et al. 8-hydroxyquinoline 1985 absorbed on a column, desorbed and digested with acid Rainwater Sample preconcentrated PIXE 0.08 μg/L No data Hansson onto polystyrene films et al. 1988 by spray-drying Seawater Sample complexed with GF-AAS with 0.0002 μg/L 90% Nakashima 8-hydroxyquinoline Zeeman back et al. 1988 absorbed on a column, ground correction desorbed and digested with acid Water and Direct aspiration of Flame-AAS (EPA 0.05 mg/L 97–98% at 0.2– EPA 1983 waste water sample method 219.1) 5.0 mg/L Direct injection GF-AAS with 1 μg/L No data EPA 1983 background correction (EPA method 219.2) Groundwater Direct aspiration Flame-AAS with 0.05 mg/L 97–98% at 0.2– EPA 1986b or leachate background 5.0 mg/L correction (EPA method 7200) Groundwater Direct injection GF-AAS with 1 μg/L No data EPA 1986b or leachate background correction (EPA method 7201)
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Table 7-3. Analytical Methods for Determining Stable Cobalt in Environmental
Samples
Sample Sample Analytical detection Percent matrix Preparation method method limit recovery Reference Food Sample digested with GF-AAS with 1.88 μg/L in 100–107% at Barbera and acid background dissolved 0.2–0.6 mg/kg Farre 1988 correction extract (leaves, liver) Milled Wheat Wet ashing (HNO 3 ), ET-AAS 20 ng/L approximately González et preconcentration and 100% al. 2000 chelation
AAS = atomic absorption spectrometry; EPA = Environmental Protection Agency; ET-AAS = electrothermal atomic absorption spectrometry; GF-AAS= graphite furnace atomic absorption spectrometry; ICP-AES = inductively coupled plasma-atomic emission spectrometry; INAA = instrumental neutron activation analysis; NIOSH = National Institute for Occupational Safety and Health; PIXE = photon induced x-ray emission
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et al. 1986), electrothermal vaporization with ICP-AES (Malinski et al. 1988) and chemiluminescence with spectrofluorimetry (Jones et al. 1989).
Analytical methods for determining cobalt radioisotopes in the environment are shown in Table 7-4. The analysis of cobalt in air is based on quantifying cobalt within aerosols or particles that become trapped on cellulose (paper) or glass fiber filters after a calibrated amount of air is passed through the filters. Since the cobalt radioisotopes do not occur naturally, but may be released as a result of nuclear weapons testing (which has been discontinued for several years), neutron-activation of specific materials (e.g., cobalt containing alloys used in piping of nuclear reactors), or a severe core damage accident in a nuclear plant, the amounts of these isotopes within the ambient environment are near or below the minimum detectable levels for these isotopes (DOE 1995). However, trace amounts of 60 Co can be detected in air, water, and sediments within or near nuclear weapons or fuel production facilities, nuclear reactors, and nuclear waste storage sites (DOE 1995; Boccolini et al. 1976; USAEC 1973). Analysis of cobalt radioisotopes in air filters, water, sediments, vegetation, and biota can be performed directly using gamma-ray spectrometry, or following some sample preparation (e.g., drying, ashing, or extraction) (Boccolini et al. 1976; Cahill et al. 1972; Cushing 1981; Hiraid et al. 1984; Windham and Phillips 1973).
The detection limits, accuracy, and precision of any analytical methodology are important parameters in determining the appropriateness of a method for quantifying a specific analyte at the desired level of sensitivity within a particular matrix. The Lower Limit of Detection (LLD) has been adopted to refer to the intrinsic detection capability of a measurement procedure (sampling through data reduction and reporting) to aid in determining which method is best suited for the required sample quantitation (USNRC 1984). Several factors influence the LLD, including background, size or concentration of sample, detector sensitivity and recovery of desired analyte during sample isolation and purification, level of interfering contaminants, and, particularly, counting time. Because of these variables, the LLDs between laboratories, utilizing the same or similar measurement procedures, will vary.
The accuracy of a measurement technique in determining the quantity of a particular analyte in environmental samples is greatly dependent on the availability of standard reference materials. Several SRMs for cobalt in environmental samples are also available. Some of these are coal, fly ash, diet, and orchard leaf SRMs available from NIST. The Community Bureau of Reference, European Communities offers SRMs for cobalt in sludges, and an SRM for cobalt in thin polymer films is available from NIST for x-ray fluorescence analysis in aerosol particle samples (Dzubay et al. 1988; Miller-Ihli and Wolf
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1986; Schramel 1989; Smith and Carson 1981; Tinsley et al. 1983). Gamma ray point sources for 60 Co (SRM 4200, 60,000 Bq [1.6 μCi] and SRM 4207, 300,000 Bq [56 μCi]) and standard solutions of 60 Co (SRM 4233, 600,000 Bq/g [16 μCi/g]) are available from NIST.
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 cobalt is available. Where adequate information is not available, ATSDR, in conjunction with the National Toxicology Program (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 cobalt.
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.
7.3.1 Identification of Data Needs
Methods for Determining Biomarkers of Exposure and Effect. Cobalt concentrations in blood or urine can serve as exposure indicator (Alexandersson 1988; Ichikawa et al. 1985; Scansetti et al. 1985). The available analytical methods are capable of determining the levels of cobalt in both the blood and urine of normal and occupationally exposed persons (Table 7-1). For the quantitation of cobalt radioisotopes, whole body counters can be used to assess radioactive cobalt body burdens that have occurred both from acute and chronic exposures to cobalt radioisotopes (Bhat et al. 1973; NCRP 1987). In vitro analytical methods are available for analyzing cobalt radioisotopes in urine, feces, and tissues obtained from normal and occupationally exposed persons (Table 7-2).
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7.3.2 Ongoing Studies
Two studies involving analytical techniques for cobalt was listed in the Federal Research in Progress database (FEDRIP 2002, 2004). N.J. Miller-Ihli and co-workers of the Agricultural Research Service in Beltsville, Maryland are developing single and multielement methods for the determination of trace elements of nutritional and health concern. This work will develop new/improved methods permitting direct analysis of solids by GF-AAS and ICP-MS, as well as methods for the determination of different chemical forms of these elements by coupling capillary zone electrophoresis with inductively coupled plasma mass spectrometry (ICP-MS). This research is supported by the U.S. Department of Agriculture (USDA) Agricultural Research Service. B.T. Jones of Wake Forest University in Winston-Salem, North Carolina along with C. Calloway of Winthrop College, South Carolina, are working to develop a novel, low-cost, portable instrument for the simultaneous determination of trace radioactive elements in nuclear forensic samples. The instrument to be developed is expected to provide analytical figures comparable to ICP-MS, but the instrument is much lower cost and more portable. The specific objectives of the project include determination of the analytical figures of merit for elements including cobalt, cesium, and strontium, and analysis of real samples such as soil, urban dust, water, and agricultural materials.
