297 HEXACHLOROBENZENE
7. ANALYTICAL METHODS
The purpose of this chapter is to describe the analytical methods that are available for detecting,
measuring, and/or monitoring hexachlorobenzene, its metabolites, and other biomarkers of exposure and
effect to hexachlorobenzene. 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
Methods for the determination of organochlorine compounds such as hexachlorobenzene generally
consist of the following steps: extraction of the analyte from the sample matrix; clean-up to remove
interfering compounds; and analysis (separation and quantitation). The primary method of analysis is gas
chromatography (GC) coupled with electron capture detection (ECD) or mass spectrometry (MS).
Analytical methods have been developed for the determination of hexachlorobenzene in blood or serum,
urine, feces, adipose tissue, and breast milk. A summary of methods is shown in Table 7-1.
Several cautions should be noted. Interferences may result from organics of biological origin that are
extracted from the sample, and from contaminated glassware, solvent, etc. Sample interferences are
usually removed using fractionation and clean-up procedures. Rigorous sample collection and
preparation methods must be followed to prevent contamination of the sample. Good quality control
procedures must be used to identify and remove interferences caused by sample contamination.
Blood (or serum) is a body fluid often utilized to assess human exposure to chlorinated organics,
including hexachlorobenzene. Blood is usually extracted with solvent (Bristol et al. 1982; Burse et al.
1990; EPA 1980b; Langhorst and Nestrick 1979; Mes et al. 1982), and the extract is cleaned up (and
sometimes fractionated) by column chromatography utilizing silica gel (Langhorst and Nestrick 1979),
Florisil (Mes et al. 1982), a combination of columns (Burse et al. 1990), or by solid-phase extraction
(Dmitrovic et al. 2002). Hexachlorobenzene may also be extracted by automated solid-phase extraction
298 HEXACHLOROBENZENE
7. ANALYTICAL METHODS
Table 7-1. Analytical Methods for Determining Hexachlorobenzene in Biological
Materials
Sample
Analytical detection Percent
Sample matrix
Preparation method
method
limit
recovery
Reference
Adipose tissue
Extraction, GPC cleanup,
Capillary GC/MS
12 ng/g
No data
EPA 1986c
Florisil fractionation, optional
additional cleanup
Adipose tissue
Maceration with sodium
GC/ECD
No data
No data
EPA 1980b
sulfate, extraction and back
extraction, Florisil
fractionation
Adipose tissue
Soxhlet extraction, cleanup
Capillary
0.001 μg/g
82
Alawi et al.
on Florisil GC/ECD; 1992
confirmation on
second column
Adipose tissue
Solvent extraction, filtration,
Capillary
0.12 ng/g
86
Mes et al.
Florisil fractionation GC/ECD; 1982
confirmation by
GC/MS
Adipose tissue
SFE with alumina (to remove
Capillary
10 μg/kg
115
Djordjevic et
lipids, purification by column GC/ECD (fatty tissue) al. 1994
chromatography
Breast milk
Separation of fat; column
cap GC/ECD
0.4 ng/g fat
No data
Abraham et al.
cleanup
1994
Breast milk
Acid treatment, elute from
GC/ECD
0.009 mg/kg
91
Stachel et al.
silica gel, concentrate
1989
Breast milk
Solvent extraction,
GC/MS/MS EI
0.068 ng/mL
63.23–
Chen et al.
concentration, SPE
83.07
2014
Blood
Solvent (hexane) extraction,
GC/ECD
No data
No data
EPA 1980b
concentration
Blood
Solvent extraction, cleanup
GC/PID
16 ng/g
79
Langhorst and
on silica gel, concentration
Nestrick 1979
Blood
Homogenization with
Capillary
0.2 ng/g
80
Mes et al.
benzene, filtration, Florisil GC/ECD; 1982
fractionation confirmation by
GC/MS
Blood
Hexane extraction,
GC/ECD;
0.16 ng/g
72
Bristol et al.
concentration confirmation by 1982
GC/MS
Serum
Solvent extraction of
GC/ECD
1 ppb
58–76
Burse et al.
denatured serum, 1990
fractionation on micro-Florisil
column, acid treatment/silica
gel cleanup
Serum
Solvent extraction, cleanup
GC/NICI MS
0.05 ng/mL
~100
Dmitrovic et al.
with solid-phase solvent 2002
extraction cartridges,
concentration
299 HEXACHLOROBENZENE
7. ANALYTICAL METHODS
Table 7-1. Analytical Methods for Determining Hexachlorobenzene in Biological
Materials
Sample matrix
Preparation method
Analytical
method
Sample
detection
limit
Percent
recovery
Reference
Serum
Solid-phase extraction,
cleanup on acid/silica gel
column
GC/MS
0.1–56 pg/g
93
Thomsen et al.
2007
Serum
Rapid headspace solid-phase
GC/MS EI
micro-extraction
0.79
103–104
R. Flores-
Ramírez et al.
2014
Plasma
SPE, cleanup on small
multilayer silica gel columns
HRGC/HRMS
89.1 pg/mL
98.6
Salihovic et al.
2012
Urine
Solvent extraction, cleanup
on silica gel, concentration
GC/PID
4.1 ng/g
84
Langhorst and
Nestrick 1979
Semen
Solvent extraction, cleanup
on Florisil, concentration
Capillary
GC/ECD;
confirmation by
NICI
~0.3 ng/mL
80
Stachel et al.
1989
Feces
Boiling with solvent, cleanup
on alumina
Capillary
GC/ECD
No data
No data
Abraham et al.
1994
ECD = electron capture detector; EI = electron ionization; GC = gas chromatography; GPC = gel permeation
chromatography; HRGC = high resolution gas chromatography; HRMS = high resolution mass spectrometry;
MS = mass spectrometry; NICI = negative ionization chemical ionization; PID = photoionization detector;
SFE = supercritical fluid extraction; SPE = solid-phase extraction
300 HEXACHLOROBENZENE
7. ANALYTICAL METHODS
using a polystyrene-divinylbenzene sorbent, with additional cleanup by column chromatography utilizing
a sulfuric acid-silica column (Thomsen et al. 2007). Analysis is usually by GC/ECD (Bristol et al. 1982;
Burse et al. 1990; EPA 1980b; Mes et al. 1982), although GC/MS (Thomsen et al. 2007), and GC coupled
with photoionization detection (PID) (Langhorst and Nestrick 1979), high resolution gas chromatography
(HRGC)/high resolution mass spectrometry (HRMS) (Salihovic et al. 2012) or MS with negative
chemical ionization (NICI) (Dmitrovic et al. 2002) may be used as well. Confirmation by GC/MS is
recommended (Bristol et al. 1982; Mes et al. 1982). Recovery for all methods is acceptable (≈70–90%)
(Bristol et al. 1982; Burse et al. 1990; Dmitrovic et al. 2002, Langhorst and Nestrick 1979; Mes et al.
1982; Thomsen et al. 2007); precision is also acceptable (≤20% relative standard deviation [RSD])
(Bristol et al. 1982; Burse et al. 1990; Langhorst and Nestrick 1979; Mes et al. 1982; Thomsen et al.
2007). Detection limits are in the low-ppb (ng/g) range (Bristol et al. 1982; Burse et al. 1990; Dmitrovic
et al. 2002; Langhorst and Nestrick 1979; Mes et al. 1982; Thomsen et al. 2007) and the ppt (pg/g) range
(Thomsen et al. 2007).
Adipose tissue is usually solvent extracted (EPA 1980b; Mes et al. 1982), and the hexachlorobenzene is
separated from the extracted fat by Florisil column fractionation (Mes et al. 1982). Analysis is by
GC/ECD (EPA 1980b; Mes et al. 1982). Confirmation by GC/MS (Mes et al. 1982) or a second GC
column is recommended. Recovery is good (82–86%) (Mes et al. 1982); precision is very good (<10%
RSD) (Mes et al. 1982). Solvent extraction followed by gel permeation chromatography (GPC) clean-up
and Florisil column fractionation was utilized for a large adipose tissue monitoring study (EPA 1986c).
Additional clean-up measures may be required if fractions are not clean enough for capillary GC/MS
analysis (EPA 1986c). Supercritical fluid extraction (SFE) and treatment with alumina for lipid removal
have been combined; additional purification was carried out by column chromatography (Djordjevic et al.
1994). Recovery was 115%, precision 10.5% RSD. Detection limits for all methods are in the low-ppb
(ng/g) range (Alawi and Ababneh 1991; Djordjevic et al. 1994; EPA 1986c; Mes et al. 1982).
Few methods are available for monitoring other tissues and fluids. Breast milk has been analyzed with a
combination of fat separation, column clean-up, and capillary GC/ECD (Abraham et al. 1994). Detection
limits were 0.4 ng/g fat; other performance data were not reported. Methods for urine (Langhorst and
Nestrick 1979) and semen (Stachel et al. 1989) have been reported. Both provide good recovery (80–
84%). A method for feces has been reported, and involves boiling with solvent and clean-up on alumina
followed by capillary GC/ECD analysis (Abraham et al. 1994). Performance data were not reported.
301 HEXACHLOROBENZENE
7. ANALYTICAL METHODS
It is well known that ingestion of hexachlorobenzene can produce porphyria (see Section 3.2.2.2).
Urinary porphyrins from humans with porphyria cuntanea tarda (PCT) can be analyzed using thin layer
chromatography (TLC). Separation and estimation of porhyrins are carried out on a TLC plate by
extraction and esterification of porphyrins, 2-dimensional development, and fluorescent scanning (Miura
and Torinuki 1977). Other analysis methods for porphyrins include spectrophotometry. Analysis by this
method is carried out by extraction of porphyrins using an anion exchange column, esterification of
porphyrins, separation by chromatography, and quantification spectrophotometrically (Grinstein 1977).
7.2 ENVIRONMENTAL SAMPLES
Most environmental analyses have been performed using multiresidue methods involving solvent extract
of the analytes from the sample matrix, clean-up to remove interfering compounds, determination by GC
with ECD, and confirmation using an ancillary method such as MS. New methods and technologies are
evolving, and this has resulted in lower detection limits. For example, detection limits are in the low ppb
to ppt range for water matrices and the low ppm to ppb range for food. Analytical methods for the
determination of hexachlorobenzene in environmental samples are given in Table 7-2.
Atmospheric hexachlorobenzene is usually sampled by pulling a volume of air through an adsorbent trap
(EPA 1988b, 1990b; Hippelein et al. 1993; Langhorst and Nestrick 1979). A filter may be included in the
sampling system in order to determine the amount of hexachlorobenzene in particulate (Atlas and Giam
1981; Brorström-Lundén et al. 1994; Farrar et al. 2006; Hippelein et al. 1993). Filters and polyurethane
foam (PUF) adsorbent are Soxhlet extracted (EPA 1990b, 1991; Hippelein et al. 1993); filters and
sorbent-impregnated polyurethane foam (SIP) adsorbent are Soxhlet extracted (Koblizkova et al. 2012);
XAD-2 adsorbent is extracted in a Soxhlet apparatus (Hippelein et al. 1993) or by solvent desorption
(Langhorst and Nestrick 1979); polymer-coated glass (POG) adsorbent is extracted by solvent extraction
(Farrar et al. 2006). Clean-up on adsorbent columns may be utilized (EPA 1988b; Farrar et al. 2006;
Hippelein et al. 1993). A variety of analytical methods are used: GC/ECD (Atlas and Giam 1981; EPA
1991), capillary GC/ECD (Brorström-Lundén et al. 1994; EPA 1990b), GC/PID (Langhorst and Nestrick
1979), and capillary GC/MS (Hippelein et al. 1993; Farrar et al. 2006; Koblizkova et al. 2012).
Confirmation on a second GC column or by GC/MS is recommended (Atlas and Giam 1981; EPA
1990b). Reported recovery is good (82–103%) (EPA 1990b; Farrar et al. 2006; Langhorst and Nestrick
1979); precision is also good (<10–20% RSD) (Farrar et al. 2006; Hippelein et al. 1993). Detection limits
depend upon the amount of air sampled, but may be in the ppb to sub-ppt range (EPA 1990b; Farrar et al.
2006; Hippelein et al. 1993; Langhorst and Nestrick 1979).
302 HEXACHLOROBENZENE
7. ANALYTICAL METHODS
Table 7-2. Analytical Methods for Determining Hexachlorobenzene in
Environmental Materials
Sample
Preparation method
Analytical
method
Sample
detection
limit
Percent
recovery
Reference
Collection on PUF; Soxhlet
extraction; cleanup on
alumina
(EPA Method
TO-10)
GC/ECD
No data
No data
EPA 1988b
≈2,200 m
3
collected on GFF
Capillary GC/MS
and XAD-2; Soxhlet
extraction; cleanup on layered
silica gel; alumina partition
0.18 pg/m
3
(calculated)
No data
Hippelein et
al. 1993
Collection on XAD-2; solvent
desorption
GC/PID
70 ppb
95
Langhorst
and
Nestrick
1979
Collection on PUF; Soxhlet
extraction; concentration
dual column
megabore
GC/ECD or
GC/ECD and
GC/MS
5 ng/m
3
82–103
EPA 1990b
Collection on SIP; Soxhlet
extraction; concentration
GC/MS
No data
No data
Koblizkova
et al. 2012
Collection on POG; solvent
extraction; clean up on silica
gel column
Capillary GC/MS
17 pg/m
3
94
Farrar et al.
2006
Modified collector; solvent
extraction; solvent exchange;
cleanup on silica gel
Capillary GC/ECD
0.4 ng/L
No data
Chan et al.
1994
Solid-phase extraction (disk
or cartridge)
(EPA Method
525.1)
capillary GC/MS
0.111 μg/L
80
EPA 1991
Solvent extraction; solvent
exchange
(EPA Method
508)
capillary
GC/ECD;
confirmation
0.077 μg/L
(estimated)
68–82
EPA
1988c
using second
column
Solvent extraction
(EPA Method
505)
GC/ECD,
confirmation
0.003 μg/L
91–100
EPA 1989b
using second
column
pH adjustment; concentration
(Master Scheme)
0.1 μg/L
on XAD-4; cleanup on silica capillary GC/MS (target)
gel
73
Garrison
and
Pellizzari
1987
303 HEXACHLOROBENZENE
7. ANALYTICAL METHODS
Table 7-2. Analytical Methods for Determining Hexachlorobenzene in
Environmental Materials
Sample
Sample Analytical detection Percent
Preparation method
method
limit
recovery
Reference
Functionalized polysulfone
GC/MS
1.3 ng/L
72.9
Nuhu et al.
membrane extraction 2011
(membrane); solvent
desorption
Solvent extraction; solvent
(National
0.12 μg/L
96
Munch et al.
exchange Pesticide Survey 1990
Method 2)
capillary
GC/ECD,
confirmation
using second
column
Centrifugation; chromic acid
Capillary GC/ECD
No data
97.5
Driscoll et
digestion; extraction
al. 1991
DLLME; phase separation
Capillary GC/ECD
0.0005–
71.1–81.3
Kozani et al.
and river 0.05 μg/L 2007
Add CH
3
OH 5% (v/v) and
GC/ECD
0.08 μg/L
84±2%
Canas and
NaCl 5% (w/v) to sample; (C18 Richter
sorptive extraction by rotation phase); 2012
of C18 extraction disk and 85±3%
Teflon disk (PDMS
phase)
Solvent extraction; solvent
(EPA Method
0.05 μg/L
95
EPA 2012a
industrial exchange; optional cleanup 612)
on Florisil
GC/ECD
pH adjustment; solvent
(EPA Method
1.9 μg/L
79
EPA 1984
industrial extraction; concentration 625)
GC/MS
Solvent extraction
(EPA Method
20 μg/L
Not
EPA 1986a
soil, 8410) applicable
sediments, capillary GC/FTIR
Various extraction; cleanup
(EPA Method
660 μg/kg
72.6 (auto-
EPA 1994
soils, solid methods 8270B) (soil, mated
wastes capillary GC/MS sediment); Soxhlet
10 μg/L extraction)
(groundwater)
Liquid-liquid extraction
HPLC
0.3 μg/L
97
Khan et al.
sediment, 2011
Solvent extraction; liquid-
GC/ECD
No data
98
Waliszewski
liquid partition; cleanup by and
sulfuric acid treatment Szymczyn-
ski 1985
304 HEXACHLOROBENZENE
7. ANALYTICAL METHODS
Table 7-2. Analytical Methods for Determining Hexachlorobenzene in
Environmental Materials
Sample
Sample Analytical detection Percent
Preparation method
method
limit
recovery
Reference
Soxhlet and sonication
dual column
No data
83–106
Ojala 1993
extraction; acetylation; capillary GC/ECD
solvent extraction;
fractionation on silica gel
Homogenization with solvent;
GC
No data
27–115
de Andrea
microwave-assisted solvent et al. 2001
extraction
Microwave extraction
Capillary GC/ECD
No data
91.7
Onuska and
centrifugation; filtration
Terry 1993
Grind with sodium sulfate;
GC/ECD
No data
No data
Oliver and
extract with hexane/acetone
Nicol 1982b
Homogenization; Soxhlet
Capillary GC/MS
12.5 ng/g
96
Tiernan et
extraction; GPC fractionation; al. 1990
silica gel fractionation
Maceration; Soxhlet
Dual capillary
5 ng/g (lipid
95
Rahman et
extraction; cleanup with GC/ECD basis) al. 1993
sulfuric acid/silica gel
Solvent extraction; column
GC/IDMS
3.7 ng/g
90–110
Majoros et
clean-up
al. 2013
Homogenization with solvent;
Capillary
0.01 mg/kg
~94
Miskiewicz
biota solvent exchange; cleanup on GC/ECD, and Gibbs
Florisil confirmation on 1994
second column
Homogenization; Soxhlet
(USGS method)
No data
50–75
Shan et al.
organisms extraction; GPC fractionation; capillary GC/ECD 1994
SPE fractionation; solvent
exchange
Isolation on Florisil column;
GC/ECD
No data
95–98
Bong 1975
solvent partition; partition on (fish), 99–
Florisil 104
(butterfat)
SFE/SFC (on-line cleanup)
Capillary GC/ECD
4 ppb
85
Nam and
King 1994
Extraction and pretreatment;
(DFG Method S9)
0.01 mg/kg
90
Thier and
Florisil cleanup GC/ECD; Zeumer
confirmation by 1987b
TLC
Extraction; silica, alumina,
GC/HRMS
1.86 pg/g fat
No data
Kim et al.
and carbon column cleanup
2013
Solid-phase extraction
GC/ECD
No data
88–94
Manes et al.
1993
305 HEXACHLOROBENZENE
7. ANALYTICAL METHODS
Table 7-2. Analytical Methods for Determining Hexachlorobenzene in
Environmental Materials
Sample
Preparation method
Analytical
method
Sample
detection
limit
Percent
recovery
Reference
Solvent extraction; solvent
partition; solvent exchange;
GPC cleanup; optional
alumina cleanup
GC/ECD,
confirmation on
second column
<0.5 ppb
88–91
Trotter and
Dickerson
1993
Sandwich-type extraction
fractionation
GC/ECD
1–2 ppb
80–100
Seidel and
Linder 1993
vegetables
Chop and blend; blend with
solvent; partition with water;
dry
GC/ECD,
confirmation by
GC/MS
0.002 ppm
93
Pylypiw
1993
foods
Solvent extraction; GPC
cleanup; optional silica gel
cleanup
(DFG Method
S19)
dual GC/ECD
No data
>70
Thier and
Zeumer
1987a
Solvent extraction; SPE
GC/MS/MS
5 μg/kg
72–95
Chen et al.
2014
Dry and mince;
homogenization; Soxhlet
extraction; sulfuric acid
Capillary GC/ECD
0.1 ng/g (dry
weight)
80–100
Calamari et
al. 1994
cleanup; fractionation on
Florisil
DLLME = dispersive liquid-liquid microextraction; ECD = electron capture detector; EPA = Environmental Protection
Agency; FTIR = Fourier transform infrared spectrometry; GC = gas chromatography; GFF = glass fiber filter; GPC =
gel permeation chromatography; HPLC = high performance liquid chromatography; HRMS = high resolution mass
spectrometry; IDMS = isotope dilution mass spectrometry; MS = mass spectrometry; PDMS = polydimethylsiloxane;
PID = photoionization detector; POG = polymer-coated glass; PUF = polyurethane foam; SFC = supercritical fluid
chromatography; SFE = supercritical fluid extraction; SIP = sorbent-impregnated polyurethane foam; SPE = solid
phase extraction; TLC = thin-layer chromatography; USGS = U.S. Geological Survey
306 HEXACHLOROBENZENE
7. ANALYTICAL METHODS
Hexachlorobenzene is usually extracted from water with organic solvents for analysis (EPA 1988c,
1989b; Kozani et al. 2007; Munch et al. 1990). Hexachlorobenzene may also be extracted and
concentrated by adsorption on adsorbent cartridges, disks, or membranes, with subsequent solvent
desorption (EPA 1988a; Nuhu et al. 2011). Clean-up of the extracts is usually not necessary; however,
methods are available for samples that contain interfering compounds (Chan et al. 1994; Driscoll et al.
1991; Garrison and Pellizzari 1987). Analysis is usually by capillary GC/ECD (Chan et al. 1994; Driscoll
et al. 1991; EPA 1988c; Kozani et al. 2007; Munch et al. 1990). Confirmation using a second method is
recommended (EPA 1988c, 1989b; Munch et al. 1990). Capillary GC/MS and GC/MS are also utilized
for analysis (EPA 1991; Garrison and Pellizzari 1987; Nuhu et al. 2011). Accuracy ranges from
acceptable (≈60–80%) (EPA 1988a; Garrison and Pellizzari 1987; Kozani et al. 2007; Nuhu et al. 2011)
to excellent (>90%) (Driscoll et al. 1991; EPA 1989b, 1991; Munch et al. 1990). Precision is rarely
reported; 16% RSD was reported for the Master Scheme (Garrison and Pellizzari 1987), 0.52–2.8% RSD
was reported for dispersive liquid-liquid microextraction (DLLME) (Kozani et al. 2007), and 9.2% RSD
was reported for functionalized polysulfone membrane extraction (Nuhu et al. 2011). Detection limits are
in the low- to sub-ppb range (EPA 1991; Garrison and Pellizzari 1987; Kozani et al. 2007; Munch et al.
1990). Detection limits in the ppt range have been achieved by methods utilizing solvent extraction and
functionalized polysulfone membrane extraction with capillary GC/ECD and GC/MS analysis,
respectively (Chan et al. 1994; EPA 1989b; Nuhu et al. 2011). Waste water is solvent extracted with
analysis by GC/ECD (EPA 2012a) or GC/MS (EPA 1989b). Reported recovery is good (79–95%) (EPA
1984, 2012a). Detection limits are in the low-ppb range, with lower detection limits reported for the
GC/ECD analysis (EPA 2012a).
Soxhlet or sonication extraction is most commonly used to extract hexachlorobenzene from solid matrices
such as soils and sediments, and wastes (EPA 2012a; Ojala 1993). Solvent extraction (Waliszewski and
Szymczynski 1985) and microwave extraction techniques (de Andrea et al. 2001; Onuska and Terry
1993) may be used as well. Clean-up is usually required for the extracts (EPA 1994; Ojala 1993;
Waliszewski and Szymczynski 1985), with subsequent analysis by GC (de Andrea et al. 2001), GC/ECD
(Waliszewski and Szymczynski 1985), capillary GC/ECD (Ojala 1993; Onuska and Terry 1993), or
capillary GC/MS (EPA 1994). Reported recovery is good (73–106%) (de Andrea et al. 2001; EPA 1994;
Ojala 1993; Onuska and Terry 1993; Waliszewski and Szymczynski 1985). Precision, where reported, is
acceptable (≤20% RSD) (de Andrea et al. 2001; EPA 1984, 2012a; Ojala 1993). Little information is
available on detection limits. Detection limits of 660 μg/kg (ppb) have been reported for automated
Soxhlet extraction with capillary GC/MS analysis (EPA 1994). A high performance liquid
chromatography (HPLC) based method for the determination of hexachlorobenzene and its possible
307 HEXACHLOROBENZENE
7. ANALYTICAL METHODS
metabolites, including chlorophenolic and chloroquinolic intermediates, by liquid-liquid extraction has
been developed (Khan et al. 2011). Sample cleanup or derivatization is not needed. Analysis is done by
HPLC with a reported recovery of 97%. Precision is acceptable (2.1% RSD). A detection limit of
0.3 μg/L is reported (Khan et al. 2011).
Fish and aquatic organisms are homogenized, then extracted with solvent (Miskiewicz and Gibbs 1994;
Oliver and Nichol 1982b), isolated on Florisil columns (Bong 1975), or Soxhlet extracted (Rahman et al.
1993; Shan et al. 1994; Tiernan et al. 1990). Clean-up is usually necessary to remove lipids and
interfering substances (Bong 1975; Miskiewicz and Gibbs 1994; Rahman et al. 1993; Shan et al. 1994;
Tiernan et al. 1990). Capillary GC/ECD analysis is used most often (Miskiewicz and Gibbs 1994;
Rahman et al. 1993; Shan et al. 1994). Capillary GC/MS (Tiernan et al. 1990) and GC/ECD (Bong 1975;
Oliver and Nichol 1982b) are also utilized. Reported recovery ranges from moderate (50–75%) (Shan et
al. 1994) to excellent (>90%) (Bong 1975; Miskiewicz and Gibbs 1994; Rahman et al. 1993; Tiernan et
al. 1990). Precision is usually not reported; however, 4–6% RSD has been achieved (Shan et al. 1994).
Detection limits, where reported, are in the low-ppb range (ng/g) (Miskiewicz and Gibbs 1994; Rahman
et al. 1993; Tiernan et al. 1990).
Fatty foods, including milk, have been extracted with solvent to remove the fat, and then cleaned up to
separate the hexachlorobenzene from the fat (Bong 1975; Thier and Zeumer 1987b; Trotter and Dickerson
1993). Newer methods for combined separation and clean-up are supercritical fluid techniques (Nam and
King 1994), solid-phase extraction (SPE) (Manes et al. 1993), and a sandwich system (Seidel and Linder
1994). Analysis is by GC/ECD (Bong 1975; Manes et al. 1993; Seidel and Linder 1993; Thier and
Zeumer 1987b; Trotter and Dickerson 1993). Confirmation on a second GC column (Trotter and
Dickerson 1993) or by TLC (Thier and Zeumer 1987b) is recommended. Capillary GC/ECD has also
been utilized (Nam and King 1994). Reported recoveries are good (>80%) (Bong 1975; Manes et al.
1993; Nam and King 1994; Seidel and Linder 1993; Trotter and Dickerson 1993). Precision, where
reported, is very good (<15% RSD) (Bong 1975; Nam and King 1994; Thier and Zeumer 1987b; Trotter
and Dickerson 1993). Limit of detection, where reported, is in the low-ppb (ng/g) range (Nam and King
1994; Seidel and Linder 1993; Thier and Zeumer 1987b; Trotter and Dickerson 1993).
Fruits, vegetables, and crops are blended, solvent extracted, and then cleaned up and fractionated
(Pylypiw 1993; Thier and Zeumer 1987a). Capillary GC/ECD is the analytical method. Recovery is
acceptable (>70%) (Pylypiw 1993; Thier and Zeumer 1987a). Precision was not reported. The reported
detection limit is 2 ppb (Pylypiw 1993).
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7. ANALYTICAL METHODS
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 hexachlorobenzene 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 hexachlorobenzene.
7.3.1 Identification of Data Needs
Methods for Determining Biomarkers of Exposure and Effect. Methods exist for measuring
hexachlorobenzene in blood (Bristol et al. 1982; Burse et al. 1990; Langhorst and Nestrick 1979; Mes et
al. 1982) and adipose tissue (Alawi and Ababneh 1991; Djordjevic et al. 1994; EPA 1980b, 1986c; Mes et
al. 1982). The methods for blood and adipose are sensitive (low-ppb range), but improved accuracy is
needed for blood analysis. The data on determination of hexachlorobenzene in urine, breast milk, and
tissues are limited, and the methods may not be sufficiently sensitive. Methods that could be used to
measure low levels in human tissues would be useful for determining the relationship between chronic
low-level exposure and the effects observed in specific tissues. Improved methods to detect phenolic
metabolites are not needed since these metabolites are not unique to hexachlorobenzene. Representative
methods for determining pentachlorophenol and other phenolic metabolites using GC/ECD and GC/MS
are shown in Table 7-3.
T
he 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.
Biomarkers for effects of hexachlorobenzene are porphyric symptoms and increased gamma-glutamic
transferase activity. Since these effects are also indicative of exposure to other toxicants, additional
studies are needed for more specific biomarkers for effects of hexachlorobenzene exposure.
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7. ANALYTICAL METHODS
Table 7-3. Analytical Methods for Determining Biomarkers of Hexachlorobenzene
Sample matrix
Preparation
method
Analytical
method
Sample
detection
limit
Percent
recovery
Reference
Blood
(pentachloro-
phenol)
pH adjustment,
solvent extraction,
derivatization
GC/ECD
10 ppb
92
EPA 1980b
Urine (chlorinated
phenol
metabolites)
Hydrolysis; solvent
extraction,
derivatization
GC/ECD,
confirmation
by GC/MS
No data
>90 (PCP); most
other metabolites
>80
EPA 1980b
ECD = electron capture detector; GC = gas chromatography; MS = mass spectrometry; PCP = pentachlorophenol
310 HEXACHLOROBENZENE
7. ANALYTICAL METHODS
Methods for Determining Parent Compounds and Degradation Products in Environmental
Media. Methods for determining hexachlorobenzene in air (EPA 1988b, 1990b; Hippelein et al. 1993;
Langhorst and Nestrick 1979) and water (Chan et al. 1994; EPA 1988c, 1989b, 1991; Garrison and
Pellizzari 1987), the media of most concern for human exposure, are reliable, but may not be sensitive
enough to measure background levels in the environment. Limited performance data are available for
methods for soil and other solid media. In addition, there is insufficient performance information for
methods for determining hexachlorobenzene in media such as shellfish, fish, and plants. Some exposure
to hexachlorobenzene may occur via ingestion of food and standardized methods for foods are needed.
Methods with sufficient sensitivity for measuring background levels in foods would be helpful as well.
7.
3.2 Ongoing Studies
No ongoing studies regarding analytical methods sponsored by NIH or EPA were identified for
hexachlorobenzene.