Introduction
Mussels and mussel shells have increasingly gained importance as bioarchives
of proxies that record physicochemical changes in their marine or
freshwater habitat (Bau et al., 2010; Gillikin et al., 2006a; Merschel and
Bau, 2015; Puente et al., 1996; Vander Putten et al., 2000; Scourse et al.,
2006; Sturesson, 1976; Wanamaker et al., 2008). The chemical composition of
bivalve shells is known to contain a record of their past growth, based on
the sequential deposition of layers of mineralized material during their
lifetime (Lindh et al., 1988; Weiner, 2008; Wilbur and Saleuddin, 1983).
Hence, mussel shells may be valuable high-resolution bioarchives of past
marine, estuarine, fluviatile and limnic conditions.
Trace elements such as the rare earths and yttrium (REY) have been shown to
be useful indicators of environmental change (Bau and Dulski, 1996; Bau et al., 2010; Bolhar et al., 2004; Kulaksız and Bau, 2013; Lee et al.,
2003; Möller et al., 2000; Murray et al., 1990; Nothdurft et al., 2004; Tepe et al., 2014; Viehmann et al., 2014; Webb and Kamber, 2000; Wyndham et al., 2004).
The REY are a group of elements that are similar in atomic structure and
chemical properties and hence behave coherently in natural systems. Their
speciation in seawater and the distinct REY patterns exhibited by different
geological materials make them very useful as geochemical proxies of oceanic
change (Byrne, 2002; Byrne and Miller, 1985).
Other trace elements have also been shown to be incorporated into the shells
during annual layer formation and are assumed to be essentially immobile
(Lindh et al., 1988). Stable isotope studies of mussel shells and
particularly of Mytilus edulis corroborate the use of shells as paleoceanographic
bioarchives (Gillikin et al., 2006b; Wanamaker et al., 2006, 2007).
Various mussel species have already been used as environmental indicators to
monitor pollution and bioavailability of (micro)contaminants (Liang et al., 2004; Lindh et al., 1988; Merschel and Bau, 2015; Puente et al.,
1996; Sturesson, 1976; Wagner and Boman, 2004; Zuykov et al., 2013). However, the
focus so far has often been on major and minor elements such as Mg and Sr,
while REY data for mussel shells are still rather scarce and
underrepresented. Given the growing importance of mussels in proxy
development for the ocean–climate system including ocean acidification,
there is ample need to better understand these organisms and how they may
be used as bioarchives of trace elements and their isotopes.
Numerous studies have provided insights into the composition of ancient
seawater and the evolution of the environmental system based on the
distribution of rare earth elements (REEs) in chemical sediments
(Alexander et al., 2009; Alibert and McCulloch, 1993; Bau et al., 1997; Bau and
Alexander, 2006, 2009; Derry and Jacobsen, 1990; Nothdurft et al., 2004;
Viehmann et al., 2014, 2015; and references therein). Similarly, other
studies have demonstrated the potential of bivalve shells to track the
environmental conditions they were exposed to (Bau et al., 2010; Dunca
et al., 2005; Heinemann et al., 2011; Klein et al., 1996; McCoy et al.,
2011; Thébault et al., 2009; Weiner, 2008). Bau et al. (2010) have shown that
the positive Eu anomalies in the REY distribution patterns of the shells of
marine Bathymodiolus mussels can be used as tracers for hidden or fossil high-temperature
hydrothermal systems, while Merschel and Bau (2015) demonstrated that shells
of freshwater Corbicula mussels may be used to study the bioavailability of
anthropogenic REE microcontaminants. This already suggests that mussel
shells archive certain REY signatures of the environment in which they grow.
However, not much has been done to evaluate how accurately mussel shells
reflect REY patterns of seawater and what impacts their partitioning
behavior.
REYSN patterns of the ODAS seawater and of all pools of
Mytilus edulis shells from the ODAS site (ODAS I to III shell pools were treated with
NaOCl; ODAS IV to VIII shell pools were heated and had their periostracum
manually removed). Note the similarity between all REYSN patterns.
In this study, we approached this issue via an in situ culture experiment using
blue mussels, Mytilus edulis, which belong to the family Mytilidae and the phylum Mollusca.
This species is endemic in the Northern Hemisphere and can be found in
littoral and sublittoral zones. Blue mussels are tolerant to wide
temperature and salinity ranges (Seed, 1992), making them good model
organisms to study the aquatic environment. The M. edulis used in our study were
cultured offshore with no contact to the ocean floor, hence avoiding any
contamination from porewater or resuspended sediment.
We compared the REY distribution in M. edulis mussel shells and ambient seawater with
the aim to (i) establish a sample preparation and analytical protocol that
allows for the reliable and reproducible determination of the ultralow REY
concentrations in mussel shells, (ii) investigate which REY species is
incorporated into the shells of M. edulis, and (iii) illustrate and provide suggestions as
to how such shells may reflect the REY characteristics of ambient seawater
and how they can be used as environmental proxies. Results from this study
show that mussel shells can serve as bioarchives of the REY distribution in
their habitat and can thus provide the basis for using mussel shells as
bioarchives that host geochemical proxies for paleoceanographic
environmental reconstructions.
Materials and methods
Samples and sites
The mussels for this study originate from three locations in the North Sea
along the coast of the German Bight, namely (a) nearshore in the inner Jade in the Jadebusen (JD; 53∘35′05′′ N, 008∘09′14′′ E), (b) offshore at the
lighthouse Roter Sand (RS) near the entrance of the Weser estuary
(53∘51′00′′ N, 008∘04′20′′ E) and (c) offshore west of the island of
Sylt at the oceanographic data acquisition station (ODAS) site (OD; Messpfahl Süd/southern measurement pole:
54∘59′36′′ N, 007∘54′46′′ E; Fig. 1). The mussels at Jadebusen and Roter Sand settled on
suspended artificial spat collectors (harnesses made from polypropylene
ropes and plastic binders), while those at the ODAS site grew wild on a steal
pile from a 25-year-old research platform of the Federal Maritime and
Hydrographic Agency (BSH), formerly used to fix oceanographic measuring
instruments. The testing areas were specifically selected and designed to
have the mussels grow on suspended artificial substrate, which eliminates
any potential contribution of porewater- or sediment-derived REY. The
mussels from Jadebusen and Roter Sand were approximately 18 months old, while those from the
ODAS site were approximately 24 months old.
Shell preparation
Eight to eleven mussels from each site were pooled together based on the
sampling site and date. The mussels were lyophilized and the soft tissue was
removed leaving the shells intact. The obtained shell sizes ranged from 40
to 55 mm.
REYSN patterns of the four replicate pools of Mytilus edulis shells determined
in our study and of international reference standard JLs-1 (a Permian
limestone from Japan; data from our study and from Dulski, 2001) used for
analytical quality assessment during method development. Note that except
for La and Y, error bars (1 σ) are smaller than the symbol size.
The shells obtained from the ODAS site were categorized in different sample
pools (ODAS I-III and ODAS IV-VIII) to evaluate two different protocols for
the removal of the periostracum, i.e., of the outer organic layer that covers
the shell surface (Bellotto and Miekely, 2007). For the ODAS I-III shell
pools, the shells were soaked in NaOCl overnight before the shells were
rinsed several times with deionized water to remove remaining NaOCl and
then air-dried. For the ODAS IV-VIII pools, the shells were put in an oven
and the periostracum was then removed using a spatula. This difference in
sample preparation does not affect the analytical results (Fig. 2), and hence the
method using NaOCl is strongly recommended because it is much more
convenient and efficient, less time consuming and minimizes potential
contamination.
The bulk carbonate shells of each individual pool were then crushed in an
agate mortar and homogenized. One and a half grams of each shell powder were
digested for 2 h at 90 ∘C in 30 mL of 5 M Suprapur®
HNO3 (Carl Roth GmBH + Co.KG, Germany) in precleaned Teflon beakers
covered by small Teflon plates. After 2 h, the beakers were uncovered
and the sample solutions were evaporated to incipient dryness. The residues
were dissolved in 25 mL of 0.5 M HNO3 (Carl Roth GmBH + Co.KG,
Germany) and filtered into small polyethylene bottles using an
acid-cleaned 0.2 µm cellulose acetate filter and syringe. The
international reference standard JLs-1 (a Permian limestone from Japan) was
used as the certified reference material because it is similar in
composition to the carbonate shell matrix and contains low REY
concentrations.
A separation and preconcentration procedure (Bau et al., 2010), adapted from
a method used to determine REY in seawater and freshwater (Shabani et al.,
1992; Bau and Dulski, 1996), was utilized owing to the low REY concentrations
and potential matrix problems that may occur due to the Ca-rich shell
matrix. A 12 mL aliquot of the digested sample solution was diluted in 500 mL deionized water, acidified to a pH value between 1.8 and 2.0 with
Suprapur® HCl (Merck KGaA, Germany), and subsequently spiked
with 0.5 mL of a 100 ppb Tm solution to monitor the recovery rates of the
REY during the preconcentration procedure, while the remaining solution was
set aside and labeled as “digest”. A 15 mL aliquot of each filtered,
acidified and spiked sample was labeled as “original” and set aside to be
used to determine the reference concentration of the Tm spike.
REYSN patterns of Mytilus edulis shells from different sites in the North
Sea compared to seawater from the ODAS site. Note the close similarity
between all REYSN patterns regardless of their origin.
Each shell sample solution was then passed through a C18 cartridge
(Waters, Sep-Pak® Classic C18, single use) preloaded
with a 2-ethylhexyl phosphate ester (Merck KGaA, Germany) in order to
quantitatively retain the REY. Each cartridge was then “washed” with 0.01 M
Suprapur® HCl (Merck KGaA, Germany) to remove remaining matrix
elements, such as major alkali and alkali earth elements, before the REY were
eluted using 40 mL of 6 M Suprapur® HCl (Merck KGaA, Germany).
The eluate was then evaporated in a Teflon beaker to incipient dryness, and
the residue was eventually dissolved in 10 mL of 0.5 M
Suprapur® HNO3 (Carl Roth GmBH + Co.KG,
Germany).
A 100 ppb internal standard consisting of Ru, Re and Bi was added to each
sample solution. Procedural blanks and the JLs-1 reference standard were
processed along with the samples for analytical quality control.
Water sample preparation
A 1000 mL sample of North Sea water from the ODAS area was filtered using a
filter tower mounted with 0.2 µm membrane filters (Sartorius AG,
Germany). The pH of the filtrate was adjusted to 2.0 using
Suprapur® HCl (Merck KGaA, Germany). Furthermore, 0.4 mL of a
100 ppb Tm solution was added to monitor the recovery rates of the REY
during the subsequent separation and preconcentration procedure. 20 mL of
this solution was set aside as “original” to determine the Tm reference
concentration. A separation and preconcentration procedure similar to the
one used for the shell samples was then employed (see Sect. 2.2).
REY speciation in the North Sea water at 25 ∘C for (a) pH
8.2 and (b) pH 7.6 (as modeled using HySS2009).
Analysis
The sample solutions were analyzed for REY using an inductively coupled
plasma mass spectrometer, ICP-MS (Perkin-Elmer/Sciex ELAN DRC-e), at Jacobs
University Bremen. As 0.5 g of 100 ppb Tm had been added as an internal
standard to each sample solution to evaluate REY recovery during
preconcentration, Tm data for the shells are not reported.
Analytical quality assessment
To validate the method, the analytical precision was determined by applying
our analytical procedure of sample digestion, REY separation and
preconcentration, and measurement by ICP-MS to multiple aliquots (n= 4) of
homogenized M. edulis shells that were milled together to form a single large sample
pool and to an aliquot of the international reference standard JLs-1, which
is a REY-poor Permian limestone from Japan.
It is common practice to present REY data normalized to Post-Archean
Australian Shale, PAAS (as first done by McLennan, 1989; indicated here by subscript “SN”). The REYSN patterns for the multiple aliquots of homogenized M. edulis shells
for the quality assessment are presented in Fig. 3. Anomalies of CeSN,
GdSN and YSN have been quantified using Eq. (1a–c).
CeSNanomaly=CeSN/(2PrSN-NdSN)GdSNanomaly=GdSN/(0.33SmSN+0.67TbSN)YSNanomaly=YSN/HoSN
Precision (Fig. 3), expressed as relative standard deviation (RSD) from the
average, is < 4 % for Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Mo, Er and
Yb, < 9 % RSD for Lu, and < 34 % RSD for La. The reason
for the high RSD for La is unclear. Except for La, however, the
reproducibility is excellent and falls within the symbol size used in Fig. 3. The
analytical accuracy of the applied method was established from the JLs-1
values obtained and compared to published reference values from Dulski (2001), and it did not show any significant systematic difference between our
data and published values (Fig. 3).
Modeling of the speciation of REY
To get a better insight as to how REY behave during their incorporation into the shells of M. edulis, the inorganic speciation of REY in North Sea seawater at the
ODAS site was modeled complementing previous work on the REE speciation in
seawater by Byrne et al. (1988) by including Y. Following Byrne et al. (1988) and Millero (1992), modeling was done for pH 8.2 and pH 7.6 at
25 and 5 ∘C using the Hyperquad Simulation and
Speciation 2009 (HySS2009) modeling software. The inorganic speciation of
REY was modeled for REY3+ (as the free uncomplexed REY),
REY(OH)2+, REY(F)2+, REY(Cl)2+, REY(SO4)+,
REY(CO3)+, REY(CO3)2- and REY(HCO3)+.
Stability constants and ligand concentrations were obtained from Byrne
et al. (1988) and Millero (1992). The percentage of the REY complexes
relative to the total REY concentration was calculated using Eq. (2) (where the
brackets denote the dissolved concentration in seawater).
%Complex of Total REY={REY
Complex}{Total REY}×100
Discussion
Partitioning of REY
Apparent bulk distribution coefficients of REY between M. edulis shells and ambient
seawater, appDTot.REYshell/seawater, may be calculated from
Eq. (3):
appDTot.REYshell/seawater=[REY]/[Ca]Shell{Total REY}/{Ca}Seawater,
where Ca in seawater is 0.01 mol L-1 and Ca in shells is 10 mol L-1.
This equation has previously been used to calculate the distribution
coefficients of trace elements between the two major polymorphs of calcium
carbonate, namely calcite and aragonite, and ambient seawater (e.g., Akagi
et al., 2004; Sholkovitz and Shen, 1995). Shells of M. edulis are known to be
bimineralic, i.e., composed of the two polymorphs of Ca carbonate: calcite
and aragonite (e.g., Lorens and Bender, 1980). The apparent distribution
coefficients of REY between the shells and seawater from the ODAS site are shown
in Fig. 6. The appDTot.REYshell/seawater of our shells reveal
fractionation with a preferential uptake of LREY as compared to HREY from
seawater into the carbonate shell.
Certain differences and similarities are observed when published field and
experimental apparent bulk partition coefficients are compared with our
results (Fig. 7). A clear separation with regard to partition coefficients for
calcite and aragonite can be observed in the field observations, showing
that appDTot.REYshell/seawater values for corals of
aragonitic composition vary between approximately 1 and 10 (Akagi et al.,
2004; Sholkovitz and Shen, 1995), while those of calcitic composition such as
foraminifera, microbialites and other marine calcites, are much higher and
range between 70 and 1656 (Palmer, 1985; Parekh et al., 1977; Webb and
Kamber, 2000). Laboratory experiments exploring the partitioning of REE and
Y between calcite or aragonite and aqueous solutions have also been carried
out to elucidate the incorporation process. Terakado and Masuda (1988)
obtained values ranging between 2.5 and ∼ 10 for calcite and ∼ 2.5 and ∼ 5 for aragonite. Zhong and Mucci (1995) on the other hand
found much higher partition coefficients for their experimental calcite,
ranging from 4169 (Pr) to 794 (Lu), and fractionation between the LREY and
HREY. This pattern is quite similar to that of the M. edulis shells and ambient North
Sea seawater determined in our study, despite much lower absolute values
(between 4.23 for La and 0.17 for Lu). A study of Bathymodiolus puteoserpentis mussels that lived close
to a high-temperature hydrothermal system (Bau et al., 2010) indicate
partition coefficient values which define a pattern showing
preferential incorporation of the MREY and a decrease towards the HREY with
a maximum at Eu.
REY partition coefficients for different marine carbonates and
ambient seawater (field studies and laboratory experiments).
Comparisons of the appDTot.REYshell/seawater pattern of our
M. edulis shells shown in Fig. 6 with the REY speciation in North Sea seawater shown in
Fig. 5a and of the REYSN patterns of the ODAS shells with the free
REYSN3+ patterns of North Sea seawater (Fig. 8) suggest that (i)
free REY3+ may be the REY species which are actually removed from
seawater and incorporated into the M. edulis shell and (ii) scavenging of REY
carbonate complexes and formation of ternary surface complexes may only play
a minor role. Based on this hypothesis, we recalculated the apparent
partition coefficients following Eq. (4), using only the concentrations of free
REY3+ in North Sea seawater instead of the total REY concentrations:
modDFree REY3+shell/seawater=[REY]/[Ca]Shell{Free REY3+}/{Ca}Seawater.
The resulting new pattern of distribution coefficients (modeled mean modDFreeREY3+shell/seawater; Fig. 6) shows preferential incorporation of the MREY and suggests that Ce is not
taken up to the same extent as its redox-insensitive REY neighbors. However,
all other REY anomalies that are present in the shells and in ambient
seawater have disappeared, indicating only minor fractionation of
neighboring REY during removal from seawater.
Incorporation of REY into CaCO3 is assumed to occur through the coupled
substitution of a REY3+ plus a charge-balancing monovalent cation for
two Ca2+ ions in the calcite's crystal structure (Elzinga et al.,
2002; Zhong and Mucci, 1995), due to the similarity of the ionic radii of
REY3+ and Ca2+. Since the ionic radius of Nd3+ is most
similar to that of Ca2+, it may be expected that patterns of REY
partition coefficients show a maximum at Nd and decrease slightly towards
the lighter and heavier REY. However, the maximum in Fig. 6 for the modeled partition coefficient, modDFreeREY3+shell/seawater, occurs at
Tb, suggesting that additional factors besides ionic size also
affect the incorporation of REY into the carbonate shells of M. edulis.
Judging from Fig. 7 and considering that M. edulis shells are bimineralic, it would be
desirable to study REY distributions and partition coefficients for calcite
and aragonite individually and not for bulk mussel shells. However, the
ultralow REY concentrations and the intimate association of the two
carbonate minerals are severe limitations that prevent such data to be
determined. Thus, we have to accept that we are restricted to REY data for
bulk shell carbonate until microanalytical techniques such as laser ablation
ICP-MS have become more sensitive.
Total REYSN and free REYSN3+ patterns of North Sea
water at the ODAS site and the average REYSN pattern of the ODAS
shells.
In any case, the calculation of an apparent partition coefficient between a
mussel shell and ambient seawater is a severe simplification, of course.
From what is currently known, it appears that the shell of a bivalve does
not directly precipitate from seawater, but from the extrapallial fluid
(EPF) of the mussel, which is secreted from the epithelial cells of the
bivalve's mantle (Wilbur, 1972). The speciation of the REY in the EPF,
therefore, will also affect REY incorporation into Ca carbonate. Although
the exact chemical composition of the EPF is not known, the combination of
elevated concentrations of organic compounds such as (amino)carboxylic
acids, in the EPF (Misogianes and Chasteen, 1979; Weiner, 1979) and high
stability constants of REY complexes with such carboxylic acids (e.g.,
Martell and Smith, 1974) renders it very likely that REY speciation in the
EPF is rather different from REY speciation in seawater and that this
“vital” effect will affect REY incorporation into the shell. Available
thermodynamic data, however, suggest that carboxylic acids are often
characterized by REY stability constants that strongly increase from the
LREY to the HREY (Byrne and Kim, 1990; Martell and Smith, 1974) and thus
may produce similar LREY–HREY fractionation between the available REY3+
species in the EPF as the (di)carbonate complexes produced in seawater.
As the exact chemical composition of the EPF is not known, it is currently
impossible to decide whether the decrease in the REY partition coefficients
between M. edulis shells and ambient seawater with decreasing REY ionic radius is
controlled by the REY speciation in seawater or by the REY speciation in the
EPF. Nevertheless, in the following we will address the impact of seawater
pH and temperature on REY partitioning, assuming that REY speciation in the
EPF is of minor importance, because this will reveal whether or not the REY
distribution in M. edulis shells a priori has any potential to serve as a pH or temperature
proxy.
Modeled REYSN patterns of hypothetical M. edulis shells from the ODAS
site for different pH and temperature conditions in ambient seawater.
Impact of temperature and pH on REY patterns in Mytilus edulis
shells
Environmental parameters such as pH and temperature affect the speciation of
REY in seawater via their impact on the activity of CO32-, and
hence they impact the amount of free REY3+ available for uptake by the mussels
(Fig. 5). The REY signature of mussel shells, therefore, may be an indicator
of pH and/or temperature changes in a mussel's habitat, unless this proxy is
obliterated by the vital effects discussed earlier.
Using the partition coefficients calculated from Eq. (4) and the concentrations of
free REY3+ in seawater at pH 7.6 and 8.2 (as calculated using
the HySS software), we modeled, from Eq. (5), the REY concentrations and REYSN
pattern of a hypothetical M. edulis shell that grew in seawater of pH 7.6 and 8.2 and
at temperatures of 25 and 5 ∘C, respectively (Fig. 9).
[REY]Shell=modDFreeREY3+shell/seawater×{FreeREY3+}/{Ca}Seawater×[Ca]Shell,
where Ca in seawater is 0.01 mol L-1 and Ca in shells is 0.01 mol L-1.
At a given temperature, the shape of the resulting REYSN patterns of
such hypothetical shells are very similar at both pH values (Fig. 9), but due to
higher availability of free REY3+ in seawater at pH 7.6, more REYs are
incorporated into the Ca carbonate at pH 7.6 as compared to pH 8.2. In
contrast, at a given pH, a temperature change results in slightly different
REYSN patterns (particularly between the MREY and HREY) but only has a minor impact on overall REY concentrations (Fig. 9). Hence, it appears that
absolute REY concentrations may have the potential to be used as a pH proxy,
whereas REY distribution patterns are more sensitive to temperature changes.
Conclusions
A new and efficient protocol for sample preparation and determination of REY
concentrations in bivalve shells was established. This quick and clean
method includes sample treatment with NaOCl followed by REY separation and
preconcentration.
The shells of Mytilus edulis used in our study demonstrate the potential of using bivalve
shells as bioarchives of proxies for changes in the physicochemical
conditions in the bivalve's habitat. All shells from three different sites
in the southern North Sea show distinct REYSN distribution patterns
that increase from the LREY to the MREY and decrease from the MREY to the
HREY. Despite the REYSN patterns of the shells being different to that
of general seawater, the shells still exhibited the distinct signatures of the
seawater they grew in, such as small positive YSN and GdSN
anomalies and a negative CeSN anomaly. Apparent partition coefficients
appDTot.REYshell/seawater of REY between the shells and
seawater are low and decrease strongly from the LREY to the HREY. A comparison
of appDTot.REYshell/seawater patterns to the REY speciation
in the North Sea seawater suggests that the free REY3+ may be the most
likely REY species to actually be removed from seawater and incorporated
into the M. edulis shell and that scavenging of REY carbonate complexes and formation
of ternary surface complexes may only play a minor role.
Although the impact of vital effects and particularly that of REY speciation
in the extrapallial fluid from which the carbonate minerals precipitate
cannot be quantified yet, we demonstrate in this study that mussel shells
like those of M. edulis can still be used as bioarchives of some REY features of
seawater. Following our assumptions that only the free REY3+ are
incorporated into the carbonate's crystal lattice, further modeling of
the REYSN patterns of a hypothetical mussel shell grown at pH of 8.2
and 7.6 and at temperatures of 25 and 5 ∘C reveals
that, with lower pH, REY concentrations in shells increase, but with little
effect on the shape of the REYSN patterns, while a temperature change
has an impact on the REYSN pattern, but only minor effects on absolute
REY concentrations. The absolute REY concentrations in shells of M. edulis may thus
have the potential to be used as a pH proxy, whereas REYSN distribution
patterns of the shells may rather be used as a temperature proxy.
Our findings open up ways of better understanding how bivalves incorporate
trace elements like REY into their shells and how these shells can be used
to extract information about their habitat. Since changes in physicochemical
conditions like pH and temperature affect REY speciation in seawater due to
the impact of these parameters on the activity of CO32-, further research to
calibrate these changes may turn the REY distribution into a valuable proxy
for paleo-pH and past ocean acidification. However, in order to successfully
develop REY systematics into a quantitative temperature and/or pH proxy, the
impact of the EPF and other vital effects needs to be assessed, for example
by studying M. edulis mussels cultured under controlled pH and temperature
conditions. Additionally, before applying the REY distribution in fossil
shells as a paleoproxy, future studies need to investigate the potential
impact of diagenesis.