Journal of Paleolimnology 31: 383–390, 2004.

383
# 2004 Kluwer Academic Publishers. Printed in the Netherlands.

A microwave digestion technique for the extraction of fossil diatoms from

coastal lake and swamp sediments

J.F. Parr1,*, K.H. Taffs1 and C.M. Lane2

1
Centre for Geoarchaeology and Palaeoenvironmental Research, School of Environmental Science and
Management, Southern Cross University, P.O. Box 157 Lismore, NSW 2480 Australia; 2Institute of
Antarctic and Southern Ocean Studies (IASOS), University of Tasmania, GPO Box 252-80, Hobart
Tasmania, 7001 Australia; *Author for correspondence (e-mail: mailto:jparr@scu.edu.au)

Received 7 February 2003; accepted in revised form 18 November 2003

Key words: Diatom, Methods, Microwave digestion, Sediments

Abstract

This study provides an introduction to a microwave digestion technique for the extraction of fossil diatoms from
sediments. The microwave technique is compared with the standard diatom extraction technique of Battarbee
(Diatom analysis. In: Berglund B.E. (ed.), Handbook of Holocene Palaeoecology and Palaeohydrology. John
Wiley and Sons) that uses a combination of dilute hydrochloric acid and hydrogen peroxide and the advantages
and disadvantages of their use are discussed. The results suggest that the microwave technique is fast,
inexpensive and most importantly produces replicable fossil diatom assemblage data. Small samples sizes are
used (0.3 g) for the microwave method thus lower quantities of chemicals are required (6 ml), which significantly
decreases the cost of sample processing. Our results show that the microwave digestion technique is a viable
alternative that will produce similar results within a shorter period of time.

Introduction comparisons between the different methods have

A wide variety of approaches have been used to
clean fossil diatom frustules. Two such techniques
that are commonly used by diatom researchers are
discussed here. The first technique pioneered by
Battarbee (1986) uses a dilute hydrochloric bath
followed by a hydrogen peroxide bath. This
method appears to be preferred by most diatom
researchers and is widely used (Battarbee and
Kneen 1982; Renberg 1990; Dokulil et al. 1997;
Gell 1997). The second method uses a standard
acid sediment digestion technique with 50 : 50 con-
centrated sulphuric and nitric acid (Dixit and Smol
1994; Wilson et al. 1996; Reavie et al. 1998; Dixit
et al. 1999; Laing and Smol 2000; Reavie and Smol
2001). While the method pioneered by Battarbee
(1986) appears to be the preferred method used in
most fossil diatom studies, to date, no critical
been performed (Kelly et al. 1998).
Renberg (1990) identified that an obvious limita-
tion for both of the above-mentioned methods is
that they are only suitable for small sets of samples
(20–40). This reported limitation was due to the
need for large quantities of chemicals, fume cup-
board space and the time it took to process large
sets of samples. As a result, Renberg (1990) pre-
sented a method for processing very large numbers
of samples (120). However, this technique is only
useful for studies that require large numbers of
samples to be processed. Thus the Battarbee
(1986) method remains the most commonly used,
and yet can be quite expensive in use of chemicals,
laboratory space and time.
An alternative technique for the processing of
diatom frustules is microwave digestion. This tech-
nique was pioneered by Acker and Russell (1999)
384

and mentioned in Dixit et al. (1999). Generally, Table 1. Sediment samples used for the comparative analysis
microwave sample preparation systems are used and their depth in cm.
to digest samples for isotope analysis of soils by Lake Hiawatha Tuckean Swamp
Inductively Coupled Plasma Mass Spectrome-
try (ICP/MS) and, element analysis by Atomic Sample Depth (cm) Sample Depth (cm)
Absorption Spectrometry (AAS) (Parr and lh1 0–1 ts1 0–1
Farrugia 2003). However, its potential use in lh2 6–7 ts2 10–12.5
microfossil extraction is gradually being realised lh3 13–14 ts3 20–22.5
and suitable protocols are being developed. For lh4 19–20 ts4 27.5–30
lh5 28–30 ts5 40–42.5
example, microwave digestion has been assessed lh6 38–40 ts6 50–52.5
for the retrieval of fossil pollen from geological
samples (Jones 1994) and has been successfully
used for the extraction of phytoliths from herbar-
ium specimens and sediments (Parr et al. 2001; Parr Lake Hiawatha is a fresh water dune contact
2002; Krull et al. 2003; Parr and Carter 2003; Parr water body located within Yuraygir National
and Farrugia 2003). Park (Timms 1982). It is a humic lake with low
Two types of microwave digestion systems are salinity, a dominance of Na+ and Cl
ions and
available – focused and pressurised. Pressurised has a high pH (>7.0) (Timms 1982). Lake
microwave digestion differs in operation somewhat Hiawatha has an area of 316 ha, is 16.8 m asl and
to focused microwave digestion. This system com- has a maximum depth of 11 m (Timms 1969).
prises six to twelve pressurised vessels that require a Tuckean Swamp is a backwater of approxi-
relatively uniform sample weight, processing time mately 5000 ha of coastal floodplain on the lower
and temperature setting. However, chemical Richmond River near Broadwater in northern
combinations may be varied for each sample. The New South Wales. Prior to European influence,
basic advantages and disadvantages of pressurised Tuckean Swamp was linked tidally to the
microwave digestion in comparison to focused Richmond River by the Tuckean Broadwater and
microwave digestion have been outlined in detail was fed by freshwater runoff from the upper catch-
by Jones and Ellin (1998). One major criticism of ment. However, the Baggotville Barrage (com-
the pressurised microwave digestion systems has pleted in 1971) prevents tidal waters moving
been that they are limited to a very small sample upstream into the swamp to protect Tuckean
size of around 0.25 g (Jones 1994) and this is parti- Swamp’s grazing and agricultural areas from salt-
cularly critical if it affects the interpretation of water intrusion. The soils of Tuckean Swamp
microfossil assemblages. Therefore, to assess the include potential and actual acid sulphate soils
ability of the microwave technique for producing developed during the last Holocene.
diatom assemblage data it was compared with the The aim of this study was thus to determine if the
method of Battarbee (1986) using sediment samples microwave digestion method could produce com-
from two coastal swamps as a case study. parable diatom assemblage data to that of a
conventional method commonly used by diatom
Description of sites researchers.

Two coastal wetlands were selected to provide

material for the comparison of the two diatom
processing techniques. These lakes were chosen
because the material was readily available and
was undergoing fossil diatom analysis for another
project. They also provided a comparison between
a pristine lake environment with sandy sediments
and a lake that has been heavily modified by
human activities and has clay based sediments
affected by acid sulphate soil runoff.
Methods
Sediment cores were extracted using a D-section
corer from Lake Hiawatha and Tuckean Swamp
in northern New South Wales. Six samples
were selected from each sediment core, each repre-
sentative of a different sediment layer (Table 1).
Each sample was processed according to the
conventionally used diatom processing technique

(Battarbee 1986) and the microwave digestion
technique.
The sediment of the Lake Hiawatha core was
predominantly sand based with four distinct soil
horizons. The surface horizon (0–14 cm) was com-
posed of a highly organic silt material. The second
horizon (14–30 cm) was composed of sandy silt
with a high organic component. Horizon three
(30–40 cm) was predominantly sand material,
with a high iron content. Horizon four (40–70 cm)
was a heavy orange clay with frequent grey/green
mottles.
The sediment of the Tuckean Swamp sediment
core had three distinct soil horizons and was
predominantly silt/clay based. Soil horizon one
(0–5 cm) was a silty loam with a high organic
component. Horizon two (5–20 cm) was a silty
clay and horizon three (20–55 cm) was a heavy,
organic clay.
Conventional digestion technique
Following the conventional (c) digestion procedure
of Battarbee (1986) approximately 0.3 g of sedi-
ment material was weighed into a beaker and 10%
hydrochloric acid was added. The beaker was
placed on a hot plate, simmering, for approxi-
mately three 3 hours (until effervescence ceased)
in order to remove the carbonates from the sample.
The samples were then thoroughly rinsed with dis-
tilled water three times to remove solutes. At all
stages, the material was left to settle for at least 6 h
before the supernatant was decanted. The samples
were then resuspended in 10% hydrogen peroxide
in order to remove the organic material, simmer-
ing, for approximately 3 hours. Following cleaning
the material was allowed to settle. The supernatant
was then removed, and the sample washed three
times (as above). The sample was then resuspended
in high-grade ethanol and stored in a vial, ready for
mounting. Mounting of the cleaned diatoms was
conducted using 700 m of sample. The sample was
allowed to dry on a coverslip and mounted using
the mounting medium Depex.
Microwave digestion
A Perkin–Elmer, Multiwave Microwave Sample
Preparation System was used in this study. The
microwave is controlled by an in-built computer
385
system. For diatom extraction, the microwave
sample preparation system was programmed to
the following settings:
a. 8 min ramp up to a maximum of 120  C.
b. 8 min digestion at 120  C.
c. 15 min cooling time.
The chemical component of the microwave
digestion protocol is one adapted from a standard
Aqua Regia sample preparation technique used
in the Environmental Analysis Laboratory at
Southern Cross University. Around 0.30-g por-
tions of sediment sample was weighed into Teflon
digestion tubes followed by 3 ml of concentrated
nitric acid and 3 ml of concentrated hydrochloric
acid. The Teflon tubes were placed into microwave
bombs and processed for 31 min. After viewing
the sample and determining the size fraction of
the diatom frustules, samples were washed through
300 and 5 m filters simultaneously collecting the
diatom fraction. The sample was then suspended in
high-grade ethanol and stored in a vial, ready for
mounting. Mounting of the cleaned diatoms was
conducted using 700 m of sample, allowed to dry
on a coverslip and mounted using the mounting
medium Depex.
Microscopy and counting assemblages
Diatom slides were examined under a microscope
at 1000 magnification. Diatoms were identified
using Krammer and Lange-Bertelot (1991a, b,
1997a, b). Around 300 diatom frustules were
counted for each slide along random transects.
The relative abundance of each species was
calculated. Diatom species with <2% relative
abundance were discarded from the analysis.
Statistical analysis
Statistical analysis was carried out using multidi-
mensional scaling (MDS; Young and Householder
1938; Coxon and Davies 1982; Borg and Groenen
1997) with SPSS software. Dissimilarities between
pairs of diatom assemblages were calculated as 2
measures over their diatom frequency profiles
for each species. These were plotted into a two-
dimensional space to display the dissimilarities
between variables as Euclidean distances.
386
Results
All samples produced adequate numbers of dia-
toms for analysis. The relative abundance of dia-
toms for each species from Lake Hiawatha and
Tuckean Swamp are summarised in Table 2.
MDS statistical analysis
Dissimilarities that do occur in diatom assemblages
between methods are shown in two-dimensional
plots (Figures 1 and 2). The corresponding codes
for each sample (Table 2) are displayed within the
associated plot areas (Figures 1 and 2). For Lake
Hiawatha stress values ¼ 0.11149 and plotted
R-squared values shown in Figure 1 explain 0.93
of the total variance of diatom assemblages
between methods. Samples 1, 3 and 4 have a very
close diatom assemblage relationship between the
conventional (c) method and the microwave diges-
tion method (md), however, some variation occurs
in the diatom assemblages for samples 2, 5 and 6
(Figure 1). For Tuckean Swamp, stress values ¼
0.01244 and plotted R-squared values shown in
Figure 2 explain 0.99 of the total variance of dia-
tom assemblages between methods. The two-
dimensional plot for Tuckean Swamp show that
the conventional and microwave digestion methods
produce very closely related assemblages for all
samples (Figure 2).
Discussion
For both the Lake Hiawatha and Tuckean Swamp
samples, MDS analysis shows strong levels of con-
fidence with the plotted R-squared values explain-
ing 0.93 and 0.99 respectively of the total variance
in diatom assemblages between methods (Figures 1
and 2). Some contrasts were found in the Lake
Hiawatha samples 2, 5 and 6 (Figure 1). The var-
iance in the diatom assemblages for samples 2 (lh2
(c), lh2 (md)) and 6 (lh6 (c), lh6 (md)) is minor
in comparison to that for sample 5 (Figure 1). For
samples 1, 3 and 4, frequency counts for the
Cymbella, Cyclotella and Anomoeoneis species are
relatively comparable. However, the lh2 (md)
assemblage has around 35% more Cymbella dia-
toms than occurs in counts for its counterpart lh2
(c). Similarly, lh6 (md) has around 24% more
Cymbella sp., however, 56% less Cyclotella than
occurs in counts for its counterpart (lh6 (c)).
Thus, samples 2 and 6 appear to have a marginally
greater separation on the plot than do those for
samples 1, 3 and 4 (Figure 1). For sample 5, the
lh5 (c) top left and lh5 (md) bottom right of the
plot shows a greater degree of variance (Figure 1).
The lh5 (md) assemblage bottom right of the plot
contains counts for Cymbella and Cyclotella
species of around 44% and 67% respectively
less than lh5 (c), however, has about 57% more
Anomoeoneis species in comparison to lh5 (c).
Nevertheless, the nature of variance found in
these diatom assemblages is clearly not a result of
selective preservation during cleaning protocols.
Rather, this type of variance can be best explained
as an artefact of random species frequencies
encountered during the counting procedure.
Although, the samples used were limited to two
coastal sediment sites they varied in composition
including sand-, silt- and clay-based materials.
During the counting of diatom assemblages, it
was found that a higher number of clay particles
occurred in some of the microwave-digested sam-
ples, nevertheless, the clarity of slides from both
methods were comparable. It was noted that in the
microwave digested samples the density of non-
diatom material was higher hence an extra transect
had to be counted to reach a total number of 300
diatom frustules. However, we suggest that care
taken during slide preparation appears to be far
more important in determining the quality of slides
produced, than the method of digest used.
The time taken for the conventional method of
diatom extraction took up to 6 days due to the time
required for settling between rinses. Hence this
method can be very time consuming and limiting
if laboratory space, fume hood space and time are
critical factors for the efficient operating of the
laboratory. Alternatively, the microwave digestion
method enables up to 40 samples to be completely
processed within 1 day. However, the PerkinElmer
Multiwave Microwave Sample Preparation System
used in this study is limited to batches of six sam-
ples, therefore, making this method more labour
intensive during the actual processing operation.
The microwave digestion technique also uses
considerably less volume of chemical per sample,
therefore, lowering the processing cost and the use
of consumables.
Table 2. Relative abundance of diatom species from Lake Hiawatha and Tuckean Swamp after processing with the conventional (c) method of Battarbee (1986) and the microwave
digestion method (md).
Lake Hiawatha LH1 (c) LH1 (md) LH2 (c) LH2 (md) LH3 (c) LH3 (md) LH4 (c) LH4 (md) LH5 (c) LH5 (md) LH6 (c) LH6 (md)
Anomoeoneis brachysira 45.00 50.51 41.11 44.79 43.00 45.95 66.08 65.57 33.53 58.75 56.52 53.74
Cyclotella strategera 13.00 12.46 15.56 12.68 7.63 7.57 7.60 12.02 8.75 3.44 9.24 4.01
Cymbella pusilla 20.00 26.60 19.44 29.01 30.28 25.85 14.04 14.21 42.86 23.75 24.73 32.62
Eunotia soleirolii 4.00 3.37 3.89 0.56 1.78 1.57 1.75 1.64 2.04 0.31 1.90 2.14
Frustulia rhomboides 7.00 4.38 8.06 7.04 9.67 10.70 8.77 3.28 8.45 4.69 4.89 5.08
Navicula radiosa 0.00 1.68 0.83 2.25 4.83 5.48 0.58 1.64 0.29 2.81 0.54 0.27
Stauroneis pachycephala 1.00 0.00 1.67 0.00 0.76 0.00 0.00 0.55 2.92 4.06 0.54 0.27
Tuckean Swamp TS1 (c) TS1 (md) TS2 (c) TS2 (md) TS3 (c) TS3 (md) TS4 (c) TS4 (md) TS5 (c) TS5 (md) TS6 (c) TS6 (md)
Actinocyclus normanii 0.00 0.97 4.42 2.96 3.72 2.07 4.41 4.24 16.44 17.57 23.61 24.51
Amphora veneta 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 2.35 0.00 2.95 2.29
Cyclotella meneghiana 4.64 1.94 82.02 79.56 78.02 73.96 71.63 76.27 21.48 31.76 9.84 9.15
Diploneis smithi 0.00 1.62 8.52 9.85 8.36 10.95 7.44 7.63 22.82 10.81 27.87 30.07
Eunotia flexuosa 42.90 27.83 0.32 0.25 0.00 0.00 0.55 0.00 1.34 0.00 0.00 0.00
Eunotia soleirolii 16.94 17.15 0.00 0.25 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
Fragilaria construens 0.00 0.00 0.00 0.00 0.00 0.00 3.86 0.00 9.40 11.82 6.56 7.19
Hantzschia amphioxys 27.32 34.95 0.63 0.74 0.00 0.30 1.10 0.00 0.34 0.00 0.66 0.00
Melosira granulata 1.09 2.91 0.00 0.00 0.00 0.00 0.55 0.28 2.68 4.73 1.31 1.96
Nitzschia sigma 1.91 3.24 0.00 0.49 0.31 0.59 0.55 0.00 0.67 0.68 0.00 0.65
Pinnularia gibba 2.46 2.59 0.00 0.25 0.00 0.00 0.28 0.00 1.01 0.00 0.00 0.00
Pleaurosigma angulatum 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.34 5.90 4.90
Rhopalodia musculus 1.09 0.97 2.84 4.19 7.74 10.06 7.16 10.17 19.80 20.61 18.36 15.36
387
388

Figure 1. Summary plots for MDS analysis of Lake Hiawatha showing 0.93 of total variance and differences between the lh3 and lh6
samples of each method.

Figure 2. Summary plots for MDS analysis of Tuckean Swamp showing 0.99 of total variance and the close relationship of diatom
assemblages from both methods.

The results show that processing by the micro-
wave digestion technique is faster than the con-
ventional method for extracting diatoms from
sediments. There are fewer steps in the microwave
digestion protocol, thus limiting the opportunity
for accidents and contamination of samples during
processing. Samples do not need monitoring dur-
ing digestion, freeing up time for the operator to
pursue other tasks. Importantly, smaller quantities
of chemicals are required for diatom extraction
making it a significantly cheaper process to use.
Finally, the digested component of the residue is
suitable for analysis by ICP/MS or AAS for the
interpretation of environmental and sedimentary
changes over time (Parr and Farrugia 2003). The
main disadvantage of the microwave digestion pro-
tocol is that the initial outlay for the system is
expensive. Although, many universities and com-
mercial laboratories involved in the chemical anal-
ysis sediments already have these systems as
standard preparation equipment by ICP/MS and
AAS.

Conclusions
This study has reported on an alternative technique
for the extraction of diatoms from lake and swamp
sediments. The results of this comparative assess-
ment demonstrated that both the conventional and
microwave digestion methods produced acceptable
numbers of diatoms for the analysis of assem-
blages. More importantly, it was found the micro-
wave technique is fast, inexpensive and most
importantly results in comparable assemblage
data to that of conventional diatom extraction
methods. Therefore, depending upon the personal
preference of the practitioner, the number of sam-
ples to be processed, and the demand for fume
hood space, the microwave digestion technique is
a viable alternative that will produce similar results
within a shorter period of time.
Acknowledgements
This research was conducted under a New South
Wales National Parks and Wildlife Service permit
(permit no. G99/01 and A3004) to Taffs. Parr is
supported by an Australian Postgraduate Award
and a Postgraduate Award Scholarship from
the Australian Institute of Nuclear Science and
Engineering. The Environmental Analysis
Laboratory, Southern Cross University, has sup-
ported this project by providing access to equipment.
In addition, we wish to acknowledge the advice
and assistance of John Arthur, Barbara Harrison,
Graham Lancaster, Greg Luker, Jenny Nolan,
Ken O’Brien and Loraine Watson of Southern
Cross University. Finally, we wish to thank resident
statistician Lyndon Brooks, also of Southern Cross
University for advice on statistical procedures.
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