Carbon Radiochemicals (¹⁴C) and Stable Isotopes (¹³C): Crucial Tools to Study Plant-Soil Interactions in Ecosystems

Abstract

The study of plant-environment interactions has grown steadily during the past two decades. This trend will continue as many environmental changes impact the functioning of ecosystems. One aspect of studying the plant-environment interactions is to focus on the way plants react to abiotic or biotic stresses including chemical mediation between plants or plants-microorganisms (allelopathy) and plant reaction to pollutants. This chapter proposes to focus on carbon radiochemicals (¹⁴C) and stable carbon isotope tracers (¹³C). Indeed, they are powerful techniques in plant ecophysiological researches to describe the transfer and effects of allelochemicals and pollutants in plants and their environment.

These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

1 Introduction

The study of plant-environment interactions has grown steadily during the past two decades. This trend will continue as many environmental changes impact the functioning of ecosystems. One aspect of studying the plant-environment interactions is to focus on the way plants react to abiotic or biotic stresses including chemical mediation between plants or plants-microorganisms (allelopathy) and plant reaction to pollutants. This chapter proposes to focus on carbon radiochemicals (¹⁴C) and stable carbon isotope tracers (¹³C). Indeed, they are powerful techniques in plant ecophysiological researches to describe the transfer and effects of allelochemicals and pollutants in plants and their environment.

¹⁴C radiochemicals are widely used in agronomy to understand pesticide’s translocation into plants and so they offer promising research perspectives in ecology. Because ¹⁴C is not present in natural environment, this is a powerful technique that offers the clue of studying where, when, how many target radiochemicals and their related metabolites are transferred into plants and in the surrounding soils. As example, the study of plant secondary metabolites (PSMs) represents an important area of research in plant ecophysiology. PSMs served to defend plants against herbivores, pathogens and abiotic stress but also as chemical mediators of interactions with competitors (Chiapusio et al. 2005). A single compound can influence multiple components within an ecological system and can have effects at different scales, from physiological to structural community roles. ¹⁴C radiochemical techniques offer then a good way to discover PSM fate in plants (Chiapusio and Pellissier 2001; Chiapusio et al. 2004).

Natural abundance of ¹³C is currently used for a diverse range of applications in environmental and plant sciences since this isotope is naturally occurring in all compartments of ecosystems. As reviewed by Brüggemann et al. (2011), isotopic signature of C varies according to plant photosynthesis strategy (C3 vs C4 vs CAM), plant water use efficiency, plant organs and its constituting compounds (e.g. lipids are ¹³C-depleted whereas cellulose and carbohydrates are ¹³C-enriched). Pulse labeling plants with stable carbon isotope ¹³CO₂ has a great efficiency to trace in situ recently assimilated C in plant organs, soil, and microorganisms including its release through respiration (Kuzyakov and Gavrichkova 2010; Epron et al. 2012). Indeed, leaves incorporate CO₂ into carbohydrates to then “feed” physiological processes of all organs such as growth, respiration, maintenance, storage, osmotic regulation and defense. C allocation among plant organs and rhizospheric microorganisms is then related to the activity of C sources (leaves) and C sinks (roots, trunk, fruits, symbiotic microorganisms). C allocation is furthermore affected by environmental variations, such as shading (Warren et al. 2012; Bahn et al. 2013), drought (Ruehr et al. 2009; Zang et al. 2014; Hartmann et al. 2015), nitrogen supply (Högberg et al. 2010), atmospheric carbon dioxide enrichment (Johnson et al. 2013) and atmospheric pollutants such as Polycyclic Aromatic Hydrocarbons (PAHs) (Desalme et al. 2011a) or ozone (Andersen 2003; Kasurinen et al. 2012).

To explore various applied examples of ¹⁴C and ¹³C techniques in an ecological context, we propose to focus on the transfer and effects in the environment of naturally occurring plant secondary metabolites (PSMs) and organic pollutants (PAHs) on plants. Among the numerous PSMs produced by plants, we focused on allelochemicals such as phenolic compounds, involved in plant-plant and plant-microorganisms interactions. PAHs are ubiquitous organic pollutants emitted by incomplete combustion of biomass or fossil fuel and recovered in all compartments of ecosystems (air, soil, water, plants). From an ecophysiological point of view, challenges still concern the way allelochemicals and organic pollutants are transferred in plants and affect their metabolism but also their surrounding microbial communities.

This chapter aims to present (1) basic aspects of extraction and quantification procedures of radiochemical (¹⁴C) and stable isotopes (¹³C) techniques in ecophysiological experiments and (2) some practical examples using labeled organic molecules (¹⁴C-PSMs and ¹³C-PAH) to trace their fate in plants and their surrounding soil, and labeled ¹³CO₂ to study the way carbon allocation is changed in plant-soil systems exposed to atmospheric pollution by PAHs.

2 Overview of Some ¹⁴C Radiochemical and ¹³C Stable Isotope Techniques

2.1 ¹⁴C Radiochemical Techniques (Table 25.1)

These powerful techniques allow to follow the ¹⁴C compound into seedlings, plants or in the soil in a quantitative and/or qualitative way to describe the transfer of radiolabeled molecules in mesocosms. Such techniques prove the absorption, the distribution, the metabolisation and/or degradation of the ¹⁴C compound in plants and their surrounding soils. Time course of the¹⁴C compound from the soil to the plant, including remanence in the soils is then well described. The identification of crucial environmental factors influencing its kinetic can also be evaluated. Once in plants, the absorbed ¹⁴C compound can be chemically transformed by oxidation, glycosylation or polymerization to be completely or partially metabolized and stocked into the vacuoles or strongly linked into cell walls. The choice for using a molecule partially or fully marked with ¹⁴C, its specific activity (mCi mmol⁻¹) and its radiochemical purity depends of the goal of the study (global signal vs precise chemical fate in inner tissues of plant or soil).

Table 25.1 Some comparisons between usual ¹⁴C radiolabeled techniques

2.1.1 Extraction Procedure

Currently, ¹⁴C tissues are extracted by grinding or by oxidizing. The grinding extraction gives part of the recovered plant radioactivity because it is function of the selected solvent (from polar to non-polar solvent). The oxidizer extraction (oxidative combustion of the plant) gives the total recovered radioactivity in the target organ or in the plant (¹⁴C is transformed in ¹⁴CO₂). Comparisons between the two extractions within a same sample offer then complementary information.

2.1.2 Quantification Procedure

¹⁴C extracts are diluted with a liquid of scintillation to be quantified by a Liquid Scintillation Counter (LSC). More specifically analyses can be performed to discover the ¹⁴C metabolites by Thin Layer Chromatography (TLC) or by Ultra High Phase Liquid Chromatography (UHPLC- UV with radioactive counter detector with continuous flux) or by Gas Chromatography Mass Spectrometry. The use of such techniques will depend of the chemical structure of the selected ¹⁴C compound. The autoradiography technique is also a qualitative technique, which gives the full image of the ¹⁴C labeled plant with a specific imager (phosphor-imager). It allows visualizing the distribution of the radioactivity into the plant. The plant is usually contaminated in a solution into the soil or in the air through ¹⁴C chemical deposition in the leaves.

2.1.3 Warning

Manipulating such extracts needs specific room and material devoted to this. Glass materials including beakers to grow plants are strongly recommended to recover the entire radioactivity including material rinsing. Therefore a trap of ¹⁴CO₂ (NaOH for example), coming from the degradation of the initial ¹⁴C molecule, is often added in the microcosms. For any experimentation, radiochemical measurements are validate only if the total recovered ¹⁴C in all compartments (including culture medium, plants, soil, rinsing wall beaker, etc.) is at minimum 80% of the initial radioactive solution. Under 80%, experiments are considered as non-valuable and must be redone.

2.2 Stable ¹³C Isotope Techniques (Table 25.2)

Enriched isotope methods involve applying huge amounts of a labeled substance and permit one to follow the flows and fates of an element without altering its natural behavior. Because the substances are enriched relative to the background, tracer studies remove or minimize problems of interpretation brought about by fractionation among pools that mix. The signal (the label) is amplified relative to the noise (variation caused by fractionation). Manipulation of stable isotopes requires special room and material devoted to this to prevent the contamination of samples mostly during their preparation.

Table 25.2 Some comparisons between usual ¹³C tracers techniques

2.2.1 Pulse-Labeling with CO₂

Pulse-labeling consists in submitting the foliage to ambient concentration of CO₂ having a different signature of ambient atmospheric CO₂ (average ∂¹³C = −8‰) during few minutes or hours. Injected CO₂ is generally highly enriched in ¹³C (up to 50–100 times background levels) but ¹³C-depleted CO₂ can also be used (∂¹³C = −47‰) (Plain et al. 2009; Streit et al. 2013; Zang et al. 2014). The main objective of ¹³CO₂ labeling is to study how fast and where the assimilated C is allocated among competing organs and partitioned among pools of C compounds. Labeling can be useful to study C allocation and partitioning in any plant or organ collected. The continuous-labeling is also possible. The choice between pulse or continuous depends on the process(es) investigated and whether the organs and C compounds involved turnover rapidly (from hours to months; pulse-labeling) or slowly (over several months to years; continuous-labeling) (Epron et al. 2012; Hartmann and Trumbore 2016).

Warning

Note that the injection of ¹³CO₂ can stress the plant and can alter its natural behavior. It is possible to perform labeling under controlled conditions as well as in the field even though it is more difficult to handle and requires several persons, especially in the case of adult trees.

2.2.2 Purification of Samples

In plant samples, total organic matter is analyzed directly but some purification with solvents can be performed to separate different target biomolecules. For example, soluble sugars, starch, lipids and structural C compounds can be purified from various organs in order to get the dynamics of C partitioning in these pools of molecules (Streit et al. 2013; Blessing et al. 2015; Hartmann et al. 2015; Heinrich et al. 2015; Desalme et al. 2017). The objective of such purification is then to get the purest fraction of target molecules rather than the biggest quantity.

Warning

Caution must be taken to do not add more C in the different fractions during purification. It is recommended to use volatile solvent and carbon-less chemicals.

2.2.3 Quantification of ¹³CO₂/¹²CO₂ Ratio

¹³CO₂/¹²CO₂ ratio in plant and soil samples (solid, liquid and gas) is analyzed by an Isotopic Ratio Mass Spectrometer (IRMS). The principle of IRMS is to ionize the CO₂ issued from the oxidation of the sample (mainly 2 isotopologues, ¹²C¹⁶O¹⁶O and ¹³C¹⁶O¹⁶O), to separate the 2 isotopologues in an electromagnet according to their mass/charge ratio (¹²CO₂ = 44 and ¹³CO₂ = 45), and to collect each one in separated faraday cups, allowing the calculation of a ¹³CO₂/¹²CO₂ ratio. Because the IRMS gives a ratio, total C content of the sample or concentration of the molecules must be determined by different techniques. Solid samples containing organic C are analyzed by an elemental analyzer (EA) in which all the organic C is oxidized into CO₂ in a combustion reactor at >1000 °C coupled with an IRMS (EA-IRMS). Liquid samples (e.g., phloem sap extract, latex, exudates) can be analyzed in EA-IRMS after being dried. Specific compound analysis can also be performed with a chromatography coupled with an IRMS. Molecules in the samples are first separated and quantified by chromatography, then oxidized to produce CO₂, and finally injected in the IRMS. Chromatographic separation of the specific molecules is performed by gas or liquid chromatography by following the same procedure as a simple chromatographic analysis. In liquid chromatography, the eluent phase must be inorganic and solutions have to be degassed to prevent CO₂ contamination.

2.2.4 In Situ Quantification of ¹³CO₂/¹²CO₂ Ratio

High temporal resolution and near-continuous measurements of ¹³CO₂/¹²CO₂ ratio are also possible in situ by using Isotope Ratio Infrared Spectrometers (IRIS) that are more portable than IRMS, which use remains limited to the lab. The principle of IRIS is to emit a radiation by a laser, and to measure the radiation absorbed or emitted by the target gas species with a spectrometer (Kerstel and Gianfrani 2008; Griffis 2013).

3 Measurement of ¹⁴C-Labeled Compounds

The way to obtain target plants with incorporated radiolabeled allelochemicals will influence the selection of the technique to detect the radioactivity. Thus, this section will explain the way to prepare radioactive solution, will indicate how to express results and will give two practical examples of using ¹⁴C allelochemicals illustrating grinding and oxidizing extractions. This part will be illustrated by the study of the effect of ¹⁴C p-hydroxybenzoic acid (POH) from the sample preparation, its extraction by grinding and oxidizing to its quantification in seedlings (Chiapusio and Pellissier 2001).

3.1 Sample Preparation (Fig. 25.1)

Preparation of the Radiochemical: The « Mother Solution » (Step 1)

Most of the time, radiochemicals are sold in a powder form. Adding solvent is then necessary to obtain a solution. The adequate quantity will depend on the radiochemical specific activity. In general, the solubilisation in the appropriate solvent is made to obtain a convenient dilution to prepare the further radiochemical solutions. For example, ethanol or methanol are commonly used with radiolabeled phenols. This so-called « mother solution», must be prepared carefully because it will be the base of preparation of all further radiochemical solutions. This solution must be kept in freezer until further use.

Fig. 25.1

Sample preparation for ¹⁴C compounds: the example of ¹⁴C p-hydroxybenzoic acid (POH) translocation in radish seedlings (non-sterile culture)

Radiochemical Solution (Step 2)

This solution is a mix of (1) the accurate tested concentration of non-radioactive chemical, called «the cold solution» and (2) an adequate quantity of the «mother solution» to get sufficient radioactivity to remain upper the threshold measurement of the radioactivity detector.

Plant Growth Conditions (Step 3)

No special culture conditions are required for the radiochemical experiments compared to usual cultures. The choice of sterile or non-sterile culture depends on the objective of the work but attention must be paid for the potential release of ¹⁴CO₂, which can be trapped in a NaOH solution if necessary.

Samples Preparation (Step 4)

No special sample preparation is required. At the end of each selected incubation period, seedlings from each beaker are washed three times in adequate solvent (e.i. ethanol/water for phenolics) to remove adsorbed compounds. Then, seedling parts are separated. Organs of the same type removed from the same beaker are weighed and frozen together (−25 °C). These constitute one sample of one organ type.

3.2 Expression of Results

Results of the radiochemicals having penetrated into the seedlings can be either expressed as concentration (for example μmol g⁻¹ FW) or as quantity (% of applied ¹⁴C chemical) for one beaker. Quantity is the ratio between the Disintegration Per Minute (DPM) of the initial ¹⁴C allelochemical solution, which was added to each beaker, and the recovery obtained from separated organs of seedlings. Allelochemical concentration is calculated at the equivalence of the cold solution moles, represented by x DPM detected in organs. Concentration pattern reflects the distribution of allelochemical in the plant organs. This unit is widely used to obtain a ratio between the allelochemical content and the plant biomass, which allows then comparisons between different studies. However, using only concentration could lead to erroneous conclusion because seedlings biomass per se is necessary to understand distribution of allelochemical in the organs of target plants (i.e. radish cotyledon biomass is about three times more than roots in 96 h seedlings). Thus, allelochemical content is used to detect preferential accumulation organ.

3.3 Practical Example of ¹⁴C Allelochemical Quantification: Extraction by Grinding and Quantification by Liquid Scintillation (Table 25.3)

Principle

Samples are ground with a mortar and pestle and soluble radiolabeled allelochemical is extracted with the adequate solvent. Homogenates are centrifuged (3000 rpm) for 3 min at 20 °C. The supernatant is set aside for further analysis after each extraction. The ground residue is extracted twice in the mortar and pestle, supernatants being combined.

Table 25.3 p-hydroxybenzoic acid (POH) translocation in radish seedlings in non-sterile condition of incubation

Counting

Supernatants and pellets are mixed with liquid scintillation cocktail. The choice and the quantity of the added liquid scintillation cocktail are made according to manufacturer recommendations. Radioactivity within samples is counted using a Liquid Scintillation Counter and express as Disintegration Per Minute (DPM).

Conclusion

p-hydroxybenzoic acid (POH) is absorbed by radish seedlings. POH concentration recovered in each organ is depending of the time and the type of organ. Focusing on the % of applied ¹⁴C POH, cotyledons are the POH sink organ at any time (Chiapusio and Pellissier 2001).

3.4 Practical Example of ¹⁴C Allelochemical Quantification: Extraction by Oxidizing and Quantification by Liquid Scintillation (Table 25.4)

Table 25.4 p-hydroxybenzoic acid (POH) translocation in radish seedlings in sterile condition of incubation

Principle

Frozen radioactive samples are wrapped in a paper (Germaflor) for oven-drying at 80 °C for 48 h or lyophilized. Each sample is then combusted in a biological oxidizer at 900 °C for about 3 min. ¹⁴CO₂ released by samples is directly trapped into a vial containing liquid scintillation cocktail.

Counting

Radioactivity within samples is counted using a Liquid Scintillation Counter and results are expressed as DPM.

Conclusion

p-hydroxybenzoic acid (POH) is absorbed by radish seedlings. Cotyledons and roots represent the radish organ having the highest POH concentrations. Focusing on the % of applied ¹⁴C allelochemical, cotyledons are the POH sink organ. Comparing results of Tables 25.3 and 25.4, the observed decreased of POH in total seedlings (Table 25.3) is due to a fraction of non-extractable POH in plant tissue (Chiapusio and Pellissier 2001). Indeed, such decrease is not observed when using the oxidizer for POH quantification (Table 25.4).

4 Measurement of ¹³C-Labeled Compounds

This section will explain the way to label plants with ¹³CO₂, to collect the samples and how to express results. Two practical examples are selected, both focusing on plants exposed to a phenanthrene (PHE) atmospheric pollution. The first example aims at considering the way carbon allocation is changed in plant-soil systems exposed to PHE by labeling plants with ¹³CO₂ and the second one the way PHE is transferred in plants-soil systems exposed to ¹³C-labeled PHE.

4.1 Labeling and Sample Collection

Plant Growth Conditions

No special culture conditions are required for plants prior to be labeled with ¹³CO₂.

Labeling of the Plants

A transparent plastic chamber must be specifically designed to label the whole crown of the plants. It means that the design and size of the chamber is related to the dimensions of the target plant. As the chamber modifies the microclimate around the crown, attention must be paid to avoid disturbance. Air temperature and hygrometry inside the chamber are maintained at those of the outside thanks to an air conditioner. ¹³C-enriched or ¹³C-depleted CO₂ is injected through pipes from a bottle of gas into the chamber at a controlled flow rate. The adequate volume of ¹³CO₂ delivered to the plant depends on plant assimilation and the studied process (which molecule? in which compartment? which turnover of the target molecule?) and so is attained by adjusting the duration of the labeling. During the labeling, it is preferable to follow the ¹²CO₂ and ¹³CO₂ assimilation rate of the labeled plant using an infrared gas analyzer (e.g. S710, SICK/Maihac). If not available, ¹²CO₂ assimilation can be measured with a gas analyzer before the labeling and the amount of ¹³CO₂ needed to label correctly the plant can be calculated. At the end of labeling period, the bottle is closed, and after around 15 min (the time for plants to assimilate ¹³C remaining in the chamber) the chamber is removed.

Samples Collection

Samples are collected before the labeling to determine the natural abundance of ¹³C in each studied compartment and at different selected dates after the labeling to follow the fate of ¹³C in each studied compartments. It is important to know the total quantity of ¹³C assimilated by the labeled plant. For trees, total assimilated ¹³C is generally considered as the ¹³C recovered in leaves collected just after the end of labeling. However, for herbaceous and small plants, ¹³C can have been already exported out of leaves to other organs, and total assimilated ¹³C corresponds rather to the total of ¹³C recovered in all the organs of the plants collected just after labeling. If possible, it is helpful to make some preliminary tests to determine the time needed to recover the ¹³C in the different parts of the plant (stems/trunks and roots) and in the soil.

Samples Preparation

Samples are either frozen in liquid nitrogen or at −20 °C and then rapidly freeze-dried or dried with microwave (useful when only ¹³C of total organ is needed). They are ground to obtain a fine powder like flour. According to the aim of the study, samples are directly injected in EA-IRMS, or treated with solvent for purification of biomolecules and dried before injection in EA-IRMS, or injected in liquid phase in LC or GC-IRMS.

4.2 Expression of Results

The formula of the ¹³C isotope composition (∂¹³C), the relative abundance of ¹³C (x(Ai)), the ¹³C label recovered (LRi), the fraction of total assimilated ¹³C (FLR_i,t) and the partitioning of LR (PLR_i,t,) are described below.

• EA-IRMS gives the signature of ¹³C (∂¹³C, expressed in ‰) of each studied organ. By convention, ¹³C isotope composition was expressed relative to Vienna Pee Dee Belemnite standard (R_PDB = 0.011802).

$$ {\delta}^{13}C=\frac{\frac{{}{}^{13}C{O}_2}{{}{}^{12}C{O}_2}}{R_{PDB}}-1 $$

• Relative abundance of ¹³C in any compartment i (x(¹³C)_i, expressed in atom %):

$$ x{\left({}{}^{13}C\right)}_i=100\times \frac{{}{}^{13}C}{\left({}{}^{13}C+{}{}^{12}C_i\right)}=100\times \frac{\left(\frac{\delta 13{C}_i}{1000}+1\right)\times {\mathrm{R}}_{\mathrm{VPDB}}}{\left[\left(\frac{\delta 13{C}_i}{1000}+1\right)\times {\mathrm{R}}_{\mathrm{VPDB}}\right]+1} $$

• The ¹³C label recovered in any compartment i (LR_i, mg ¹³C):

$$ L{R}_i=\frac{\left(x{\left({}_{\;}{}^{13}C\right)}_{lp}-x{\left({}_{\;}{}^{13}C\right)}_{up}\;\right)}{100}\times {Q}_i $$

Q_i the quantity of C in the compartment i (mg C); lp and up denote labeled and unlabeled pots.

• Because C assimilation differs between plants and treatments, data can be expressed in fraction of total assimilated ¹³C (FLR_i,t) by taking into account the total assimilated ¹³C (i.e., quantity of ¹³C recovered in the total plant or in leaves at day 0).

$$ FL{R}_{i,t}=\frac{L{R}_{i,t}}{\sum \limits_iL{R}_{i,t=0}}\times 100 $$

• The partitioning of LR in each compartment at a given harvest-time (PLR_i,t, %) is expressed in percentage of the total ¹³C recovered in the system at the respective harvest-time t (i.e. the total ¹³C recovered at T1, T2 or T3)

$$ PL{R}_{i,t}=\frac{L{R}_{i,t}}{\sum \limits_iL{R}_{i,t}}\times 100 $$

4.3 Practical Example of ¹³C Quantification in the Different Compartments of a Plant-Soil System Submitted to PHE Atmospheric Pollution: ¹³CO₂ Pulse Labeling and Quantification by EA-IRMS (Figs. 25.2 and 25.3)

Fig. 25.2

Kinetics of the fraction of ¹³C label recovered (FLR, %) in total plant, cumulative respiration, and microbial biomass after 1 month of red clover (Trifolium pratense) exposure to atmospheric PHE (exposed, closed circle) and to ambient air (control, open circle)

Data are expressed in percentage of ¹³C label recovered in each compartment at each harvest-time according to the total ¹³C recovery in the soil-plant system just after the end of labeling. Each point represents the mean ± standard deviation (n = 3). Overall differences across time, between treatments (PHE) and their interaction for the FLR in total plant, cumulative respiration and microbial biomass were tested by a linear mixed effect test (P < 0.05, *; P < 0.01, **; P < 0.001, ***; NS: non significant)

Fig. 25.3

Partitioning of ¹³C (PLR, %) among red clover organs (leaves, stems and roots) after 1 month of exposure to atmospheric PHE (plants exposed to atmospheric PHE) and to ambient air (control plants). Each fraction of the bar represents the mean percentage of ¹³C recovery (n = 3) in leaves, stems, and roots of red clover (Trifolium pratense) according to the total ¹³C recovery in the plant at the respective harvesting time

Principle

Plant-soil microsystems were subjected to a PHE atmospheric pollution during 28 days in specific chambers (Desalme et al. 2011a). Each chamber (3 dedicated to polluted atmosphere and 3 to control atmosphere) contains four red clover (Trifolium pratense L.) cultivated pots.

Labeling

At the end of PHE exposure, pure ¹³CO₂ was injected in chambers containing the plant-soil systems during 1.5 h. The chambers were opened and vented for 10 min to remove all the ¹³CO₂ in excess then the light were turned off.

Sample Collection

Samples of leaves, stems, roots and soil were sampled over a 4 days-period: 2 h, 14 h, 38 h and 86 h after the beginning of labeling. Samples of unlabeled pots were collected just before the labeling for each treatment (polluted and control). After being weighted, all the samples were frozen in liquid nitrogen, freeze dried and ground into fine powder. Microbial biomass was extracted from each fresh soil sample by chloroform fumigation of soils during 24 h to solubilize microflora, and extraction with K₂SO₄ of fumigated and non-fumigated samples.

¹³C abundance Determination in the Different C Pools

C content and C isotopic composition of plants, soil and microbial biomass were determined using an elemental analyzer coupled with an isotopic ratio mass spectrometer (EA-IRMS). Isotope composition of respired CO₂ was analyzed for 3 consecutive days on the 3 polluted and 3 control pots collected at 86 h by introducing them into a 2.4 L tight chamber and analyzing the accumulation of ¹³CO₂ and ¹²CO₂ during 30 min in the dark.

Expression of Results

Results of ¹³C amounts recovered in plant organs, soil, microbial biomass and cumulative respiration were expressed in fraction of total assimilated ¹³C recovered (FLR, %) (Fig. 25.2). The quantity of label recovered in each compartment of the plant was also expressed as the partitioning of the total label recovered at each harvest time (PLR) (Fig. 25.3).

Conclusion

The amount of ¹³C recovered in plants and in cumulative respiration was lower in polluted than in control systems (Fig. 25.2). In terms of ¹³C partitioning among plant organs at each harvest-time, ¹³C was preferentially retained in the aboveground organs in both polluted and control systems (Fig. 25.3).

Exposure to PHE had a significant overall effect on the carbon partitioning among the clover organs. More ¹³C are retained in the leaves (51% versus 39% on average in polluted and control plant soil systems, respectively) at the expense of the other plant sinks (roots).

4.4 Practical Example of ¹³C-PHE Quantification in Different Compartments of a Plant-Soil System Submitted to PHE Atmospheric Pollution (Table 25.5)

Table 25.5 Phenanthrene (PHE) concentrations and ratio ¹³C-PHE/¹²C-PHE recovered in red clover shoot and soil after 8 days to exposure to atmospheric ¹²C PHE or ¹³C + ¹²C PHE

Principle

Recent studies showed that using ¹³C-labeled chemical compounds is a relevant way to trace the fate of organic pollutants in plant-soil mesocosms (Cebron et al. 2011; Cennerazzo et al. 2017). We propose here to use the ¹³C-phenanthrene/¹²C-phenanthrene ratio (¹³C-PHE/¹²C-PHE) as a tool to identify the respective contribution of atmospheric and soil exposure pathways for red clover (Trifolium pratense) contamination.

Labeling

Red clovers were exposed to PHE atmospheric pollution during 8 days in specific chambers (Desalme et al. 2011b) (3 dedicated to polluted atmosphere and 3 to control atmosphere). Before reaching the chambers, air passed through an evaporator filled with PHE pills. In three exposure chambers, evaporators were filled with PHE pills (50 mg) from ¹³C PHE (10 mg) and ¹²C PHE (40 mg) powder (Sigma–Aldrich). A control exposure chamber was performed with an evaporator filled with PHE pills from 50 mg of ¹²C PHE powder.

Sample Collection

Leaves were collected after 8 days of exposure. Leaflets and petioles were separated, lyophilized and grinded with a ball crusher. Soils were collected and dried at room temperature.

¹³C-PHE/¹²C-PHE Extraction and Analysis

Extractions from leaflet, petiole and soil samples were performed twice with 15 mL of hexane/acetone (v/v: 2/1) by accelerated solvent extraction(ASE100^®; Dionex, France) during 8 min (120 °C, 100 bar). Leaflet and petiole extracts were concentrated to 0.8 mL under N₂ flux at 35 °C (TurboVap) and purified using a SPE column 10 mL (SILICA GEL, Sigma). PHE quantification was performed with gas chromatography coupled with a mass spectrometer (GCMS-QP5050A; Shimadzu, France).

Conclusion

The ¹³C-PHE/¹²C-PHE ratios recovered in leaflets and in petioles were in accordance with the ¹³C-PHE/¹²C-PHE ratio of the pills filled in the evaporators but were different from ¹³C-PHE/¹²C-PHE ratio of the soil (Table 25.5). These results confirmed that the ¹³C-PHE/¹²C-PHE ratio can be used to identify the major contributing pathway for plant contamination.

5 General Conclusion

This chapter gave basis to set up experiments using ¹³C labeling tracers and ¹⁴C radiochemicals. The ¹³C is now currently used for a diverse range of applications in environmental and plant sciences and the ¹⁴C remains a powerful technique to trace specifically ¹⁴C molecule translocation into plants and their environment. Specific methodology, equipment and a quite high budget (i.e. the cost of the ¹³C or ¹⁴C molecules) are required for such experiments but they remain crucial tools to understand plant-environment interactions.