Several methods are available for the analysis of chromium in different biological media. Some of the

more recent methods for the determination of chromium are reported in Table 6-1. Several other reviews

on the subject provide a more detailed description of the available analytical methods (EPA 1984a;

Fishbein 1984; IARC 1986a, 1990; Torgrimsen 1982; WHO 1988).

The determination of trace quantities of chromium in biological materials requires special precautionary

measures, from the initial sample collection process to the final analytical manipulations of the samples.

Contaminations including dust contamination or losses of the samples during collection, transportation,

and storage should be avoided. Biological samples collected with stainless steel scalpels, trays, and

utensils are unacceptable for chromium analysis. Similarly, contamination or loss arising from sample

containers should be avoided. Chromium-containing grinding and homogenizing equipment should not

be used for preparation of biological samples. Reagents of the highest purity should be used to avoid

contamination. The possible loss of chromium due to volatilization during wet and dry ashing should be

minimized (EPA 1984a).

The determination of chromium in most biological samples is difficult because of the matrix interference

and the very low concentrations present in these samples. Prior to 1978, numerous erroneous results were

reported for the chromium level in urine using electrothermal atomic absorption spectrometry (EAAS)

because of the inability of conventional atomic absorption spectrometry systems to correct for

CHROMIUM 316

6. ANALYTICAL METHODS

Sample matrix Preparation method

Analytical

method

Sample

detection limit

Percent

recovery Reference

residue complexed with APDC and

extracted with MIBK; evaporated

residue dissolved deposited in

polycarbonate foil

PIXE 0.3 µg/L 87% at 4.5 µg/g Simonoff et al. 1984

Blood, serum Sample after wet digestion converted

to a volatile chelate usually with

fluorinated acetylacetone

GC/ECD 0.03 pg

0.5 pg

1.0 ng

No data Fishbein 1984

Lyophilization, ashed, and dissolved

in 0.1 N HCI

GFAAS 0.005 µg/L 103% at 0.30 µg/L Randall and Gibson 1987

Blood Diluted with 0.1% EDTA and 5%

isopropanol

GFAAS-Zeemaneffect

background

correction

0.09 µg/L No data Dube 1988

0.2 µg/g tissue

114% recovery at

10 µg/sample

NIOSH 1994a

(Method No. 8005)

Erythrocytes Dilution with Triton X100 GFAAS No data No data Lewalter et al. 1985

pyrolytic graphite

tube and Zeeman

background

correction

0.02 µg/L (serum)

0.1 µg/L (urine)

No data Sunderman et al. 1989

Body fluids (milk,

urine, etc.)

Dried sample ashed by oxygen

dilution in 1N HCl

GFAAS with

tungsten iodide or

deuterium arc or

CEWM

background

correction

<0.25 µg/L 91% at 0.55 µg/L Kumpulainen 1984

CHROMIUM 317

6. ANALYTICAL METHODS

Sample matrix Preparation method

Analytical

method

Sample detection

limit Percent recovery Reference

Urine None GFAAS 0.05 µg/L 91% at 0.22 µg/L Randall and Gibson 1987

Urine None GFAAS with

CEWM

background

correction and

WM-AES

0.09 µg/L (CEWMAAS)

0.02 µg/L

(WM-AES)

No data Harnly et al. 1983

Urine No sample preparation other than

addition of yttrium internal standard

ICP-AES 12 µg/L 77% at 13 µg/L Kimberly and Paschal

1985

Urine Sorption onto polydithiocarbonate

resin, ash sorbate in low temperature

oxygen plasma and dissolve in

ICP-AES 0.1 µg/sample 100% recovery at

1 µg/50mL urine

NIOSH 1994b

(Method 8310)

Urine None GFAAS 0.0052 µg/L No data Kiilunen et al. 1987

Urine Sample spiked with standard

chromium (standard addition)

GFAAS 0.03–0.04 µg/L No data Veillon et al. 1982

Urine Diluted with water GFAAS-Zeemaneffect-

background

correction

0.09 µg/kg No data Dube 1988

Milk powder Mixed with water GFAAS 5 µg/kg 134–141% at

17.7 µg/kg

Wagley et al. 1989

Tissue

(Chromium(V))

Injection of sodium dichromate EPR 0.1 mmol/kg No data Liu et al. 1994

AAS=atomic absorption spectrophotometry; APDC=ammonium pyrrolidine dithiocarbonate; CEWM=continuum source echelle monochromator wavelength-modulated;

ECD=electron capture detector; EDTA=ethylenediaminetetraacetic acid; EPR=electron paramagnetic resonance spectroscopy; GC=gas chromatography;

PIXE=proton-induced X-ray emission spectrometry; XRF=X-ray fluorescence analysis; WM-AES=wavelength-modulated atomic emission spectrometry

CHROMIUM 318

6. ANALYTICAL METHODS

the high nonspecific background absorption. Similarly, the reported serum and plasma chromium

concentrations of normal subjects have varied more than 5,000-fold since the early 1950s. The chromium

levels in human serum or plasma as reported in the mid-1980s ranged from 0.01 to 0.3 µg/L, and the daily

urinary excretion rate of chromium in healthy and nonoccupationally exposed humans is <1 µg/day

(Anderson 1987; Harnly et al. 1983; Sunderman et al. 1989; Veillon 1989). The four most frequently

used methods for determining low levels of chromium in biological samples are neutron activation

analysis (NAA), mass spectrometry (MS), graphite spark atomic emission spectrometry (AES), and

graphite furnace atomic absorption spectrometry (GFAAS). Of these four methods, only the GFAAS is

readily available in conventional laboratories, and this method is capable of determining chromium levels

in biological samples when an appropriate background correction method is used (Greenberg and Zeisler

1988; Plantz et al. 1989; Urasa and Nam 1989; Veillon 1989).

The problem of developing accurate data for chromium in biological samples is further complicated by

the lack of Standard Reference Materials (SRM). Only recently have chromium certified materials, such

as brewer's yeast (SRM-1569), bovine liver (SRM-1577), human serum (SRM-909), urine (SRM-2670),

orchard leaves (SRM-1571), spinach leaves (SRM-1570), pine needles (SRM-1575), oyster tissue

(SRM-1566), and tomato leaves (SRM-1573) been issued by the National Institute of Standards and

Technology (formerly the National Bureau of Standards). Because of the lack of SRMs, the less recent

data should be interpreted with caution (EPA 1984a), unless the data are verified by interlaboratory

studies.

Another difficulty with the analytical methods used to detect chromium is the ability of the applied

analytical method to distinguish between chromium(III) and chromium(VI). However, in biological

samples where chromium is generally present as chromium(III), the choice of a particular method is

dictated by several factors including the type of sample, its chromium level, and the scope of the analysis.

These factors, in combination with the desired precision and accuracy and the cost of analysis, should be

considered in selecting a particular analytical method. Although the methods reported in Table 6-1 are

some of the more recent methods, they are not necessarily the ones most commonly used. A comparison

of the various commonly used methods and the methods for the avoidance of contamination during

sampling, sample handling, and analysis are provided by Kumpulainen (1984).

CHROMIUM 319

6. ANALYTICAL METHODS

Analytical methods for determining chromium in environmental samples are reported in Table 6-2.

Chromium may be present in both the trivalent and hexavalent oxidation states in most ambient

environmental and occupational samples, and sometimes the distinction between soluble and insoluble

forms of chromium(VI) is necessary. The quantification of soluble and insoluble chromium is done by

determining chromium concentrations in aqueous filtered and unfiltered samples. However, soluble

chromium(VI) may be reduced to chromium(III) on filtering media, particularly at low concentrations,

reduction (Sawatari 1986). Routine analytical methods are not available that can quantify the

concentration of both chromium(VI) and chromium(III) in air samples when present at a total

(CARB 1990). The three commonly used methods that have the best sensitivity for chromium detection

in air are GFAAS, instrumental neutron activation analysis(INAA), and graphite spark atomic emission

spectrometry (Schroeder et al. 1987). Measurements of low levels of chromium concentrations in water

have been made by specialized methods, such as inductively coupled plasma mass spectrometry

(ICP-MS), capillary column gas chromatography (HRGC) of chelated chromium with electron capture

detection (ECD), and electrothermal vaporization inductively coupled plasma mass spectrometry

(Henshaw et al. 1989; Malinski et al. 1988; Schaller and Neeb 1987). A method using high performance

liquid chromatography interfaced with direct current plasma emission spectrometer has been used for the

determination of chromium(III) and chromium(VI) in water samples (Krull et al. 1983). An alkaline

digestion procedure followed by UV-VIS spectroscopy has been developed which can quantify

chromium(VI) in soil, sediment, and sludge (EPA 1997).

As in the case of biological samples, contamination and chromium loss in environmental samples during

sample collection, storage, and pretreatment should be avoided. Chromium loss from aqueous samples

due to adsorption on storage containers should be avoided by using polyethylene or similar containers and

acidifying the solution to the proper pH. The preferred methods for digestion of environmental samples

have been discussed by Griepink and Toelg (1989).

CHROMIUM 320

6. ANALYTICAL METHODS

Sample matrix Preparation method

Analytical

method

Sample detection

limit Percent recovery Reference

Air (total

chromium)

Air particulate matter

collected on filter is cut out

and irradiated with X-ray

photons

Air (total

chromium)

The collected particulates in

and redissolved in acidified

water

Air (total

chromium)

Particulate matter collected

on cellulose ester filter,

digested with aqua regia

0.5–100 µg

Lo and Arai 1988

Air (total

chromium)

Air particulate collected on

cellulose ester filter, wet

Flame atomic

absorption

0.06 µg/sample 98% at 45–90 µg/sample NIOSH 1994c

(Method 7024)

Air (total

chromium)

Sample collected on

cellulose ester membrane

filter dissolved in acid

mixtures

ICP-AES 1 µg/sample 98% at 2.5 µg/filter NIOSH 1994d

(Method 7300)

Air

(chromium(VI))

Sample collected on sodium

carbonate-impregnated

cellulose filter leached with

sodium bicarbonate

Ion chromatography/

coulometric

89–99% at 100 ng CARB 1990

Air

(chromium(VI))

Sample collected in filters

containing sodium

bicarbonate buffer at 15

L/minute

Ion chromatography/

coulometric

94% Sheehan et al. 1992

CHROMIUM 321

2. HEALTH EFFECTS

Sample matrix Preparation method

Analytical

method

Sample detection

limit Percent recovery Reference

Occupational air

(welding fumes)

The particular matter on filter

chromium(III) oxidized to

chromium(VI) by addition of

solution was acidified with

HCl and reduced to

solution was complexed with

HPLC-UV 10 pg No data Maiti and Desai 1986

Occupational air

(chromium(VI))

Extract with 0.05M

FIA-UV/VIS 0.11 ng >90% Wang 1997a

Welding fumes

(total

chromium(VI))

Air particulate collected on

PVC filter is extracted with

and complexed with diphenyl

carbazide

Spectrophotometry at

540 nm

0.05 µg/sample No data NIOSH 1994e

(Method 7600)

Zatka 1985

Welding fumes

(total

chromium(VI))

Air particulate collected on

PVC filter, extracted with

diphenylcarbazide

Chromatography at

540 nm

Spectrophotometry at

540 nm

3.5 µg/sample No data NIOSH 1994f

(Method 7604)

Simultaneous

determination of

chromium(III) and

chromium(VI) in

water extract from

metal fumes

Sample solution at pH 5

reacted with disodium

ethylenediamine tetraacetic

acid

at 50 °C for 1 hour

HPLC on anion

exchange column

solution and

simultaneous UV and

AAS detection

0.2 ng by UV for

chromium(VI) 2.0 ng

by UV 5.0 ng by AAS

for chromium (IV)

5 ng by AAS for

chromium (III)

95–105% at

0.002–2.0 µg

Suzuki and Serita 1985

Sample matrix Preparation method

Analytical

method

Sample detection

limit Percent recovery Reference

CHROMIUM 322

2. HEALTH EFFECTS

Atomospheric

deposition (snow);

determination in

soluble

(chromium(VI))

and particulate

(chromium(III))

part

The melted snow filtered

through Nucleopore filter; the

and dried by freeze-drier;

this preconcentrated solution

placed in plastic tubes; both

plastic tube and Nuclepore

filter irradiated with protons

PIXE 2 µg/L (soluble

portion)

26 µg/L (snow

particle)

No data Jervis et al. 1983;

Landsberger et al. 1983

Either the above Nucleopore

filter or the preconcentrated

liquid placed in plastic vial is

irradiated by thermal neutron

INAA 5 µg/L (soluble

portion)

115 µg/g (snow

particle)

No data Jervis et al. 1983;

Landsberger et al. 1983

Drinking water,

surface water,

and certain

domestic and

industrial effluents

(dissolved

chromium(VI))

Complex chromium(VI) in

water with APDC at pH 2.4

and extracted with MIBK

Furnace AAS 2.3 µg/L No data EPA 1983

(Method 218.5)

Drinking water,

groundwater and

water effluents

(chromium(VI))

Buffer solution introduced

into ion chrom. Derivitized

with dipenylcarbazide

Ion chromatography

spectrophotometry at

530 mm

0.3 µg/L 100% at 100 µg/L EPA 1996a

(Method 7199)

Waste water and

industrial effluent

for chromium(VI)

only

Buffered sample mixed with

separated by centrifugation

or filtration

DPPA at pH 10–12 30 µg/L 90% at 0.2 mg/L Harzdorf and Janser

1984

Sample matrix Preparation method

Analytical

method

Sample detection

limit Percent recovery Reference

CHROMIUM 323

2. HEALTH EFFECTS

Waste water 1986

(chromium(VI))

Sample mixed with a

masking agent and

cetyltrimethyl-ammonium

bromide solution at pH

4.7–6.6, heated in water bath

Spectrophotometry at

583 nm

Lower than

diphenylcarbazone

method

No data Qi and Zhu 1986

Water (total

chromium)

Calcium nitrate added to

water and chromium is

converted to chromium(III) by

GFAAS or ICP/AES 1.0 µg/L

(GFAAS) 7.0 µg/L

97–101% at 19–77 µg/L EPA 1983, 1986a

(Method 218.2 and

7191)

Industrial wastes,

soils, sludges,

sediments, and

other solid wastes

(total chromium)

Digest with nitric

acid/hydrogen peroxide

ICP-AES 4.7 µg/L 101% at 3.75 mg/L EPA 1996b

(Method 6010)

Oil wastes, oils,

greases, waxes,

crude oil (soluble

chromium)

Dissolve in xylene or methyl

isobutyl ketone

AAS or GFAAS 0.05 mg/L 107% at 15 µg/L EPA 1986b

(Method 7190)

Groundwater,

domestic and

industrial waste

(chromium[VI])

Chromium(VI) is

coprecipitated with lead

sulfate, reduced, and

resolubilized in nitric acid

AAS or GFAAS 0.05 mg/L (AAS)

2.3 µg/L (GFAAS)

93–96% at 40 µg/L EPA 1986c

(Method 7195)

Groundwater-EP

extract, domestic,

and industrial

waste

(chromium[VI])

Chelation with ammonium

pyrrolidine dithiocarbonate

and extraction with methyl

isobutyl ketone

AAS No data 96% at 50µg/L EPA 1983, 1986d

(Method 218.4 and

7197)

Water, waste

water, and EP

extracts

(chromium(VI))

Direct DPPA 10 µg/L 93% at 5 mg/L EPA 1986e

(Method 7198)

Sample matrix Preparation method

Analytical

method

Sample detection

limit Percent recovery Reference

CHROMIUM 324

2. HEALTH EFFECTS

Soil, sediment

and sludges

(chromium(VI))

Alkaline digestion extraction

UV-VIS No data 85–115% EPA 1997

(Method 3060A and

7196A)

DPPA=differential pulse polarographic analysis; EAAS=electrothermal atomic absorption spectrometry; EP=extraction procedure (for toxicity testing); FIA/uv/vis=flow injection analysisultraviolet/

UV=ultraviolet; XRF=X-ray fluorescence analysis

CHROMIUM 325

6. ANALYTICAL METHODS

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 chromium 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 chromium.

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.

chromium in urine (Gylseth et al. 1977; Kilburn et al. 1990; Lindberg and Vesterberg 1983a; McAughey

et al. 1988; Minoia and Cavalleri 1988; Mutti et al. 1985b; Sjogren et al. 1983; Tola et al. 1977), blood

(Kilburn et al. 1990; Lewalter et al. 1985; McAughey et al. 1988; Wiegand et al. 1988), hair (Randall and

Gibson 1987, 1989; Takagi et al. 1986), nails (Takagi et al. 1988) and erythrocytes (Lukanova et al.

1996) to occupational exposure levels. Since chromium is an essential element, levels of chromium

compounds have to be relatively high in humans before they signify an increase due to exposure. Hair

has been useful in determining chronic occupational exposure to chromium in high concentrations

(Randall and Gibson 1989); the usefulness of this method for detecting prior exposures is limited to a

timespan of months (Simpson and Gibson 1992). Analytical methods to detect chromium concentrations

in urine (Randall and Gibson 1987), whole blood (Dube 1988), serum/plasma (Simonoff et al. 1984), and

tissue (Liu et al. 1994) have been reported. Generally, the detection limits are in the subppb to ppb range,

and recoveries are good (>70%). These methods are sensitive enough to measure background levels in

the general population.

Chromium induced DNA-protein complexes may be used as a biomarker of exposure as discussed in

Section 2.12.2. These complexes can be detected by potassium chloride-sodium dodecyl sulfate mediated

CHROMIUM 326

6. ANALYTICAL METHODS

precipitation. These methods have a number of inherent limitations including being tedious and subject

to considerable interindividual and interlaboratory variations (Singh et al. 1998b). Only one study has

attempted to utilize this biomarker, and it was found that volunteers exposed to chromium in drinking

water showed no increase in protein-DNA crosslinking in blood cells (Kuykendall et al. 1996). This

suggests that this procedure may not be sensitive enough for use in environmental monitoring unless an

individual has received a potentially toxic level of exposure. Chromium forms chromium-DNA

complexes inside of cells and these complexes constitute a potential biomarker for the assessment of

environmental or occupational exposure. Recently, a novel method has been described for the sensitive

detection of chromium-DNA adducts using inductively coupled plasma mass spectrometry (Singh et al.

1998b). The detection limits of this method are in the parts per trillion range and allows for the detection

of as few as 2 chromium adducts per 10,000 bases, which coupled with the low DNA sample

requirements, make this method sensitive enough to measure background levels in the population. There

are no data to determine whether there are age-specific biomarkers of exposure or effects or any

interactions with other chemicals that would be specific for children.

concern. Methods have been developed that can determine low levels of total chromium and

chromium(VI) in the air (Barrie and Hoff 1985; CARB 1990; NIOSH 1994c, 1994d; Sheehan et al. 1992).

methods are sufficient to determine background chromium levels in the environment and levels at which

health effects may occur. Chromium can be detected in water at concentrations in the ppb range (EPA

1983, 1996a; Harzdorf and Janser 1984) with recoveries of 90% or greater being reported. Methods are

available that can differentiate chromium(VI) from chromium(III) in water samples (EPA 1986c). A

reliable analytical method for extracting and quantifying chromium, including chromium(VI), from soil

surfaces has also been reported (EPA 1997). Current analytical methods exist that are sufficient for

measuring background levels of chromium in soil (EPA 1996b, 1997) and water (EPA 1983, 1986a,

1996a).

No ongoing studies regarding the determination of different speciated forms of chromium (as opposed to

total chromium content) in biological or some environmental media (e.g., soil, sediment) were found.

CHROMIUM 327