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Environmental Processes
Uptake of soil-derived carbon into plants: implications for disposal of nuclear waste Soroush Majlesi, Jukka Juutilainen, Anne Kasurinen, Promise Mpamah, Tatiana Trubnikova, Markku J. Oinonen, Pertti Martikainen, and Christina Biasi Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b06089 • Publication Date (Web): 27 Mar 2019 Downloaded from http://pubs.acs.org on March 27, 2019
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Uptake of soil-derived carbon into plants: implications for disposal of nuclear waste
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Authors: Soroush Majlesia*, Jukka Juutilainena, Anne Kasurinena Promise Mpamaha, Tatiana Trubnikovaa,
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Markku Oinonenb, Pertti Martikainena, Christina Biasia*
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aUniversity
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70211 Kuopio, Finland
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bLaboratory
of Eastern Finland, Department of Environmental and Biological Sciences, P.O. Box 1627, FI-
of Chronology, Finnish Museum of Natural History, P.O. Box 64, FI-00014 Helsinki, Finland
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Abstract
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Radiocarbon (14C) is potentially significant in terms of release from deep geological disposal of
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radioactive waste and incorporation into the biosphere. In this study we investigated the transfer of soil-
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derived C into two plant species by using a novel approach, where the uptake of soil-derived C into newly
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cultivated plants was studied on 8000-year leftover peat in order to distinguish between soil-derived and
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atmospheric C. Two-pool isotope mixing model was used to reveal the fraction of soil C in plants. Our
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results indicated that although the majority of plant C was obtained from atmosphere by photosynthesis,
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a significant portion (up to 3-5%) of C in plant roots was derived from old soil. We found that uptake of
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soil C into roots was more pronounced in ectomycorrhizal Scots pine than in endomycorrhizal reed
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canary grass, but nonetheless, both species showed soil-derived C uptake in their roots. Although plenty
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of soil-derived C was available in canopy air for re-assimilation by photosynthesis, no trace of soil-derived
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C was detected in aboveground parts, possibly due to the open canopy. The results suggest that the
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potential for contamination with 14C is higher for roots than for leaves.
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Key words: Radiocarbon; Soil-derived C; Two-pool isotope mixing model; radioecology; radioactive
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waste
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*Corresponding
authors:
[email protected],
[email protected] 34 1 ACS Paragon Plus Environment
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Introduction
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Radioactive waste is considered as one of the major concerns of the nuclear power
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industry. Safe disposal of spent fuel is a crucial step to ensure the protection of humans
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and the environment in the long term. Countries such as Finland and Sweden have
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selected deep geological disposal for management of long-lived radioactive waste. The
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above method is considered by many as the best option to guarantee the long-term
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safety of radioactive wastes (1-3) and a deep geological repository is currently being
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constructed in Finland. However, the long-lived radioactive waste must be isolated from
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humans and the environment for a very long time, and understanding of the processes
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and pathways of radionuclide behavior still needs to be improved for adequate
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assessment of possible risks in the distant future.
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Among the radionuclides possibly released into the environment, radiocarbon (14C) is of
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great significance because of its high potential to enter the biosphere. About one fifth of
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the body mass of all living organisms consists of carbon (C), and
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from nuclear waste mixes with common stable isotopes of C found in nature (12C, 13C).
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This radionuclide can thus, theoretically, move from deep geological disposals to the
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biosphere and be incorporated into plants, animals and to the human food chain.
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Radiocarbon has a relatively long half-life of 5730 years, which additionally contributes
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to the risk of significant effects from this radionuclide in the food chain.
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The releases of 14C from repositories could be in the form of gases (14CO2, 14CH4) or as
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aqueous species (4). Both gaseous and aqueous 14C species can end up in living matter
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through, e.g., photosynthesis by plants (CO2 uptake), oxidation and assimilation of CH4
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by methanothrophs or direct uptake of water and inorganic and organic C species by
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plants and animals. The 14C taken up by organisms can also re-enter the soil since plant
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litter and debris of soil animals and microorganisms are the major sources of soil
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organic matter. Exposure of humans could results from uptake by contaminated plants
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and consequent transfer of 14C into the food chain.
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Traditionally it is believed that C uptake from soil or belowground sources into plants
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or plant roots is low and therefore of less significance compared to stomatal
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atmospheric CO2 uptake from the air (4). However, even small portions of soil-derived
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14C
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Thus, it is important to accurately assess the amount of C that plants utilize from the
ending up in plants could significantly increase transfer of
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14C
14C
possibly released
into the biosphere.
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soil. Particularly the potential uptake from old native soil is relevant for studies on
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geological disposal of nuclear waste. Direct incorporation of soil CO2 (gaseous and/or
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dissolved) and other species of dissolved inorganic C (e.g. bicarbonate) by roots is
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possible (5-7). In addition, plants are known to be able to take up organic carbon and
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nitrogen forms from the soil, with mycorrhizal fungi possibly facilitating the uptake of
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organic substrates (8, 9). Carbon uptake by the roots is believed to be only a few percent
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of the total C in the plant (10, 11), but the uncertainty of the estimates is large.
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Furthermore, indirect uptake of soil-derived CO2 via leaf-level photosynthesis might
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play a role. The rate of re-assimilation of soil-derived CO2 by plant photosynthesis is
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poorly known, but it may be a more important source of soil-derived C than root uptake.
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Up to a few percent of soil-derived C in plant canopy atmosphere has been assumed to
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be reassimilated by plants (11).
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The key problems with analyzing the possible transfer of soil-derived C into plants are
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how to distinguish soil-derived C from other sources of C, and how to determine the
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contribution of soil-derived C in the foliar uptake of C. The most reliable methods to
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distinguish the C sources in plants are isotope methods (12). Continuous and pulse
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labelling methods are frequently used but these techniques are intrusive (12).
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Additionally, experimental labelling of soil with
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Soil–plant systems with natural differences in isotope signature overcome the
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limitations of labelling studies but such systems are rare.
14C
poses risks to the environment.
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This paper addresses transfer of soil-derived C into plants by using a new approach to
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distinguish between soil-derived and atmospheric C (13). The principal idea of this
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approach is to study uptake of soil C into plants in cultivated cutaway peatlands, where
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a distinct natural
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between the “normal” 14C levels in air and the up to 8000-year-old leftover peat, which
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is strongly depleted in 14C (due to decay of 14C with time). This large difference in 14C is
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due to the fact that the top layers of the peat have been removed by peat extraction,
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leaving peat that is depleted in
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opportunity to calculate, with high accuracy, the contributions of atmospheric C and
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soil-derived C in the plants growing on the leftover peat. Furthermore, it is possible to
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quantify the proportion of soil-derived CO2 in canopy air and thus estimate uptake of 14C
14C
pattern exists: there is a very large difference in
14C
compared to the modern
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14C
14C
content
level. This gives an
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via leave photosynthesis. This approach allows us to introduce a robust tool to follow
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the fate of soil C, here with a natural distinct 14C signal. The aims of this study were to
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investigate 1) uptake of soil C via roots, 2) foliar uptake of C released from soil as CO2
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and 3) differences in C uptake of soil-derived C between two plant species. Soil C acts
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here as a proxy of
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incorporated into soil organic matter. All results are also relevant for basic studies on C
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cycling since more information on internal re-cycling of C is needed for the scientific
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community dealing with C dynamics and e.g. climate change research. The study was
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carried out in Linnansuo (Eastern Finland) and the experiments were performed on a
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cutaway peat soil cultivated with reed canary grass (Phalaris arundinacea L.) and Scots
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pine (Pinus sylvestris L.). The field studies were complemented with laboratory
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incubation studies with plant seedlings under controlled conditions.
14C
possibly released during disposal of nuclear waste and
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Materials and Methods
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Field experiments - study site
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The study was carried out in Linnansuo on an 8000-year cutover peatland complex (62◦
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30_ N, 30◦ 30_ E) located in the rural area of the city of Joensuu in eastern Finland in
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2009. According to climatic data available for the region, the mean annual temperature
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and precipitation in the region is 2.1 ◦C and 669 mm, respectively (14). In its pristine
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state, the peatland was classified as an ombrotrophic Sphagnum fuscum bog (14). Peat
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extraction, including drainage, began in 1978 (14). After the end of peat extraction, in
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2001, when the thickness of the remaining peat was 20-85 cm, the cutover peatland was
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cultivated with reed canary grass (RCG, a species used for bioenergy production). The
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carbon content of the residual peat at the surface was 39 ± 18% (15). The variability of
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soil C and the organic matter content was rather high (42-63%), because sand was
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mixed into the surface peat when digging the drainage ditches, in order to improve soil
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aeration and thus plant growth. The C/N ratio of the peat was 40.3 and its pH was 4.3-
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6.3 (14). The site served as the primary research site in this study and was chosen to
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study transfer of C from soil into grass roots, leaves and the canopy air. The dense
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growth of RCG generates canopy air, which might contain a considerable amount of soil-
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derived CO2.
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The second site used for the study was a smaller, forested cut-away peatland adjacent to
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the RCG cultivation. There, Scots pine (Pinus sylvestris L.) were planted in 2001. The
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trees were three to at maximum five meters tall with rather low average stand density,
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so the canopy formed by the trees was relatively open. The plants of both species had
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been growing for 8 years at the time of the study.
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Both plant species are common in Finland and are adapted to the Finnish climatic
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conditions; these plants are thus also likely to grow on repositories in the future.
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Importantly, RCG is a perennial grass species whereas Scots pines are woody plants.
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Scots pines form ectomycorrhizas in their fine root systems whereas RCG are
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endomycorrhizal plants (16) and might thus behave differently with respect to C
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transfer from belowground sources.
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Field Sampling
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Soil and Biomass
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Soil samples were taken to the depth of 15cm from two different strips on the RCG and
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Pine sites, and divided into 0-5 cm, 5-10 cm and 10-15 cm sections, by using a metal soil
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corer with a diameter of 7 cm (n=3 for RCG and n=2 for Pine). We sampled sections
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based on depth, since no horizons were present in the residual peat. One soil core taken
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from the RCG site extended to the bottom of the peatland, down to 45 cm, and was also
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divided in 5 cm increments. In the laboratory, roots were carefully removed before 14C
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dating. For analysis of mycorrhizal colonization in roots, intact soils cores (n= 4 for each
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site) were sampled separately and roots were not removed until analysis (see below).
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Plant samples were taken on two different strips of each site. Samples from
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aboveground parts of RCG were once pooled (n=6) and once, in a single replicate, also
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taken at several heights (8, 40 and 80cm). The Scots pine samples were only taken at
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different heights (55, 70, 110, 115, 190 and 260cm; n=6). Only single needles from the
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tips of the pine twigs were sampled, to ensure analysis of 5 ACS Paragon Plus Environment
14C
in young needles. Stems
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and leaves of the plants were dried and root samples were additionally carefully
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washed before drying and sent to the Radiocarbon Dating Laboratory in Poznan, Poland,
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for 14C dating.
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Mycorrhizal colonization level
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The total mycorrhizal colonization level was calculated as total number of mycorrhizal
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short roots divided by total number of all short roots in the sample x 100. Samples came
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from Linnansuo as soil core samples (n=4). The roots were washed for mycorrhizal
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status analyses. For RCG roots, root clearing before staining was performed: roots were
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kept in 10 % KOH for 24 hours at room temperature, then rinsed in 1% HCl to make
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them acidic again before staining. The samples were stained for 24 hours at room
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temperature with lactophenol blue stain (Diagnostica Merck, E. Merck, 64271
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Darmstadt Germany) and then rinsing overnight (12 h) at room temperature in
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deionized water to remove excess stain before microscopy. Measurement was done
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with 10 x 1 cm long root pieces on a glass slide. Samples were examined by Motic AE21
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inverted research microscope (Motic Microscopes, China).
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In Scots pine root analysis, 1-m-long fine root (≤ 2 mm in diameter) samples were
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analysed for total mycorrhizal colonization level. Pine fine roots were studied with a
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stereomicroscope under a 7.5x-50 x magnification (Motic SMZ-168, Motic Microscope,
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China). All short roots (mainly 1st order roots) were examined for the presence of
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mycorrhizal stuctures on the basis of their gross morphological features. In general,
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non-mycorrhizal roots were mainly monopodial, and rarely dichotomous in their
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ramification pattern, the visibility of the cortical cells was clear, and there could be root
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hairs emanating from the short root surface. In mycorrhizal short roots, the ramification
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pattern was usually dichotomous/coralloid/tubercle-like and rarely monopodial, roots
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did not have root hairs, but sometimes hyphae/extramatrical mycelium/rhizomorps
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emanating from the fungal mantle covering the short roots, and only in some thin-
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mantled mycorrhizas, root cortical cells could be seen through mantle.
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Carbon dioxide
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Carbon dioxide in canopy air was sampled at the heights of 8, 40 and 80cm from RCG
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cultivations, and at 55, 70, 110, 115, 190 and 260cm from pine cultivations (the same
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heights that were used for sampling leaves). Carbon dioxide was sampled with the
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molecular sieve technique (17), where CO2 molecules are trapped (adsorbed) in pores
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in the sieve. Type 13X zeolite sieves (Merck 1.05703.1000) were used in this study, and
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they were packed in glass containers with approximate volume of 100 ml.
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sampling, air was pumped through the molecular sieves for about five minutes. After
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sampling, CO2 was released in the Laboratory of Chronology in Helsinki, Finland for
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14CO 2
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than 95%. Amount of fractionation by the molecular sieves is fairly limited (17) but this
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is, nevertheless, taken into account when post-processing the raw 14C data as mentioned
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below (by using the 13C value).
For
analysis under very high temperature and under-pressure, with efficiency greater
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Laboratory studies
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To complement the field studies, laboratory experiments were carried out in a
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greenhouse at the Research Garden of the University of Eastern Finland in Kuopio using
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RCG and Scots pine seedlings. All plants (RCG and Scots pine) were grown from seeds
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sown into pots in the greenhouse. The C uptake experiment started in April 2009 and
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ended in September 2009 (5 months in 2009). During the experiment, all plants were
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kept in two separate rooms with equal size of 68.2 m2. In total, the temperature differed
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between these two rooms (by 3 ͦC max.; on average 1.5 ͦC). However, statistical tests did
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not detect any effect of temperature on C transfer, so we consider all samples as one
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treatment. Both species were grown in flower pots on old peat collected from an
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adjacent cut-away peatland (Linnansuo) which was still under peat harvesting, and thus
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not influenced by modern plants. A total of 400 plastic pots (OS Plastic A/S Danmark,
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11mm in diameter), as well as equivalent number of small flat plates were carefully
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washed and dried for the experiment.
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Peat and fine sand (0.5-1.2mm) were carefully mixed at the ratio of 1:1 to ensure
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homogeneous soil material. The sand was added to increase aeration of the peat and to
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aid root penetration. Pots filled with 450 g of fresh peat-sand mixture in each pot were 7 ACS Paragon Plus Environment
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kept in the research garden from sowing the seeds to the end of the experiment. Both
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plant species were grown from seeds collected from the Natural Resources Institute of
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Finland (LUKE, Finland).
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In total, 154 and 112 pots, respectively, were planted with RCG and pine in the two
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chambers. We planted about 300 RCG seeds per square meter (similar to the field
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density) and four Scots pine seeds per pot. Scots pine was later thinned to one seedling
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per pot. The pots were watered once a day. One week after the pots were thinned, the
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addition of fertilizer (about 0.5dL of 0.1% Taimi-Superex fertilizer, NPK 19-4-20,
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Kekkilä, Finland) was started. The fertilization was done once a week.
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Throughout the experimental period, temperature and air humidity were monitored.
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The average minimum/maximum temperatures in the two rooms were 18/20 and
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18/23oC (on average 19 and 20.5 oC, respectively). The minimum/maximum relative
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humidities in the chambers were 75/80% respectively. Artificial light in the
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greenhouses turned off when the solar radiation exceeded 400 W/m2; the photoperiod
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was 16 hours.
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Soil and biomass
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All RCG leaves and pine needles from the pots were collected at the end of the
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laboratory experiment. Like in the field experiment, they were dried at 65 ○C, weighed
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and a subsample was sent for 14C analysis to the Poznan Radiocarbon Dating laboratory
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in Poland. After cutting off the shoots, the residual content of the pots (roots and soil)
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was shaken carefully to remove as much soil as possible for soil analysis while roots
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were carefully washed over a sieve (mesh size 8) to remove the remaining soil
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materials. Finally, the washed roots were dried before 14C analysis.
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Radiocarbon analysis
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All radiocarbon dating of solid samples (soil, plants) were carried out by accelerator
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mass spectrometer (AMS) in the Poznan Radiocarbon Dating Laboratory in Poland, and
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of gas samples by AMS in the Laboratory of Chronology, Helsinki, Finland (17,18). AMS
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is a technique for measuring the concentration of a single isotope (14C) that is rare in the 8 ACS Paragon Plus Environment
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presence of more abundant isotopes. With this method, it is possible to analyze the
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proportion of 14C with high precision (
