Sources and sinks of chloromethane in a salt marsh ecosystem: constraints from concentration and stable isotope measurements of laboratory incubation experiments

Frank Keppler *ab, Amelie Ninja Röhling a, Nicole Jaeger a, Moritz Schroll a, Simon Christoph Hartmann ac and Markus Greule a
aInstitute of Earth Sciences, Heidelberg University, Im Neuenheimer Feld 234–236, D-69120 Heidelberg, Germany. E-mail: frank.keppler@geow.uni-heidelberg.de
bHeidelberg Center for the Environment HCE, Heidelberg University, D-69120 Heidelberg, Germany
cMax Planck Institute for Chemistry, Hahn-Meitner-Weg 1, 55128 Mainz, Germany

Received 20th November 2019 , Accepted 11th February 2020

First published on 12th February 2020


Abstract

Chloromethane (CH3Cl) is the most abundant long-lived chlorinated organic compound in the atmosphere and contributes significantly to natural stratospheric ozone depletion. Salt marsh ecosystems including halophyte plants are a known source of atmospheric CH3Cl but estimates of their total global source strength are highly uncertain and knowledge of the major production and consumption processes in the atmosphere-halophyte-soil system is yet incomplete. In this study we investigated the halophyte plant, Salicornia europaea, and soil samples from a coastal salt marsh site in Sardinia/Italy for their potential to emit and consume CH3Cl and using flux measurements, stable isotope techniques and Arrhenius plots differentiated between biotic and abiotic processes. Our laboratory approach clearly shows that at least 6 different production and consumption processes are active in controlling atmospheric CH3Cl fluxes of a salt marsh ecosystem. CH3Cl release by dried plant and soil material was substantially higher than that from the fresh material at temperatures ranging from 20 to 70 °C. Results of Arrhenius plots helped to distinguish between biotic and abiotic formation processes in plants and soils. Biotic CH3Cl consumption rates were highest at 30 °C for plants and 50 °C for soils, and microbial uptake was higher in soils with higher organic matter content. Stable isotope techniques helped to distinguish between formation and degradation processes and also provided a deeper insight into potential methyl moiety donor compounds, such as S-adenosyl-L-methionine, S-methylmethionine and pectin, that might be involved in the abiotic and biotic CH3Cl production processes. Our results clearly indicate that cycling of CH3Cl in salt marsh ecosystems is a result of several biotic and abiotic processes occurring simultaneously in the atmosphere-plant-soil system. Important precursor compounds for biotic and abiotic CH3Cl formation might be methionine derivatives and pectin. All formation and degradation processes are temperature dependent and thus environmental changes might affect the strength of each source and sink within salt marsh ecosystems and thus considerably alter total fluxes of CH3Cl from salt marsh ecosystems to the atmosphere.



Environmental significance

Chloromethane contributes significantly to natural stratospheric ozone depletion. Understanding the origin and fate of chloromethane in the environment is important in order to predict its contribution to future stratospheric ozone destruction. In this study we investigated a halophyte plant and soil samples from a coastal salt marsh site for their potential to emit and/or consume chloromethane and differentiated between biotic and abiotic processes using flux measurements, stable isotope techniques and Arrhenius plots. We found these processes are temperature dependent and thus environmental changes might affect the strength of each source and sink within salt marsh ecosystems and thus considerably alter total fluxes of chloromethane from salt marsh ecosystems to the atmosphere.

Introduction

Chloromethane (CH3Cl) is one of the most abundant chlorinated organic compounds in the atmosphere, with a global mean mole mixing ratio of around 550 to 560 parts per trillion by volume (pptv) observed in 2016.1 These values are around 2 to 3% higher than the CH3Cl mixing ratios measured in 2012.2 Chloromethane with an estimated global life time in the atmosphere of 0.9 years is the largest carrier of natural chlorine to the stratosphere and responsible for almost 17% of the chlorine-dependent destruction of stratospheric ozone.1,2 Global emissions of CH3Cl to the atmosphere have been estimated to be in the range of 4 to 5 Tg (1 Tg = 1012 g) per year with many sources occurring in terrestrial environments.1–3 Initially the oceans were considered to be the major emitter of CH3Cl to the atmosphere4 but in the past three decades it has become clearly evident that other sources must dominate fluxes of CH3Cl to the atmosphere.5,6 Hence numerous natural terrestrial sources of CH3Cl have been discovered such as CH3Cl emissions from fungi,7 tropical and subtropical plants,8–11 halophyte plants and salt marshes,12–15 rice paddies,16 aerated and flooded soils,17–20 saline soils,21,22 mangroves,23 senescent leaves and leaf litter24–26 and wild fires.24,27 Anthropogenic CH3Cl release to the atmosphere comes from the combustion of coal and biomass27 and from the chemical industry28 and negligible emissions from cattle29 and humans.30

The dominant removal process for atmospheric CH3Cl currently estimated at 2.5 to 3.4 Tg per year2 is degradation by photochemically formed OH radicals31 and to a minor degree by Cl radicals. Another potentially important global sink for CH3Cl is microbial degradation in soil, which has been estimated with large uncertainties to range from 0.1 to 1.6 Tg per year.2,32,33 Several methylotrophic bacteria capable of utilizing CH3Cl as sole carbon and energy source have been isolated from various environments,34 including soils,35,36 sludge,37–39 seawater,40,41 as well as plant leaves.42,43 Moreover, stable carbon and hydrogen isotope studies44–46 have confirmed biogenic degradation of CH3Cl in soil and plant matter. The ubiquitous occurrence of methylotrophic bacteria in the biosphere implies that microorganisms in soils and on plants may play a more important role as a sink for CH3Cl than previously thought.47

In principle, CH3Cl formation in plants and soil can be divided into several biotic and abiotic processes as conceptually outlined in Fig. 1. Biotic CH3Cl production has been suggested to involve an enzymatic reaction, where methyl halide transferase catalyzes the formation of CH3Cl via the reaction of S-adenosyl-L-methionine (SAM) with chloride ion.48 This process might be widespread among vascular plants49 but also likely occurs in other eukaryotes such as fungi, algae, animals and humans. However, CH3Cl is also produced by abiotic processes, where methoxy groups of plant structural components such as pectin and lignin can be chlorinated.24 This ubiquitous process occurring in terrestrial ecosystems at ambient temperatures25,26,50,51 is much more efficient – up to 100% conversion of plant chloride content to CH3Cl – under elevated temperatures such as biomass burning.24,52 Furthermore, compounds containing methyl groups bonded to sulfur (methyl-thiol groups) such as in SAM, S-methylmethionine (SMM) or other methionine derivatives might also be involved in abiotic CH3Cl formation.53,54


image file: c9em00540d-f1.tif
Fig. 1 Schematic overview of CH3Cl cycling in a salt marsh ecosystem including all currently known biotic and abiotic sources and sinks.14,19,45,46,57 So far six formation and degradation processes of CH3Cl have been described to occur in plant-soil systems: (1) enzymatic formation by plants; (2) abiotic formation by plants; (3) biodegradation by microbes on leaf surface; (4) biodegradation by soil microbes; (5) abiotic formation in soil and (6) biotic formation in soil.

So far several studies have investigated the role of salt marsh ecosystems in the release of CH3Cl to the atmosphere.12–15 Even though they cover only 0.1% of the global surface layer55 salt marshes were initially estimated to provide up to 10% of the total atmospheric CH3Cl source strength.12 However, subsequent reports2,13 have substantially down-scaled net-emissions from this source and are now thought to range from 1.1 to 170 Gg (1 Gg = 109 g) per year which accounts for only 0.02 to 3.4% of global CH3Cl emissions. Furthermore, it has recently been suggested that tropical and sub-tropical salt marshes emit much more CH3Cl to the atmosphere than temperate salt marshes and thus low latitude salt marshes might be considered as significant sources of atmospheric CH3Cl.14 Within these ecosystems the primary CH3Cl source seems to be above-ground vegetation, typically halophytic plants rather than the soil.56 However, most field studies that have investigated salt marsh ecosystems and report net emissions of CH3Cl to the atmosphere do not differentiate between the different processes occurring in halophytic plants and the surrounding saline soils controlling these emissions to the atmosphere.

It is currently not possible to conduct research on a field scale and differentiate between all the likely formation and degradation processes as schematically shown in Fig. 1. Therefore, we decided to undertake a laboratory investigation to study in considerable detail all known CH3Cl formation and degradation processes thought to occur in a saltmarsh ecosystem. For this we collected several fresh halophyte plant and soil samples from Sardinia/Italy and measured the fluxes of CH3Cl from fresh and dried plant and soil matter at different temperatures and employed stable isotope techniques to distinguish between consumption and formation processes. In addition, we performed similar experiments with model compounds (natural analogues) namely pectin, SAM and SMM which were supplemented with chloride ion to provide more information about methyl donor compounds likely involved in the biotic and abiotic formation processes of CH3Cl. When discussing our results we outline how CH3Cl fluxes from saltmarsh ecosystems to the atmosphere might alter, particularly during global change.

Material and methods

Plants and soil material

The glasswort Salicornia europaea is a halophytic annual dicot which grows in various zones of intertidal salt marshes. Whole plants and soil were sampled in June 2016 at a salt marsh (Fig. 2) near Puntaldia, Sardinia/Italy (40°48′33.5′′N; 9°40′48.6′′E). The climate of this region is Mediterranean, with mild and partly rainy winters and hot and dry summers. Average daily mean temperatures range from around 10 to 27 °C in December and August, respectively.
image file: c9em00540d-f2.tif
Fig. 2 Salt marsh near Puntaldia (Sardinia/Italy) with S. europaea as the major halophyte species.

Three whole plants of S. europaea were collected including roots. In addition, two surrounding soil samples (sandy loam) differing in their organic matter content, were sampled from the upper soil horizon (0 to 2 cm depth) (Fig. 3). Samples were transported within two days to the laboratory and incubation experiments followed subsequently. Please note that soil sampling and further processing in the laboratory might have affected the redox state of soil components. Soil samples were sieved to <2 mm and their pH determined potentiometrically in 0.01 M CaCl2 with a soil to solution ratio of 1[thin space (1/6-em)]:[thin space (1/6-em)]2.5. Total carbon (TC) of soil samples was measured by a SC analyser (144DR element analyser, Leco) and total inorganic carbon (TIC) was determined using the “carbonate bomb” method according to Müller and Gastner.58 Total organic carbon (TOC) was calculated by the difference of TC and TIC (Table 1).


image file: c9em00540d-f3.tif
Fig. 3 Soil samples (left soil 1 and right soil 2) collected from the upper horizon of the salt marsh.
Table 1 Water, chloride and organic carbon content and pH of plant and soil samples
Sample species Water content [%] TOC [%] TIC [%] pH Cl-content [%]
a Air dried sample; remaining water content was determined by drying at 105 °C for 24 h. n.d. = not determined.
S. europaea fresh 77.3 ± 2.5 n.d. n.d. n.d. 8.3
S. europaea drieda 7.1 ± 1.5a n.d. n.d. n.d. 8.3
Soil 1 fresh 59 6.0 2.4 9.1 n.d.
Soil 2 fresh 20 3.7 0.3 8.1 n.d.
Soil 1 drieda 5.8a 6.0 2.4 9.1 n.d.
Soil 2 drieda 1.7a 3.7 0.3 8.1 n.d.


Chloromethane model precursor compounds

Three compounds were selected as methyl precursor compounds(Fig. 4) and in the presence of chloride investigated for their potential to produce CH3Cl. The stable isotope composition of the CH3Cl released was also determined.
image file: c9em00540d-f4.tif
Fig. 4 Structural forms of the three model precursor compounds pectin (upper), S-(5′-adenosyl)-L-methionine (middle) and S-methylmethionine (lower). The methyl group bonded to oxygen or sulfur is highlighted in red.

S-(5′-Adenosyl)-L-methionine chloride dihydrochloride, DL-methionine methylsulfonium chloride and pectin (apple) and sodium chloride (99.5%) were all sourced from Sigma Aldrich (Seelze, Germany or Gillingham, UK). Pectin was dissolved in water and sodium chloride added, and then following freezing was lyophilised to produce a pectin powder containing 18[thin space (1/6-em)]000 ppmw chloride ion.

Incubation of samples

To monitor the biotic and abiotic pathways of CH3Cl production and consumption, both fresh and dried plant and soil samples were incubated in 170 mL gastight incubation flasks at different incubation temperatures for time periods of 24 to 48 h. Fresh plant samples were incubated at temperatures ranging from 20 to 40 °C, whereas dried plant and soil samples were incubated at temperatures ranging from 20 to 70 °C.

Prior to abiotic emission experiments all plant and soil samples were air dried for 8 weeks at room temperature (∼22 °C) and relative humidity of ∼60 to 65%. Around 20 g of plant sample, 40 g of soil 1 sample (with higher TOC content) and 80 g of soil 2 sample (with lower TOC content) were incubated in 170 mL incubation flasks at temperatures ranging from 20 to 70 °C (10 °C steps with maximal temperature variations of ±2 °C). The incubation time was usually between 24 and 48 h, during which the headspace of the incubation flask was sampled at least five times.

For incubation experiments with fresh samples, 25 g of fresh plant material (only 1–3 cm intact green and reddish stems without the lignified parts) were incubated in triplicate at 20, 30 and 40 °C and 50 g of fresh soil samples were incubated in triplicate at 20, 30, 40, 50, 60, and 70 °C. In addition to headspace sampling for CH3Cl quantification, at the end of each incubation period a 25 mL sample was removed for stable isotope measurements and stored in 12 mL Exetainer® (Labco Limited, Lampeter, UK) until analysis. For incubation experiments of model precursor compounds 0.5 g of dry material was used.

Analysis of chloromethane

Determination of chloromethane emission rates

CH3Cl quantification was performed using a Hewlett Packard HP 6890 gas chromatograph coupled to a MSD 5973 mass spectrometer (GC-MS, Agilent Technologies, Palo Alto, CA). Headspace samples (200 μL) were injected into a flow of helium (1 mL min−1) that then passed through a GasPro column (60 m × 0.32 mm i.d., Agilent Technologies). A split ratio of 5[thin space (1/6-em)]:[thin space (1/6-em)]1 was employed and the GC oven was held isothermally at 150 °C. The MSD was operated in the single ion monitoring mode measuring ion currents at m/z = 50 (12CH335Cl) and m/z = 52 (12CH337Cl). To quantify CH3Cl, the peak area for ion m/z 50 at the expected retention time of this analyte was compared to a calibration curve prepared using four CH3Cl standards at different mixing ratios in the range of 0.5 to 45 parts per million by volume (ppmv). The limit of quantification for CH3Cl was 0.25 ppmv. Chloromethane production rates (per hour) were calculated by the slope of linear increase of CH3Cl concentration normalized to dry weight (dw) biomass, respectively. In the manuscript units of the rates are reported in ng gdw−1 h−1.

Determination of chloromethane degradation rates

A stable carbon isotope tracer approach was used to differentiate between CH3Cl production and consumption. Hence fresh plant (25 g) and fresh soil (50 g) samples were incubated in triplicate in 170 mL gastight incubation flasks. Following the addition of 5 ppmv of 13CH3Cl (99 atom%, Campro Scientific), plant material was incubated at 20, 30 and 40 °C whilst soils were incubated at 20, 30, 40, 50, 60 and 70 °C. Aliquots of the headspace (200 μL) from each incubation were sampled 3–5 times until the remaining 13CH3Cl fraction was <50% or the 12CH3Cl production balanced the 13CH3Cl uptake (max. 48 h for soil and max. 96 h for plant sample).

The simultaneous measurement of 12CH3Cl and 13CH3Cl concentrations at different timepoints were used to calculate gross production and uptake rates. Four stable isotopologues of CH3Cl were measured by employing the MSD in the single ion monitoring mode measuring ion currents at m/z = 50 (12CH335Cl), m/z = 52 (12CH337Cl), m/z = 51 (13CH335Cl) and m/z = 53 (13CH337Cl) and corrected for 12CH3Cl/13CH3Cl fragmentation ratios according to recent studies.46,59 To quantify 12CH3Cl and 13CH3Cl, the corrected peak areas for ions m/z 50 and 51 at the expected retention time of these analytes were compared to a standard response curve. Chloromethane production and consumption rates (per hour) were calculated by the slope of linear increase and decrease of CH3Cl concentration normalized to dry weight biomass, respectively, and rate units are reported in ng gdw−1 h−1.

Stable isotope values of chloromethane

The conventional delta notation, expressing the isotopic composition of the sample relative to that of V-SMOW standard (Vienna Standard Mean Ocean Water) for hydrogen (δ2HV-SMOW) and V-PDB standard (Vienna Pee Dee Belemnite) for carbon (δ13CV-PDB) on per mil basis, was used.

Stable hydrogen and carbon isotope ratios of CH3Cl were measured by an in-house cryogenic pre-concentration unit coupled to a Hewlett Packard HP 6890 gas chromatograph (Agilent Technologies, Palo Alto, CA) and an isotope ratio mass spectrometer (IRMS) (Isoprime, Manchester, UK), as previously described.60,61 For stable hydrogen isotope analysis, a ceramic tube reactor without chromium pellets at 1400 °C was used for high-temperature conversion. The CH3Cl working standard was calibrated against IAEA standards NBS 22, LVSEC (carbon), VSMOW and SLAP (hydrogen) using TC/EA-IRMS (elemental analyzer-isotopic ratio mass spectrometer, IsoLab, Max Planck Institute for Biogeochemistry, Jena, Germany), yielding the following values: δ13C: −32.84 ± 0.06‰ (n = 11, 1σ) and δ2H: −140.1 ± 1.0‰ (n = 10, 1σ). The H3+ factor, determined daily during this investigation (1 month period), was in the range 7.6–8.3.

Determination of stable isotope values of plant methoxy groups and model precursor compounds

Stable carbon and hydrogen values of the methyl pool containing methoxy groups (OCH3) and methyl-thiol groups (SCH3) were measured, using the “HI method” described in previous investigations.62–64 Briefly, hydriodic acid (250 μL) was added to dried and milled S. europaea samples (40 mg and 75 mg for carbon and hydrogen isotopes, respectively) in crimp-top glass vials (1.5 mL). The vials were sealed with crimp caps containing PTFE-lined butyl rubber septa (thickness 0.9 mm) and incubated for 30 min at 130 °C. After equilibration to room temperature aliquots of the generated iodomethane (CH3I) in the headspace (carbon: 15 μL; hydrogen: 50 μL) were directly injected into a Hewlett Packard HP 6890N gas chromatograph (Agilent Technologies, Palo Alto, CA) coupled to a DeltaPLUS XL isotope ratio mass spectrometer (ThermoQuest Finnigan, Bremen, Germany).

Calculation of activation energy

The Arrhenius eqn (1) was used to determine the temperature dependence between the observed production rates of CH3Cl by S. europaea during the incubation experiments at different temperatures (20 °C to 70 °C).
 
image file: c9em00540d-t1.tif(1)
k is the reaction rate coefficient (here k represents the CH3Cl gas flux in ng g−1 h−1), A is the pre-exponential factor, Ea is the activation energy, R is the universal gas constant and T is the temperature.

Calculation of the activation energies enables the produced CH3Cl to be better characterized as either biotic or abiotic. By approximating a pseudo-first order reaction the reaction rate coefficient k is proportional to the production rates (PR) of CH3Cl. Ea can then be determined from the slope of the Arrhenius plot (−Ea/R), using a plot of ln(PR) as a function of the inverse temperature.

To quantify the temperature sensitivity of CH3Cl formation rates from plants and soil the temperature coefficient Q10 was calculated for each incubation experiment, using eqn (2):

 
image file: c9em00540d-t2.tif(2)
where PR1 and PR2 are the production rates of CH3Cl at temperatures T1 and T2.

The Q10 temperature coefficient describes the change in emission rates due to a temperature change of 10 °C.

Statistics, uncertainties and data presentation

The results of the individual experiments (usually three replicates, n = 3) including emission and consumption rates and stable isotope values of CH3Cl are presented as the mean value with the standard deviation (SD) at the 1σ level. In instances where only two replicates could be conducted, mean values without errors are reported. For isotope analysis of the methyl precursor pool of plants, soils and model compounds, 3 to 15 replicate samples were measured. For linear regression analysis and the calculation of mean values and SD, Microsoft Excel was used. During heating experiments maximal temperature variations of ±2 °C were measured and this value is provided as the error shown in the figures.

Results and discussion

In this section we firstly present the results obtained from our investigations with the plant and soil samples collected from the field which were investigated for the 6 different production and consumption processes as outlined in Fig. 1. This includes observed release rates of CH3Cl from fresh and dried samples as well as the consumption rates when 13CH3Cl was added at an initial mixing ratio of 5 ppmv. We then present the respective activation energies and the stable carbon and hydrogen isotope patterns of CH3Cl emissions from plant and soil samples at temperatures between 20 and 70 °C. For comparison and to provide more information about potential methyl donor compounds involved in biotic and abiotic formation processes of CH3Cl we show emission rates, activation energies and stable isotope patterns of CH3Cl released from chemical model compounds (pectin, SAM and SMM) that were supplemented with chloride when exposed to the same heating regime.

Chloromethane production and consumption by fresh S. europaea

The CH3Cl production rates of fresh S. europaea increased exponentially with temperature increase from 0.2 ± 0.04 ng g−1 h−1 at 20 °C to 2.1 ± 0.8 ng g−1 h−1 at 40 °C (Fig. 5A) and are in a similar range to those reported for S. europaea from field experiments in a coastal area of the Baltic Sea15 and for Salicornia virginica from a coastal salt marsh in California.13 In contrast much higher emission rates (>100 ng g−1 h−1) were reported for field studies with other halophytic plants.9,23,59 However, we would point out that particular attention should be paid when comparing emission rates from different studies, since (i) production rates strongly depend on the plant species,9 meteorological conditions and soil properties, and (ii) high intra-species variations have been reported for tropical plants9 and salt marsh vegetation.13,14,56,65 Due to visible plant decomposition above 45 °C, we did not conduct any temperature incubation experiments for fresh S. europaea above 40 °C. Emissions observed in our investigation for S. europaea is clearly different when compared with emissions rates of CH3Cl from fresh fern leaves of Osmunda regalis incubated under similar conditions46 where it was shown that highest emissions rates of CH3Cl occurred at 20 or 30 °C and substantially decreased at 40 °C. Thus, we presume that CH3Cl production by fresh S. europaea includes both biotic and abiotic CH3Cl formation processes. This is further supported by the calculated Ea energies of 84 ± 17 kJ mol−1 (Table 2), since activation energies reported for enzyme-catalyzed reactions are usually within the range of 21–63 kJ mol−1.66
image file: c9em00540d-f5.tif
Fig. 5 Chloromethane production (A), related Arrhenius plots (B) and degradation rates with added 13CH3Cl (5 ppmv initial mixing ratio) (C) of fresh S. europaea at 20, 30 and 40 °C. Emission and degradation rates were normalized to sample dry weight. Error bars for (A and C) indicate SD of triplicate experiments. Error bars in (B) indicate maximal temperature variations of ±2 °C.
Table 2 E a and Q10 temperature coefficients for different temperature ranges for CH3Cl production from plants and soils
Sample E a kJ mol−1 Q 10 (20–30 °C) Q 10 (30–40 °C) Q 10 (40–50 °C) Q 10 (50–60 °C) Q 10 (60–70 °C) Q 10 (mean total range)
a Calculated for temperature range 40 to 60 °C. b Excluded due to decomposition of SAM at 70 °C. n.d. = not determined.
S. europaea fresh 84.0 ± 16.7 2.80 ± 0.12 3.80 ± 1.31 n.d. n.d. n.d. 3.28 ± 0.70
S. europaea dried 105.9 ± 2.4 3.63 ± 0.19 7.08 ± 0.69 1.38 ± 0.87 5.48 ± 0.18 3.03 ± 0.08 3.75 ± 0.07
Soil 1 fresh 54.5 ± 3.2 2.67 ± 0.05 1.10 ± 0.31 2.22 ± 0.41 1.81 ± 0.12 3.55 ± 0.65 2.09 ± 0.02
Soil 2 fresh 64.1 ± 0.5 5.08 ± 2.12 2.41 ± 1.28 1.35 ± 0.28 4.34 ± 1.21 0.79 ± 0.10 2.17 ± 0.09
Soil 1 dried 110.2 ± 1.4 n.d. 6.60 ± 0.27 2.27 ± 0.05 3.38 ± 0.03 3.28 ± 0.08 3.57 ± 0.06
Soil 2 dried 109.1 ± 0.7 n.d. 6.61 ± 0.34 2.11 ± 0.04 3.27 ± 0.22 3.57 ± 0.22 3.57 ± 0.05
Pectin 83.5 ± 3.8 n.d. 7.98 ± 0.19 1.49 ± 0.02 3.31 ± 0.69 1.69 ± 0.02 2.71 ± 0.10
SMM 110.5 ± 0.1 n.d. 5.34 ± 0.63 4.74 ± 0.24 1.87 ± 0.02 2.61 ± 0.10 3.50 ± 0.02
SAM 105 ± 3.7a n.d. n.d. 6.58 ± 2.14 2.51 ± 0.54 (0.69 ± 0.10)b 3.40 ± 0.07a


By applying the stable carbon isotope tracer technique46,59 we identified overlying CH3Cl uptake and emission processes in fresh S. europaea, a phenomenon previously reported for some ferns and tropical trees.46,67 Chloromethane consumption rates (at a starting mixing ratio of 5 ppmv 13CH3Cl) showed slight increase during temperature increase from 1.1 ± 0.3 ng g−1 h−1 at 20 °C to 1.3 ± 0.4 ng g−1 h−1 at 30 °C and then decreased with further temperature increase to 0.8 ± 0.2 ng g−1 h−1 at 40 °C (Fig. 5C). A decrease of uptake rates at higher temperatures indicating a biotic process was previously also shown for ferns,46 even though the temperature optimum of ferns was somewhat higher at 40 °C. Please note that this experimental approach cannot be directly transferred to environmental field conditions as initial mixing ratios of 5 ppmv are about three to four orders of magnitude higher than those observed in the environment and thus only indicate the maximum potential of S. europaea to biologically degrade CH3Cl. The reason for using this relatively high concentration is because current state-of-the-art tools were not available to allow measurements at environmental CH3Cl concentrations for the degradation experiments. Interestingly, measured CH3Cl gross consumption rates were similar to those observed for plants (mean of 4 ± 3 ng g−1 h−1) in a tropical rain forest in Malaysia.67

Chloromethane production and consumption by dried S. europaea

For air-dried S. europaea increasing incubation temperature from 20 to 70 °C led to an exponential increase in CH3Cl production rates from 1.0 ± 0.3 ng g−1 h−1 at 20 °C to 600 ± 220 ng g−1 h−1 at 70 °C (Fig. 6A). In comparison to the incubation of fresh samples (Fig. 5) these rates are approximately an order of magnitude higher (e.g. 25.4 ± 4.7 ng g−1 h−1versus 2.1 ± 0.8 ng g−1 h−1 at 40 °C). Wishkerman et al.51 reported comparable emission rates of 18 ng g−1 h−1 and 224 ng g−1 h−1 at 30 °C and 50 °C, respectively, for another halophyte, Batis maritima. However, in that study finely ground material (after freeze-drying) was used for the heating experiments whilst in our investigation whole leave material (air-dried) was utilised. The calculated mean Ea value for CH3Cl formation from dried S. europaea was 106 ± 2 kJ mol−1 (Table 2) and is close to the Ea values presented by Wishkerman et al.51 for the saltwort plant B. maritima (107 kJ mol−1) and Derendorp et al.50 for halophyte leaf litter (103 kJ mol−1, mean value for 12 species). Although our experiments with air-dried plant samples do not rule out the involvement of biological processes in CH3Cl formation, the observed high Ea values strongly support abiotic production processes as the main source of CH3Cl formation by dry plant matter since activation energies reported for enzyme-catalyzed reactions are usually much lower. Furthermore, we found average Q10 values (range 20 to 70 °C) of around 3.8 – emissions almost double with every 5 °C rise in temperature – which is almost in the same range as those found for abiotic CH3Cl formation from B. maritima51 and other halophyte species50 (finely milled samples in the temperature range 30 to 50 °C).
image file: c9em00540d-f6.tif
Fig. 6 Chloromethane production rates (A) of dried S. europaea in a temperature range of 20 to 70 °C. Error bars indicate SD of triplicate experiments. Arrhenius plots (B) for CH3Cl emissions of each replicate experiment. Error bars in (B) indicate maximal temperature variations of ±2 °C.

Several studies have previously reported abiotic production of CH3Cl from plant material, and suggested that methoxy groups of plant structural components, such as lignin and pectin, can be chlorinated, leading to the release of CH3Cl.22,24,25,50,51,68 This ubiquitous process acting in terrestrial ecosystems24,69 under ambient temperatures is much more efficient under elevated temperatures such as those attained during biomass burning.24,52

It is important to note that some subtropical and tropical ferns have been shown to be large CH3Cl emitters most probably via an enzymatic biotic process and for those ferns no measureable abiotic CH3Cl formation was detectable after drying.46 Whilst the reason for this might be due to the lower chloride content of ferns, it suggests the importance of different biotic and abiotic CH3Cl formation pathways in halophytic and non-halophytic plant species.

Please note that no measureable consumption of CH3Cl was observed for incubation experiments with dried S. europaea when 5 ppmv 13CH3Cl were added.

Chloromethane production and consumption in fresh soils

Two different surface soil samples of S. europaea (Fig. 3) were used for the incubation experiments and their typical geochemical parameters are shown in Table 1. The first soil sample (soil 1) was higher in TOC (6.0%) and pH (9.1) whilst the second sample (soil 2) was lower in TOC (3.7%) and pH (8.1). Fresh soil 1 showed production rates from 0.2 ± 0.02 ng g−1 h−1 at 20 °C to 7.4 ± 0.3 ng g−1 h−1 at 70 °C, an exponential increase with respect to increasing temperature (Fig. 7, left upper panel). These rates are considerably higher (by a factor of ∼4) when compared with the emission rates observed from soil 2 (0.03 ± 0.01 to 1.6 ± 0.2 ng g−1 h−1, at 20 and 70 °C (Fig. 7, right upper panel)). The calculated mean Ea values for CH3Cl formation from fresh soils 1 and 2 were 54.5 ± 3.2 and 64.1 ± 0.5 kJ mol−1, respectively. These values are considerably lower than those observed for dried soils and plants (Table 2) suggesting that both biotic and abiotic factors were involved in the release of CH3Cl from the fresh samples. The consumption rates (when 5 ppmv 13CH3Cl were added) of both soils were highest at 50 °C and decreased upon further temperature increase (Fig. 7, lower panel). This might be in agreement with results obtained for fresh S. europaea (Fig. 5). However, the temperature optimum (∼50 °C) for biotic CH3Cl consumption in the two fresh soils was higher than that measured for fresh S. europaea (∼30 to 40 °C, Fig. 5) possibly suggesting that the microorganisms responsible for CH3Cl degradation in plants and soils are different. We would highlight that detailed microbial investigations were beyond the scope of this study. Soil 1 showed ∼40 times higher maximum uptake rates (42.3 ± 1.5 ng g−1 h−1) compared to soil 2 (1.1 ± 0.1 ng g−1 h−1). Interestingly, at 20 °C measured consumption rates of both soils were in a similar range to those previously published for forest soils.70 However, as previously mentioned it is difficult to compare the observed consumption rates in this study with those obtained in previous field studies as initial mixing ratios of CH3Cl are about three to four orders of magnitude higher than those typically observed in the environment. Assuming a linear relationship between the observed consumption rates in fresh soil and S. europaea and CH3Cl air mixing ratio (∼500 to 600 pptv CH3Cl in the field) might result in very low consumption rates in the range of around 0.001 to 0.0001 ng g−1 h−1 and thus difficult to identify in the field.
image file: c9em00540d-f7.tif
Fig. 7 Chloromethane production (upper panel), Arrhenius plots (middle panel) of each replicate experiment and consumption rates (lower panel) at 5 ppmv initial mixing ratio of fresh soil 1 (panel A) and soil 2 (panel B) incubated from 20 to 70 °C (±2 °C). Error bars for CH3Cl production and 13CH3Cl consumption experiments indicate SD of triplicate experiments. Error bars for Arrhenius plots show maximal temperature variations of ±2 °C.

Chloromethane production and consumption in dried soils

Emissions of CH3Cl from dried soil 1 increased exponentially with increasing temperature, and thus was highest at 70 °C with 38.9 ± 2.3 ng g−1 h−1 (Fig. 8A) and were about 5 times higher than those from dried soil 2 (7.1 ± 0.6 ng g−1 h−1; Fig. 8B). The exponential CH3Cl emission patterns of both soils over the temperature range 30 to 70 °C are comparable to the emission profile of dried S. europaea although emission rates for S. europaea were 1 to 2 orders of magnitude higher. According to Keppler et al.18 and Hamilton et al.,24 the main factors influencing abiotic CH3Cl production from soil and plants are chloride concentration and organic carbon content and this likely explains the differences in emission rates observed here. In addition iron oxides in soils have been shown to also play a role in formation of CH3Cl.18,71 However, the content of iron in the two different soils was not determined in this study. Furthermore Ea values for CH3Cl (Fig. 8, lower panel) formation from dried soil 1 and 2 were 110 ± 1 kJ mol−1 and 109 ± 1 kJ mol−1, respectively, and thus almost identical with the Ea calculated for CH3Cl release from dried plant matter (112 ± 3 kJ mol−1) (Table 2), which are typical for abiotic reaction pathways. Please note that no measureable consumption of CH3Cl was observed for incubation experiments with dried soils when 5 ppmv 13CH3Cl were added and thus no comparison can be made between fresh and dry soils. However, these results suggest that consumption of CH3Cl in saline dry soils does not play a relevant role.
image file: c9em00540d-f8.tif
Fig. 8 Chloromethane production rates of dried soil 1 (A) and soil 2 (B) from 30 to 70 °C. Please note that CH3Cl production rates of dried soils for 20 °C were below the limit of quantitation and therefore were not presented. Error bars for CH3Cl production indicate SD of triplicate experiments. Arrhenius plots (lower panel) for CH3Cl emissions of each replicate experiment with error bars showing maximal temperature variations of ± 2 °C.

Chloromethane production from chemical model compounds

For our model experiments we chose three chemical compounds (please refer to material and methods including Fig. 4) that have previously been regarded as methyl precursors of biotic and abiotic CH3Cl formation processes in plants and soils.24,53,72,73

The three chemical model substances containing either methoxy or methyl thiol groups showed similar profiles at temperatures from 30 to 70 °C with exponentially rising (R2 ranging from 0.90 to 0.99) CH3Cl release rates (Fig. 9). Lowest rates of around 8 ng g−1 h−1 at a temperature of 30 °C reaching ∼440 ng g−1 h−1 at 70 °C were observed for pectin. Highest rates were measured for SAM with a maximum value of almost 10 μg g−1 h−1 at 60 °C. Please note that at 70 °C SAM started to decompose which was clearly visible by colour change and liquefaction. This was most likely the reason for the somewhat lower rates observed at 70 °C so consequently we did not consider this value for calculation of Q10 and Ea values. Emission rates of CH3Cl observed for SMM were lower (∼one order of magnitude) than for SAM but higher (∼one order of magnitude) than for pectin. The respective mean Q10 and Ea values (range 30 to 70 °C, except for SAM 40 to 60 °C, Table 2) for pectin, SAM and SMM are 2.7, 3.5 and 3.4 and 83 ± 5, 105 ± 4 and 111 ± 0.1 kJ mol−1, and are in the same range as those Q10 and Ea values measured for S. europaea and dried soils. However, CH3Cl release rates of the model components were one to three orders of magnitude higher when compared with those from the plant and soils indicating that each of the single tested compounds might strongly contribute to CH3Cl formation depending on the compound concentration and amount of chloride available in the plant and soils.


image file: c9em00540d-f9.tif
Fig. 9 Chloromethane production rates (A) of chemical model compounds in a temperature range of 30 to 70 °C (except for SAM at 30 °C). Error bars indicate SD of triplicate experiments. Please note that y-axis is on a logarithmic scale. Arrhenius plots (B), error bars on x-axis show maximal temperature variations of ±2 °C.

Application of stable isotope techniques

Stable carbon and hydrogen isotope values of methyl precursors for CH3Cl formation

In a first step we measured both the stable hydrogen (δ2H) and carbon (δ13C) isotope values of the bulk methyl pool (OCH3 or SCH3 groups) of the three chemical model compounds and S. europaea collected from the field. Unfortunately, the δ2H and δ13C values for the methyl pool for soils 1 and 2 could not be determined, but we consider them to be in the same range as those measured for S. europaea because the major fraction of soil organic matter is expected to be derived from S. europaea (also refer to discussion below). δ2H values spanned a range of −257‰ to −78‰ (Table 3). The methyl pool of S. europaea showed a mean δ2H of −185 ± 18‰, indicating a large apparent hydrogen fractionation compared to the δ2H value of saltmarsh water assuming that this is similar to seawater (∼0‰). This in good agreement with previous results from halophytic plants where an average apparent hydrogen fractionation between the methyl pool of halophytic plants (9 species) and source water of around −190 ± 25‰ was reported by Greule et al.61 In that study,61δ2H values of thermally (50 to 300 °C) emitted CH3Cl were only marginally affected by varying temperatures but correlated well with the δ2H values of the plant source water (meteoric water) and the plant methoxy pool. The respective δ13C values of the methyl pool span a range of −49.1‰ to −23.8‰ with S. europaea and SAM showing lowest and highest values (Table 3).
Table 3 Stable carbon and hydrogen isotope values of methyl moieties (OCH3 or SCH3) of model compounds and S. europaea
Compound Origin δ 2HOCH3/SCH3 [‰] δ 13COCH3/SCH3 [‰]
Pectin (from apple) Sigma-Aldrich −257 ± 3 n = 10 −39.52 ± 0.25 n = 15
S-Adenosyl-L-methionine chloride dihydrochloride Sigma-Aldrich −168 ± 3 n = 8 −23.78 ± 0.29 n = 6
DL-methionine methylsulfonium chloride Sigma-Aldrich −78 ± 2 n = 10 −46.55 ± 0.33 n = 15
S. europaea Field sample −185 ± 18 n = 3 −49.13 ± 3.1 n = 3


Stable carbon and hydrogen isotope patterns of CH3Cl emissions from dried plants and soils

Stable hydrogen isotope values of CH3Cl (δ2HCH3Cl) released from dried S. europaea decreased slightly from −165 ± 8‰ when heated at 20 °C to −189 ± 15‰ when heated at 70 °C (Fig. 10). A similar trend towards more negative δ2HCH3Cl values were observed at these temperatures for soils 1 and 2, with δ2HCH3Cl values from −157 ± 8‰ to −207 ± 19‰ and from −149 ± 9‰ to −194 ± 4‰ for soil 1 (Fig. 11) and soil 2 (Fig. 12), respectively. Most of the δ2HCH3Cl values from the two dried soil samples and dried S. europaea correspond well with the measured value of the methyl pool of S. europaea (−185 ± 18‰). On the one hand, this supports the assumption that the methyl pool of the soil samples is similar to that of S. europaea. On the other hand, these data would imply that there is little or no hydrogen isotope fractionation associated with CH3Cl production. In contrast we observed larger carbon isotope deviations from the methyl pool of S. europaea for δ13CCH3Cl values with increasing temperatures (−50‰ to ∼−75‰ at 50 to 70 °C). Similar isotope differences (−55‰ to −73‰) in δ13CCH3Cl values relative to the methyl pool have been observed on heating dried leaf tissue at 40 to 60 °C.26 Both the stable hydrogen and carbon isotope patterns fit well with previous mechanistic considerations of the reaction pathway where it was suggested that a nucleophilic substitution reaction – between chloride ion and a methyl ester or methyl thiol moiety (SN2 type) – takes place during abiotic formation from biomass.52,68 Thus the carbon atom of the methyl moiety directly reacts with chloride ion causing a so-called primary isotope effect,74 whereas hydrogen is only affected by a secondary isotope effect leading to no or only minor fractionation during formation of CH3Cl.45,46,57 Interestingly, the δ13CCH3Cl values at the lowest temperatures (30 and 40 °C) of both the plant and soil samples were close to the δ13C value of the methyl pool possibly indicating that several methyl precursor compounds with distinguished δ13C values of their methyl pool are active with varying temperatures. For further discussion see the section below which discusses the stable carbon and hydrogen isotope patterns of CH3Cl emissions from chemical model compounds.
image file: c9em00540d-f10.tif
Fig. 10 Stable hydrogen (blue circles) and carbon isotope (red diamonds) values as well as production rates (grey columns) of CH3Cl from dried S. europaea when heated at temperatures ranging from 30 to 70 °C. Blue (δ2H) and red (δ13C) horizontal bars indicate the range (mean ± SD) of stable isotope values for the methyl pool of S. europaea. Error bars indicate SD of triplicate experiments.

image file: c9em00540d-f11.tif
Fig. 11 Stable hydrogen (blue circles) and carbon isotope (red diamonds) values as well as production rates (grey columns) of CH3Cl from dried soil 1 when heated at temperatures ranging from 30 to 70 °C. Please note that δ13CCH3Cl value at 30 °C could not be determined. Blue (δ2H) and red (δ13C) horizontal bars indicate the isotope range (mean ± SD) of the methyl pool. Error bars indicate SD of triplicate experiments.

image file: c9em00540d-f12.tif
Fig. 12 Stable hydrogen (blue circles) and carbon isotope (red diamonds) values as well as production rates (grey columns) of CH3Cl from dried soil 2 when heated at temperatures ranging from 30 to 70 °C. Please note that δ2HCH3Cl and δ13CCH3Cl values at 30 °C could not be determined. Blue (δ2H) and red (δ13C) horizontal bars indicate the isotope range (mean ± SD) of the methyl pool. Error bars indicate SD of triplicate experiments.

Stable carbon and hydrogen isotope patterns of CH3Cl emissions from model compounds

The δ2HCH3Cl values measured for CH3Cl emitted from each of the tested precursor compounds pectin, SMM and SAM lie within a narrow range and do not substantially change (except for pectin at 30 °C, Fig. 13) with rising temperatures and very well reflect the δ2H values of the corresponding precursor methyl pool. This is similar to the pattern observed for the plant and soil samples (Fig. 10–12) although a slight tendency towards more negative values was observed with increasing temperatures. Again, this might support the above mentioned idea that for both plant and soils several methyl precursor compounds are involved in the production of CH3Cl with contributions from each differing with increasing temperatures.
image file: c9em00540d-f13.tif
Fig. 13 δ 2HCH3Cl values (mean of duplicate experiments) of CH3Cl emitted from pectin, SMM and SAM when heated at temperatures ranging from 30 to 70 °C (except SAM at 30 °C). Horizontal bars (blue, green and red) indicate the δ2H values of the corresponding precursor methyl pool (SMM, SAM and pectin). Error bars show maximal temperature variations of ±2 °C.

In contrast to results from stable hydrogen isotope measurements we observed much larger isotope deviations for δ13CCH3Cl values relative to the corresponding precursor methyl pool and with increasing temperatures (Fig. 14). Whilst the isotope differences between δ13CCH3Cl values and the methyl pool of the three model compounds were between −1‰ and −41‰ at 30 °C they increased to −25‰ and −60‰ at 70 °C with SAM and SMM showing the lowest and highest isotope changes, respectively. Again, the three model compounds showed similar patterns of δ13CCH3Cl values as that observed for the plant and soil samples (Fig. 10–12).


image file: c9em00540d-f14.tif
Fig. 14 δ 13CCH3Cl values (mean of duplicate experiments) of CH3Cl emitted from pectin, SMM and SAM when heated at temperatures ranging from 30 to 70 °C (except SAM at 30 °C). Horizontal bars (blue, green and red) indicate the δ13C values of the corresponding precursor methyl pool (SMM, SAM and pectin). Error bars show maximal temperature variations of ±2 °C.

Conclusion

The present study clearly reveals the immense complexity involved in the CH3Cl emission and consumption processes simultaneously occurring in a salt marsh ecosystem. We could clearly identify the six formation and degradation processes suggested in the introduction (Fig. 1): biotic and abiotic formation by S. europaea, biodegradation by plant and soil microbes and biotic and abiotic formation in soil. For both plant and soils, CH3Cl release was substantially higher by the abiotic process than by the biotic process and found to increase exponentially with temperature in the range of 20 to 70 °C. The large observed differences of CH3Cl release rates between fresh and dried soil and plant materials can be easily explained by previous studies.24,50–52 These investigations clearly showed that CH3Cl release rates by abiotic reactions are drastically increasing with decreasing water content in the sample. This observation but also taking into account the calculated activation energies fully support our suggestion of predominantly abiotic CH3Cl formation processes occurring in both S. europaea and the two saline soils. Due to high chloride availability, abiotic CH3Cl formation seems not only important for dead plant matter but also for living S. europaea, particularly at temperatures above 30 °C. Using a stable isotope tracer approach, we also observed that living S. europaea is able to consume CH3Cl. The observed consumption rates by fresh soils were much higher when compared with those from S. europaea and reached maximum values at around 50 °C whereas the maximum consumption rate for S. europaea was observed at ∼30 °C. However, we would point out that in order to perform the consumption experiment the initial mixing ratios of CH3Cl were considerably higher than those normally observed in the environment and thus CH3Cl consumption rates in the field might be expected to be much lower.

The associated stable isotope measurements including δ2HCH3Cl and δ13CCH3Cl values of emitted CH3Cl and the methyl pool measured for plant and soil as well as previously suggested methyl precursors compounds such as SAM, SMM and pectin provide a deeper insight into the compounds that are a likely important source of the methyl moiety for CH3Cl production in soils and halophytes. For example, our results would suggest that compounds such as SAM containing methyl thiol groups might be of particular importance at lower temperatures (20 to 40 °C) whilst other compounds such as pectin might gain importance at higher temperatures (>40 °C).

We are aware that our laboratory approach neglects the complexity of salt marsh ecosystems under field conditions. Nevertheless, if we use the patterns for emission and consumption we observed in our investigation this allows us to make some assumptions on how fluxes of CH3Cl from salt marshes might change in the future. In a salt marsh ecosystem net emission rates to the atmosphere depend on the interplay between release and consumption of CH3Cl from plants and soils. Based on our laboratory studies it appears that abiotic release of CH3Cl from both saline soils and halophytes might exponentially increase with increasing temperatures which is already very pronounced in the temperature range of 20 to 30 °C (Q10 up to 5). It has already been shown in numerous investigations that temperature changes and solar irradiation control emissions of CH3Cl from salt marshes12–15,23,56 and saline soils.19,21 Thus, CH3Cl emissions from salt marsh ecosystems, especially from halophytic plants, will likely increase with elevated temperatures induced by climate change. On the other hand, these higher release rates from plants and soils might be partly balanced by increasing consumption rates in soils also occurring at higher temperatures. However, under ambient mixing ratios (∼500 to 600 pptv) consumption rates of saline soils and halophytes might be insignificant and thus production could greatly exceed consumption in salt marshes under natural conditions.

For future studies we suggest combined approaches including field and complementary laboratory incubation experiments – using both concentration and stable isotope measurements – to be a promising approach for a better understanding of the atmosphere-plant-soil cycling of CH3Cl and other important atmospheric trace gases.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This study was part of the CHLOROFILTER project (German Research Foundation/DFG KE 884/10-1) and supported by DFG KE 884/8-2. We are grateful to Stefan Rheinberger for technical support and Finn and Simone Keppler for support in collecting samples in the field. We thank Thomas Klintzsch for statistical evaluation of the data and the three reviewers and the editor for carefully reading the manuscript and suggestions for improvement.

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