Download Cleaning of Marine Sediment Samples for Large Diatom Stable

Journal of Earth Science, Vol. 23, No. 2, p. 161–172, April 2012
Printed in China
DOI: 10.1007/s12583-012-0241-x
ISSN 1674-487X
Cleaning of Marine Sediment Samples for Large
Diatom Stable Isotope Analysis
Zhifang Xiong (熊志方), Tiegang Li* (李铁刚)
Key Laboratory of Marine Geology and Environment, Institute of Oceanology,
Chinese Academy of Sciences, Qingdao 266071, China
Xavier Crosta
UMR-CNRS 5805 EPOC, Université Bordeaux 1, Avenue des Facultés, Talence Cedex 33405, France
ABSTRACT: Diatom stable isotope analysis offers considerable potential in palaeoceanography, particularly where carbonate material is scarce or absent. However, extracting pure diatom frustules free
of external labile organic matter from marine sediments is an essential requirement for their applications as paleoenvironmental proxies. Here, based largely on previous work, we developed a method including physical separation and chemical oxidation steps to concentrate and clean pure large diatoms
from laminated diatom mat and diatomaceous clay sediment samples for their stable isotope analysis.
Using the physical separation techniques consisting of the removal of carbonate and excess organic
matter, sieving, differential settling, and heavy liquid floatation, pure diatoms can be successfully isolated from the sediment samples with opal concentration more than 10%. Subsequent time oxidation
experiment shows that labile organic matter coating pure diatom valves can be effectively removed with
30% H2O2 at 65 ℃ for 2 h. Measurements of δ13C after every step of physical separation demonstrate
that contaminants and lost diatoms can influence the original diatom stable isotope signal, highlighting
the importance of a visual check for dominant diatom size in the initial sample and purity in the final
sample. Although the protocol described here was only applied to diatom mats or diatom oozes containing large diatoms (Ethmodiscus rex), we believe that this method can be adapted to common diatoms of general marine sediment samples.
KEY WORDS: large diatom, stable isotope, physical separation, chemical oxidation, Parece Vela basin,
palaeoceanography.
This study was supported by the National Natural Science
Foundation of China (No. 40776031) and the National Fundamental Research and Development Planning Project (No.
2007CB815903).
*Corresponding author: [email protected]
© China University of Geosciences and Springer-Verlag Berlin
Heidelberg 2012
Manuscript received June 2, 2011.
Manuscript accepted September 22, 2011.
INTRODUCTION
Marine diatoms have been widely used as key
archives to trace paleoenvironmental conditions due to
their important role in global carbon fixation and consequent significant contribution to total ocean primary
production (Nelson et al., 1995). Among the geochemical proxies regarding diatoms, palaeoceanographic application of four stable isotope ratios is a
rapidly expanding field, including carbon and nitrogen
isotopes of diatom-bound organic matter (δ13Cdiatom
and δ15Ndiatom) and silicon and oxygen isotopes of
diatom silica (δ30Sidiatom and δ18Odiatom). These
162
opal-based isotopic proxies have great potential to reconstruct palaeoceanographic parameters, such as
dissolved CO2 concentration in seawater, oceanic primary productivity, nutrient (i.e., nitrate and silicic acid)
relative utilization patterns, sea-surface temperature,
and oxygen isotopic composition of seawater (Leng et
al., 2009; Swann and Leng, 2009; Crosta and Koç,
2007; De La Rocha, 2006; Sigman et al., 1999;
Shemesh et al., 1993). However, the basic prerequisite
for successfully interpreting the four isotope ratios in
palaeoceanography is the extraction of pure diatoms
from bulk marine sediments and subsequent removal
of labile organic matter coating isolated diatom valves.
This is because a small amount of contaminants (e.g.,
silt, clay, tephra, radiolarian, and excess and labile organic matter) can have a large effect on the diatom
stable isotopic compositions (Tyler et al., 2007; Morley et al., 2005, 2004; Crosta et al., 2002; Sigman et
al., 1999; Singer and Shemesh, 1995). Therefore, to
obtain the real signal of diatom stable isotopes, physical separation and chemical oxidation techniques are
used to separate the diatom fraction from bulk sediments and remove labile organic matter on the exterior
of diatom frustules, respectively.
Some attempts have been made to develop the
physical separation methods (see review in Swann and
Leng, 2009; Leng and Barker, 2006), which involve a
range of chemical and physical preparation steps, such
as excess organic matter and carbonate removal, sieving, differential settling, heavy liquid separation, and,
more recently, gravitational split-flow thin fractionation (SPLITT), an alternative approach to heavy liquid
separation (Swann et al., 2006; Crosta et al., 2005;
Morley et al., 2004; Rings et al., 2004; Sigman et al.,
1999; Shemesh et al., 1995, 1988; Juillet-Leclerc,
1986; Labeyrie and Juillet, 1982). Although Morley et
al. (2004) devised a series of structured clean-up
stages to isolate diatom silica from lake sediments (see
Morley et al., 2004 and Fig. 3 therein), at present, the
diatom physical separation technique for marine
sediment samples has not yet to be standardized.
Several oxidative reagents, including perchloric/
nitric mixture (Shemesh et al., 1993), hydrochloric/
peroxidic solution (Crosta et al., 2002; Singer and
Shemesh, 1995), perchloric acid (72% or 55%) (Bruelle et al., 2007; Sigman et al., 1999), 30% peroxide
Zhifang Xiong, Tiegang Li, Xavier Crosta
(Robinson et al., 2005, 2004), and sulfochromic solution (Hatté et al., 2008), have been routinely employed
to remove labile organic matter coating diatom frustules during the chemical oxidation procedures. Nevertheless, there are some uncertainties and debates
about the effect of a given oxidative reagent, suggesting that a single chemical oxidation treatment may not
be suitable for all sediment types. For example, Sigman et al. (1999) suggested that nitric acid represented
a potential source of contamination for δ15Ndiatom;
conversely, the time oxidation experiment performed
by Crosta et al. (2002) demonstrated that nitric acid
did not bias the δ15Ndiatom record. Having demonstrated that 72% perchloric acid in the published protocol of Sigman et al. (1999) could go beyond removing the external organic material and began to attack
diatom-intrinsic organic matter, Robinson et al. (2005,
2004) adopted 30% peroxide instead. However,
Brunelle et al. (2007) illustrated that the 30% peroxide
cleaning protocol was not be sufficient for clay-rich
sediments and therefore replaced it with 55% perchloric acid. To sum up, the optimal chemical oxidation
conditions should be confirmed for each set of samples by a combination of trial and error.
Laminated diatom mats (or oozes) formed by
large or giant diatoms are known from some sporadic
sites in the world ocean and they play an important
role in ocean export production (Kemp et al., 2006
and references therein). However, their significance in
global carbon and silicon cycles has not heretofore
been sufficiently investigated. The use of four stable
isotope ratios of individual large diatom species as indicators of past palaeoceanographic change will evidently enhance our understanding of this issue. Here,
based largely on previous work, we describe the development of such an approach for the cleaning of
diatom frustules from marine sediments as a preliminary step for paleoceanographic interpretations. Although discussed here in association with large diatoms, the method has the potential to be applied to
common diatom species.
EXPERIMENTS
Sample Descriptions
Eight sediment samples were chosen from the
northwest (15°N–21°N, 136°E–140°E) of Parece Vela
Cleaning of Marine Sediment Samples for Large Diatom Stable Isotope Analysis
basin of the eastern Philippine Sea, with an average
water depth of 4 800 m. These samples were selected
on the basis of their different compositions and origins
to assess the applicability of our method to general
marine sediment samples. These samples, with opal
concentration ranging from ~10% to ~70%, could be
classified into two groups: diatom mat samples and
diatomaceous clay samples (Table 1). The diatom mat
samples are dominated by fragmented valves of large
(and mat-forming) diatom Ethmodiscus rex in
near-monospecific assemblages with extremely low
abundances of other diatom species and radiolarian
Table 1
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(Zhai et al., 2009). The diatomaceous clay samples are
characterized by fewer diatoms and more detrital materials and radiolarian. The diatom assemblages in
diatomaceous clay samples are more heterogeneous
than those in diatom mat samples.
All samples exhibit distinct sizes for diatoms and
contaminants, with >50 µm of E. rex valve fragments,
<60 µm of other diatoms, <140 µm of radiolarian, and
>30 µm of detrital grains. AMS14C dates indicate the
investigated samples formed during the late Wisconsinan glacial stage (mainly during the last glacial
maximum) (Table 1).
Characteristics of sample used to carry out the experiment of diatom cleaning from
marine sediment samples of Parece Vela basin, eastern Philippine Sea
Depth (m)
Lithology
Opal (%)
TOC (%)
Age (14C a B.P.)
WPD-03-40
2.03
Diatom mats
71.4
0.249
24 117
WPD-03-70
2.63
Diatom mats
61.7
0.195
25 725
WPD-03-22
1.28
Diatom mats
53.0
0.285
20 671
WPD-03-48
2.19
Diatom mats
51.7
0.231
24 546
WPD-03-99
3.21
Diatomaceous clays
40.9
0.209
27 279
WPD-03-62
2.47
Diatom mats
26.9
0.203
25 296
WPD-03-88
2.99
Diatomaceous clays
17.3
0.223
26 689
WPD-03-104
3.31
Diatomaceous clays
9.0
0.149
27 546
Sample
Cleaning Procedures
Physical separation
Large diatom shells were physically separated
from the sediment samples described above using a
four-step protocol adapted from the combination of
techniques described by Shemesh et al. (1995, 1988),
Sigman et al. (1999), Morley et al. (2004), Swann et al.
(2006), and Hatté et al. (2008). Remarkably, the individual diatom species (E. rex) was successfully isolated from the samples with our physical separation
technique.
Step 1 (Removal of carbonate and excess organic matter) Approximately 10 g of bulk wet
sediments (named Step 0) were weighed into 300 mL
polypropylene bottle and then oxidized with 100 mL
of 10% H2O2 at 60 ℃ in bath for 4 h. Subsequently,
50 mL of 1 mol/mL HCl were added to the bottle and
settled overnight. The supernatant was decanted and
the slurry was subsequently rinsed two times with distilled and deionized water (DDW).
Step 2 (Sieving) The sediment slurry was
wet-sieved to obtain the fractions of 63–154 and >154
µm using 63 and 154 µm steel meshes, which were
chosen by comparing the size of large diatoms with
that of nondiatom material (e.g., silt, clay, and radiolarian) under the light microscope. The other diatoms,
clay, and smaller detrital grains (e.g., silt) were removed in this step.
Step 3 (Differential settling) The two size
fractions (63–154 and >154 µm) were placed into two
separate centrifuge tubes and then centrifuged at 1 500
rpm for 4 min to remove the detrital grains whose
sizes are close to those of large diatoms. Afterwards,
the top diatom layer was carefully removed with a pipette, while the bottom remainder of each fraction was
repeatedly mixed and centrifuged until the majority of
large diatoms were concentrated and retrieved with a
pipette.
Step 4 (Heavy liquid flotation) The two subsamples from Step 3 were placed into two centrifuge
164
tubes with 6 mL of 2.3 g/mL sodium polytungstate
(SPT), mixed, and centrifuged at 2 500 rpm for 20 min.
This resulted in large diatoms predominantly suspending onto the heavy liquid, while denser detrital
grains sank to the bottom of the centrifuge tubes. The
floating diatoms were decanted into a second tube,
while the remainder was repeatedly separated using
the SPT procedure until the majority of the large diatoms were removed. As the diatom fraction decreases
in the subsamples during repetitions of Step 4, the
density discrimination between large diatoms and
contaminants is gradually reduced. Consequently, this
step removed detrital grains with density approaching
that of large diatoms and should be carefully inspected.
The purified 63–154 and >154 µm diatom fractions
were then sieved at 10 µm to remove SPT and finally
dried at 45 ℃ for 24 h.
Chemical oxidation
To compare and assess the efficiency of chemical
oxidation treatment, we tested three different oxidative
Zhifang Xiong, Tiegang Li, Xavier Crosta
solutions: 30% H2O2, HClO4+HNO3, and 55% HClO4.
Due to large sample requirements, several purified
samples of >154 µm fraction were mixed and homogenized well as starting material. The starting material was further divided into 24 subsamples, from
which each 8 subsamples were treated by the three
acid solutions mentioned above, respectively, at 65 ℃
for a time period spanning 0 to 10 h. After chemical
oxidation, each subsample was rinsed with DDW,
dried, and analyzed for its carbon stable isotope composition.
Estimation of Sample Purity
The technique’s efficiency was assessed at each
step of the protocol described above (including physical separation and chemical oxidation) by visual inspection of diatom size fractions under both light microscope and scanning electron microscopy (SEM).
Photos of the subsamples were taken at each step under light microscope and SEM to assess subsample
purity.
Figure 1. Light microscope photos showing the identification of diatoms and contaminants and the calculation of their surface area. The blue and red colors mark the contaminants (b) and (d) and diatoms (e). Their
surface area was calculated using the image processing software (Leica Qwin pro) described in the text.
Cleaning of Marine Sediment Samples for Large Diatom Stable Isotope Analysis
The estimation of sample purity is based on the
surface area of diatoms and contaminants. Ten representative photographs (515×386 µm) were taken of
each epoxy-coated slide at ×200 magnification using
the imaging system connected to a Leica DM4000 B
light microscope. These photographs were simplified
into two types to favor the determination of sample
purity: (1) off-white large diatom (E. rex) valve fragments fill the whole photograph as matrix, while
gray-brown or black contaminants are embedded in
them as grains (Fig. 1a), and (2) both off-white large
diatom valve fragments and gray-brown or black contaminants disperse in the photograph with grain forms
(Fig. 1c). The surface area of diatoms and contaminants in the photographs were calculated by the image
processing software (Leica Qwin pro). Their calculation methods are dependent on the photograph types.
For the first type of photographs, the contaminants
were identified by the “color detection” tool and their
area (or the percentage of their area in the whole photograph area seen as the summation of diatom and
contaminant areas) was further calculated with the
“area measurement” tool in the software Leica Qwin
pro (Fig. 1b). For the second type of photographs, the
contaminant area was similarly obtained by the “color
165
detection” and “area measurement” tools (Fig. 1d).
The diatoms were marked on the photograph using the
computer mouse and their area was calculated by the
“interactive measurement” tool (Fig. 1e). Finally, all
the sample purity can be calculated following
the formula: sample purity=diatom area/(diatom area+
contaminant area).
Analysis of Sample δ13C
The carbon isotope measurements of dry and
homogenized samples were performed on a Euro
Vector EA3000 elemental analyzer in line with a GV
IsoPrime isotope ratio mass spectrometer. Carbon isotope data were reported in the standard notation (δ13C),
relative to the Pee Dee Belemnite standard, expressed
in units of ‰, with an analytical precision (SD) better
than 0.1‰.
RESULTS AND DISCUSSION
Assessment of Physical Separation Efficiency
Two attempts are made to assess the efficiency of
every step to remove contaminants during the physical
separation, including (1) taking light microscope
and/or SEM photographs (Figs. 2–4) and (2) calculating the sample purity (Fig. 5) before and after every
Figure 2. Light microscope and SEM photos of sample (WPD-03-99) after each step of physical separation
showing the successful diatom isolation from marine sediments with the first three-step protocol. S0, the
initial sample; S1–S3, the sample after steps 1 to 3 of physical separation.
166
Zhifang Xiong, Tiegang Li, Xavier Crosta
Figure 3. Light microscope photos of sample (WPD-03-88) after each step of physical separation exhibiting
the successful diatom isolation from marine sediments with the four-step protocol. S0, the initial sample;
S1–S4, the sample after steps 1 to 4 of physical separation.
Figure 4. Light microscope photos of sample (WPD-03-104) after each step of physical separation showing
the unsuccessful diatom isolation from marine sediments with the four-step protocol. S0, the initial sample;
S1–S4, the sample after steps 1 to 4 of physical separation.
step of physical separation for both diatom size fractions. These photographs and sample purity data, in
combination with our ongoing diatom-cleaning work,
suggest that samples with opal concentration >20%
can be successfully purified from marine sediments
using the first three-step protocol (steps 1–3) (Figs. 2
and 5a–5f), while samples with opal concentration
ranging from 10% to 20% can be isolated by the
four-step protocol (steps 1–4) (Figs. 3 and 5g). Conversely, the samples with opal concentration <10%
cannot be totally purified by the physical separation
technique described here (Figs. 4 and 5h). All the
samples successfully separated by our method have
final diatom content in excess of 95% with almost no
visible detrital grains in the final samples. In addition,
the sample purity of purified >154 µm diatom fraction
is slightly higher than that of 63–154 µm fraction
(Figs. 5 and 6) due to the presence of unremoved trace
radiolarian in the 63–154 µm fraction.
For diatom-rich samples purified by three-step
protocol, our results indicate that sieving (step 2) is
the more efficient step of the protocol whereby accounting for ~50% to 90% of total diatom content increase (Figs. 5a–5f). For diatom-poor sample isolated
by the four-step protocol (i.e., WPD-03-88), the separation efficiency of every step depends on the sieved
fraction sizes. Steps 2 and 4 in the >154 µm fraction
and steps 3 and 4 in the 63–154 µm fraction are the
most effective, respectively (Fig. 5g). An interesting
and puzzling issue is that Step 4 in the diatom-poorest
Cleaning of Marine Sediment Samples for Large Diatom Stable Isotope Analysis
sample WPD-03-104 slightly decreased the sample
purity (Fig. 5h). We speculate that Step 3 made the
density difference between remaining contaminants
and large diatoms so little that it could result in the
(a)
96
92
88
0
1
2
3
appearance of relatively more contaminants (mainly
radiolarian and detrital grains, see Fig. 4) in both size
fractions after Step 4, which would finally depress the
sample purity.
12
100
8
96
4
92
88
0
100
0
0
1
2
3
20
95
(e)
85
10
75
0
0
1
2
2
3
(d)
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8
4
0
0
1
2
3
20
95
(f)
10
85
75
0
0
1
2
3
80
100
(g)
(h)
70
50
0
1
88
3
40
90
Diatom contents (%)
80
0
92
Contaminant contents (%)
Diatom contents (%)
8
8
4
96
(c)
88
(b)
12
16
96
12
Contaminant contents (%)
100
167
1
2
Steps
3
20
60
0
20
40
0
0
4
1
2
Steps
3
Diatoms (bulk or >154 µm )
Diatoms (63-154 µm )
Contaminants (bulk or >154 µm )
Contaminants (63-154 µm )
4
Figure 5. The purity of sample (i.e., percentage diatom and contaminant content) after each step of physical
separation using the first three- or four-step protocol. (a) WPD-03-40 (71.4% opal); (b) WPD-03-70 (61.7%
opal); (c) WPD-03-22 (53.0% opal); (d) WPD-03-48 (51.7% opal); (e) WPD-03-99 (40.9% opal); (f)
WPD-03-62 (26.9% opal); (g) WPD-03-88 (17.3% opal); (h) WPD-03-104 (9.0% opal).
95
90
85
25%
10%
Diatom contents (%)
100
Diatoms (>154 µm )
Diatoms (63-154 µm )
80
75
0
20
40
Opal (%)
60
80
Figure 6. Comparisons of physical separation efficiency for the sample with different opal concentrations.
The physical separation efficiency is not solely
dependent on the opal concentration of the initial
sample. Separation efficiency in the samples with opal
concentration between 10% and 25% clearly depends
on the diatom and radiolarian relative contents. In that
vein, the smaller fraction (63–154 µm) presents a better separation efficiency than the larger fraction as a
result of fewer radiolarians present in the former fraction. For the samples with opal concentration >25%,
our results indicate that diatoms represent the main siliceous fraction in agreement with radiolarian being a
minute component of plankton. In that case, the separation efficiency is nearly identical in the 63–154 and
>154 µm size fractions (Fig. 6).
Zhifang Xiong, Tiegang Li, Xavier Crosta
168
Our success of separating large diatoms from
marine sediment samples also indicate that the
four-step protocol described by Morley et al. (2004) is
not only valid for lacustrine samples but also suitable
for marine samples. Swann et al. (2006) suggested that
the first three-step protocol is particularly ideal for the
marine samples dominated by large diatoms and containing low diatom species diversity. The successful
physical separation for diatom mat samples with the
first three-step protocol, assessed here visually
through the calculation of sample purity over each
step of the protocol, supports the observation of
Swann et al. (2006).
Influence of Physical Separation on Diatom δ13C
The carbon of initial samples (in the Step 0)
comprises inorganic carbon, excess organic carbon
(unrelated to diatom), labile organic carbon coating
diatom frustules, and organic carbon occluded within
the matrix of diatom. The δ13C of samples after Step 1
generally shows a moderate decrease compared to that
of initial samples (Fig. 7), consistent with the result of
time oxidation experiments carried by Singer and
Shemesh et al. (1995) and Crosta et al. (2002). This
decrease in δ13C may be due to the removal of marine
inorganic carbonate and diagenetically altered excess
organic matter, both enriched in 13C relative to fresh
marine organic matter (Coplen et al., 2002). The oxidation step (H2O2 and HCl in Step 1) is assumed to
completely remove excess organic carbon and inorganic carbon, leaving the sample with some labile organic carbon and diatom-bound organic carbon.
(a)
d 13C (‰)
d 13C (‰)
-20
-18
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-22
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(c)
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d 13C (‰)
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(b)
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(f)
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d 13C (‰)
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(h)
-24
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1
2
Steps
3
4
Sediments or fractions (＞154 µ m )
0
1
2
Steps
3
4
Sediments or fractions ( 63 - 154 µ m )
Figure 7. The δ13C after each step of physical separation using the first three- or four-step protocol. Note
that several data in Figs. 7d and 7f–7h are absent due to sample mass limitations for δ13C determination. (a)
WPD-03-40 (71.4% opal); (b) WPD-03-70 (61.7% opal); (c) WPD-03-22 (53.0% opal); (d) WPD-03-48
(51.7% opal); (e) WPD-03-99 (40.9% opal); (f) WPD-03-62 (26.9% opal); (g) WPD-03-88 (17.3% opal); (h)
WPD-03-104 (9.0% opal).
Cleaning of Marine Sediment Samples for Large Diatom Stable Isotope Analysis
Indeed, we observed the greatest δ13C decrease after
Step 1 (Fig. 7), although the decrease measured here is
much less than the 10‰ drop observed for the Southern Ocean sediments (Crosta et al., 2002) possibly
because of the investigation of better preserved
near-monospecific diatom assemblages.
The carbon content of all subsamples in steps 2
to 4 is mainly related to diatoms, suggesting that any
change in δ13C in steps 2 to 4 may be linked to processes affecting diatom size distribution and/or preservation. Furthermore, considering that diatom assemblages are here near-monospecific (i.e., E. rex) in steps
2 to 4, changes in δ13C cannot be related to species-specific vital effects. Conversely, providing that
the size of E. rex fragments presents a positive correlation with the size of intact E. rex specimens, we believe that changes in δ13C result from size-related
carbon isotope fractionation ascribed to the size variation of diatoms by the sieving and settling effects.
This is further supported by different δ13C values between the two diatom size fractions analyzed here (Fig.
7). Size-related vital effects are also known to impact
the δ18Odiatom of lacustrine diatoms (Swann et al., 2008,
2007). For the >154 µm fraction, the samples can be
compiled into three groups in terms of opal concentration range, within each of which they present a similar
change trend of δ13C: group 1 (WPD-03-40,
WPD-03-70, and WPD-03-22), group 2 (WPD-03-48
and WPD-03-99), and group 3 (WPD-03-62 and
WPD-03-88). Unlike the >154 µm fraction, the samples of 63–154 µm fraction do not exhibit systematic
change trend of δ13C in relation to opal content, indicating that the composition of 63–154 µm fraction is
less stable than that of >154 µm fraction, possibly in
relation to greater contaminant content (Fig. 5).
Evaluation of Chemical Oxidation Efficiency
The large diatoms purified by physical separation
are still coated by labile organic matter (Figs. 8b and
8c). This coating interacts with the marine environment during settling and burial and is therefore prone
to early diagenesis enrichment in 13C and 15N, which
has a substantial impact on the interpretation of diatom stable isotopes. Three oxidative solutions are
proposed here to test whether they can effectively remove the labile organic matter without altering the
169
silica matrix and the organic matter therein. The oxidation time-course experiment shows that the δ13C of
purified diatom samples treated with H2O2,
HClO4+HNO3, and HClO4 at 1 h have similar values,
which are still maintained until 3 h (Fig. 8a). This observation suggests that the H2O2 in the Step 1 of
physical separation not only completely removed excess organic matter but also removed a majority of labile organic matter. After 3 h, the δ13C of samples oxidized by three oxidative solutions show similar
time-dependent patterns but with a large scatter in the
absolute values. A slight increase in δ13C is measured
at 4 h and is followed by a rapid decrease until 10 h.
The decrease is less pronounced when H2O2 is the
oxidative reagent and δ13C stabilizes at around the
early oxidation stage value (Fig. 8a). Conversely, there
is a strong decrease in δ13C with the other oxidative
reagents possibly related to their strong corrosion capacity. At 8 h, the SEM photographs of samples
treated with the three oxidative solutions reveal that
the diatom frustules are characterized by “corrosional
holes” (Figs. 8e–8g), indicating that the three oxidative solutions attack the diatom frustules, which results in partial release of diatom-bound organic matter
(Hatté et al., 2008) and consequently leads to carbon
isotope fractionation (Robinson et al., 2004). It here
seems that H2O2 presents a lesser corrosion capacity
than the other reagents. Based on the above discussion,
the reasonable oxidation time should be fixed within 3
h. At 2 h, the three oxidative solutions yield similar
results of δ13C and can effectively remove all of the
labile organic matter (Figs. 8a and 8d), suggesting that
they are appropriate oxidative reagents for chemical
oxidation. Considering the use of more hazardous
HClO4 and HNO3 than H2O2, and the potential influence of HNO3 on δ15Ndiatom (Sigman et al., 1999), we
recommend the following condition of chemical oxidation: 30% H2O2 at 65 ℃ for 2 h.
Significance of the Cleaning Method
Our method, when applied to specific diatom
mats, allows for the extraction of individual diatom
species from sediment samples, which will greatly
improve the use of diatom stable isotopes as palaeoceanographic proxies. It is the first attempt to obtain
monospecific diatom (E. rex) samples. Because
170
monospecific diatoms are much less impacted by differential species-specific vital effects, this will provide
a direct picture of the surface signal. For example, we
measured the δ13Cdiatom of >154 and 63–154 µm diatom (E. rex) fractions extracted from a sediment core
consisting of laminated E. rex diatom mats (LDM),
diatomaceous clay (DC), and pelagic clay (PC), located in the Parece Vela basin of the eastern Philippine
Sea. The downcore δ13Cdiatom records of the two diatom (E. rex) size fractions display an abrupt ~6‰ to
Zhifang Xiong, Tiegang Li, Xavier Crosta
~7‰ enrichment at the transition from DC to LDM,
while the δ13Corg record from bulk sediment demonstrates a gradual ~2‰ increase (Xiong et al., unpublished data). We suggest that δ13Cdiatom better captures
environmental changes than δ13Corg during the DC to
LMD transition because of reduced different vital effects in near-monospecific E. rex fractions. The paleoceanographic implications will be discussed in another study.
Figure 8. δ13C curves showing the effect of oxidation time on carbon isotope composition of diatom-bound
organic matter using three different oxidative solutions and SEM photos showing the efficiency of chemical
oxidation with three different oxidative solutions.
Visual inspection was routinely undertaken to
ensure sample purity in previous studies (Crosta et al.,
2005; Morley et al., 2005; Shemesh et al., 1995), but
SEM control was seldom applied. We here show that
sample purity is never 100% even when the bulk material is near-monospecific diatom mats of E. rex and
when a very strict protocol is applied (Fig. 5), which
impacts the δ13Cdiatom values (Fig. 7). Additionally, we
here show that the size fraction of near-monospecific
assemblages yields different δ13C values. As such, two
critical outputs are obvious: (1) the contaminants can
considerably influence the original δ13Cdiatom signal
and (2) the diatom size fraction and the diatom preservation state have impact on the bulk δ13Cdiatom signal.
In that vein, one may question the influence of contaminants on the δ13Cdiatom signal of diversified assemblages of small diatoms (10–30 µm) presented in
previous investigations. As shown here, the smaller
the diatom fraction is, the greater the contaminant
content is (Fig. 5). Additionally, such assemblages are
composed of different species at different life stages
that present different fractionation factors. As shown
Cleaning of Marine Sediment Samples for Large Diatom Stable Isotope Analysis
here, even a given species yields different δ13Cdiatom
signals when different size fractions are investigated
(Xiong et al., unpublished data). Further tests are
needed to check whether and how contaminants and
diatom loss/alteration alter the original signals of
δ15Ndiatom, δ18Odiatom, and δ30Sidiatom.
It is noteworthy that the method described here
works best for sediment samples dominated by large
diatoms and is not directly transferable to other samples especially characterized by small diatoms with
high species diversity. However, our constructed
method framework should be applicable to common
diatoms in case of reconfirming the experiment conditions, such as reagent concentration, sieve size, centrifuge rate, settling time, and SPT density.
171
HClO4+HNO3 or HClO4 and an oxidation time of 2 h.
ACKNOWLEDGMENTS
Special thanks are owed to Rebecca Robinson
and George Swann for discussions on the diatomcleaning method and comments on an early draft.
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