Can a red wood-ant nest be a trap for fault-related CH4 micro-seepage? A case study from continuous short-term in-situ sampling

Methane (CH4) is common on Earth, forms the major commercial natural gas reservoirs, and is a key component of the global carbon cycle, but its natural sources are not well-characterized. We present a geochemical dataset acquired from a red wood-ant (RWA; Formica polyctena) nest in the Neuwied Basin, a part of the East Eifel Volcanic Field (EEVF), focusing on methane (CH4), stable carbon isotope of methane (δ13C-CH4), RWA activity patterns, earthquakes, and earth tides. Nest gas and ambient air were continuously sampled in-situ and analyzed to detect microbial, thermogenic, and abiotic fault-related micro-seepage. Methane degassing was not synchronized with earth tides. Elevated CH4 concentrations in nest gas appear to result from a combination of microbial activity and fault-related emissions moving via through fault networks through the RWA nest. Two δ13C-CH4 signatures were identified in nest gas: −69‰ and −37‰. The −69‰ signature of δ13C-CH4 within the RWA nest is attributed to microbial decomposition of organic matter. This finding supports previous findings that RWA nests are hot-spots of microbial CH4. Additionally, the −37% δ13C-CH4 signature is the first evidence that RWA nests also serve as traps for fault-related emissions of CH4. The −37‰ δ13C-CH4 signature can be attributed either to thermogenic/fault-related or to abiotic/fault-related CH4 formation originating from e.g. low-temperature gas-water-rock reactions in a continental setting at shallow depths (microseepage). Sources of these micro-seeps could be Devonian schists (“Sphaerosiderith Schiefer”) with iron concretions (“Eisengallen”), sandstones, or the iron-bearing “Klerf Schichten”. We cannot exclude overlapping micro-seepage of magmatic CH4 from the Eifel plume. Given the abundance of RWA nests on the landscape, their role as sources of microbial CH4 and traps for abiotically-derived CH4 should be included in estimation of methane emissions that are contributing to climatic change.


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Methane (CH4) is common on Earth, forms the major commercial natural gas reservoirs, and is a key 52 component of the global carbon cycle [1][2]. This second-most important greenhouse gas currently has 53 an average atmospheric concentration of 1.82 ppm, and continues to increase [3]. Today, most natural 54 occurrences of CH4 are associated with terrestrial and aquatic processes. In the shallow subsurface, 55 CH4 is produced on geological time scales mainly by thermal conversion of organic matter resulting from 56 heat and pressure deep in the Earth's crust or by microbial activity. This biotic CH4 includes the formation 57 of thermogenic CH4 and microbial aceticlastic methanogenesis [4][5]. In contrast, abiotic CH4 is produced 58 in much smaller amounts on a global scale and is formed by either high-temperature magmatic 59 processes (Sabatier-type reactions) in volcanic and geothermal areas, or via low-temperature (<100 °C) 60 Fischer-Tropsch-Type (FTT) gas-water-rock reactions in continental settings, even at shallow depths. It 61 is found in specific geologic environments, including volcanic and geothermal systems; fluid inclusions 62 in igneous intrusions; crystalline rocks in Precambrian Shields; and submarine, serpentinite-hosted 63 hydrothermal fields or land-based serpentinization fluids [2,4]. 64 In most geologic environments, biotic and abiotic gases occur simultaneously. Both thermogenic 65 and abiotic CH4 reach the atmosphere through marine and terrestrial geologic gas (micro-)seeps, and 66 during the exploitation and distribution of fossil fuels. To identify whether locally elevated CH4 67 concentrations in the atmosphere are due transportation via fault networks, a determination of possible 68 methane source(s) is required. At the land surface, CH4 is produced by methanogenic Archaea in 69 anaerobic soil environments or through oxidation by methanotrophic bacteria in aerobic topsoils [6]. Faults and fracture networks act as preferential pathways of lateral and vertical degassing, creating 74 linear fault-linked anomalies, irregularly-shaped diffuse or "halo" anomalies and irregularly-spaced 75 plumes or "spot anomalies" [e.g. 11-12].
[10] showed that faults had ẟ 13 C-CH4 = −37‰ and a significantly 76 higher CH4 flux (11.5±6.3 t CH4 km −1 yr −1 ) than control zones. In Europe, micro-seeps occur both 77 onshore and offshore, with estimated CH4 flux in Europe of 0.8 Tg yr −1 and total seepage of 3 Tg yr −1 78 [12,5]. 79 Recent research has revealed close relationships between the spatial distribution of red wood-80 ant nests (Formica rufa-group; henceforth RWA) and tectonic fault zones [13][14][15][16]. Exploratory testing of 81 fault-zone gases revealed that helium (He) and radon (Rn) in RWA nests exceeded atmospheric and 82 background concentrations [13][14]. RWA mounds also have been found to be "hot spots" for CO2 83 emissions in European forests [17][18][19][20]. [21] showed that ant mounds (Lasius flavus, Lasius niger and 84 Formica candida) contributed measurable amounts to soil gas emissions from wetlands (CO2: 7.02% 85 and N2O: 3.35%), but act as sinks with regard to the total soil CH4 budget (-4.28%). In contrast to that, 86 higher net CH4 emission (3.5 µg m²h -1 ) were found in fire ant mounds situated in natural pasture soils 87 We used a combination of geochemical, geophysical, and biological techniques; state-of-the-art 94 image analysis; and statistical methods to identify associations between RWA activity, continuous in-95 situ CH4 degassing, earth tides, and tectonic processes. We aimed to test from a 96 geochemical/geophysical point of view three different hypothesis: a) whether a RWA nest can indicate 97 actively in-situ degassing faults trapping migrating CH4 from the deep underground; b) whether RWA 98 activity changes during the CH4 (micro)-seepage process; and c) whether CH4 (micro)-seepage process 99 is affected by external agents (earth tides, earthquake events, or meteorological conditions). 100 Specifically, we tested the null hypotheses that, in the field, in-situ concentrations of both CH4 and ẟ 13 C-101 CH4 and RWA activity are independent. We found a RWA nest appears to trap fault related micro-102 seepage of CH4, and that degassing pattern are independent from earth tides and meteorological 103 conditions. 104

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We explored associations between RWA activity, in-situ methane concentrations in an ant nest 106 and ambient air, tectonic events, and weather processes and earth tides at the Goloring site near 107   RWA activity using an "AntCam": a high-resolution camera system (Mobotix MX-M12D-Sec-DNight-142 D135N135; 1,280 × 960 pixels) installed ≈5 m from a RWA nest (Fig 1B). During the 192-hr CH4 143 sampling campaign, which ran from 4-11 August, 2016, ant activities were recorded and time-stamped 144 continuously (12 Hz). The network-compatible AntCam was connected to a network-attached storage 145 (NAS) system for data storage via a power-over-Ethernet (POE) supply. A computer connected to the 146 NAS evaluated the RWA activities on-site and in real time using C++ code to accelerate image 147 evaluation. Image analysis extended the system of [36] and was based on the difference image 148 technique (Fig 2). To reduce negative influences caused by, e.g., moving blades of grass, we used a 149 mask to restrict analysis to only the visible top of the mound. To compensate for slight movements of 150 the camera, e.g., due to wind, an image registration of the current image relative to the previous image 151 was done based on mutual information before the determination of the absolute difference image [37]. 152 Results of RWA activity were written to a file. Every hour, this file was sent via email (mobile data 153 transfer, LTE router) to a mail server. Since two different sensors were used for the day and night, 154 respectively, we computed different polynomials to map the sum of absolute differences onto manually 155 designed activity categories in a follow-up procedure. The coefficients of the polynomials were obtained 156 from a minimization of the sum of squared differences between the polynomial model and the manually 157 assigned category for two selected weeks. A first-order polynomial was adapted to the daytime data and 158 a third-order polynomial was adapted to the nighttime data. To avoid numerical difficulties, we first 159 centered and scaled the data by subtracting the mean of the data during the target time and dividing by 160 the standard deviation. Both values were computed for day-and nighttime, respectively.

Gas sampling and geochemical analyses
170 Field measurements of CH4 were taken from 4-11 August 2016. A stainless-steel probe (inner 171 diameter 0,6 cm; Fig 1C) was inserted into the F. polyctena nest to a depth of 80 cm and remained 172 there, unmoved, during the entire 192-hour sampling campaign. The probe was used for continuous 173 CH4/ẟ 13 C-CH4 measurements. The probe was equipped with a flexible tip attached to a pushable rod 174 and a sealable outlet for docking sampling equipment. The closed probe was inserted into the nest. 175 After opening by pushing the rod, the probe was evacuated twice, using a 20-ml syringe. After this, the 176 outlet was closed to prevent atmospheric influence. The outlet was only opened after docking the 177 sampling unit to it. 178 Concentrations of CH4 and ẟ 13 C-CH4 in nest gas (NG) and ambient air (AA) were monitored 179 using a portable CRDS analyser (G2201-i; Picarro, USA) that measured 12 CH4, 13 CH4 and H2O quasi-180 simultaneously at 1 Hz, and provided ẟ 13 C values relative to the Vienna Pee Dee Belemnite standard. 181 The G2201-i uses built-in pressure and temperature control systems, and automatic water-vapor 182 correction to ensure high stability of the portable analyzer. Effects of water vapor on the measurement 183 were corrected automatically by the Picarro ® software. The manufacturer guarantees concentration 184 precision for the analysis of CH4 in the "high precision mode" of 5 ppbv ± 0.05 % ( 12 C) and 1 ppbv ± 185 0.05% ( 13 C) within a concentration range of 1.8-1000 ppm. The guaranteed precision of ẟ 13 C-CH4 is 186 The CRDS analyzer was deployed in a dry, wind-sheltered location near the RWA nest. Nest 188 gases were pumped from the aforementioned probe into the CRDS analyzer for analysis of CH4 and 189 ẟ 13 C-CH4 values. Ambient air was measured 2 m away from the nest for 15 min every four hours during 190 the operation using a 3-way-valve, avoiding disturbance of the nest or the position of the steel probe. 191 All gases passed through a chemical trap filled Ascarite ® (sodium hydroxide coated silica; 192 www.merckgroup.com) before entering the system to remove carbon dioxide (CO2) because the high 193 concentrations of CO2 in the nest samples could interfere with the measurements of CH4 and ẟ 13 C- CH4. 194 Gas samples were dried by a Nafion® drying tube (Nafion MD110, PermaPure LLC, USA) before 195 measurements to ensure higher accuracy and subsequently analyzed for CH4 concentration and ẟ 13 C-196 CH4. To assure quality of the CH4 and ẟ 13 C-CH4 values, reference gas measurements were taken every 197 8 h during the operation. Fluctuations in atmospheric CH4 and ẟ 13 C-CH4 values were validated against 198 a single, 4-h measurement of ambient air. Carbon isotope ratios are expressed using standard delta (ẟ) We examined associations between the six measured meteorological variables and RWA 221 activity and CH4 concentrations. As many of these variables were correlated with one another, we used 222 principal components analysis (R function prcomp) on centred and scaled data to create composite 223 "weather" variables (i.e., principal axes) that were used in subsequent analyses. 224 We used the "median+2MAD" method [41] to separate true peaks in CH4 concentrations from 225 background or naturally-elevated concentrations: any observation greater than the overall 226 median+2MAD (2.31 ppm CH4 in nest gas and 2.11 ppm CH4 in ambient air) was considered to be a 227 peak concentration. Background and elevated CH4 concentrations were separated based on the 90% 228 quantile of the CH4 concentration [42]. For interpreting the significance of the correlation coefficient, we 229 followed [43]. For ẟ 13 C-CH4, we considered concentrations < −35‰ or > 0‰ to be peak concentrations. 230 Only peaks occurring in both data sets at the same time were considered to be true peaks. The Keeling 231 plot method [44] was applied to determine the carbon-isotope composition of the found peaks to obtain 232 insights into the processes that govern the distinction between isotopes in the ecosystem. 233

Meteorological conditions
238 During the one-week field campaign in August 2016, air temperatures ranged from 5.7-29.1 °C 239 (mean = 16.2 °C), with only 2.1 mm rainfall overnight between 9 and 10 August. Variation in atmospheric 240 pressure (mean 988 ± 2.24 hPa) and wind speed (1.67 ± 1.72 km/h) were small. The first three axes 241 derived by the principal components analysis accounted for nearly 80% of the variance in the data (Table  242 1). The first axis represents temperature and humidity, the second axis represents atmospheric pressure 243 (with additional contributions of humidity and windspeed), and the third axis represents rainfall and 244 windspeed (with a minor contribution of temperature). 245

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Median RWA activity and the three principal axes of weather were modestly associated, and 251 accounted for only 8% of the variance in ant activity ( Table 2). The ant activity increased slightly at lower 252 temperatures (PC-1) and slightly decreased when rainfall (PC-3) was present. PC-2 was not associated 253 significantly with RWA activity. 254 Table 2 Summary ANOVA table of  Weather conditions explained 10% of the variation in CH4 (ppm) ( Table 3), but explained 22% 261 of the variation in ẟ 13 C-CH4 (‰), which decreased with all measured weather variables (Table 4). 262 Table 3 Summary ANOVA table of   Ants were most active during the late afternoon and early evening hours (Fig 3A; 4A). The 275 video streams showed that the ants went on foraging, building and maintaining the nest as they had 276 done since the start (on March, 18 th ) of our longer 7-month field campaign. Decomposition of the time-277 series into its additive components (Fig 3B-D) illustrated that during the one-week gas-sampling 278 campaign, there was a trend towards increasing activity over the first four days, followed by a sharp 279 decline towards the end of the week (Fig 3B). There were two noticeable peaks of activity, at mid-day 280 and early afternoon, followed by sharp spikes in activity near 16:30 hours (Fig 3C).  showed that mean nest gas emissions are of the same order, although we had 20× more observations. 313 317 ẟ 13 C-CH4 in the nest ranged from −58.48 to −49.54‰ (Fig 4C). Eight significant peaks (red and 318 blue marks in Fig 5A, B) in nest gas were found for CH4 and ẟ 13 C-CH4 (Fig 5a, b). These peaks occurred 319 between 17:39 (UTC) and 06:54 (UTC) the following day, but were otherwise not temporally predictable. 320 Results of the Keeling plots [44] revealed two signatures for ẟ 13 C-CH4 at −37‰ (blue markers and dots 321 in Fig 5A, B and C) and −69‰ (red markers and dots in in Fig 5A, B and C) in nest gas (Fig 5C).  Fig 4). This micro-earthquake neither influenced degassing nor RWA activity. 342 Comparison of ẟ 13 C-CH4 nest gas signatures with published data suggests that it can be 359 attributed to two different sources (Fig 7). The ẟ 13 C-CH4 signature of −69‰ in nest gas indicates a 360 microbial source, such as decomposing organic matter that is high in nutrients [8]. This result supports 361 the findings of [22] that the aboveground parts of ant nests are hot-spots of CH4 production. 362 The second isotope signature, −37‰ ẟ 13 C-CH4, can be attributed either to thermogenic/fault-363 related [10] or to abiotic/fault-related CH4 formation [45]. This result provides the first evidence that RWA 364 nests may serve as traps for fault-related emissions of CH4.
[10] found a ẟ 13 C-CH4 signature of −37‰ 365 for fugitive emission of CH4 via migration along fault zones in the United Kingdom. Our result of -37‰ 366 ẟ 13 C-CH4 is of the same order (Fig 7) and can be attributed to fault-related CH4 emission moving through 367 the RWA nest. 368 Because the largest quantities of abiotic gases found on Earth's surface are produced by low-418 temperature gas-water-rock reactions [54] we attribute the −37‰ ẟ 13 C-CH4 signature in RWA nests to 419 fault-related emissions of abiotically formed CH4 by gas-water-rock reactions occurring at low-420 temperatures in a continental setting at shallow depths (micro-seepage). Probable sources might be 421 Devonian schists ("Sphaerosiderith Schiefer") with iron concretions ("Eisengallen") sandstones and/or 422 the iron-bearing "Klerf Schichten". However, we cannot exclude the possibility of overlap by magmatic 423 CH4 micro-seepage from the Eifel plume. 424 In summary, we suggest that RWA nests can indicate actively degassing faults trapping 425 migrating CH4 from the deep underground, but that future work should seek to determine if the −37‰ 426 signature can be attributed to a purely abiotic source, or a combination of abiotic/thermogenic source. 427 Such a study should use additional measurements of ẟ 13 H and run long enough to determine the 428 influence of irregularly timed earthquake events on patterns of methane degassing. 429

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Neither our second or third hypotheses were supported by the data. During the investigation 440 period, ant activity was higher than we had observed in 2009-2012, although an "M-shaped" pattern in 441 daily activity was still identifiable [36]. Relatively high RWA activities during the late afternoon and early 442 evening hours could be attributable to direct sun hitting the nest during that time or with activities 443 associated with rebuilding damage to the nest that had occurred on 18 March. We did not find any 444 evidence that ant activity changed during the CH4 (micro)-seepage process, or that there were strong 445 effects of weather (see also 36]), or methane seepage. Additional external agents, including mice and 446 "anting" birds, or micro-earthquakes did not influence ant activities during the sampling week. We 447 conclude that during our 8-day sampling period, RWA activity was independent from external agents. 448 Nest gas CH 4 and ẟ 13 C-CH 4 and external parameters 449 We also did not find strong support for a relationship between CH4 in the nest and external 450 variables during our 8-day sampling period. Atmospheric CH4 concentrations were always lower than 451 CH4 in the RWA nest and there seemed to be little influence of atmospheric CH4 on CH4 in the nest. 452 Less than 25% of the variance in CH4 and ẟ 13 C-CH4 were accounted for by weather conditions (cf. [57]). 453 Earth tides also were not correlated with methane degassing in the nest. The −37‰ ẟ 13 C-CH4 signature 454 in nest gas was detected only once. The micro-earthquake on August 9 did not influence CH4 degassing 455 because of its far distance (20 km). On August 13, there was another earthquake (ML: 0.7; D = 13 km) 456 only 2.3 km away from the nest. It might be, that the −37‰ ẟ 13 C-CH4 signature in nest gas was a 457 precursor to the August 13 earthquake, promoting degassing due to an increase in compressive stress 458 [9,10]. But this remains unanswered as the CH4 measurement campaign was terminated at August 11. 459

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For the first time, both CH4 and ẟ 13 C-CH4 in a RWA nest was continuously recorded in situ. 461 Methane degassing nor RWA activity was synchronized with earth tides, micro-earthquakes, or weather 462 conditions. Elevated CH4 concentrations in nest gas appear to result from a combination of microbial 463 activity and fault-related emissions moving via through fault networks through the RWA nest. Two ẟ 13 C-464 CH4 signatures were identified in nest gas: -69‰ and -37‰. The -69‰ signature of ẟ 13 C-CH4 within the 465 RWA nest is best attributed to microbial decomposition of organic matter. This finding supports previous 466 findings that RWA nests are hot-spots of microbial CH4. Additionally, the -37‰ ẟ 13 C-CH4 signature is 467 the first evidence that RWA nests also may serve as traps for fault-related emissions of CH4. The -37‰ 468 ẟ 13 C-CH4 signature can be attributed either to thermogenic/fault-related or to abiotic/fault-related CH4 469 formation originating from, e.g., low-temperature gas-water-rock reactions in a continental setting at 470 shallow depths (micro-seepage). Future work on the −37‰ signature should use additional 471 measurements of ẟ 13 H and run long enough to determine the influence of irregularly timed earthquake 472 events on patterns of methane degassing. 473