JAEA-Technology
2020-012
DOI:10.11484/jaea-technology-2020-012

Practical Guide on Soil Sampling, Treatment,
and Carbon Isotope Analysis for Carbon Cycle Studies
Jun KOARASHI, Mariko ATARASHI-ANDOH, Hirohiko NAGANO
Untung SUGIHARTO, Chakrit SAENGKORAKOT, Takashi SUZUKI
Yoko SAITO-KOKUBU, Natsuko FUJITA, Naoki KINOSHITA
Haruyasu NAGAI, Naishen LIANG, Hiroyuki MATSUZAKI
and Genki KATATA

Environment and Radiation Sciences Division
Nuclear Science and Engineering Center
Nuclear Science Research InstituteSector of Nuclear Science Research

October 2020
Japan Atomic Energy Agency

日本原子力研究開発機構

本レポートは国立研究開発法人日本原子力研究開発機構が不定期に発行する成果報告書です。
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なお、本レポートの全文は日本原子力研究開発機構ホームページ（https://www.jaea.go.jp）
より発信されています。
国立研究開発法人日本原子力研究開発機構 研究連携成果展開部 研究成果管理課〒319-1195 茨城県那珂郡東海村大字白方 2 番地4
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Tel +81-29-282-6387, Fax +81-29-282-5920, E-mail:ird-support@jaea.go.jp© Japan Atomic Energy Agency, 2020

JAEA-Technology 2020-012

Practical Guide on Soil Sampling, Treatment, and Carbon Isotope Analysis
for Carbon Cycle Studies
Jun KOARASHI, Mariko ATARASHI-ANDOH, Hirohiko NAGANO*1, Untung SUGIHARTO*2,
Chakrit SAENGKORAKOT*3, Takashi SUZUKI, Yoko SAITO-KOKUBU+1,
Natsuko FUJITA+1, Naoki KINOSHITA+2, Haruyasu NAGAI,
Naishen LIANG*4, Hiroyuki MATSUZAKI*5 and Genki KATATA*6
Environment and Radiation Sciences Division,
Nuclear Science and Engineering Center, Nuclear Science Research Institute,
Sector of Nuclear Science Research
Japan Atomic Energy Agency
Tokai-mura, Naka-gun, Ibaraki-ken(Received July 30, 2020)
There is growing concern that recent rapid changes in climate and environment could have a significant
influence on carbon cycling in terrestrial ecosystems (especially forest ecosystems) and could
consequently lead to a positive feedback for global warming. The magnitude and timing of thisfeedback remain highly uncertain largely due to a lack of quantitative understanding of the dynamics oforganic carbon stored in soils and its responses to changes in climate and environment. The tracing ofradiocarbon (natural and bomb-derived 14C) and stable carbon (13C) isotopes through terrestrial
ecosystems can be a powerful tool for studying soil organic carbon (SOC) dynamics. The primary aimof this guide is to promote the use of isotope-based approaches to improve our understanding of the
carbon cycling in soils, particularly in the Asian region.
The guide covers practical methods of soil sampling; treatment and fractionation of soil samples;
preparation of soil samples for 13C (and stable nitrogen isotope, 15N) and 14C analyses; and 13C, 15N, and14
C measurements by the use of isotope ratio mass spectrometry and accelerator mass spectrometry(AMS). The guide briefly introduces ways to report 14C data, which are frequently used for soil carbon
cycling studies. The guide also reports results of a case study conducted in a Japanese forest ecosystem,
as a practical application of the use of isotope-based approaches. This guide is mainly intended forresearchers who are interested but are not experienced in this research field. The guide will hopefully
encourage readers to participate in soil carbon cycling studies, including field works, laboratory
experiments, isotope analyses, and discussions with great interest.
Keywords: Radiocarbon (14C), Stable Carbon and Nitrogen Isotopes (13C and 15N), Global Carbon Cycle,
Soil, Forest Ecosystem, Sampling, Accelerator Mass Spectrometry (AMS)This guide was prepared through the project “Research on Climate Change using Nuclear and Isotopic
Techniques” organized by the Ministry of Education, Culture, Sports, Science and Technology, Japan(MEXT), under the framework of the Forum for Nuclear Cooperation in Asia (FNCA).
+1 Tono Geoscience Center+2 Aomori Research and Development Center*1 Post-Doctoral Fellow until March 2020
*2 National Nuclear Energy Agency of Indonesia*3 Thailand Institute of Nuclear Technology*4 National Institute for Environmental Studies*5 The University of Tokyo*6 Ibaraki UniversityiJAEA-Technology 2020-012

炭素循環研究のための土壌採取、処理、炭素同位体分析の実践ガイド

原子力科学研究部門

日本原子力研究開発機構
原子力科学研究所 原子力基礎工学研究センター
環境・放射線科学ディビジョン

小嵐 淳、安藤 麻里子、永野 博彦*1、Untung SUGIHARTO*2、Chakrit SAENGKORAKOT*3、
鈴木 崇史、國分(齋藤) 陽子+1、藤田 奈津子+1、木下 尚喜+2、永井 晴康、
梁 乃申*4、松崎 浩之*5、堅田 元喜*6（2020 年 7 月 30 日受理）

近年急速に進行する温暖化をはじめとした地球環境の変化は、陸域生態系（とりわけ森林生態系）における炭素循環に変化をもたらし、その結果、温暖化や環境変化の進行に拍車をかける
悪循環が懸念されている。しかしながら、その影響の予測には大きな不確実性が伴っており、その主たる要因は、土壌に貯留する有機炭素の動態とその環境変化に対する応答についての定
量的な理解の不足にある。放射性炭素（ 14C）や安定炭素（ 13C）同位体の陸域生態系における動きを追跡することは、土壌有機炭素の動態を解明するうえで有力な研究手段となりうる。本
ガイドは、同位体を利用した土壌炭素循環に関する研究を、特にアジア地域において促進させることを目的としたものである。
本ガイドは、土壌の採取、土壌試料の処理、土壌有機炭素の分画、13C の同位体比質量分析法
による測定及びその試料調製、ならびに 14C の加速器質量分析法による測定及びその試料調製に関する実践的手法を網羅している。本ガイドでは、炭素循環研究において広く用いられる 14C
分析結果の報告方法についても簡単に紹介する。さらに、同位体を利用した研究手法の実際的応用として、日本の森林生態系において実施した事例研究の結果についても報告する。本ガイドによって、同位体を利用した炭素循環研究に興味を持って参画する研究者が増加し、地球環境の変化の仕組みについての理解が大きく進展することを期待する。

このガイドは、アジア原子力協力フォーラムの枠組みを活用して文部科学省が主催する「気候変動科学プロジェクト」における研究活動の一環として作成されたものである。
原子力科学研究所：〒319-1195 茨城県那珂郡東海村大字白方 2 番地 4
+1 東濃地科学センター+2 青森研究開発センター*1 2020 年 3 月まで博士研究員*2 National Nuclear Energy Agency of Indonesia*3 Thailand Institute of Nuclear Technology*4 国立環境研究所*5 東京大学*6 茨城大学

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Contents
1.

Introduction ------------------------------------------------------------------------------------------------------ 1

2.

Soil Sampling ---------------------------------------------------------------------------------------------------- 4

3.

4.

5.

6.

7.

8.

2.1

Pit-digging Method ----------------------------------------------------------------------------------------- 4

2.2

Core-sampling Method ------------------------------------------------------------------------------------- 7

2.3

Advantages and Disadvantages of the Different Methods -------------------------------------------- 8

Soil Sample Treatment ----------------------------------------------------------------------------------------- 9
3.1

Drying Soil Samples --------------------------------------------------------------------------------------- 9

3.2

Cutting Soil Core Samples -------------------------------------------------------------------------------- 9

3.3

Sieving Soil Samples --------------------------------------------------------------------------------------- 9

3.4

Removing Carbonate Minerals (Inorganic Carbon) from Alkaline Soils ------------------------- 10

3.5

Treatment for Litter Samples ---------------------------------------------------------------------------- 10

Fractionation of Soil Samples ------------------------------------------------------------------------------- 12
4.1

Density Fractionation ------------------------------------------------------------------------------------ 12

4.2

Aggregate-size Fractionation --------------------------------------------------------------------------- 14

4.3

Chemical Fractionation ---------------------------------------------------------------------------------- 16

Analysis of Stable Isotopes ---------------------------------------------------------------------------------- 18
5.1

Stable Isotopes of Carbon and Nitrogen --------------------------------------------------------------- 18

5.2

Configuration of the Measurement System ----------------------------------------------------------- 18

5.3

Sample Preparation --------------------------------------------------------------------------------------- 20

5.4

Practical Procedure of the Measurement -------------------------------------------------------------- 21

5.5

Data Processing ------------------------------------------------------------------------------------------- 22

Sample Preparation for Radiocarbon Analysis ------------------------------------------------------------ 23
6.1

Combustion of Soil Sample to Generate CO2 --------------------------------------------------------- 23

6.2

Purification of CO2 --------------------------------------------------------------------------------------- 23

6.3

Conversion of CO2 into Graphite ----------------------------------------------------------------------- 25

6.4

Preparation of Graphite Target -------------------------------------------------------------------------- 26

6.5

A Recent Advance in the Sample Preparation Method ---------------------------------------------- 27

Radiocarbon Analysis by Accelerator Mass Spectrometry ---------------------------------------------- 28
7.1

Radiocarbon ----------------------------------------------------------------------------------------------- 28

7.2

Accelerator Mass Spectrometry ------------------------------------------------------------------------ 28

7.3

Reporting Radiocarbon Data ---------------------------------------------------------------------------- 31

Practical Application ------------------------------------------------------------------------------------------ 34
8.1

Soil Sampling and Treatment --------------------------------------------------------------------------- 34

8.2

Soil Organic Carbon Fractionation --------------------------------------------------------------------- 35

8.3

Stable Isotope Analysis ---------------------------------------------------------------------------------- 36

8.4

Radiocarbon Analysis ------------------------------------------------------------------------------------ 36iii

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8.5

Results and Interpretations ------------------------------------------------------------------------------ 37

8.6

Summary --------------------------------------------------------------------------------------------------- 44

Acknowledgements -------------------------------------------------------------------------------------------------- 46
References ------------------------------------------------------------------------------------------------------------ 47iv

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目

次

1.

序論 --------------------------------------------------------------------------------------------------------------- 1

2.

土壌採取 --------------------------------------------------------------------------------------------------------- 4

3.

4.

5.

6.

7.

8.

2.1

土壌掘削法 -------------------------------------------------------------------------------------------------- 4

2.2

コアサンプリング法 -------------------------------------------------------------------------------------- 7

2.3

異なる土壌採取法の長所と短所 ----------------------------------------------------------------------- 8

土壌試料の処理 ------------------------------------------------------------------------------------------------ 93.1

土壌試料の乾燥 -------------------------------------------------------------------------------------------- 93.2

土壌コアの切断 -------------------------------------------------------------------------------------------- 93.3

土壌試料の篩がけ ----------------------------------------------------------------------------------------- 9

3.4

アルカリ性土壌からの炭酸塩鉱物（無機炭素）の除去 --------------------------------------- 10

3.5

リター試料の処理 --------------------------------------------------------------------------------------- 10

土壌試料の分画 ---------------------------------------------------------------------------------------------- 12
4.1

比重分画 --------------------------------------------------------------------------------------------------- 12

4.2

団粒サイズ分画 ------------------------------------------------------------------------------------------ 14

4.3

化学分画 --------------------------------------------------------------------------------------------------- 16

安定同位体分析 ---------------------------------------------------------------------------------------------- 185.1

炭素と窒素の安定同位体 ------------------------------------------------------------------------------ 18

5.2

測定システムの構成 ------------------------------------------------------------------------------------ 185.3

試料調製 --------------------------------------------------------------------------------------------------- 20

5.4

測定の実施手順 ------------------------------------------------------------------------------------------ 21

5.5

データ処理 ------------------------------------------------------------------------------------------------ 22

放射性炭素分析のための試料調製 ---------------------------------------------------------------------- 236.1

土壌試料の燃焼による CO2 生成 -------------------------------------------------------------------- 23

6.2

CO2 精製 --------------------------------------------------------------------------------------------------- 23

6.3

CO2 のグラファイトへの転換 ------------------------------------------------------------------------ 25

6.4

グラファイトターゲットの作製 --------------------------------------------------------------------- 26

6.5

試料調製における最近の進展 ------------------------------------------------------------------------ 27

加速器質量分析法による放射性炭素分析 ------------------------------------------------------------- 287.1

放射性炭素 ------------------------------------------------------------------------------------------------ 28

7.2

加速器質量分析 ------------------------------------------------------------------------------------------ 287.3

放射性炭素データの報告方法 ------------------------------------------------------------------------ 31

実際的応用 ---------------------------------------------------------------------------------------------------- 34
8.1

土壌採取と処理 ------------------------------------------------------------------------------------------ 348.2

土壌有機炭素の分画 ------------------------------------------------------------------------------------ 358.3

安定同位体分析 ------------------------------------------------------------------------------------------ 368.4

放射性炭素分析 ------------------------------------------------------------------------------------------ 36v

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8.5

結果と解釈 ------------------------------------------------------------------------------------------------ 37

8.6

まとめ ------------------------------------------------------------------------------------------------------ 44

謝辞 ------------------------------------------------------------------------------------------------------------------ 46
参考文献 ------------------------------------------------------------------------------------------------------------ 47vi

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1. Introduction
The world’s soils store more than twice the amount of carbon (C) present in the atmosphere. They
produce the dominant emission flux of C to the atmosphere through microbial decomposition of soilorganic carbon (SOC), which currently balances the net flux of C entering terrestrial ecosystems
through photosynthesis in plants (i.e., the difference between the influx of C by photosynthesis and theoutflux of C by plant respiration, Fig. 1.1)1,2). The gross C fluxes through microbial decomposition of
SOC are estimated to be about 10 times that of the C emission flux due to the use of fossil fuels2). Hence,even a small change in the dynamics (stock and turnover) of SOC could have a significant impact on theatmospheric CO2 concentration, the global C cycle, and consequently the Earth’s climate system3,4).

Fig. 1.1. Simplified schematic of the terrestrial carbon cycle.
White numbers indicate reservoir mass (i.e., carbon stocks in PgC) and yellow numbers indicate annual
carbon fluxes (in PgC y−1). Diagram is from U.S. Department of Energy, Office of Science. Data arefrom IPCC1,2).

There are a number of natural and artificial processes that can potentially affect the SOC dynamics.
Increase in soil temperature due to global warming can accelerate microbial decomposition of SOC andenhance the release of CO2 from soil to the atmosphere5–8). This could cause positive feedback that-1-

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further accelerates global warming. Studies using coupled carbon cycle–climate models suggest that thisfeedback could lead to a doubling of the projected warming by the end of this century9,10). Soil moistureis also known as a major factor controlling SOC decomposition11,12). Fluctuations of soil water content
in the Asian monsoon region have reportedly been increasing since the 20th century. This is owing to
the decreased frequency and increased year-by-year variation of precipitation, accompanied by the
progression of climate change13). Recent studies indicate that repeated dry–wet cycles of soils canenhance the microbial decomposition of SOC through alteration in substrates available for soil
microorganisms14–16). Land-use change is one of the most important artificial processes to change thestock and turnover of SOC. Significant losses of SOC have been observed following the conversion offorests to agricultural land in many regions over the world17). Management practices, such as thinning,
clear-cutting, and planting for forests18) and tilling and plowing for agricultural land19) can also affect
the SOC dynamics.
The tracing of radiocarbon (14C) through terrestrial ecosystems is a powerful tool for quantitatively
understanding SOC dynamics on two different time scales: centuries to millennia, and years to
decades4,20,21). Natural 14C is produced at a relatively constant rate in the upper atmosphere by cosmic
ray interactions. Therefore, 14C content in SOC can be a useful indicator of how long SOC has resided
in the soil on a time scale of centuries to millennia, because it decreases with time as a result of theradioactive decay of 14C (with a physical half-life of 5730 years) after the addition of 14C as organic C
into the soil. Atmospheric weapons testing in the 1950s and early 1960s injected a large amount of“bomb-14C” into the atmosphere with a peak value in 1964 that almost doubled the natural 14C
concentration22). Since the nuclear test ban treaty, the amount of 14C in atmospheric CO2 has gradually
decreased as the bomb-14C has moved into the ocean and terrestrial C reservoirs (and as the atmospheric
CO2 concentration has become diluted by the burning of 14C-free fossil fuels). This global bomb-14C
spike has proven to be useful as a tracer for studying SOC dynamics on a time scale of years to decades,
because it enables us to evaluate how much organic carbon recently fixed via photosynthesis has
incorporated into SOC in the soil3,4,20,23–26).
Recent developments of the 14C measurement techniques by the use of accelerator mass spectrometry(AMS) have opened a new range of possible uses of 14C because of their analytical capability for asmall quantity of sample, and thus have great potential to facilitate studies to constrain the complicateddynamics of SOC. The availability and quality of 14C measurements have recently been increasing, andas we know well, 14C measurements have been vigorously carried out for the purpose of 14C dating (i.e.,age determination), which contribute to the progress of a diverse range of research fields such asarchaeology and geology27,28). Radiocarbon has also proven very useful in SOC dynamics studies;
however, applications of 14C to these studies are still limited, and therefore currently no satisfactory andstandard approach to quantifying SOC dynamics is established29–31).

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This guide aims at introducing ways to study SOC dynamics using 14C (and stable carbon and nitrogenisotopes) and describing its practical procedures including: soil sampling, treatment, and carbon isotope
analysis. The guide also reports results of a case study conducted in a Japanese forest ecosystem, as apractical application of the use of isotope-based approaches. This guide is mainly intended forresearchers who are interested but, inexperienced in the field of climate change science and in theapplications of nuclear and isotope techniques. We hope this guide will contribute to a rapid expansionof the application of 14C to studying SOC dynamics, particularly in Asian countries, and as a result,contribute to an improved understanding of the SOC dynamics and its interactions with climate andenvironmental changes in these regions.

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2. Soil Sampling
The objective of soil sampling is to collect samples that will best represent the average physicochemical
properties (including organic carbon content) of an investigated site. To achieve this, an undisturbed
area should be selected. Avoid heavily populated areas. In general, soils, forest soils in particular, havespatial variability. These variabilities are caused by multiple factors, such as bedrock type and parent
material, climate, vegetation, disturbances, and their combined effects. One must take into consideration
all these sources of spatial variability to systematically collect soil samples and describe soil properties
at the site. This is why sampling strategies and methodologies must be selected with care.
There are two widely used methods for soil sampling: (1) the pit-digging method and (2) the
core-sampling method. Each of the methods have their advantages and disadvantages. Therefore, achoice of the soil sampling method depends on the purpose of the study and precision required.

2.1.

Pit-digging Method

In the pit-digging method, a soil pit is dug, and soil samples are collected from a face of the soil pit atan arbitrary interval of soil depth (Fig. 2.1). Use a spade to dig a large rectangular hole (soil pit), about
60 cm deep (in the case of collecting soil samples down to the depth of 50 cm, for example) and an areaof 100 cm × 100 cm. Please note that the area where soil samples are collected should be undisturbed
during the digging.

Fig. 2.1. Schematic diagram (a) and photos (b, c) of a soil pit prepared for soil sampling.

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Soil samples are then collected layer by layer from a face of the pit as shown in Fig. 2.2. In forest soils,
mineral soil is generally covered by forest-floor organic (litter) layers. Therefore, the litter layers are
first removed completely, and then the soil samples are collected. Fig. 2.2 shows the case of soil
sampling of 10-cm interval layers up to a total of 50 cm. Soil samples are collected using a hand shovel
and clean trays. Place the tray at the bottom of the target layer, and slide down the face of each layerfrom top to bottom using shovel, after removing a thin surface of the face of the soil for avoiding
contamination with soils that were tumbled down from upper soil layers25,32). The tools should always
be kept clean by wiping, for example with paper towels, to avoid cross-contamination of soil samples
with samples of different soil layers. If the litter layer is also a research target, it should be collectedfrom a defined area (e.g., 30 cm × 30 cm, this is to determine the amount of litter materials per unit
area) and kept as a sample33). The samples collected are put into plastic bags.

Fig. 2.2. Schematic diagram (a) and photos of soil sampling: collection of litter samples fromforest-floor organic layer (b) and collection of soil samples at the depth interval of 10 cm (c).

In the pit-digging method, soil samples are collected from an arbitrary volume by grab sampling, andthe bulk density of the soil cannot be determined. Therefore, we need to collect soil samples separately
for the purpose of determining the bulk density (Fig. 2.3). Bulk density is a measure of how dense andtightly packed a sample of soil is, which is necessary to evaluate the inventory of soil organic carbon in
the soil (see Section 3.3 for details). Bulk density is determined by measuring the mass of dry soil per-5-

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unit of volume (e.g., in g cm−3). The bulk density of soil depends not only on the soil type but also thedepth of soil even in a single soil profile. Soils dominated by minerals have a different bulk densityfrom those rich in organic materials.
A known-volume (100 cm3) soil sampler is often used to collect soil samples for bulk density
determination (Fig. 2.3). The following is the operation procedure of soil sampling utilizing a soil
sampler Model DIK-1601 (Daiki Rika Kogyo Co., Ltd., Japan): (1) Unscrew the lock pin to open thesampler window; (2) Load the sample cylinder (100 cm3 in volume) into the sampler and screw the lockpin back on; (3) Push the sampler into the soil until the ground surface reaches the level with the top of
the cylinder; (4) Pull the sampler out of the soil and unscrew the lock pin to open the window sampler;(5) Cleanly cut the soil sample at the bottom of the cylinder with a cutter to separate the cylinder fromthe sampler; (6) Carefully pull the cylinder out of the sampler vertically; (7) Clean soil from the top of
cylinder using trowel until the surface reaches the level with the top of the cylinder; and (8) Recover thesoil samples from the cylinder and put into a plastic bag.

Fig. 2.3. Schematic diagram of a soil sampler for bulk density determination (a) andphotos of soil sampling: sampling (b) and sample recovery (c, d).

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2.2.

Core-sampling Method

A core sampler is used to collect soil samples in this method (Fig. 2.4)4,33,34). As in the pit-digging
method, forest-floor organic (litter) layers are removed (if sites are in forests). Then, the core sampler isplaced on the ground surface and driven into the soil approximately 25 cm in depth (in the case of a coresampler with 10-cm diameter and 30-cm long: Model HS-25, Fujiwara Scientific Company, Japan).
Before pulling the core sampler out of the soil, it is better to check, by opening the lid of the coresampler, whether the level of soil surface in the area isolated by the core sampler is almost the same asthat in the surrounding area. This is to ensure that a significant compaction of soil-core sample does notoccur during the sampling. Afterwards, slowly pull the core sampler up and take the plastic bag
previously set inside the sampler, which now contains a soil core sample.

Fig. 2.4. Schematic diagram of a soil core sampler (a) and photos of soil core sampling:sampling (b,c) and sample recovery (d).

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2.3.

Advantages and Disadvantages of the Different Methods

Sampling of forest-floor litter layers varies among soil scientists and, also, on the purpose of theresearch; therefore, there are no accepted standards for how horizons should be sampled.
The advantages of the core-sampling method are efficiency and practicality. A corrugated knife
equipped on the outside edge of the core-sampling frame will generally cut through surface soil layerswith no difficulty, even if vegetation has spread large amount of fine roots in the soil. Therefore, oncethe sample is cut on all sides, it is easy to partition it from the soil. This method provides us with ahopefully undamaged soil core retrieved from a precisely identified location with minimizing
contamination of each layer in the soil profile. More importantly, this method enables us to collect soil
samples at a very thin depth interval as needed (see Section 3.2); this can sometimes be an essential
factor to achieve your research goal4). The main disadvantage of this method may be related to therepresentativeness of the core sample. The area of the core sample collected is normally very small
compared with the site-scale target area to be investigated. To overcome this issue, one may collectthree or more replicated core samples within the target area to consider the spatial variability of soil
samples33,34). Another disadvantage may be the relatively high cost of the equipment.
The advantages of the pit-digging method don’t require any specific equipment (you only need spade,hand shovel, tray, knife, ruler, plastic bags, skewers, and so on) and can cover a large area. This method
is also convenient if you want to obtain large amounts of soil samples from each of the soil layers.
However, the strongest advantage of this method is that it enables us to collect soil samples from deeper
layers compared with the core-sampling method; this can also sometimes be the key requirement foryour research objectives25). The disadvantage of this method may be that soil sample in a target soil
layer can easily be contaminated with other layers of the soil profile during sampling; therefore, carefulsampling procedure is required32). This may also be noted as a disadvantage, digging soil pits arelabor-intensive.

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3. Soil Sample Treatment
Pretreatment is generally required for soil and litter samples prior to soil carbon analysis. It includes
drying and sieving of soil samples, both of which are also essential for evaluating SOC stock in the soil.
In this chapter, practical methods for pretreatment of soil and litter samples are briefly described. You
can find more detailed procedures for soil sample handling and storage35,36) and for soil
physicochemical, mineralogical, and microbiological analyses37–41) in well-established protocol guides.

3.1.

Drying Soil Samples

Soil samples are dried immediately at room temperature (or 50°C in an oven) in a laboratory. If this is
not possible then, the samples should be stored in a freezer (at −30°C, for example) to prevent
decomposition of organic materials in the samples. Measuring the weight of the soil sample before andafter drying allows us to determine the water content of the soil.

3.2.

Cutting Soil Core Samples

According to the research purpose, the soil core samples (e.g., 20 cm long) can be divided into several
sections (thin-layered samples) in terms of the depth intervals (e.g., 0–2 cm, 2–4 cm, 4–6 cm, …15–20cm) before drying (Fig. 3.1a-c). The core cutting should be conducted for core samples that are in afrozen state by using a saw. This will enable us to precisely obtain thin-layered samples without any
cross-contamination4,33). An important point that should be kept in mind is that, before cutting each of
the thin-layered samples, an outer part of the soil core is peeled away, and only the inner part of the soilcore should be used for analysis. This procedure is done to eliminate any possible contamination fromthe upper to lower soil layers during the sampling42). Please note that the weight of the outer part of thesoil core should also be measured and considered in the calculation of soil bulk density.
Soil bulk density of each of the separated layers can be estimated as the dry weight of the soil samples(including both inner and outer parts) divided by the volume of the layer (i.e., the cross-section area ofthe core sample multiplied by the thickness of the layer).

3.3.

Sieving Soil Samples

The dried soil samples are sieved through a 2-mm mesh to remove gravel and roots (Fig. 3.1d). This
procedure is required not only to obtain a homogenized soil sample for analysis, but also to quantify the
carbon stock in the soil layer. Weigh both fractions (i.e., > 2 mm and < 2 mm fractions) to calculate thegravel (> 2 mm in size) content in the soil sample. SOC stock in a given soil layer, IS (kgC m−2), can becalculated as:
IS = BD ∙ CS ∙ d ∙ (1−g)(Eq. 3.1)-9-

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where BD is the bulk density (kg m−3), CS is the carbon content of the soil (< 2 mm) sample (kgC kg−1soil), d is the thickness of the layer (m), and g is the gravel content (kg kg−1)25).

Fig. 3.1. Photos of soil-core cutting (peeling an outer part of the soil core (a), cutting (b), and athin-layered sample obtained (c)) and sieving of a dried soil sample (d).

3.4.

Removing Carbonate Minerals (Inorganic Carbon) from Alkaline Soils

Alkaline soils with high pH values (pH > 7) may contain various soluble carbonate minerals such as
sodium bicarbonate and carbonate. These inorganic compounds should be removed before analysis, if
the focus of the research is on organic carbon in soils. Treatment of soil samples with hydrochloric acid(HCl) solution can remove such inorganic compounds from the samples because HCl reacts withcarbonate to release C as CO2.

3.5.

Treatment for Litter Samples

A different procedure is required for the treatment of litter samples collected from forest-floor litter
layers. To obtain a homogenized sample for analysis, litter samples are pulverized by using a blender
after removing roots, branches, and stones (Fig. 3.2). SOC stock in the litter layer, IL (kgC m−2), can becalculated as:
IL = C L ∙ M L(Eq. 3.2)- 10 -

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where CL is the carbon content of the litter sample (kgC kg−1) and ML is the amount of litter materials
per unit area (kg m−2) in the litter layer.

Fig. 3.2. Photos of the treatment for litter samples: separation of a litter sample into leaf-litter fraction
and the other (roots, branches, and stones) fraction (a); pulverization of the leaf-litter fraction (b); andhomogenized leaf-litter sample (c).

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4. Fractionation of Soil Samples
Soil organic carbon (SOC) is a complex mixture of organic compounds with heterogeneous physical,
chemical, and biological properties, from undecomposed and partially decomposed plant materials to
substances synthesized by soil microbes43). SOC also consists of various functional pools that arestabilized by physical and chemical mechanisms in soil44). These SOC compounds and pools greatly
differ in degradability, and the overall dynamics (stock and turnover) of soil C is thus largely regulated
by such a heterogeneous nature of SOC. This demonstrates that SOC modeled as a homogeneous single
pool (i.e., only represented as a bulk soil) causes a misunderstanding of the real SOC dynamics.
Therefore, quantitative understanding of the heterogeneity in SOC dynamics is a key to accurately
predicting the response of soil C to future changes in climate and environment4).
Fractionation of SOC, as a potentially effective, operational method, has been attempted to separate soil
C into SOC fractions with different properties and turnover times30). Fractionation methods include: (1)physical separation of SOC into particle- and/or aggregate-size fractions and density fractions25,45–50);
and (2) chemical extractions to fractionate SOC according to solubility and chemical reactivity4,25,51–53).
Combinations of some of these physical and chemical fractionation methods have also been
proposed29,30,54). However, a successful and standard method to identify SOC pools according toturnover time and thus to quantify the SOC dynamics remains unexplored.
The objective of this chapter is to introduce some basic methods for SOC fractionation and to describe
practical procedures for the methods.

4.1.

Density Fractionation

A density fractionation method is used to separate a soil sample into soil fractions with different
densities; this is based on contrasting densities between soil mineral particles (typically 2.5–3.0 g cm−3)and organic materials (< 1.4 g cm−3) in the soil45,48,55). Here, as an example, a simple method using aheavy liquid (density of 1.6 cm−3) to separate a soil sample into two fractions: organic matter-dominant
low-density fraction (LF, < 1.6 g cm−3) and mineral-dominant high-density fraction (HF, > 1.6 g cm−3),
is described. The flowchart of the physical fractionation method is shown in Fig. 4.1.
Soil samples (approximately 5 ml, after removing roots by hand) are weighed into 50 ml centrifuge
tubes and mixed with 35 ml of 1.6 g cm−3 sodium polytungstate (SPT) liquid (Fig. 4.2). The tubes areshaken gently and centrifuged at 3000 rpm for 30 min, and the floating materials (LF) are aspirated ontoa pre-baked glass microfiber filter (pore size: 0.7 μm). The cycle of gentle shaking, centrifugation, andaspiration is repeated until no LF remained floating (typically three times). The LF is then rinsed at least
three times with ultrapure water (a minimum of 45 ml) on the filter under vacuum using a separate
collector, and dried at 50°C in an oven50).

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Fig. 4.1. Flowchart of the density fractionation method.

After floating off the LF materials completely, the residue (HF) in the centrifuge tubes is rinsed morethan 7 times with ultrapure water (45 ml) by shaking and centrifuging at 3000 rpm for 15 min to remove
SPT from the HF samples, and then dried at 50°C in an oven50).
Mass balance (the initial weight of the sample vs. the sum of the weights of LF and HF samples) is used
to assess the completeness of recovery. Note that dissolved organic carbon lost to SPT solution and- 13 -

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ultrapure water during the fractionation is not recovered; the carbon loss during fractionation may
sometimes reach more than 10% of the total soil C for some soils25,31,55,56). The fractions obtained arethen homogenized by grinding using a mortar and pestle (after removing roots again if necessary), andthen are used for analysis.

Fig. 4.2. Photos of the physical fractionation procedure: addition of a heavy liquid (SPT) into acentrifuge tube with soil sample (a); separation of soil samples into low-density (LF) and high-density(HF) fractions (b); filtration of LF sample (c); collection of LF sample (d); grinding of HF sample (e);and the obtained LF and HF samples (f).

4.2.

Aggregate-size Fractionation

An aggregate-size fractionation method is used to separate a soil sample into soil fractions withdifferent size classes of aggregates in which primary soil particles (clay, silt, and sand) are bound toeach other with the help of soil organic matter as binding agent49,57–59). Here, as an example, a simple
aggregate-size fractionation method to separate a soil sample into two fractions: macroaggregate
fraction (> 250 m in size) and microaggregate fraction (< 250 m), is described. The flowchart of theaggregate-size fractionation method is shown in Fig. 4.3.
Soil sample (approximately 10 g, after removing roots by hand) is put on a polyethylene mesh sheet
having 250 m pores (approximately 30 cm × 30 cm in size, Nichika, Kyoto, Japan) attached to a

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sieving set (Mini-sieve, Merck, Germany). Both the mesh sheet and the sieving set should be cleaned
with detergent before fractionation. The sieving set is submerged in 200 ml of ultrapure water in a
500-ml beaker for 10 minutes, and is then slowly moved in and out of the water repeatedly for 2 min(approximately 50 times in 5 cm strokes) (Fig. 4.4). The particulate materials retained on the mesh sheet
are dried at 50 ºC for 24 h, and recovered as macroaggregates (> 250 m in size). The solution in the
beaker is filtrated with a 0.45-m pore-sized nitrocellulose membrane filter (Merck Millipore, MA,
USA) under vacuum condition. The particulate materials retained on the membrane filter are dried at
50ºC for 24 h, and recovered as microaggregates (< 250 m in size). Each of the aggregate-size
fractions is then homogenized by grinding before analysis. Note that, as in the physical fractionation,organic carbon dissolved in ultrapure water (< 0.45 m) is lost during fractionation, if the dissolved
fraction is not recovered.

Fig. 4.3. Flowchart of the aggregate-size fractionation method.

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Fig. 4.4. Photos of the aggregate-size fractionation procedure: submersible sieving set with a cleaned
sieve with mesh size of 250 m (a); the top view of submersible sieving set (b); pulling up and down thesieving set 50 times for 2 min (c); filtration of microaggregate samples that were passed through thesieve and contained in the water (d); grinding both microaggregate and macroaggregate (collected on
the sieve) samples (e); and the homogenized microaggregate and macroaggregate samples (f).

4.3.

Chemical Fractionation

A chemical fractionation method is used to separate a soil sample into soil fractions according todifferent solubility and chemical reactivity of SOC. For example, acid hydrolysis is believed to remove
compounds that are readily available to microorganisms, such as carbohydrates and proteins, whileleaving more biologically recalcitrant materials43,51). Here, as an example, a simple method using acidand base solutions is described4). The flowchart of the chemical fractionation method is shown in Fig.
4.5.
Before starting fractionation, plant leaf fragments (PLF) are removed from soil samples by using
tweezers, and are collected as a component of SOC for analysis. Approximately 2–7 g of the soil
samples are hydrolyzed with 60 ml of 1.2M HCl at 80°C for 2 h. The suspension is centrifuged at 2000
rpm for 3 min and the supernatant is discarded. This treatment is repeated three times. The residue isthen repeatedly washed with deionized water until the pH is > 4 to obtain acid-insoluble SOC fraction.

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Subsample (~1–3 g) of the acid-insoluble fraction is further extracted with 60 ml of 1.2M NaOH at
80°C for 2 h, then the supernatant is decanted and replaced with fresh solution after centrifugation at2000 rpm for 3 min. The extraction process is repeated until the supernatant become colorless. The
residue is then washed with deionized water until the pH is < 10, treated three times with HCl to remove
all carbonate contaminants, and repeatedly washed with deionized water as before to obtain insoluble
SOC fraction4).
Carbon content and 14C isotope ratio are measured for the bulk (unfractionated) and two insoluble SOC
fractions (Fig. 4.5). Using the measured values, C inventories and 14C isotope ratios for the acid- andbase-soluble SOC fractions can be quantified through mass balance calculations.

Fig. 4.5. Flowchart of the chemical fractionation method4).

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5. Analysis of Stable Isotopes
5.1.

Stable Isotopes of Carbon and Nitrogen

Carbon and nitrogen have two stable isotopes each: 12C and 13C, and 14N and 15N. Their average naturalabundances are 98.89% for 12C and 1.11% for 13C, respectively, for carbon isotopes, and 99.64% for 14N
and 0.36% for 15N, respectively, for nitrogen isotopes. As the variation in the isotope composition innature is exceedingly small for both carbon and nitrogen isotopes, the isotope composition is reported asa value relative to an internationally accepted standard and is generally expressed as the per mil (‰)
deviation of the 13C/12C (or 15N/14N) ratio of the sample from that of the standard, as follows:δ = [(RSample/RStd) − 1] × 1000

(Eq. 5.1)

where RSample and RStd are the ratios of the heavy to light stable isotopes (13C/12C and 15N/14N) in thesample and standard, respectively. The internationally accepted standards are Pee Dee Belemnite (PDB)
for stable carbon isotopes and the atmosphere for stable nitrogen isotopes. Usually, working standards
calibrated against the internationally accepted standards are used in routine measurements. Examples of
the commercially available working standards are glycine (Lot No. AZ300M9R2283, Nacalai Tesque,Inc., δ15N vs. air: 1.12 ± 0.2‰ and δ13C vs. PDB: −32.3 ± 0.2‰) and L-alanine (Lot No.
AZ100M6R397405, Nacalai Tesque, Inc., δ15N vs. air: 5.0 ± 0.2‰ and δ13C vs. PDB: −19.6 ± 0.2‰).
Here, an analytical method for the stable isotope ratios using isotope ratio mass spectrometry combinedwith elemental analysis is described; it has been used in the environmental science laboratory in theJapan Atomic Energy Agency (JAEA).

5.2.

Configuration of the Measurement System

The carbon and nitrogen contents are measured using an elemental analyzer (EA, vario PYRO cube,
Elementar, Germany), and the stable isotope ratios are measured using an isotope ratio mass
spectrometer (IRMS, IsoPrime 100, IsoPrime, UK). The measurement system is shown in Fig. 5.1.
Soil samples are burned in a combustion furnace heated to 920°C in the vario PYRO cube, and the
combustion products are passed through an oxidation catalyst by a constant flow of helium as a carriergas (Fig. 5.2). The oxidation products are then passed through a reduction reactor. Copper granules
within the reduction reactor reduce nitrogen oxides (NO, N2O, and N2O2) to N2. The CO2 and N2 arepassed through a water trap and a CO2 absorption column where CO2 is separated from N2 bycontrolling the temperature of the column. The N2 and CO2 are then passed through a thermal
conductivity detector (TCD) sequentially and are sent to the IRMS. Generally, carbon content in soil

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samples (soil organic matter) exceeds nitrogen content and therefore a diluter is used to reduce theamount of CO2 to balance it with N2. The CO2 flow from the EA is injected into a helium flow in the
diluter, and then a part of the CO2 flow is introduced into the IsoPrime 100.

Fig. 5.1. The measurement system for stable carbon and nitrogen isotopes: IRMS connected to EA.

Fig. 5.2. The configuration of the vario PYRO cube.

The reference N2, sample gas, and reference CO2 can be introduced into the IsoPrime 100 in this orderby switching the valve in the reference gas injector box (“Ref Gas Box” in Fig. 5.1). Then, stable
isotope ratios of carbon and nitrogen are measured against a pulse of reference gas of known isotopic
composition. The carbon and nitrogen isotopes in the sample gas are ionized at an electron impact
source. The ions generated by the ion source are deflected by an electromagnet, separated by the mass,and finally detected simultaneously by a multi-collector.

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5.3.

Sample Preparation

Prepare what you need before work: aluminum foil on the work bench, several tweezers andmicro-spatulas, tin boats of appropriate size, working standards, tungsten oxide powder, soil samples,and a 96-hole tray for wrapped samples. The samples must be dried and homogenized by grinding using
a mortar and pestle. The powdered samples are stored in a closed container to prevent it from absorbing
moisture. For the measurement, each of the samples is weighed and wrapped in a tin boat (Fig. 5.3).
Several sizes of tin boat exist; the size of tin boat used here is 12 mm × 6 mm × 6 mm (height), and it
can be changed depending on the carbon and nitrogen contents of the sample. To avoid contamination of
the samples, the weighing area and all tools (tweezers, micro-spatulas, sample trays, and aluminum foilon the balance weighing pan) used for the weighing procedure must be kept clean. Wear gloves to avoid
contamination from natural greases on the hands.

Fig. 5.3. Sample preparation procedure for stable isotope analysis: (a) a tin boat; (b) putting soil
samples in the tin boat; (c) weighing; (d-f) enfolding the sample by the tin boat; and (g) a tin-wrappedsample for analysis.

First, as a blank sample, three new tin boats with no soil samples are crushed with tweezers to remove
any air. Next, place a new tin boat on the pan of the microbalance and press the TARE button. Remove
the tin boat from the balance pan and place a small amount of sample into the boat. Weigh the sample. If- 20 -

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necessary, adjust the sample weight by adding or removing some sample materials. Do not forget torecord the weight. For soil samples, powdered tungsten oxide (equivalent to 1–3 times the sample
weight) must be added as a combustion catalyst. Using the tweezers, close the top of the tin boat tightly
and fold it over. Fold it several times in all directions and form it in a ball-like form. To avoid potential
sample loss, take care not to damage the tin foil when folding. The resulting ball should be tightly
packed to ensure that the sample is completely sealed inside the tin-foil ball to optimize the combustion
process. The final geometry should be as spherical as possible to prevent any jamming inside theautosampler of the EA. A working standard is needed for every five or six unknown samples. Put the
tin-wrapped ball-like sample in a tray and record the tray position number. Wrap the tray in Parafilm if
it will take a long time from the sample preparation to measurement. Clean your tools and the work
surface with Kimwipes and alcohol before moving to the next sample.
For the isotope ratio measurements, it is desirable to adjust the amounts of carbon and nitrogen inworking standards and samples to be as equal as possible. It is, therefore, better to measure the amountsof carbon and nitrogen in samples before measuring the isotope ratios. If the IRMS is optimized andtuned well, the isotope ratios can be stably measured when the peak height is 2–10 nA. Since therelationship between the amounts of carbon and nitrogen and the peak height changes depending on thetuning, the condition of the device, and the setting of the diluter, it is better to determine the required
sample weight after confirming the relationship by a preliminary measurement of the working standard.

5.4.

Practical Procedure of the Measurement[When returning from Sleep mode] Check the amount of helium remaining in the helium gas cylinder.
Click the “Wake up now” button in the vario PYRO cube software (vario software) (Option > Settings >Sleep and Wake up) to make the EA standby and confirm that the furnace temperature and helium
carrier flow rate have reached the specified values. Open the helium gas valve behind the diluter. After
turning off the ion source in the Tune Page window of the IonVantage software (Option > Settings >Source On), open the Nupro valve. Wait until the vacuum gauge stabilizes at 2–6 × 10−6 mbar and thecolor of the bar in the display turns green. Then, turn on the ion source again. Confirm that the source
status becomes “Operational” (green). To tune the acceleration voltage, click the “Run Peak Center”
button in the Peak Center Results window for each reference gas and check the peak shape in the Peak
Display window. Then, save the tuning file. Check the stability and linearity of the mass spectrometer.
Place the tin-wrapped sample in the sampler tray of the autosampler (Fig. 5.4). Open the document (orcreate a new one and name it) to be used for the measurement in the vario software. Open the sample
list (or create a new one) in the IonVantage software. Enter the sample information in the sample list.
Save the sample list. Select the line you want to run, and click the “Run” button. In the window thatappears, confirm that the boxes before “Acquire Sample Data” and “Auto Process Samples” are checked,- 21 -

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and click the “OK” button. After the measurement, be sure to close the Nupro valve and then close thehelium gas valve behind the diluter. Click the “Sleep now” button in the vario software.

Fig. 5.4. Photos of the EA-IRMS system (a), putting the tin-wrapped sample in the autosampler of theEA (b), and operating softwares to measure stable isotope ratios of the samples (c).

5.5.

Data Processing

Export the data of the TCD peak area in the EA measurement from the vario software. To calculate theamounts of nitrogen and carbon, calibration curves (to evaluate the amounts of nitrogen and carbon
from the peak areas) are created based on the measurement results of a set of the working standard
samples.
Data of the isotope measurements are collected from the data folder or batch file folder in the project
file folder. The δ values in the data file need correction using the measurement results of the working
standard samples. Confirm that the isotope ratio of the working standard does not vary largely before
using these data.

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6. Sample Preparation for Radiocarbon Analysis
Sample pretreatment is required for radiocarbon (14C) measurement with the use of accelerator mass
spectrometry (AMS). It includes: combustion of solid samples into gas samples; purification of CO2 in
the gas samples; conversion of CO2 into graphite; and preparation of graphite targets for AMS. In thischapter, practical methods of sample preparation for AMS are briefly described.

6.1.

Combustion of Soil Sample to Generate CO2

Soil samples (equivalent to about 3–4 mg of carbon, but this depends on the AMS system to be used for14

C measurement) are weighed into pre-combusted quartz tubes (6 mm in diameter, Fig. 6.1).

Approximately 1 g of copper oxide (CuO) wire, 0.5 g of reduced copper wire, and Ag foil
(approximately 5 cm × 4 mm) are also added into the tubes. The tubes are inserted into pre-combusted
quarts tubes (9 mm in diameter), which are then attached to a multipurpose vacuum line. The vacuum
line is equipped with pressure gauges and a vacuum gauge to continuously monitor the pressure in theline. The tubes are evacuated to a pressure of about 10−3 mbar and then flame-sealed. The samples in thesealed tubes are combusted at 500°C for 30 min and 850°C for 2 h4).

Fig. 6.1. Schematic diagram of the sample preparation for combustion (a) andphoto of the combusted samples in the quartz tubes (b).

6.2.

Purification of CO2

The gas generated in the tubes by combustion is introduced into the vacuum line and CO2 in the gas is
cryogenically purified and then recovered (Fig. 6.2). The gas introduced into the line is passed throughtwo multi-loop traps cooled with liquid nitrogen, in which CO2 in the gas sample is completely trapped.

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Molecular nitrogen (N2), O2 and other gaseous compounds in the gas sample are not trapped in thesetraps and pumped away. The CO2 trapped in the first multi-loop trap is then liberated by changing thecooling agent from liquid nitrogen into liquid nitrogen-ethanol mixture (approximately −90°C) whilewater remains trapped in this trap. The CO2 is then transferred to the second multi-loop trap thatremains cooled with liquid nitrogen, and consequently, all CO2 in the sample is trapped in the second
trap. The CO2 trapped in the second trap is liberated as before, and is transferred to a known-volume
reservoir to quantify the amount of carbon by measuring the CO2 pressure.

Fig. 6.2. Photos of the start-up of the vacuum line (a), the transfer of CO2 to a trap cooled with liquid
nitrogen (b), and flame-sealing of the purified CO2 in the Pyrex glass tube (c).

Finally, the purified CO2 is separated into two fractions: one is for 13C analysis with the IRMS
(approximately 1 mg of carbon as CO2) and the other is for 14C measurement with the use of AMS. Thisis conducted by expanding the CO2 in two known-volume areas in the line for 5 min. Each of thefractions (CO2 in the two known-volume areas) is transferred to a pre-combusted Pyrex glass tube
cooled with liquid nitrogen and flame-sealed. Note that each of the gas transfer processes requires
approximately 3 minutes to allow the CO2 transfer to complete.

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6.3.

Conversion of CO2 into Graphite

For 14C measurement with the use of AMS, it is necessary to prepare a graphite target. A number ofgraphitization methods have been developed and they rely on reduction of CO2 in the presence of acatalyst. The hydrogen reduction method is the most widely used method for graphite production from
CO260–62). The net reaction is:
CO2 + H2 → H2O + C (graphite)(Eq. 6.1)

The CO2 reduction is achieved in a reaction tube made by Pyrex glass (Fig. 6.3). A small cup (made by
Vycor glass with high-temperature resistance) is placed inside the reaction tube; the small cup contains
iron powder which works as a catalyst (Fig. 6.3). The iron powder in the cup is pre-cleaned and reducedwith H2 gas at 450°C. Part of the purified CO2 and H2 gases (CO2 : H2 = 1 : 2.5) are transferred into thereaction tube using a vacuum system (Fig. 6.4). After that, the reaction tube (and a water trap tube
directly connected to the reaction tube) is sealed and separated from the vacuum system, and is set to anelectric heating furnace for CO2 reduction (Fig. 6.5). The electric heating furnace is run at 650°C within
10–12 h including a cooling time. Water generated during the reaction in the tube is removed by thewater trap tube which is cooled at the upper part of the electric heating furnace.

Fig. 6.3. Photos of the vacuum system (a) and a small cup with iron powder placed inside the reaction
tube (b).

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Fig. 6.4. Photos of the procedure of filling H2 gas in a reaction tube containing CO2 sample and ironpowder (a) and a vacuum gauge to quantify the amount of H2 gas transferred to the reaction tube (b).

Fig. 6.5. Photos of the reduction of CO2 to graphite in a reaction tube using an electric heating furnace(a) and a water trap to remove water generated during the reduction (b).

6.4.

Preparation of Graphite Target

At the AMS facility of the Aomori Research and Development Center of JAEA, for example, the target
piece is set in a target press tool and then the graphite sample goes through the funnel to the top of thetarget piece (Fig. 6.6). A back pin is inserted into the target piece from the back side and then the backpin is pressed by a press machine. As a result, the graphite sample is set to the surface of the target piece.
After confirming that the surface of the target piece is clean, target pieces are arranged in the order ofmeasurement. These processes are carried out under a clean bench to avoid any contamination.

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Fig. 6.6. Photos of a target piece (a), schematic diagram of setting graphite sample in the target piece (b),and photo of the loading of graphite into a target piece by using a press machine (c).
This target piece is one used at the JAEA-AMS-MUTSU.

6.5.

A Recent Advance in the Sample Preparation Method

A new graphitization system has recently been developed and employed to rapidly and efficiently
prepare samples for AMS-14C measurement. The graphitization system relies on an AutomatedGraphitization Equipment (AGE3, Ionplus AG, Switzerland) directly coupled to an elemental analyzer(EA). AGE3 uses a molecular sieve trap, instead of a cryogenic trap, to isolate CO2 from the combustion
gas63). The samples (wrapped in a tin boat as in the stable isotope analysis, see Section 5.3) are
combusted in the EA, and the resulting CO2 in the combustion gas is adsorbed in a column filled withzeolite material. Afterwards, the CO2 is thermally released from the column and transferred to agraphitization reactor in the AGE3. This system has now started operation at the AMS facility of the
Tono Geoscience Center of JAEA64).

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7. Radiocarbon Analysis by Accelerator Mass Spectrometry
7.1.

Radiocarbon

Radiocarbon (14C), the radioactive isotope of carbon, is a cosmogenic radionuclide and is therefore
constantly being generated by the interaction of cosmic rays with nitrogen isotope (14N) in theatmosphere. The 14C nuclei is unstable and decays back to 14N by emitting a β particle, with the physicalhalf-life of 5730 years. The 14C nuclei generated in the atmosphere is oxidized to 14CO2 (the form ofcarbon dioxide) in a matter of a few months. The 14CO2 mixes into the troposphere and enters the global
carbon cycle that continuously exchanges carbon between atmosphere, terrestrial, and ocean carbon
reservoirs. The natural abundance of 14C is roughly one in every trillion (1012) carbon atoms (anabundance of ~10−10%) in the environment65).

7.2.

Accelerator Mass Spectrometry

Accelerator mass spectrometry (AMS) is an excellent technique to measure 14C in environmental
samples (including soil samples and soil organic carbon fractions), because of the low detection limit,
good precision, small sample volume required, and short measurement time. Here, we introduce three ofthe AMS systems operating in Japan: JAEA-AMS-MUTSU, JAEA-AMS-TONO-5MV, and
MALT-AMS.

7.2.1. JAEA-AMS-MUTSU
The accelerator mass spectrometer (AMS) at the Aomori Research and Development Center of JAEA
(JAEA-AMS-MUTSU) is based on a 3 MV Tandetron system (Model 4130, High Voltage Engineering
Europe). JAEA-AMS-MUTSU has two beamlines: one is optimized for 14C measurement and the otheris for 129I measurement. The AMS is equipped with a simultaneous injection system for high-precision
14

C measurement60). Photo and schematic layout of JAEA-AMS-MUTSU are shown in Fig. 7.1. At thenegative ion source for the 14C measurement, negative carbon ions (C−) are produced by the cesium (Cs)sputtering. Molecular ions (12CH2−, 13CH−) which are isobar of 14C are also produced. Accelerated
negative carbon ions and molecular ions collide with Ar gas which is installed at the center of theaccelerator. Outermost electrons of negative carbon ions are stripped by this collision and molecularions are broken and also stripped by this collision. Therefore, negative carbon ions and molecular ionsbecome positive ion (12Cn+, 13Cn+, 14Cn+). After passing through the accelerator, positive carbon ions canbe separated by the analyzing magnet. Finally, only the target ion (14C) can be introduced into theionization detector and then counted, whereas 12C and 13C ions can separately be detected by Faraday
cup detectors.

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Fig. 7.1. Photo (a) and schematic layout (b) of the JAEA-AMS-MUTSU system.
A beamline on red is optimized for 14C measurement.

7.2.2. JAEA-AMS-TONO-5MV
The AMS at the Tono Geoscience Center of JAEA (JAEA-AMS-TONO-5MV) is a multi-nuclide system
based on a 5 MV tandem Pelletron type accelerator (Model: 15SDH-2) by National Electrostatics
Corporation, USA64,66). One beamline of the AMS enables us to measure not only 14C but also othernuclides such as 10Be, 26Al, and 129I. Schematic layout of the JAEA-AMS-TONO-5MV is shown in Fig.
7.2. The AMS has two ion sources: a 40-target MC-SNICS ion source and a 12-target MGF-SNICS.
Currently, only the MC-SNICS is used, and the MGF-SNICS is not used. Low energy side of the AMS
beamline is equipped with a 90° double focusing injection magnet producing fast sequential isotope
injection for multi-nuclide measurement. High energy side of the AMS beamline is equipped with a 90°
double focusing analyzing magnet, multi-Faraday cup detector system, a 20° electrostatic cylindricalanalyzer, and an ionization detector. The two Faraday cup detectors and the ionization detector are used
for measurements of 12C,13C, and 14C, respectively. The ionization detector consists of a cathode- 29 -

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electrode, grid and anodes of five ΔE electrodes67). This has a beneficial effect on discrimination
between isobars and enables us to detect multi nuclides by using the single beamline.

Fig. 7.2. Schematic layout of the JAEA-AMS-TONO-5MV system.

7.2.3. MALT-AMS
The AMS at the Micro Analysis Laboratory, Tandem accelerator, the University of Tokyo (MALT-AMS)
is based on a Pelletron 5UD tandem accelerator by National Electrostatics Corporation, charged by apellet chain up to 5 MV68). MALT-AMS comprises a Cs-sputter ion source, a sequential injection
system, and multi-Faraday cup system for high precision AMS measurements. There are several beamcourses such as NRA (nuclear reaction analysis) and PIXE (particle induced X-ray emission), in
addition to AMS course, at MALT-AMS (Fig. 7.3).
A new method for 14C-AMS has been used at MALT-AMS in which only beams with mass 13 and 14
are injected into the accelerator. In this method, 14C/13C is measured by 14C counts at the final detector
and 13C4+ current by MFC 04-2 (one of multi Faraday cups). 13C/12C is evaluated from the current ratioof 13C4+ generated at the terminal from 13CH− (mass 14) and 12C4+ generated from 12CH− (mass 13)
measured by MFC 04-1 and MFC 04-4, respectively. All three stable isotope beams, 13C4+ from 13C−,
13

C4+ from 13CH− and 12C4+ from 12CH−, come to different positions after the analyzing magnet so thatthree offset Faraday cups are needed for the simultaneous measurements with sequential injection of- 30 -

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mass 13 and 14. Using this method, 13C/12C can be measured reasonably69).

Fig. 7.3. Photo of the accelerator (a) and schematic layout (b) of the MALT-AMS system.

7.3.

Reporting Radiocarbon Data

There are several ways to report radiocarbon (14C) data depending on the application65,70). As with stable
isotopes, 14C data are reported based on comparing the 14C/12C of the sample to that of a universal
standard. Two factors are to be considered, however, to report 14C data: corrections of mass-dependentisotope fractionation and radioactive decay. Regarding the mass-dependent isotope fractionation, the14

C/12C ratios are corrected for mass-dependent isotope fractionation based on stable carbon isotope

ratios (13C/12C), assuming that the mass-dependent fractionation of 14C in relation to 12C is twice aslarge as that of 13C during carbon cycling (biochemical and physical) processes. Regarding theradioactive decay, the 14C/12C ratios in the standard used for the AMS measurement decrease over timeas the 14C in the standard is subjected to radioactive decay. This is corrected by selecting a specific time(1950 in the case of 14C) and decay to take into account the 14C lost between 1950 and the year the
standard was measured65).
Two ways to report 14C data are briefly described below, which are frequently used for carbon cycle
studies.

7.3.1. Radiocarbon age
Like stable carbon isotopes (12C and 13C), 14C enters the terrestrial ecosystems by uptake of atmospheric
CO2 through photosynthesis. During its lifetime, organisms remain in isotopic equilibrium with the- 31 -

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atmosphere due to the quick exchange of C between them. Immediately after the organisms die,however, the 14C isotope ratio (14C/12C) in the organisms (organic matter such as plant tissues in thiscase) starts to decrease as a result of the radioactive decay of 14C with a physical half-life of 5730 years.
Therefore, measuring the 14C isotope ratio of a sample enables us to estimate the age of the organic
matter in the sample by comparing it with the 14C isotope ratio of a standard with known age. This iswell known as 14C dating. The 14C isotope ratio of the atmospheric CO2 in 1950 is generally used as astandard for this purpose.
Conventional 14C age (in years BP; BP means before present with present defined as 1950) is reportedas:
14

C age = −8033∙ln(A0/A)(Eq. 7.1)

where 8033 is the mean life of 14C (= 5568/ln2 in years) based on the Libby half-life (5568 years, thisvalue is still used by convention for reporting the 14C age, although the half-life value has been updated
as before), A0 is the initial 14C/12C ratio of the sample which is defined by reference to the 1950 standard,and A is the 14C/12C ratio of the sample. If A is smaller than A0, then A can give an estimate of the time
elapsed since the sample was isolated from exchange of 14C (and 12C) with the atmosphere. Note thatthe 14C dating assumes that there has been no loss (other than radioactive decay) or addition of 14C tothe sample since it ceased exchanging with the atmosphere (i.e., since death of the organism).

7.3.2. Δ14C
The absolute amount of 14C in a sample in the year it was measured can be more informative
particularly for geochemical applications. In this case, a correction for radioactive decay undergone by
the standard between 1950 and the year the sample was measured must be done, and 14C value for the
sample is reported as:
Δ14C = [Rsample,−25/{0.95×ROXI,−19×exp((y−1950)/8267)}−1] × 1000

(Eq. 7.2)

where Rsample,−25 is the 14C/12C ratio of the sample normalized to the common δ13C value of −25‰ toaccount for the mass-dependent isotopic fractionation, ROXI,−19 is the 14C/12C ratio of the OX-I standard
normalized to δ13C value of −19‰, and y is the year the sample was measured. The term 0.95×ROXI,−19
represents the internationally accepted 14C reference value in 1950, and the following exponential term
is for decay correction for change in the OX-I standard since 1950. Note that in this case of decay
correction, the half-life of 5730 years, not the Libby half-life, is used, so that 8267 is the mean life of14

C (= 5730/ln2 in years).

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The notation Δ14C is per mil (‰, or parts per thousand) deviation of the sample from the standard.
Positive Δ14C value indicate that the sample has more 14C than the atmospheric CO2 in 1950, indicating
the presence of bomb-14C4,20,65). A Δ14C value of +1000‰ would be equivalent to a doubling of the
14

C/12C ratio in the atmosphere, which is roughly the value reached in 1964 for atmospheric CO2 owing

to the atmospheric weapons testing22).

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8. Practical Application
This chapter demonstrates an application using 14C and stable carbon and nitrogen isotopes to studying
soil carbon dynamics, which was conducted in a Japanese temperate forest. The chapter includes: soil
sampling and treatment; soil organic carbon fractionation by two methods (density and aggregate-size);stable isotope and 14C analysis; and results obtained and their interpretations.

8.1.

Soil Sampling and Treatment

Soil sampling was conducted at a 30-year-old evergreen broadleaved forest site in Tsukuba (in thepremise of the National Institute for Environmental Studies, NIES) in November 2018 (Fig. 8.1). The
method used for soil sampling was the soil-digging method (see Chapter 2). This is because the soil at
this forest site has been classified as Andosol and is assumed to accumulate a significant amount of
SOC even in deeper soil layers; therefore, we want to evaluate it throughout the soil profile. Litter
samples was collected from O horizon (forest-floor litter layer) in the area of 30 cm × 30 cm, andthereafter, soil samples of five soil layers (0–10, 10–20, 20–30, 30–40, and 40–50 cm) were collectedfrom a face of the pit. To determine the bulk density of the soil, we also collected soil samples fromeach of the soil layers, by using a known-volume (100 cm3) soil sampler.

Fig. 8.1. Photos of the forest site in Tsukuba (a), soil sampling (b),and the face of the soil pit after sampling (c).

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The litter and soil samples were immediately transported to the laboratory and then dried to a constantweight at room temperature. The litter samples were finely chopped using a mixer to obtain
homogenized samples after removing coarse woody debris (fallen branches and twigs). The soil samples
were sieved through a 2-mm mesh, and for analysis, the < 2 mm fractions were ground into powder in amortar after carefully removing fine roots in the samples.
The soil samples of the 100-cm3 volume were also dried, weighed, and sieved through a 2-mm mesh.
Both fractions obtained by sieving (i.e., > 2 mm and < 2 mm fractions) were weighted to evaluate thebulk density and the gravel (> 2 mm) content of the soil.

8.2.

Soil Organic Carbon Fractionation

To investigate the physical status of SOC and its relation to the SOC dynamics in the soil profile, weapplied two fractionation methods. The density and aggregate-size fractionation methods, described in
Sections 4.1 and 4.2, were applied to the soil samples. Through the density fractionation method, we
separated the soil samples into two SOC fractions: a low-density fraction (LF: < 1.6 g cm−3 in density)and a high-density fraction (HF: > 1.6 g cm−3) (Fig. 8.2). Through the aggregate-size fractionation, we
separated the soil samples into two SOC fractions: macroaggregate fraction (MAA: > 250 m in size)
and microaggregate fraction (MIA: < 250 m) (Fig. 8.3).

Fig. 8.2. Examples of LF (a) and HF (b) fraction samples isolated from the topmost (0–10 cm) soil layer
observed using a stereomicroscope (SZX7, OLYMPUS, Japan). The LF fraction was dominated by plant
detritus or particulate organic matter (POM), whereas the HF fraction was dominated by soil mineralparticles.

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Fig. 8.3. Examples of MAA (a) and MIA (b) fraction samples isolated from the topmost (0–10 cm) soil
layer observed using a stereomicroscope (SZX7, OLYMPUS, Japan). The MAA fraction was dominatedby macroaggregates (> 250 m in size), whereas the MIA fraction was dominated by microaggregates(< 250 m).

8.3.

Stable Isotope Analysis

A portion of each of the samples (litter, bulk soil, and SOC fractions) was analyzed for their total C and
N contents using an elemental analyzer (vario PYRO cube, Elementar, Germany). Stable carbon andnitrogen isotope ratios (δ13C and δ15N, respectively) were analyzed using an isotope ratio mass
spectrometer (IsoPrime100, Isoprime Ltd., UK) connected with an elemental analyzer (vario PYRO
cube), with an analytical uncertainty of < 0.1‰ (1SD). See Chapter 5 for detailed procedures of thestable isotope analysis.

8.4.

Radiocarbon Analysis

Radiocarbon analyses were made with the JAEA-AMS-MUTSU for the bulk soil samples and the
JAEA-AMS-TONO-5MV for the SOC fraction samples, respectively. The bulk soil samples wereprepared for AMS-14C measurements in the manner described in Sections 6.1–6.4. Briefly, the samples
were combusted to generate CO2 in evacuated quartz tubes with CuO, and the resulting CO2 wascryogenically purified. Graphite targets for AMS were formed on fine iron power by reducing thepurified CO2 with hydrogen gas. To prepare the SOC fraction samples for AMS-14C measurements, weused the automated graphitization system at the Tono Geoscience Center of JAEA (see Section 6.5).

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8.5.

Results and Interpretations

8.5.1. Carbon content and SOC stock
Results of the soil sampling and treatment and the measurement of C content in the soil samples areshown in Table 8.1. The SOC stock in the O horizon (litter layer) was evaluated using the equation (Eq.
3.2), based on the measured data of the litter inventory and C content of the litter sample. The SOC
stock in each of the soil layers was evaluated using the equation (Eq. 3.1), based on the measured dataof the bulk density, gravel content, and C content of the soil sample.

Table 8.1. Properties and SOC stocks in the O horizon and soil layers.
Layer

Bulk densityb

Litterinventorya−3(g cm )(kg m−2)

Gravelcontent

C content
c

(gC g

−1

soil)

SOC stock(kgC m−2)(%)

O (litter)

2.93

NA

NA

224

0.65

0–10 cm

NA

d

0.52

2.5

86

4.30

10–20 cm

NA

0.53

0.7

52

2.74

20–30 cm

NA

0.48

0

42

2.04

30–40 cm

NA

0.47

0

31

1.45

40–50 cm

NA

0.52

0

22

1.13

aLitter inventory was evaluated from the dry weight of the litter sample in the collection area of 30 cm × 30 cm.
bBulk density was evaluated from the dry weight of soil sample collected by the 100-cm3 volume sampler.

cGravel content was determined as a percentage by mass of the > 2 mm fraction of the 100-cm3 volume sample.
dNA: Not available.

The bulk density of the soil was very low and fairly uniform throughout the soil profile. There wasvirtually no coarse particles (> 2 mm in diameter) in all soil layers. The litter sample showed a highcontent of C (224 gC g−1), indicating a relatively small contribution of soil mineral particles in the O
layer. The C content of the soil decreased with depth, and as a result, the SOC stock decreased withdepth. The total stock of SOC in the first 20 cm of soil was determined to be 7.0 kgC m−2 for this forest
site, which was approximately 1.4 times greater than the global average of SOC stock (5.1 kgC m−2)71).
These properties are attributable to the unique physical and chemical properties of Andosol, whichdevelops from parent materials of volcanic origin (volcanic ash). The large accumulation of SOC in thissoil is probably due to the formation of organometallic complexes (Al-humus complexes) that resist
microbial decomposition72).

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Carbon contents of the density and aggregate-size fractions are given in Table 8.2, together with that of
the bulk soil. Regarding the desntity fractionation, the C content of the LF fraction was higher
compared with that of the bulk soil, whereas the C content of the HF fraction is lower. The results
indicate that the density fractionation is useful to separate the soil into two different SOC pools withdifferent properties: the LF fraction is a particulate organic matter (POM)-dominant fraction withrelatively free of mineral materials (soil particles), and the HF fraction is a mineral-dominant fraction. It
is interesting to see that the C content of the LF fraction does not change so much with soil depth, eventhough the C contents of the bulk soil and HF fraction decrease with soil depth.

Table 8.2. Carbon content (in gC g−1 soil) of the bulk soil, density fractions, and aggregate-size
fractions.
Layer

Bulk

Density fractionsa

Aggregate-size fractionsb

LF

HF

MAA

MIA

O (litter)

224

NAc

NA

NA

NA

0–10 cm

86

150

67

84

76

10–20 cm

52

140

45

45

50

20–30 cm

42

150

40

41

44

30–40 cm

31

160

29

30

31

40–50 cm

22

133

20

20

22

aLF and HF represent low-density (< 1.6 g cm −3) and high-density (> 1.6 g cm−3) fractions, respectively.

bMAA and MIA represent macroaggregate (> 250 m) and microaggregate (< 250 m) fractions, respectively.
cNA: Not available.

In contrast to the density fractionation, the two MAA and MIA fractions obtained through theaggregate-size fractionation were very similar in the C content and its decreasing trend with soil depth.
These fractions significantly differed in the size of soil aggregates, but seemed to consist of bothorganic and mineral materials at a similar percentage by mass in each of the soil layers at this forest site.
In general, C content in soil aggregates increases with the size of aggregates because of a relatively
large contribution of POM and fine roots in macroaggregate formation. However, a lack of aggregate
size-dependent change in the C content has been reported for Andosols59,73).
SOC stocks in the density and aggregate-size fractions are summarized in Table 8.3. The density
fractionation revealed that approximately 80% of the SOC was present in the HF fraction, i.e., in theform of organo-mineral associations. Only in the top 0–10 cm layer of the soil, the LF fraction
represented a significant fraction to accumulate SOC. This is probably because of a continuous input of
POM from the upper O layer through microbial decomposition of litter materials. The aggregate-size
fractionation revealed that approximately two-thirds of the total SOC was present in association with- 38 -

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microaggregates. The result indicates that microaggregates play an important role in SOC accumulationin this soil. Note that the SOC stocks in the separated fractions (LF and HF fractions for the density
fractionation, or MAA and MIA fractions for the aggregate-size fractionation) do not sum up to 100% insome soil layers due to a loss of SOC (mostly as dissolved organic carbon) during the fractionation
procedure.

Table 8.3. SOC stocks (in kgC m−2) in the density and aggregate-size fractions.
Density fractionsa

Layer

Aggregate-size fractionsb

LF

HF

MAA

MIA

0–10 cm

1.04 (24.2%)c

2.83 (65.7%)

0.98 (22.7%)

2.81 (65.3%)

10–20 cm

0.24 (8.8%)

2.29 (83.6%)

0.93 (34.0%)

1.56 (56.9%)

20–30 cm

0.16 (7.8%)

1.84 (90.2%)

0.74 (26.2%)

1.27 (62.3%)

30–40 cm

0.12 (8.5%)

1.33 (91.5%)

0.26 (17.8%)

1.17 (80.8%)

40–50 cm

0.10 (8.6%)

1.05 (91.4%)

0.23 (20.6%)

0.88 (78.0%)

Total

1.66 (14.2%)

9.34 (79.9%)

3.14 (26.9%)

7.69 (65.9%)

aLF and HF represent low-density (< 1.6 g cm −3) and high-density (> 1.6 g cm−3) fractions, respectively.
bMAA

and MIA represent macroaggregate (> 250 m) and microaggregate (< 250 m) fractions, respectively.

cNumber in parentheses indicates percentage of the total SOC stock in the soil layer.

8.5.2. Radiocarbon signatures of SOC
Conventional 14C ages determined for the bulk soil, density fractions, and aggregate-size fractions areshown in Table 8.4. In general, 14C age of SOC in the bulk soil increased with soil depth, ranging from
Modern to more than 2000 years BP. This indicates that SOC in deeper soil layers is, on average, older
than that in shallower soil layers. A question here is why the litter materials in the O horizon showed 14C
age of 50 years BP (or a negative Δ14C value, also see Table 8.5); that is too old when considering thatturnover times (as a result of the addition via litter fall and the decomposition) of litter materials arenormally several years to a decade in O horizons of temperate forests4,74). A possible explanation for thisis that the current atmospheric CO2 in this region has a negative Δ14C value with a contribution of CO2
emission from 14C-depleted fossil fuel combustion75). Thus the recently added litter materials in the O
horizon might show an older 14C age. To test this, we collected atmospheric samples at this site in 2018
and obtained Δ14C values of around −16‰ for the atmospheric CO2.
In contrast to the O horizon, SOC in the topmost 0–10 cm soil layer had a modern 14C age, indicating asignificant incorporation of 14C-enriched organic materials that have been fixed since 1950 (i.e., theatmospheric nuclear weapons testing) in this soil layer. In the soil layers deeper than a depth of 10 cm,
however, SOC was found to have resided in the soil long enough (several hundred to several thousand- 39 -

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years) for significant radioactive decay to occur.

Table 8.4. Conventional 14C ages (in years BP) of the bulk soil, density fractions, and aggregate-size
fractions.
Layer
O (litter)

Density fractionsa

Bulk
50c
d

Aggregate-size fractionsb

LF

HF

MAA

MIA

NAe

NA

NA

NA

ModernModernModernModern0–10 cmModern10–20 cm

210

Modern

160

140

80

20–30 cm

1010

70

1000

1070

910

30–40 cm

1740

—f

1630

1740

1530

40–50 cm

2330

—

2400

2490

2230

aLF and HF represent low-density (< 1.6 g cm −3) and high-density (> 1.6 g cm−3) fractions, respectively.

bMAA and MIA represent macroaggregate (> 250 m) and microaggregate (< 250 m) fractions, respectively.
cThe analytical uncertainty of the 14C age was less than ± 50 years BP.
dModern indicates that C in the sample contains post-1950, bomb-14C.
eNA: Not available.

fThe amount of sample obtained is small and not enough for 14C analysis.

Soil organic carbon in the LF and HF fractions differed in 14C age (Table 8.4). The SOC in the HF
fraction, which is the predominant pool of C in this soil (see Table 8.3), had a 14C age similar to that in
the bulk soil, whereas the SOC in the LF fraction was significantly younger compared with the HF
fraction (and the bulk soil). This result indicates that the SOC in the LF fraction represents a rapidly
cycling pool of C in the soil. It is particularly interesting to see the stark contrast in 14C age of SOC
between the two fractions in the 20–30 cm soil layer. In contrast, SOC in the MAA and MIA fractions
was relatively similar in 14C age throughout the soil profile, although SOC in the MAA fraction wasconsistently older than that in the MIA fraction. This result suggests that, in general, SOC in soil
aggregates with different size classes can turn over on a similar timescale in the same depth of this soil.
The Δ14C values determined for the bulk soil, density fractions, and aggregate-size fractions are shownin Table 8.5. The Δ14C values of SOC can be interpreted, however, as for the 14C age: the Δ14C value of
SOC in the bulk soil decreased with soil depth, reflecting more contribution of bomb-14C in the upper
soil layers and more influence of 14C radioactive decay in the deeper soil layers. The SOC in the LF
fraction are enriched in 14C (bomb-14C) and therefore dominated by SOC fixed since 1950s. On theother hand, the SOC in the HF fraction generally consists of SOC fixed prior to 1950s. Twoaggregate-size fractions show a similar Δ14C value, indicating that cycling of SOC in these fractions, on
average, occurs on a similar timescale.

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Table 8.5. Radiocarbon signatures (Δ14C in ‰) of the bulk soil, density fractions, and aggregate-size
fractions.
Layer

Bulk

Density fractionsa

Aggregate-size fractionsb

LF

HF

MAA

MIA

O (litter)

−6.1c

NAd

NA

NA

NA

0–10 cm

35.9

64.7

50.8

63.3

62.0

10–20 cm

−25.6

78.0

−19.5

−17.7

−10.5

20–30 cm

−117.6−8.4−116.9−124.3−107.430–40 cm

−194.5—−183.8−194.5−173.040–50 cm

−251.4—−258.4−266.1−242.1aLF and HF represent low-density (< 1.6 g cm −3) and high-density (> 1.6 g cm−3) fractions, respectively.

bMAA and MIA represent macroaggregate (> 250 m) and microaggregate (< 250 m) fractions, respectively.
cThe analytical uncertainty of the Δ14C value was 4–6‰.
dNA: Not available.

The SOC fractions obtained through any of the fractionation methods are still a mixture of young andold SOC; it has recently been revealed that even in deeper soil layers, decadally cycling SOC (i.e., SOC
generated via photosynthesis since the atmospheric nuclear weapons testing and thus enriched in 14C
due to the incorporation of bomb-14C) can exist25). This contradicts the assumption in the 14C dating
method that there has been no addition of 14C in SOC in the soil sample since the SOC was added into
the soil. In this respect, conventional 14C age cannot represent real dynamics of SOC in many cases,
particularly for SOC pools with a relatively young 14C age (or a less 14C-depleted Δ14C value). For suchsemi-open systems like SOC, it should therefore be considered better to estimate its turnover time ormean residence time by considering both the inflow (addition of SOC via various soil processes) andoutflow (loss of SOC via microbial decomposition (and radioactive decay for 14C)) of C in a SOC pool.
To achieve this, some modeling approaches have been proposed4,23,25,45,55,76). Details of theseapproaches are well explained and summarized in a chapter of the recently published book65). One thing
we should note here is that these modeling approaches are all still built upon several assumptions.
As an example, a time-dependent single-pool model can be used to predict the change in Δ14C value in a
SOC pool:F(t) = {I(t−1) ∙ Fatm(t−1) + F(t−1) ∙ C(t−1) ∙ (1−k−λ)} / C(t)(Eq. 8.1)

where F(t) represents the Fraction Modern of carbon (= Δ14C(t)/1000+1) in the SOC pool at year t,I(t−1) the amount of C inputs to the SOC pool at year t−1 (i.e., in the previous 1-year time step),
Fatm(t−1) the Fraction Modern of carbon in the atmospheric CO2 at year t−1, and F(t−1) the Fraction
Modern of carbon in the SOC at year at year t−1. C(t) and C(t−1) represent the amounts of C in the SOC- 41 -

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pool at years t and t−1, respectively, k represents the decomposition rate constant for the SOC pool (iny−1), and λ the radioactive decay constant for 14C (1.21 × 10−4 y−1). Fatm(t) is derived from theatmospheric 14C-CO2 record22,77). For the target SOC pool, we can predict Δ14C value by using thismodel to find a value of k to the best explain the measured Δ14C value, and then estimate the turnovertime of the SOC pool as k−1 (in year)25).
With the use of this modeling approach (Eq. 8.1), the turnover time of SOC for the O horizon (littermaterials) can be estimated as 6 years based on its Δ14C value (−6.1‰). The Δ14C value (+35.9‰) of
SOC in the topmost (0–10 cm) soil layer, which shows a contribution of bomb-14C, yields two possible
estimates for turnover time: approximately 11 and 190 years. The turnover times of SOC for the deeper
soil layers can be estimated as approximately 450, 1190, 2020, and 2770 years for the 10–20 cm, 20–30
cm, 30–40 cm, and 40–50 cm soil layers, respectively.

8.5.3. Stable carbon and nitrogen isotope ratios of SOC
Stable carbon isotope ratios (δ13C) of the bulk soil, density fractions, and aggregate-size fractions areshown in Table 8.6. The δ13C values of the O horizon was around −29‰, which was similar to δ13C
values of C3 plants, the current vegetation at this forest site. The δ13C value of the bulk soil increased
with soil depth. The δ13C value of SOC is influenced by the degree of biological transformation and thevegetation (i.e., around −29‰ for C3 plants and around −13‰ for C4 plants)78). Regarding the effect of
biological transformation, it is well documented that an enrichment of heavier isotopes (13C) relative tolighter isotopes (12C) in SOC occurs during its decomposition via biological metabolic processes of soil
microorganisms79,80). Therefore, an increase in δ13C value of SOC with depth can occur because SOC in
deeper soil layers is more biologically transformed through repeated microbial processing. However, the13

C enrichment through the biological transformation is typically within several per mil from the surface

to deeper soil layers80–82). Therefore, the large change in the δ13C value of SOC with depth (from−26.1‰ to −19.5‰) at this forest soil can be mostly attributed to change in the vegetation. The high
δ13C values (−19.5‰) observed for the deeper soil layers indicate an accumulation of SOC thatoriginated from C4 plants, and suggest that the forest site was previously dominated by C4 plants andthe vegetation had changed to current C3 plants.
For this study site, the amount of SOC of the previous C4-plant (grassland) origin in a soil can be
estimated through a mass balance equation:
SOCC4 = (δ13CSOC – δ13CC3) / (δ13CC4 − δ13CC3)(Eq. 8.2)

where SOCC4 is the proportion of C4 plant-derived SOC (%), δ13CSOC is the δ13C value of the SOC (‰),
δ13CC3 is the δ13C value of C3 plant-derived SOC (‰), and δ13CC4 is the δ13C value of C4 plant-derived- 42 -

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SOC (‰), respectively. For instance, the contribution of the previous grassland-derived SOC to the
SOC currently accumulated in the 40–50 cm soil layer can be estimated to be 59% by using the equation(Eq. 8.2) and the measured δ13C values (i.e., δ13CSOC and δ13CC3), when the value of δ13CC4 is assumed
to be −13‰78). Similarly, the contribution of grassland-derived SOC can be estimated as 18% of thetotal SOC in the 0–10 cm soil layer, which indicates that SOC in this layer is mostly of forest origin.
This finding from δ13C data is qualitatively consistent with 14C results showing older SOC in deeper soil
layers (Tables 8.4 and 8.5). In this way, using the 13C data, we may be able to separate soil C into two
different SOC pools with different origins (i.e., turnover times) without any physical and chemical
fractionations of soil samples, if the study site had experienced a land use conversion (e.g., grassland toforest and forest to agricultural land) and information about when the conversion occurred can beobtained83).

Table 8.6. Stable carbon isotope ratios (δ13C in ‰) of the bulk soil, density fractions, andaggregate-size fractions.
Layer

Bulk

Density fractionsa
LF

Aggregate-size fractionsb
HF

MAA

MIA

c

NA

NA

NA

O (litter)

−28.8

NA

0–10 cm

−26.1

−27.7

−25.2

−26.1

−25.4

10–20 cm

−22.9

−26.7

−22.7

−22.9

−22.9

20–30 cm

−20.3

−25.7

−20.2

−20.3

−20.3

30–40 cm

−19.5

−25.9

−19.4

−19.6

−19.5

40–50 cm

−19.5

−26.4

−19.4

−19.5

−19.4

aLF and HF represent low-density (< 1.6 g cm −3) and high-density (> 1.6 g cm−3) fractions, respectively.

bMAA and MIA represent macroaggregate (> 250 m) and microaggregate (< 250 m) fractions, respectively.
cNA: Not available.

The δ13C values of the SOC fractions showed contrasting patterns between the two fractionation
methods (Table 8.6). In the density fractionation, the HF fractions were similar in the δ13C value to thebulk soils, but the LF fractions were lower in the δ13C value compared with the bulk soils and HF
fractions. This indicates that the SOC in the LF and HF fractions largely differ in its origin; the LF
fractions consist mostly of SOC of the recent forest origin throughout the soil profile, whereas the HF
fractions are a mixture of previous grassland- and recent forest-derived SOC with a depth trend in the
mixture ratio. In contrast, in the aggregate-size fractionation, the δ13C values of SOC were generally
similar between the MAA and MIA fractions, indicating that both aggregate-size fractions have asimilar mixture ratio of the grassland- and forest-derived SOC throughout the soil profile. These results
are consistent with the results of 14C analysis showing that the SOC in the LF fraction is younger thanthat in the HF fraction, and that the MAA and MIA fractions are generally similar in 14C age or Δ14C
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value in each of the soil layers (Tables 8.4 and 8.5).
It has been documented that, as with 13C, 15N is also enriched relative to 14N during microbial
decomposition of SOC84). Because δ15N value is less affected by the vegetation, δ15N value may be abetter indicator of the degree of biological transformation of SOC in soils. Clear trends of increasing
δ15N value with soil depth were observed for the bulk soil and for the three SOC fractions (except the
LF fraction) (Table 8.7), indicating that SOC in these soil and fractions is transformed more in deeper
soil layers through microbial degradation. Consistent with the 14C signatures, the δ15N values suggest a
relatively less degradation of SOC in the LF fractions, compared with the other fractions, throughout thesoil profile. The concomitant increase in the δ15N value and 14C age with soil depth implies that thelonger SOC resides in soil, the higher the degree of microbial transformation85).

Table 8.7. Stable nitrogen isotope ratios (δ15N in ‰) of the bulk soil, density fractions, andaggregate-size fractions.
Layer

Bulk

Density fractionsa

Aggregate-size fractionsb

LF

HF

MAA

MIA

O (litter)

−1.2

NA

NA

NA

NA

0–10 cm

2.4

0.0

3.5

2.2

3.2

10–20 cm

7.0

2.1

7.4

7.1

7.1

20–30 cm

9.6

3.2

10.0

9.8

9.8

30–40 cm

10.4

5.2.

10.1

10.6

10.7

40–50 cm

10.0

4.5

10.3

10.7

10.5

aLF and HF represent low-density (< 1.6 g cm −3) and high-density (> 1.6 g cm−3) fractions, respectively.

bMAA and MIA represent macroaggregate (> 250 m) and microaggregate (< 250 m) fractions, respectively.
cNA: Not available.

8.6.

Summary

In this way, 14C and stable isotope analyses are useful to understand and further quantify the carbon
dynamics in soils. The isotope-based approaches enable us to compare the soil carbon dynamics indifferent terrestrial ecosystems and regions, which may offer a way to explore the diverse ecologicalroles of soils in the global carbon cycle. Studies using coupled carbon cycle–climate models suggestthat soil carbon feedback to climate change could lead to a doubling of the projected warming by theend of this century. However, there is still a great deal of mechanistic uncertainty that underlies
simulations of the responses of SOC to climate change. In these models, soils are generally
characterized by several pools of SOC with specific turnover times. Therefore, quantification of SOC
dynamics by using the 14C approach can be incorporated into the models to improve their accuracy and- 44 -

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reliability. In addition, isotope-based approaches provide a unique opportunity to investigate how soil
carbon dynamics changes along with, for example, land-use conversion and the progression of climatechanges. Such investigations will be important to assess the impacts of natural and artificial processeson the global carbon cycle. Findings obtained through the isotope-based approaches are essential to
improve our predictions of the future change in the Earth’s climate.

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Acknowledgements
This guide was prepared in collaboration between four Japanese organizations: Japan Atomic Energy
Agency (JAEA), National Institute for Environmental Studies (NIES), The University of Tokyo, andIbaraki University, through the project “Research on Climate Change using Nuclear and Isotopic
Techniques” organized by the Ministry of Education, Culture, Sports, Science and Technology, Japan(MEXT), under the framework of the Forum for Nuclear Cooperation in Asia (FNCA).
Two of the co-authors, Untung Sugiharto of Natioanl Nuclear Energy Agency of Indonesia (BATAN)
and Chakrit Saengkorakot of Thailand Institute of Nuclear Technology (TINT), acknowledge thefinancial support provided by the Nuclear Researchers Exchange Program of MEXT, to participate inthis project.
The authors thank Toshiya Yoshida of the JAEA, and Munemasa Teramoto and Hajime Tomimatsu of
the NIES for support with the fieldwork; Misuzu Kaminaga, Kikuko Yoshigaki, Mariko Ota, and Taro
Ishii of the JAEA for support with the laboratory work; the staff at the Aomori Research andDevelopment Center and the Tono Geoscience Center of JAEA for the AMS-14C measurements; andHamza El-Asaad of the JAEA for proofreading this document.

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国際単位系（SI）
表１．SI 基本単位
SI 基本単位

基本量

名称
記号
長
さメ ートル m
質
量 キログラム kg
時
間
秒
s
電
流ア ンペア A
熱力学温度 ケ ル ビ ン K
物 質 量モル mol
光度 カ ン デ ラ cd

表２．基本単位を用いて表されるSI組立単位の例
SI 組立単位
組立量
名称
記号
面
積 平方メートルm2
体
積 立方メートルm3
速 さ ， 速 度 メートル毎秒m/s
加速度 メートル毎秒毎秒
m/s2
波
数 毎メートルm-1
密 度 ， 質 量 密 度 キログラム毎立方メートル
kg/m3
面
積
密
度 キログラム毎平方メートル
kg/m2
比
体
積 立方メートル毎キログラム
電流
密
度 アンペア毎平方メートル
磁 界 の 強 さ アンペア毎メートル
量 濃 度 (a) ， 濃 度 モル毎立方メートル
質量
濃度 キログラム毎立方メートル輝
度 カンデラ毎平方メートル屈折率 (b) （数字の） １
比 透 磁 率 (b) （数字の） １

乗数
24

10
1021
1018
1015
1012
109
106
103

3

m /kg
A/m2
A/m
mol/m3
kg/m3
cd/m2
1
1

102
101

ヨ

表５．SI 接頭語
記号
乗数
名称
タ
Ｙ
シ
10-1 デ

ゼ
タ
エ ク サ

Ｚ
Ｅ

10-2

ペ
テ

タ
ラ

Ｐ
Ｔ

ギ
メ

ガ
ガ

Ｇ
Ｍ

マイクロ
ノ
10-9 ナコ
10-12 ピ
10-15 フェムト

キ
ロ
ヘ ク ト
デ
カ

ｋ
ｈ
da

10-3
10-6

記号

セ ン チ
ミ
リ

ト
10-18 ア
10-21 ゼ プ ト
10-24 ヨ ク ト

d

c
m
µ
n
p
f
a
z
y

表６．SIに属さないが、SIと併用される単位
名称
記号
SI 単位による値
分
min 1 min=60 s
時
h 1 h =60 min=3600 s（a）量濃度（amount concentration）は臨床化学の分野では物質濃度（substance concentration）ともよばれる。
（b）これらは無次元量あるいは次元１をもつ量であるが、そのことを表す単位記号である数字の１は通常は表記しない。

d
°
’

日
度
分

表３．固有の名称と記号で表されるSI組立単位
SI 組立単位
組立量
他のSI単位による SI基本単位による
名称
記号
表し方
表し方
（ｂ）
平
面
角 ラジアン(ｂ)
rad
m/m
1
（ｂ）
(ｂ)
(c)
2
2
立
体
角 ステラジアン
sr
1
m /m
周
波
数 ヘルツ（ｄ）
Hz
s-1
力
ニュートン
N
m kg s-2
圧力
応
力 パスカル,Pa
N/m2
m-1 kg s-2
エ ネ ル ギ ー , 仕 事 , 熱 量 ジュールJNm
m2 kg s-2
仕 事 率 ， 工 率 ， 放 射 束 ワット
WJ/s
m2 kg s-3
電
荷
電気量 クーロン,
CsA
電 位 差 （ 電 圧 ） , 起 電 力 ボルト
VW/A
m2 kg s-3 A-1
静電
容量 ファラドF
C/V
m-2 kg-1 s4 A2
電
気
抵
抗 オーム
Ω
V/A
m2 kg s-3 A-2
コ ン ダ ク タ ン ス ジーメンス
S
A/V
m-2 kg-1 s3 A2
磁
束 ウエーバWb
Vs
m2 kg s-2 A-1
磁
束
密
度 テスラ
T
Wb/m2
kg s-2 A-1
イ ン ダ ク タ ン ス ヘンリーH
Wb/A
m2 kg s-2 A-2
セ ル シ ウ ス 温 度 セルシウス度(ｅ)℃K
光
束 ルーメンlm
cd
cd sr(c)照度 ルクスlx
m-2 cdlm/m2
Bqs-1
放 射 性 核 種 の 放 射 能 （ ｆ ） ベクレル（ｄ）
吸収線量, 比エネルギー分与,
グレイ
Gy
J/kg
m2 s-2カーマ
線量当量, 周辺線量当量,Sv
J/kg
m2 s-2シーベルト（ｇ）
方向性線量当量, 個人線量当量酸素
活性 カタールkat
s-1 mol

1 d=24 h=86 400 s
1°=(π/180) rad1’=(1/60)°=(π/10 800) rad”1”=(1/60)’=(π/648 000) radha 1 ha=1 hm 2=104m2
L，l 1 L=1 l=1 dm3=103cm3=10-3m3t
1 t=103 kg

秒
ヘクタール
リットル
トン

表７．SIに属さないが、SIと併用される単位で、SI単位で表される数値が実験的に得られるもの
名称
記号
SI 単位で表される数値
電 子 ボ ル ト
ダ ル ト ン
統一原子質量単位

eV
Da
u

天

ua

文

単

位

1 eV=1.602 176 53(14)×10 -19J

1 Da=1.660 538 86(28)×10-27kg
1 u=1 Da

1 ua=1.495 978 706 91(6)×1011m

表８．SIに属さないが、SIと併用されるその他の単位
名称
記号
SI 単位で表される数値
バー
ル bar １bar=0.1MPa=100 kPa=10 5Pa
水銀柱ミリメートル mmHg １mmHg≈133.322Pa(a)SI接頭語は固有の名称と記号を持つ組立単位と組み合わせても使用できる。しかし接頭語を付した単位はもはや
コヒーレントではない。
(b)ラジアンとステラジアンは数字の１に対する単位の特別な名称で、量についての情報をつたえるために使われる。
実際には、使用する時には記号rad及びsrが用いられるが、習慣として組立単位としての記号である数字の１は明示されない。
(c)測光学ではステラジアンという名称と記号srを単位の表し方の中に、そのまま維持している。
(d)ヘルツは周期現象についてのみ、ベクレルは放射性核種の統計的過程についてのみ使用される。
(e)セルシウス度はケルビンの特別な名称で、セルシウス温度を表すために使用される。セルシウス度とケルビンの単位の大きさは同一である。したがって、温度差や温度間隔を表す数値はどちらの単位で表しても同じである。
(f)放射性核種の放射能（activity referred to a radionuclide）は、しばしば誤った用語で”radioactivity”と記される。
(g)単位シーベルト（PV,2002,70,205）についてはCIPM勧告2（CI-2002）を参照。

表４．単位の中に固有の名称と記号を含むSI組立単位の例
SI 組立単位
組立量
SI 基本単位による
名称
記号
表し方
-1
粘
度 パスカル秒
Pa s
m kg s-1
力 の モ ー メ ン ト ニュートンメートル
Nm
m2 kg s-2
表面
張力 ニュートン毎メートル
N/m
kg s-2角
速度 ラジアン毎秒
rad/s
m m-1 s-1=s-1
角
加速度 ラジアン毎秒毎秒
rad/s2
m m-1 s-2=s-2
熱 流 密 度 , 放 射 照 度 ワット毎平方メートル
W/m2
kg s-3
熱 容 量 , エ ン ト ロ ピ ー ジュール毎ケルビン
J/K
m2 kg s-2 K-1
比 熱 容 量 ， 比 エ ン ト ロ ピ ー ジュール毎キログラム毎ケルビン J/(kg K)
m2 s-2 K-1
比 エ ネ ルギ ー ジュール毎キログラム
J/kg
m2 s-2
熱伝
導率 ワット毎メートル毎ケルビン W/(m K) m kg s-3 K-1
体 積 エ ネ ル ギ ー ジュール毎立方メートル J/m3
m-1 kg s-2
電界の強さ ボルト毎メートル
V/m
m kg s-3 A-1
電荷密
度 クーロン毎立方メートル C/m3
m-3 s A
表面
電
荷 クーロン毎平方メートル C/m2
m-2 s A電 束 密 度 ， 電 気 変 位 クーロン毎平方メートル C/m2
m-2 s A誘
電率 ファラド毎メートル
F/m
m-3 kg-1 s4 A2
透磁率 ヘンリー毎メートル
H/m
m kg s-2 A-2
モ ル エ ネ ル ギ ー ジュール毎モル
J/mol
m2 kg s-2 mol-1
モルエントロピー, モル熱容量 ジュール毎モル毎ケルビン J/(mol K) m2 kg s-2 K-1 mol-1
照 射 線 量 （ Ｘ 線 及 び γ 線 ） クーロン毎キログラム
C/kg
kg-1 s A
吸収線
量率 グレイ毎秒
Gy/s
m2 s-3放射
強度 ワット毎ステラジアン
W/srm4 m-2 kg s-3=m2 kg s-3
放射輝
度 ワット毎平方メートル毎ステラジアン W/(m2 sr) m2 m-2 kg s-3=kg s-3
酵 素 活 性濃 度 カタール毎立方メートル kat/m3
m-3 s-1 mol

名称

オングストローム
海
里
バ
ー
ン

Å
Ｍ

１Å=0.1nm=100pm=10-10m
１M=1852m

b

ノ
ネ
ベ

ト
パ
ル

kn
Np
Ｂ

１b=100fm2=(10-12cm) 2 =10-28m2
１kn=(1852/3600)m/s

ル

dB

ッ
ー

デ

シ

ベ

SI単位との数値的な関係は、対数量の定義に依存。

エ

表９．固有の名称をもつCGS組立単位
名称
記号
SI 単位で表される数値
ル
グ erg 1 erg=10-7 J

ダ
ポ

イ
ア

ス
ス

ト ー ク
チ
ル

フ
ガ

ォ

ン dyn 1 dyn=10-5N
ズ P 1 P=1 dyn s cm-2=0.1Pa s
ス St 1 St =1cm2 s-1=10-4m2 s-1
ブ sb 1 sb =1cd cm-2=104cd m-2
ト ph 1 ph=1cd sr cm-2 =10 4lx
ル Gal 1 Gal =1cm s-2=10-2ms-2

マ ク ス ウ エ ルガ
ウス
エルステッド（ ａ）

Mx
G
Oe

1 Mx = 1G cm2=10-8Wb
1 G =1Mx cm-2 =10-4T
1 Oe (103/4π)A m-1（a）３元系のCGS単位系とSIでは直接比較できないため、等号「は対応関係を示すものである。

キ
レ
ラ

名称
ュ
リ
ン

レ
ガ

ト

表10．SIに属さないその他の単位の例
記号
SI 単位で表される数値
ー Ci 1 Ci=3.7×1010Bq

ゲ

ン

ン R
ド rad
ム rem
マ γ

フ
ェ
ル
ミ
メートル系カラット

1 R = 2.58×10-4C/kg

1 rad=1cGy=10-2Gy
1 rem=1 cSv=10-2Sv
1 γ=1 nT=10-9T
1 フェルミ=1 fm=10-15m

ト
標

準

気

1 メートル系カラット = 0.2 g = 2×10-4kg
ル Torr 1 Torr = (101 325/760) Pa
圧 atm 1 atm = 101 325 Pa

カ

ロ

リ

ー

cal

ミ

ク

ロ

ン

µ

大

」

1 cal=4.1858J（｢15℃｣カロリー），4.1868J（｢IT｣カロリー），4.184J（｢熱化学｣カロリー）

1 µ =1µm=10-6m（第8版，2006年）