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A Simple Digestion Method with a Lefort Aqua Regia Solution for Diatom
Extraction †‡

Article  in  Journal of Forensic Sciences · November 2014

DOI: 10.1111/1556-4029.12615

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 J Forensic Sci, January 2015, Vol. 60, No. S1
doi: 10.1111/1556-4029.12615
TECHNICAL NOTE Available online at: onlinelibrary.wiley.com

PATHOLOGY/BIOLOGY

Huipin Wang,1,2 M.D.; Yan Liu,3 M.D.; Jian Zhao,4 B.D.; Sunlin Hu,4 Ph.D.; Yuzhong Wang,1 M.D.;
Chao Liu,4 Ph.D.; and Yanji Zhang,5 B.D.

A Simple Digestion Method with a Lefort Aqua

Regia Solution for Diatom Extraction*,†

ABSTRACT: Presence of diatoms in tissues has been considered as a significant sign of drowning. However, there are limitations in the
present extraction methods. We developed a new digestion method using the Lefort aqua regia solution (3:1 nitric acid to hydrochloric acid)
for diatom extraction and evaluated the digestive capability, diatom destruction, and diatoms’ recovery of this new method. The kidney tissues
from rabbit mixed with water rich in diatoms were treated by the Lefort aqua regia digestion method (n = 10) and the conventional acid diges-
tion method (n = 10). The results showed that the digestive capability of Lefort aqua regia digestion method was superior to conventional acid
digestion method (p < 0.01); the structure of diatom remained almost intact; and the recovery of diatom was comparable to the conventional
acid digestion method (p > 0.05). The Lefort aqua regia reagent is an improvement over the conventional acid digestion for recovery of dia-
toms from tissue samples.

KEYWORDS: forensic science, forensic pathology, drowning, diatom test, digestion method, diatom extraction, Lefort aqua regia

A body found in water poses a challenge for the forensic
pathologist and other investigators to determine the cause and
manner of death (1–3). Presence of diatoms in tissues has been
considered as a significant sign of drowning since the beginning
of twentieth century. Diatom test has been considered as the
gold standard for the diagnosis of drowning (4). The detection
of diatoms requires the removal of both extra and intracellular
organic matter from the siliceous frustules of diatoms and other
material in the sample; so that the details of diatom structures
can be reviewed for the purpose of taxonomic identification.
Various extraction methods have been developed to successfully
isolate diatoms from water and tissues samples. Strong acid
digestion (5), enzymatic digestion with proteinase K (6), and sol-
ubilization with Soluene-350 (7,8) were commonly used world-
wide. According to Hurlimann et al. (9), the ideal digestion
method should encompass disposable equipment and leave mini-
mal residues of the digested samples.
In the conventional acid digestion method, there are large
amount of residues in the samples, which may make effect of
diatom identification. The Lefort aqua regia solution (3:1 nitric
1
Faculty of Forensic Science, Southern Medical University, Guangzhou,
China.
2
Zhongshan Forensic Science Institute, Zhongshan, China.
3 ity of contamination with exogenous diatoms.
South China University of Technology, Guangzhou, China.
4
Guangzhou Forensic Science Institute, Cangbian Road 30, Guangzhou,
China.
5 Experimental Design
Harbin Forensic Science Institute, Harbin, China.
*Presented at the 6th European Academy of Forensic Sciences (EAFS)
Conference, August 20–24, 2012, in The Hague, Netherlands.
†
Financial support provided by Grant-in-Aids for Scientific Research from
China Public Security Ministry (No.2010ZDYJGDST017).
Received 20 June 2013; and in revised form 9 Jan. 2014; accepted 20 Jan.
2014.
acid to hydrochloric acid) has been used by United States Geo-
logical Survey to digest organic matters to analyze inorganic ele-
ments in the soil for years (http://www.caslab.com/
Test-Method-I-6463-86/ [accessed January 8, 2014]). In this
study, we developed a new digestion method using the Lefort
aqua regia solution to extract diatoms in the organs tissues for
forensic diatom test.
This qualitative and quantitive study aimed to develop a sim-
ple digestion method using the Lefort aqua regia solution which
was more efficient than the conventional acid digestion method,
to reduce the amount of organic matter.
Materials and Methods
Reagents, Supplies, and Instruments
Analytical grade nitric acid, hydrochloric acid, and 30%
hydrogen peroxide (Guangzhou Chemical Reagent Factory);
MW3000 Microwave Digestion System (Anton Parr Company);
Self-designed Vacuum Filtration Device (Fig. 1); Quanta 600
Scanning Electron Microscope (FEI Company, Hillsboro, Ore-
gon, USA) equipped with Genesis7000 Energy Disperse Spec-
troscopy (EDAX Company, Mahwah, New Jersey, USA). All
the reagents and instruments were tested to exclude the possibil-
The new Lefort aqua regia digestion method was evaluated by
detecting diatoms in rabbit tissues which were mixed by water
rich in diatoms.
Kidney tissues from rabbits died of air embolism were taken
and minced. Water sample was collected below ground surface

© 2014 American Academy of Forensic Sciences S227

S228 JOURNAL OF FORENSIC SCIENCES
FIG. 1––Self-designed vacuum filtration device.
in the Pearl River in Guangzhou, China, during spring time.
Filtered formalin was added to achieve 5% final concentration
and preserved in the dark for further analysis.
Twenty samples (each sample with 2 g of kidney tissue and
2 mL of water) were analyzed by both diatom test methods (Le-
fort aqua regia digestion method, n = 10; conventional acid
digestion method, n = 10).
Four water samples (each 2 mL of water) were vacuum-fil-
tered directly and observed by SEM. The average number of
four water samples was considered as the standard number in
the samples. A negative control was also performed with no
water added to be sure that there is no contamination during the
autopsy procedures or of food intake in life occurred.
The Lefort aqua regia digestion method: Samples were put into
the 100-mL beaker. 15 mL of concentrated nitric acid and 5 mL
of concentrated hydrochloric acid were added in succession. 2 mL
of hydrogen peroxide was dripping in at the rate of 1 drop per
3 sec. The dripping of hydrogen peroxide should be stopped
immediately when a great deal of bubbles was generated fiercely
in the beaker and then resumed dripping when the amount of bub-
bles become less and steady. Samples were left without interrup-
tion for 15 min and then put into the thermostatic water tank at
85°C for 50 min, and during heating, another 3 mL of hydrogen
peroxide was added at the rate of 1 drop per 3 sec.
The above procedures should be operated in the fume hood.
The Lefort aqua regia should be made fresh and not be stored
there; make only what you need to the test and destroy the resi-
due. It can be destroyed by cautious and careful dilution with
water or neutralized and disposed of in the approved manner.
Never put the Lefort aqua regia in a closed container or near
flammables.
The conventional acid digestion method: In acid digestion,
20 mL of concentrated nitric acid and 5 mL of 30% hydrogen
peroxide were added and boiled on a water bath at 85°C for
50 min.
The digestion solution was filtered with a millipore filtration
(Pore diameter size is 0.45 lm), and the weight change of each
membrane was recorded. Then, the membranes were analyzed by
SEM after the filter membranes were coated with a layer of gold.
Statistical Analysis
Values in the text and table were expressed as the means  SD,
and statistical analyses were performed by two-way analysis of
t-test using SPSS 17.0 (Chicago, Illinois, USA), for windows.
p < 0.05 was considered as statistically significant.
Results
Digestive Capability and Diatom Destruction
The degree of digestion of the kidney tissues in the limited
time (50 min) was evaluated by the weight of the precipitate on
the filtration membrane after digestion. As demonstrated in
Table 1, the weight of the precipitate of the Lefort aqua regia
digestion method was 0.00604  0.00310 g/2 g and that of con-
ventional acid digestion method was 0.00979  0.00204 g/2 g.
The digestive capability of Lefort aqua regia digestion method
was superior to conventional acid digestion method, with the dif-
ference being statistically significant (p < 0.01).
In Fig. 2, there was a minimal amount of organic matter in
the samples digested with the Lefort aqua regia, and the struc-
ture of diatoms remained intact with a clear background and lit-
tle impurity in all the replicates performed. The fragment of
diatoms was rarely found. The results indicated that the digestive
ability of Lefort aqua regia digestion method was very strong,
and fewer residues were left.
Recovery of Diatom
There was no diatom found in the negative control sample.
The standard number of diatoms in water samples was 711.2/mL.
As shown in Table 2, the recovery of diatoms with the Lefort
aqua regia digestion method was 71.1  10.3% and that of con-
ventional acid digestion method was 66.3  9.0%. There was no
statistical difference between the two groups (p > 0.05). The
results showed that there was no more diatom loss in the Lefort
aqua regia solution.
Discussion
In 1988, Auer and Mottonen (10) developed a chemical dia-
tom extraction method with nitric acid plus hydrogen peroxide.
We developed the Lefort aqua regia digestion method which
could decrease the impurities resulting in a cleaner background
and reduce the damage of diatom comparing with the conven-
tional method.
The Lefort aqua regia solution (H2((N3O9)Cl)) (11) with
strong oxidization is composed of concentrated nitric acid and
hydrochloric acid (3:1), and its pH (<1) is lower than that of
aqua regia (1~2) because of the Lefort ionizes nearly completely.
The oxidization of hydrogen peroxide is stronger in the solution
as well. Those properties allow the organic matter digested faster
and near completion. However, due to the nature of the Lefort
aqua regia solution, the experiment must be conducted with
extreme caution. The rate of dripping must be controlled at the
TABLE 1––Weight of precipitate after digestion of 2 g rabbits’ kidney tissue
(Mean  SD).
Methods Cases Weight of precipitate (g) p
Lefort aqua regia 10 0.00604  0.00327 0.007
digestion method
Conventional acid 10 0.00979  0.00204
digestion method
p represents statistical difference between the two methods.
 WANG ET AL.
FIG. 2––Diatoms detected in the samples digested with the Lefor aqua regia digestion method/(Upper: the Lefor aqua regia digestion method; Lower: the
conventional acid digestion method).
TABLE 2––Recovery of diatoms (Mean  SD).
Methods Cases Recovery of diatoms
Lefort aqua regia 10 71.1  10.3 0.283
digestion method (%)
Conventional acid 10 66.3  9.0
digestion method (%)
p represents statistical difference between the two methods.
rate of 1 drop per 3 sec or even lower. This reaction should be
conducted in the wide-mouthed containers and in the fume hood
because of its aggressive nature.
The conventional acid digestion is the most classic and widely
used diatom extraction method in the laboratory. It is simple and
easy to operate. Some modified acid digestion method was
developed by forensic scientist, such as the disorganization can
(12) and microwave digestion (13). In this study, we developed
a new modified method of acid digestion method by adding
hydrochloric acid to improve the digestive capability for organic
matters, to verify if this method could extract diatoms from tis-
sues with less impurities and higher recovery of diatom. The
results revealed that the digestive capability of Lefort aqua regia
digestion method was superior to conventional acid digestion
method; the structure of diatom remained almost intact; and the
recovery of diatom was comparable to the conventional acid
digestion method.
. LEFORT AQUA REGIA SOLUTION FOR DIATOM EXTRACTION S229
Conclusion
p The Lefort aqua regia digestion method for diatom extraction
has been developed and tested. Diatoms were found in our
study, and the new method was helpful for diatom identification.
The digestive capability of Lefort aqua regia digestion method
was superior to conventional acid digestion method; the structure
of diatom remained almost intact; and the recovery of diatom
was comparable to the conventional acid digestion method. In
conclusion, the Lefort aqua regia digestion method is an
improvement over the conventional acid digestion for recovery
of diatoms from tissue samples.
Acknowledgments
The authors thank Dr. Ma for her assistance in preparing the
manuscript.
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Additional information and reprint requests:
Chao Liu, Ph.D.
Guangzhou Forensic Science Institute
Cangbian Road 30, Yuexiu District
Guangzhou
China
E-mails: 768594951@qq.com; liuchaogaj@21cn.com