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JOURNAL OF ANALYTICAL ATOMIC SPECTROMETRY, NOVEMBER 1994, VOL. 9 1269

Determination of Total Chromium and Chromium(vi) in Animal Feeds

by Electrothermal Atomic Absorption Spectrometry
M. Elisa Soares, M. Lourdes Bastos and Margarida A. Ferreira
laboratory of Toxicology and Laboratory of Bromatology, Faculty of Pharmacy, University of Oporto,
R. Anibal Cunha, 164, 4000 Oporto, Portugal
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An electrothermal atomic absorption spectrometric method was developed for the determination of total and
hexavalent chromium in animal feeds. For measuring total chromium the samples were digested in a mixture
of three acids (HN0,-HCI-HF). Chromium in its hexavalent state was dissolved in 0.01 mol I-' NaOH solution.
A mixture of Pd+Mg was used as a chemical modifier. Extensive validation of the proposed method was
carried out both by the standard additions method and by analysis of National Institute of Standards and
Technology Standard Reference Material 1548 Total Diet. The detection limits were 0.53 and 0.42 pg I-' for
total and hexavalent chromium, respectively. Measurements can be made over a linear range between 0.53
and 50 and 0.42 and 50 pg I-' for total and hexavalent chromium, respectively. The relative standard
deviations were 4.5 and 7.0% for total and hexavalent chromium, respectively, hence the proposed method
is satisfactory for routine analyses. In 70 samples that were analysed the mean levels found were 1.93 pg
g-' (0.20-6.87 pg g-') and 230 ng g-' (10.0-780 ng g-I), for total chromium and chromium(vi), respectively.
The large variations in the levels of metal found in the analysed feeds shows diverse contamination of the
raw materials used in the preparation of the samples.
Keywords: Chromium; chromium(vr); electrothermal atomic absorption spectrometry; animal feeds

Chromium is a biologically important element that is involved
in glucose and lipid metabolism,' but it is also considered to
be toxic. It is only toxic with excessive intake and the toxicity
is mainly dependent on the oxidation state.2 The estimated
safe and required dietary intake of Cr is 0.05-0.20 mg d-' .3
Particularly in its hexavalent oxidation form, Cr is a major
water pollutant, usually as a result of industrial effluents. It
accumulates in soils and therefore can contaminate crops and
other plants or can be incorporated into these systematically
from the Several vegetable materials such as grains, seeds
or leaves are constituents of animal feeds and pre-mixes that
are supplemented with mineral additives and other nutrients.
Hence, it is important to monitor feeds for metallic elements
that are toxic or, even if they are essential elements, to avoid
problems of toxicity when their concentrations exceed the safe
levels. The measurement of Cr levels is especially important
for the reasons of the toxicity of CrV' and the biodependence
of Cr"' .
Several methodologies for the quantification of Cr have been
described in the literature, namely in biological and
in Also, several attempts have been made to quantify
Cr both in its state as an essential element of nutrition (Cr"')
and in its toxic state (CrV'), particularly in water14-19 and
in
One of the most reliable techniques to measure metallic
species at ng 8-l and sub-ng g-' levels is electrothermal
atomic absorption spectrometry (ETAAS). However, use of
this method specifically to control Cr"' and CrV1levels in feeds
has not been reported in the literature. The proposed method
was developed to provide a viable procedure to determine Cr
in feedstuffs in order to verify contamination levels, particularly
the hexavalent content.
Initially an evaluation of the method of sample preparation
for dissolution of the total Cr using a digestion procedure,
from which good results have been obtained with another
complex matrix, was carried out.22 For extraction of CrV' a
procedure previously applied by other workers to soils was
adopted2'; this procedure was accurately evaluated in the
present work. Next, charring and atomization temperature
studies were performed as well as studies of the range of
calibration. The final phase of the work was establishing the
quality assurance of the method both by the standard additions
technique and by analysing a reference material.
Subsequently, the method was applied to the quantification
of total Cr and CrV' in 70 feedstuff samples.
Experimental
Reagents
To avoid contamination all poly( tetrafluoroethylene) (PTFE)
materials, pipettes, micropipette tips, autosampler cups and
calibrated flasks were immersed for 24 h in freshly prepared
15% v/v H N 0 3 and then rinsed thoroughly with doubly
de-ionized water before use.
All the acids and NaOH were of Suprapur grade (Merck).
Ammonium nitrate was pro analysi grade (Merck).
Chromium(II1) standards were prepared daily from a
1000mg I-' solution (Titrisol, Merck). A solution of
1000 mg 1-' chromium(v1) was prepared by dissolving 2.829 g
of K2Cr207(AnalaR, Merck) in 1 1 of de-ionized water; from
this, other diluted standard solutions were prepared daily.
The chemical modifier used was a mixture of Pd(NO,),
(Merck) and Mg(N03)2 (Merck) prepared by mixing equal
volumes of solutions containing 3 and 2 g 1-', respectively, in
15% v/v HNO3.
The reference material was National Institute of Standards
and Technology (NIST) Standard Reference Material (SRM)
1548, Total Diet obtained from The Office of Reference
Materials, Laboratory of the Government Chemist,
Teddington, UK.
Apparatus
A Perkin-Elmer HGA-700 furnace installed in a Model 1100B
atomic absorption spectrometer with deuterium arc back-
ground correction was used, equipped with an AS-60 auto-
sampler and an Epson EX-800 printer. The analyses were
carried out using platforms ( Perkin-Elmer part No.
AAPB109-324) inserted into pyrolytic graphite coated graphite
tubes (Perkin-Elmer part No. AAPB109-322). A single-
element, hollow cathode lamp (Perkin-Elmer part No. AAPC
303-6039) was operated at 25 mA, and all data were taken at
the 357.9 nm wavelength. The integration time was 5 s. The
slit-width was 0.7nm and argon was used as the purge gas,
with an internal flow rate of 300 ml min-'. Readings from the
spectrometer were taken using the peak area mode.
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1270 JOURNAL OF ANALYTICAL ATOMIC SPECTROMETRY, NOVEMBER 1994, VOL. 9

Sample Preparation
The samples used in the present study were of different
compositions, but heterogeneous with respect to particle size,
some of them being granulated feeds depending on the animal
O6
0.5
tt Standard
al
species for which they were intended. Thus, the first step
consisted of grinding the samples to a particle size ofjust 1 mm.
5 0.4
Samples of approximately 0.5 g were accurately weighed in
e
2 0.3
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duplicate. One sample was digested in a 50 ml PTFE container 2

with a mixture of concentrated H N 0 3 (10 ml), HCl(5 ml) and 0.2
H F (1 ml) and then heated just to dryness on a hot-plate.
0.1
Further portions of HNO, (10 ml) and HCl(5 ml) were added
to the residue and the mixture was again evaporated to dryness. I

This residue was dissolved in 0.5 ml concentrated HNO, and
diluted with water to 25 ml in a calibrated flask. The total Cr
was measured in this solution. The other 0.5g portion was
placed in a 15 rnl poly(propy1ene) tube, 9 ml of 0.01 mol 1-'
NaOH solution added, the cap fitted on and the tube then
shaken horizontally in an oscillating agitator for 17 h at room
temperature in order to extract the Crv', as has been described
elsewhere.20After the addition of 1 ml of 1 mol 1-' NH4N0,
solution, the sample was shaken briefly and centrifuged for
15 min (3000 rev min-'). The CrV' was measured in the
supernatant liquid.
Results and Discussion
Optimization Programme for ETAAS
The following solutions were used to optimize the ETAAS
method: an acidic CrV1standard solution (50 pg 1-'); an acidic
Cr"' standard solution (50 pg 1-'); a solution of the acid
digested samples; an alkaline CrV' standard solution (50 pg
1-'), prepared under the same alkaline conditions as the feed
samples; and a solution resulting from the alkaline extraction
of CrV' from the feeds. These solutions were used to establish
the best charring and atomization temperatures and to obtain
the most repeatable and sensitive signals in the shortest analysis
time. The autosampler was programmed to pipette sequentially
the modifier, followed by the standard solution or the sample
solution and to dispense them together onto the platform.
The charring and atomization curves obtained for total Cr
and Crvl, in acidic and alkaline medium are shown in Figs. 1
and 2, respectively. For both situations the optimum tempera-
tures were 1600 and 2500 "C for the charring and atomization,
respectively. The optimized ETAAS programme used is sum-
marized in Table 1.
As can be observed in the graphs, the temperatures chosen
correspond to the best signals for the five situations studied.
As was expected, the standard acidic solutions give the same
readings for the Cr"' and CrV' stock solutions. Nevertheless,
the absorbance signals obtained for CrV'in the alkaline medium
are lower. Thus, the furnace programme adopted was the same
0.6 -
0.5-
al
C
(c1
0.4-
e -
s
n
0.3
0.2 -
Q CrV' solutions were added to the feed samples and subjected
t d
1100 1300 1500 1700 1900 2100 2300 2500 2700 2900
Temperat u rePC
Fig. 1 Charring and atomization graphs for total Cr in acidic CrV'
and Cr"' standard solutions and in a solution of the digested samples
1100 1300 1500 1700 1900 2100 2300 2500 2700 2900
TemperaturePC
Fig. 2 Charring and atomization graphs for CrV1in an alkaline Cr"'
standard solution and in an alkaline extract of CrV1from animal feeds
Table 1 Optimized ETAAS programme
Step Temperature/" C Ramp/s Hold/s
Dry 1 130 15 15
Dry 2 300 15 15
Char 1600 30 30
Atomize 2500 0 5
Clean out 2650 2 3
for measuring total Cr and CrV', but it was necessary to use
the appropriate standard solutions, ie., in acidic and in alkaline
media, prepared for the CrV' standard.
Analytical Curve and Detection Limit
To evaluate the optimum calibration range for total Cr and
for CrV', standard solutions of CrV' in acidic and alkaline
media of from 0 to 100.0 pg I-' were used. Linearity was
observed over the concentration range 0.53-50.0 pg 1-' for
total Cr and 0.42-50.0 pg 1-' for Cr".
To calculate the detection limits, 20 determinations were
carried out in 0.2% v/v HNO, and in 0.01 mol 1-' NaOH-
1 mol I-' NH4 NO3 solution for total Cr and Crvl, respectively.
The values were calculated as the concentration corresponding
to three times the standard deviation (SD) of the background
noise and were, respectively, 0.53 and 0.42 pg 1-'.
Validity of the Method
Digesting difficult matrixes with a three-acid mixture
(HN0,-HCl-HF) has produced good results.22 The use of
high-purity grade acids and carrying out the digestion in PTFE
containers ensures that the sample pre-treatment can be carried
out without contamination. This was confirmed by including
a blank assay in each batch of samples, using the same amounts
of acids and submitting these to the digestion procedure.
To extract the Cr present in its hexavalent state, the method
of solubilization with NaOH solution that had been applied
to soils was adopted,20 after testing this procedure with the
present matrix.
Along with the wet digestion procedure and the sample
extraction procedure, the method of standard additions was
also performed for both Cr determinations. Standard acidic
to the over-all digestion procedure. Also, alkaline CrV'standard
solutions were added to the feed samples and again subjected
to the alkaline extraction procedure. The results obtained in
these studies are presented in Table 2. As can be observed, the
recovery study shows that the absorbance readings for the
samples can be compared directly with the standard analytical
curves obtained for total Cr and for Crvl.
Validation of the digestion procedure was confirmed by
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JOURNAL O F ANALYTICAL ATOMIC SPECTROMETRY, NOVEMBER 1994, VOL. 9 1271

Table 2 Statistical results for the recoveries obtained by the standard additions method

Parameter Total Cr CrV'

Concentration/pg 1- ' 5.0 10 25 2.5 5.0 10

n 6 6 6 10 10 10
Recovery & SD (YO) 96f0 95+ 1 96+3 94+3 96f3 95+ 1
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Table3 Comparison of recoveries of Cr"' and Cr'" added to one Table5 Deviations (YO)from the expected values for total Cr and
feed sample CrV' obtained in the interference studies

Cr found CrV' Cr"' Total Cr Added concentration/pg 1- '

in sample/ added/ added/ found/ Recovery
pgg-' pgml-' pgml-' 1gg-I (%I 5 10 25
3.8 5.0 0.0 8.3 90 Species (n=4) (n=4) (n = 4)
3.8 10.0 0.0 14 102 Total Cr 6.3 5.7 6.7
3.8 0.0 5.0 8.7 98 CrV' 6.0 10.0 7.5
3.8 0.0 10.0 13.8 100

Table 4 Principal mineral constituents in feedstuffs (Decreto-Lei

No. 57/1985, Diario de Republica No. 54, I Serie, 06/03/85) used in
the interference studies
Major Minor
Mineral Concentration (YO) Mineral Concentration/pg g-
Phosphates 0.8 Cobalt 10
Calcium 1.o Copper 175
Sodium 0.15 Manganese 250
Potassium 0.5 Zinc 250
Iron 0.12
analysing a reference material. Although it was impossible to
obtain a reference material with the same composition as the
animal feed samples, NIST SRM N-1548 Total Diet was used.
As the certified value was given only for total Cr (1.43 pg g-I),
the acid digestion procedure was performed on ten 0.5 g
portions and the mean value of the metal concentration
obtained was 1.39 k0.13 pg g-', signifying a recovery of
99 _+ 3% (mean & SD).
It is assumed that after digestion with strong acids Cr will
be in its hexavalent form. To compare the recoveries of Cr"'
and CrV1added to feed samples and determined as total Cr, a
sample was spiked with standards of both species, submitted
to acid digestion and the total Cr measured. The results are
summarized in Table 3. It can be concluded that after acid
digestion the determination of total Cr does not depend on
the oxidation state of the metal. As a consequence, in the
present procedure CrV1standards prepared in an acidic or
alkaline medium, whichever was convenient, were acceptable.
The precision of the analytical method was evaluated by
measuring the absorbance signals 20 times on the same digested
sample and in the same alkaline extract of the sample. For
evaluation of the precision of the over-all procedure, readings
of 20 different digested aliquots and of 20 different alkaline
extracted samples were performed. The relative standard devi-
ations (RSDs) ranged between 2.2 and 2.7% for the analytical
method and between 4.5 and 7.0% for the over-all procedure,
for total Cr and Crv', respectively. These results are fully
acceptable values, bearing in mind the digestion step and all
the manipulations involved.
To study the interferent effects of the principal mineral
constituents present in feedstuffs on the measurement of Cr,
mixed standard solutions containing the mean recommended
levels for the various species (see Table 4) were ~repared.'~For
total Cr, three aliquots of the standard mixed solution were
added to three different concentration levels of standard Cr
acidic solutions and the absorbance signals were measured
under the analytical conditions described above. For Cr",
three 1 ml aliquots of the mixed solution of minerals were
added to 0.01 moll-' NaOH solution (9 ml) and three different
concentration levels of the aqueous CrV' standards and the
mixtures shaken horizontally in an oscillating agitator for 17 h
at room temperature. After adding 1 ml of 1 mol I-' NH4N0,
' solution, the absorbance signals were obtained. The absorbance
readings were interpolated on the analytical curves established
independently for total Cr and Cr". The deviations from the
expected values were always acceptable (< lo%), and are
presented in Table 5.
Preconcentration of the metal, which is required for the
analysis of waterlg was not needed because the levels of both
total Cr and CrV' that were present in the feedstuffs were high
enough to enable quantification without any enrichment steps.
Applications
A wide range of feeds, a total of 70 samples, were analysed by
application of the ETAAS methodology developed. As would
be expected, compared with total Cr, CrV' was present at low
concentrations, the mean value found being 230 ng g-' (range
10.0-780 ng g-'). The RSD was 60.9%, which shows the great
variability of levels present in the range of samples analysed.
Nevertheless, considering the potential toxicity of the metal in
this oxidation state and its bioavailability, it is advisable to
establish where this potential danger to animal health is arising.
For total Cr, the mean value found was 1.93 pg g-'
(0.20-6.87 pg g-I), the RSD being 77.2%. Considering that
almost all of the metal is present as Cr"', and that in this form
the metal is an essential element and poorly absorbed by the
gastro-intestinal tract, these high levels do not cause a toxicity
problem for animals.
It can be assumed that these different values result from the
different degrees of contamination of the soils where the crops
had been grown. Also, the different technological procedures
used in the preparation of the animal feeds can affect the
oxidation state of the element.
Conclusions
An accurate and precise method to quantify total Cr and CrV1
in animal feeds is presented. The ETAAS conditions are the
same for both determinations which facilitates the analyses
being carried out using the same furnace programme, this
aspect being a very important criteria in routine analyses.
This work received financial support from Junta Nacional de
Investigaqiio Cientifica (Centro de Analise do Alimento) and
AssociaqZo Nacional das Farmacias.
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1272 JOURNAL OF ANALYTICAL ATOMIC SPECTROMETRY, NOVEMBER 1994, VOL. 9

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