Hypersaline tidal flats as important Blue Carbon systems: A case
study from three ecosystems

Abstract. Hypersaline tidal flats (HTFs) are coastal ecosystems with freshwater deficits often occurring in arid or semi-arid regions near mangrove supratidal zones with no major fluvial contributions. Here, we estimate that organic carbon (OC), total nitrogen (TN) and total phosphorus (TP) are being buried at rates averaging 21 (± 6), 1.7 (± 0.3), and 1.4 (± 0.3) g m−2 y−1, respectively, during the previous century in three contrasting HTFs systems, one in Brazil (eutrophic) and two in Australia (oligotrophic). Although these rates are lower than those from nearby mangrove, saltmarsh and seagrass systems, the importance of HTFs as sinks for OC, TN and TP may be significant given their extensive coverage. Despite the measured short-term variability between net air-saltpan CO2 influx and emission estimates found during the dry and wet season in the Brazilian HTF, the only site with seasonal CO2 fluxes measurements, the OC sedimentary profiles over several decades suggests efficient OC burial at all sites. Indeed, the stable isotopes of OC and TN (δ13C and δ15N) along with C : N ratios show that microphytobenthos are the major source of the buried OC in these HTFs. Our findings highlight a previously unquantified carbon as well as nutrient sink and suggest that coastal HTF ecosystems could be included in the emerging blue carbon framework.


. To date, there has been large scale destruction and degradation of these systems on a global scale as a result of anthropogenic pressures on coastal areas including infilling for urban and agricultural/aquaculture development (Halpern et 75 al., 2008). Although there has been the implementation of various laws in some parts of the world to prevent the loss of coastal vegetated systems, these legislations rarely extent to protect HTFs that are viewed as being ecological deserts with no obvious vegetation (Albuquerque et al., 2013). Furthermore, the landward encroachment of mangrove forests as a response to rising sea levels, coupled to barriers preventing landward migration of HTF (i.e. the "coastal squeeze"), may also contribute to the loss of these ecosystems (Alongi, 2008;Saintilan et al., 2014;Kelleway et al., 2017). 80 Given the substantial areal extent of these HTFs and the fact that they remain relatively undisturbed in many regions around the world, HTFs may have unrecognized ecological values (Burford et al., 2016). However, information on OC, nitrogen and phosphorus burial and sediment CO2 fluxes from these ecosystems remains scarce (e.g. Bento et al., 2017;Schile et al., 2017). Determining if HTFs are a source or sink of carbon is critical to understanding their importance and value in regards to climate change and coastal carbon sequestration (Lovelock and Duarte, 2019). Here, we quantify carbon and nutrient 85 burial and atmospheric CO2 fluxes in HTFs in Australia and Brazil. We hypothesize that microphytobenthos in HTFs sequester CO2 from the atmosphere and a portion of this organic matter (and associated nitrogen and phosphorus) is buried, similar to the traditional vegetated blue carbon systems.
In Australia, the Karumba HTF is located adjacent to the oligotrophic mouth of the Norman River estuary in the 95 southeastern coast of the Gulf of Carpentaria. The study site consists of a large continuous HTF (16.9 km 2 ) in the high upper year average maximum monthly temperatures (from 1993 to 2019) vary from 23 ºC in the winter months to 31 ºC in the summer months (Bureau of Meteorology, 2019). The sheltered strait between Curtis Island and the mainland of Australia is largely occupied by mangrove forests and large continuous expanses of HTFs. The Gladstone site contained two HTF study areas; Site 1 (2.84 km 2 ) was situated in the higher tidal area and is inundated less frequently and for shorter periods of time than Site 2 (0.95 km 2 ). 110 In Brazil, the tropical HTF was located in the Guaratiba State Biological Reserve, ~40 km south of Rio de Janeiro City which forms part of the Sepetiba Bay estuary system. This conservation area is surrounded by the urban expansion area of Rio de Janeiro City, and Sepetiba Bay receives discharges of nutrients and organic matter from its watershed dominated by agriculture, pasture and urban uses (Rezende et al., 2010). The HTF covers an area of approximately 7.4 km 2 equivalent to almost 36% of the fringing mangrove forest (Estrada et al., 2013;Soares et al., 2017) (Table 1). There is little variation in 115 topography and the tidal range is 0.1 -2.0 m (Masuda and Enrich-Prast, 2016;Bento et al., 2017). The 32-year monthly average rainfall and temperature vary from 36 mm and 21 ºC in dry winter months and 138 mm and 27 ºC in rainy summer months, reaching annual averages of accumulated rainfall of 1058 mm (Estevam, 2019) (Table 1).

Sediment core sampling and analysis
Sediment cores (one core per site for a total of four cores) were collected from the middle of HTFs by using either a 50 cm 120 long, 5 cm diameter Russian Peat Auger (Karumba core) or by inserting a PVC tube (8.7 cm diameter) into the substratum using manual percussion (Gladstone and Guaratiba cores). Only cores with no observed compaction were retained for further analysis. The sediment cores were sectioned at 1 cm intervals (with the exception of the Karumba core which was sectioned at 2 cm intervals). Dry bulk density (DBD, g cm -3 ) was determined as the dry sediment weight (g) divided by the initial volume (cm 3 ) (Ravichandran et al., 1995). From the original dry section, a non-homogenized portion was rewetted and treated with 125 30% hydrogen peroxide (H2O2) to remove organic matter without altering grainsize. A solution of sodium hexametaphosphate was used as a deflocculating agent to separate aggregates prior to grain size analysis. Grain size analyses were conducted using a CILAS 1090L diffraction laser unit or wet sieving following the methods used by Conrad et al., (2019). Total phosphorous (TP) was measured after acid digestion (H2O/HF/HClO4/HNO3, 2:2:1:1) using a Perkin Elmer ELAN DRCe ICPMS.
Organic carbon (OC) and total nitrogen (TN) stable isotope ratios of mangrove leaves, microphytobenthos, and HTF 130 sediments were measured to identify the sources of organic matter (OM) contributing the sediment column at each site. Fresh green leaves from mangrove trees (n = 3 for each dominant species: Rhizophora mangle, Avicenna shaueriana, and Laguncularia racemose) were collected at 1-2m above the soil, and washed with deionized water soon after sampling in the Brazilian HTF. Samples were then lyophilized, crushed, sieved, and ~6-8 mg encapsulated in tin capsules to determine the OC, TN and their isotopic composition (δ 13 C and δ 15 N). Microphytobenthos samples, in the form of dense algal mats, were 135 collected from the surface of HTF sediments, scrapped and thoroughly washed with deionized water to avoid sediment contamination. A total 6 microphytobenthos samples were collected and analyzed (3 from Brazil, 2 from Karumba, and 1 from Gladstone). A homogenized portion was acidified to remove carbonate material, washed in deionized water, dried (60 °C) and https://doi.org/10.5194/bg-2020-426 Preprint. Discussion started: 24 November 2020 c Author(s) 2020. CC BY 4.0 License. then ground to powder for OC and δ 13 C analyses using a Leco Flash Elemental Analyzer coupled to a Thermo Fisher Delta V IRMS (isotope ratio mass spectrometer). A non-acidified homogenized portion was also analyzed for TN and δ 15 N. Analytical 140 precision: C = 0.1%, N = 0.1%, δ 13 C = 0.1‰ and δ 15 N = 0.15 ‰. We assess whether HTFs accumulate carbon and then to compare HTF with well-established, nearby mangrove systems.
Radionuclides from the uranium-238 ( 238 U) decay series were measured in a high-purity germanium (HPGe) planar or well gamma detectors. Identical geometry was used for all samples and sample dry weights were between 20 and 30 g.
Sealed and packed samples were set aside for at least 21 days to allow for radon-222 ( 222 Rn) ingrowth and to establish secular 145 equilibrium between radium-226 ( 226 Ra) and its granddaughter lead-214 ( 214 Pb). Lead-210 ( 210 Pb) activity was determined by the direct measurement of the 46.5 KeV gamma peak. 226 Ra activity was determined via the 214 Pb daughter at 351.9 KeV. 210 Pb and 226 Ra activities were calculated by multiplying the counts per minute by a correction factor that includes the gamma-ray intensity and detector efficiency determined from standard calibrations. Excess 210 Pb was used to determine ages of sediment intervals using the Constant Initial Concentration (CIC) model (Appleby and Oldfield, 1992). Mass accumulation rates were 150 multiplied by the percent OC, N and TP to calculate burial rates. between dry and rainy seasons in the HTF in Brazil and non-monsoon months in Australia. In all sampling sites, we used 155 sediment chambers connected in a closed system with an infrared or cavity ring-down analyzer as reported in Lovelock (2008).

Air-sediment gas flux measurements
The sediment chambers were composed of transparent plexiglass (light chamber) or an opaque material such as PVC or covered by layers of aluminium foil (dark chamber) for measurements of light and dark air-sediment CO2 fluxes respectively (Leopold et al., 2015). Before each measurement, the chambers were gently pushed into the sediment (~2 cm) to form a gas tight seal.
Each short-term incubation lasted 5-15 min to achieve a linear change in CO2 concentration within the chambers. Gas 160 concentrations were measured using either a Los Gatos Research (LGR) Ultra-Portable Greenhouse Gas Analyzer (UGGA) or Picarro G4301 GasScouter recorded at 1-second intervals in the Australian sites, and a PPSystems EGM-4 or a Vaisala GMT222 at 1-minute intervals in the Brazilian sites. Equipment were previously calibrated with CO2 standards of 400 and 1000 ppm in the laboratory.
where s is the regression slope for each chamber incubation deployments (ppm sec -1 or ppm min -1 , converted to ppm h -1 ), V is 170 the chamber volume (m 3 ), R is the universal gas constant, Tair is the air temperature inside the chamber (K), A is the surface https://doi.org/10.5194/bg-2020-426 Preprint. Discussion started: 24 November 2020 c Author(s) 2020. CC BY 4.0 License. area of sediment inside the chamber (m 2 ). Negative values represent net sediment CO2 uptake while those positive represent net CO2 emission from sediments to the atmosphere. We assume that pressure in the chamber is 1 atm. To determine the net ecosystem exchange (NEE), we integrate diurnal and night fluxes from light and dark chambers for each sampling day, respectively. To test the normality of CO2 emissions data we performed a Komogorov-Smirnov test. For non-normally 175 distributed data, a Mann Whitney test (significance level; p<0.05) was undertaken to compare light and dark fluxes at the combined Brazil and Australian samples, and also to compare wet and dry season Brazil fluxes.

Organic matter source
To assess the source of organic matter (OM), sediment, HTF microphytobenthos, and nearby mangrove end member samples 195 were analysed for δ 13 C stable isotopes and cross-plotted against molar C:N ratios (Fig. 4). Microphytobenthos samples showed a small spread in δ 13 C and molar C:N ratios ranging from -13.4 to -19.0 ‰ and 7.9 to 14.8 respectively (Fig. 4). Similarly, values of δ 13 C and molar C:N ratios showed little down-core variation in both the Guaratiba (-17.7 to -18.4 ‰ and 7.6 to 9.7 respectively) and Karumba sediment cores (-15.5 to -20.5 ‰ and 10.5 to 14.6 respectively). In contrast, both the Gladstone sediment cores showed a considerable range in the δ 13 C and molar C:N values (-20.1 to -24.2 ‰ and 13.6 to 21.8 at site 1; -200 16.6 to -24.4 ‰ and 10.7 to 34.8 at site 2). Higher δ 15 N and lower C:N ratio values were noted in the Guaratiba HTF compared to other sites (Fig. 4).

C, N and P burial in HTFs versus vegetated blue carbon ecosystems
Considerable differences in OC burial rates between the two Gladstone sites were observed in this study. The likely difference between sites is due to the tidal area of each site, i.e. upper vs lower tidal areas are expected to accumulate carbon at different 215 rates . By averaging the sediment burial rates on a centennial scale (i.e. entire core) of the four sediment cores across all the study sites, we estimate that HTF ecosystems accumulate OC, TN and TP at rates of 21 (± 6), 1.7 (± 0.3), and 1.4 (± 0.3) g m -2 y -1 , respectively. These centennial scale averages reduce short term variations allowing comparisons with saltmarsh, mangrove forests, and seagrass beds which have been studied extensively using similar methodologies and timeframes (McLeod et al., 2011). The average OC accumulation rates in HTF systems were ~12, ~8 and ~7 fold lower than 220 the global averages reported for saltmarsh (245 ± 26 g m -2 y -1 ; Ouyang and Lee (2014)), mangrove forests (163 ± 40 g m -2 y -1 ; Breithaupt et al. (2012)), and seagrasses (138 ± 38 g m -2 y -1 ; McLeod et al. (2011)), respectively. These lower burial rates may be related to the lower organic matter supply (including no contribution from below ground productivity) and/or lower sediment accretion rates than the traditional blue carbon systems. Furthermore, the reduced structural complexity and ability of the microalgae to trap sediments, the lower primary production rates, the lack of underground root protection, and the fact 225 that microalgae organic material is more labile can explain the lower burial and sediment accretion rates of HTFs than traditional, vegetated blue carbon systems.
Hypersaline tidal flats can be a significant source of nutrient export to adjacent ecosystems which may potentially fuel primary productivity in nutrient-limited receiving marine ecosystems Burford et al., 2016). Here, we find that these HTF ecosystems are also sites for the long-term storage of nitrogen and phosphorus ( Table 2). The high TP 230 burial rates compared to TN observed are likely due to the lack of anthropogenic nitrogen inputs observed in other systems.
Estuary, China with TP accumulation rates reaching 48.1 g m -2 y -1 (Alongi et al., 2005). Anthropogenic activities such as urbanization and major industrial developments drive degradation and increased primary production in mangrove forests . Nutrients such as iron and phosphorus may be limiting to mangrove growth (Alongi, 2010;Reef et al., 2010), and those forests receiving high nutrient loads from highly concentrated anthropogenic nutrient discharges accumulate 240 OC, TN, and TP at rates much higher than those from the undisturbed mangrove . Nevertheless, the nitrogen and phosphorus burial in HTFs as shown here over long periods of time may play an important role in nutrient sequestration from other coastal anthropogenic activities, e.g. shrimp farming activities (Ashton, 2008;Marchand et al., 2011).
By upscaling the average OC, TN and TP accumulation results for the past century in this study to the regional areas of HTFs, we can provide a first-order estimate of the amount of OC, TN and TP being stored annually in these HTFs. Ridd 245 and Stieglitz (2002) identify the areal extent of both HTFs and mangrove forests for five estuaries in Queensland, Australia with the HTFs identified as having a ~10-fold higher areal extent (279 km 2 ) than mangrove forests (29 km 2 ) over the five estuaries. In these estuaries alone, HTFs would contribute to the annual accumulation of approximately 5.76 ± 1.57, 0.46 ± 0.09 and 0.40 ± 0.08 Gg y -1 of OC, TN and TP respectively which is similar to the contribution of mangrove forests (4.73 ± 1.16, 0.36 ± 0.06 and 0.26 ± 0.03 Gg y -1 for OC, TN and TP respectively) when based on global average accumulation rates 250 (Breithaupt et al., 2012;Breithaupt et al., 2014). In contrast to Australia, the mangrove forests of Guaratiba (20.9 km 2 ) have been identified to have a ~3 fold higher area than local HTFs (7.4 km 2 ) (Soares et al., 2017); resulting in annual OC, TN and TP accumulation in HTFs (0.15 ± 0.04, 0.01 ± 0.00 and 0.01 ± 0.00 Gg y -1 respectively) equivalent to 4-6% of those estimated for mangrove forests (3.41 ± 0.84, 0.26 ± 0.04 and 0.19 ± 0.02 Gg y -1 for OC, TN and TP respectively) when based on global average accumulation rates (Breithaupt et al., 2012;Breithaupt et al., 2014). 255 Our estimates suggest HTFs are capable of long-term storage of OC, TN and TP and given their large areal extent, have the potential to store as much OC, TN and TP as traditional coastal blue carbon systems in arid regions such as Queensland, Australia. To improve these estimates, there is clearly a need for determining carbon and nutrient accumulation rates from additional coastal HTFs and assess their areal cover in Australia, Brazil and elsewhere.

Organic matter source 260
Microphytobenthos associated with coastal HTF ecosystems were an important source of OM accumulation in each of the sediment profiles. Microscopic examinations in previous studies have identified the cyanobacteria Oscillatoria spp., Lyngbya spp., Microcoleus spp., and Phormidium spp. as the dominant microphytobenthos in HTF ecosystems (Adame et al., 2012;Burford et al., 2016;Masuda and Enrich-Prast, 2016;Bento et al., 2017). These microphytobenthos are likely to be the important species contributing to the accumulation of OM, particularly in Guaratiba and Karumba where the δ 13 C and C:N 265 ratio values were consistently similar to that of the HTF microphytobenthos end member values (Fig. 4). Therefore, we suggest that microphytobenthos are the dominate source of OM accumulating in the sedimentary substrates during the past century.
In contrast to the Guaratiba and Karumba profiles, the considerable spread in δ 13 C and molar C:N ratio values along the Gladstone sedimentary profiles suggests the OM accumulation inputs are from a combination of microphytobenthos and mangrove material (Fig. 4). These results are not surprising given the vast areal extent of mangrove systems in the Gladstone 270 https://doi.org/10.5194/bg-2020-426 Preprint. Discussion started: 24 November 2020 c Author(s) 2020. CC BY 4.0 License.
Harbour and their close proximity to the HTFs. Effective N consumption in coastal wetland sediments (Wadnerkar et al., 2019) may increase overall sedimentary C:N ratios. Sedimentary N and the relatively higher δ 15 N values observed in the Guaratiba HTF sediments (Fig. 3) may be indicative of eutrophication . Indeed, wastewater inputs typically have elevated δ 15 N values due to elevated nitrogen cycling including denitrification (Costanzo et al., 2005). Anthropogenic wastewater inputs high in N and P loads are also of growing concern across the globe, particularly in HTF areas near shrimp 275 farming (Ashton, 2008;Marchand et al., 2011). While there are no shrimp farms near our study sites, the release of high N and P loads may drive eutrophication of adjacent coastal areas (Ashton, 2008;Marchand et al., 2011) and modify carbon burial rates . In addition to the increase of the N and P release, shrimp farms would drive a reduction of the HTFs area that may remove N and P.

CO2 fluxes at the air-sediment interface 280
The great variability of air-saltpan CO2 fluxes here suggests a highly dynamic and productive metabolism along the HTFs.
The oligotrophic Gladstone's sites were net sources of CO2 to the atmosphere in the dry season (0.72 ± 0.01 g C m -2 d -1 ), while the eutrophic Guaratiba's HTF experienced net CO2 uptake and source in the dry and rainy season (-0.03 ± 0.01 and 0.71 ± 0.22 g C m -2 d -1 , respectively). These estimates of net seasonal fluxes of CO2 contribute to reduce the scarcity of studies quantifying this gas exchange at the air-sediment interface in HTFs (Table 3). The net CO2 source observed during rainy 285 compared to the net influx during dry seasons in Brazil (Mann-Whitney, p<0.05) may be attributed to higher temperature and cloud cover over sampling days in the rainy summer than the dry winter. Previous evidence indicates that the light attenuation by clouds may reduce microphytobenthos photosynthetic activity (Blackford, 2002;Barnett et al., 2020), while warmer sampling conditions on average ± SE, 26.7 ± 0.02 and 21.5 ± 0.02°C during the wet and dry season, respectively, may stimulate heterotrophy in tidal flat systems (Laviale et al., 2015;Lin et al., 2020). The CO2 source to the atmosphere found during the 290 rainy summer still contrasted with previous evidence in the same Brazilian HTF on enhanced CO2 sink after rain events in winter (Bento et al., 2017), suggesting that factors other than the occurrence of precipitation (e.g., rainfall duration and intensity) may cause the dynamic short-term changes on microphytobenthic production. In addition, higher values of airsaltpan CO2 influx during similar sunnier periods in the Brazilian than Australian HTFs may be attributed to more eutrophic conditions, which could stimulate microphytobenthic production in saltpan sediments (Xie et al., 2019). These findings 295 highlight the high temporal variability and the need for future seasonal sampling due to the short-term shifts in air-saltpan CO2 exchange, specifically considering the potential net atmospheric CO2 sink in HTFs as indicated by the autochthonous OM in found in the sedimentary profiles. As such, gaining a clearer understanding of the drivers of net primary production in HTFs during changing climatic and anthropogenic conditions is critical to determine their global relevance as atmospheric carbon sinks. 300

Can HTFs be considered "Blue Carbon" systems?
While much of the research on blue carbon systems continues to focus on mangrove forests, tidal marshes, and seagrass meadows, there are suggestions of considering other ecosystems in the blue carbon framework (Raven, 2018;Trevathan-Tackett et al., 2015;Lovelock and Duarte, 2019). Tidally influenced freshwater forests, marine macroalgae and kelp beds, and https://doi.org/10.5194/bg-2020-426 Preprint. Discussion started: 24 November 2020 c Author(s) 2020. CC BY 4.0 License.
HTFs for instance, are all ecosystems where blue carbon stocks and sequestration rates may be conceptually equivalent to 305 conventional blue carbon systems (Raven, 2018;Krause-Jensen et al., 2018;Krauss et al., 2018;Lovelock and Duarte, 2019). Lovelock and Duarte (2019) discuss several key assessment criteria for the inclusion of an ecosystem in the blue carbon framework. First, an ecosystem needs to be capable of long term-storage of CO2 resulting in significant greenhouse gas (GHG) removal from the atmosphere. The results from this study indicate that HTF ecosystems are capable of long-termstorage of fixed CO2 at rates averaging 21 ± 6 g C m -2 y -1 . Given that HTFs are extensively distributed in coastal areas showing 310 freshwater deficit such as in northern Australia and Brazil, the scale of CO2 removal can be significant and comparable to traditional blue carbon systems in some key arid regions. While this study demonstrates carbon burial in three HTF systems, accurate estimates on the magnitude of this carbon sink on national or global scales will require further studies and improved areal estimates.
The second consideration for inclusion into the blue carbon framework is that management of an ecosystem is 315 possible. Management should maintain or enhance carbon and nitrogen stocks and thereby reduce GHG emissions (Lovelock and Duarte, 2019). Over the past few decades, HTFs have experienced large scale destruction and degradation on a global scale as a result of anthropogenic pressures such as urban and agricultural/aquaculture development (Ashton, 2008;Halpern et al., 2008;Martinez-Porchas and Martinez-Cordova, 2012) which may ultimately lead to large scale release of CO2 to the atmosphere. Local, national and/or international management actions, therefore, have the potential to reduce and possibly 320 revert these losses and destruction, thereby maintaining or even enhancing C sequestration similar to adjacent mangroves and saltmarshes. These management practices include regulating urban development or the construction of shrimp farming to prevent HTF ecosystem decline (Halpern et al., 2008;Martinez-Porchas and Martinez-Cordova, 2012). Moreover, current frameworks and management strategies in place for coastal vegetated ecosystems have the potential to incorporate HTFs given their close association. Therefore, we suggest that HTF ecosystems can be classified as blue carbon systems and should be 325 included in global management and mitigation polices and are likely to be important contributors on regional scales.

Conclusions
The investigated HTF ecosystems have accumulated significant amounts of OC, TN and TP during the previous century.
Although these accumulation rates are lower than other vegetated blue carbon systems per unit area, a substantial amount of carbon and nutrients are sequestered in HTFs considering their extensive global areal extent and should not be overlooked. 330 Stable isotope analysis along with the molar C:N ratios indicate that the microphytobenthos associated with these HTFs are an important source of the organic material accumulated along the sediment columns of these systems. To improve the robustness of our observations, there is a need for determining carbon and nutrient accumulation rates and CO2 fluxes from additional coastal HTFs and to determine a more precise areal estimate of HTFs in Australia, Brazil and other parts of the world. However, our initial data implies that these coastal HTF ecosystems fit the definition of blue carbon systems and could be included in 335 global and regional management and mitigation polices.

Conflict of Interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be 345 construed as a potential conflict of interest.

Author Contributions
DRB, HRM, and CJS designed and obtained funding for this work. DRB, CJS, HRM, RBP, DTM and LSM contributed to acquisition of data, and contributed to the analysis and interpretation of data. All of the authors made contributions to the drafting of the article and revisions critically for important intellectual content. All authors gave the final approval of the 350 version to be published.      https://doi.org/10.5194/bg-2020-426 Preprint. Discussion started: 24 November 2020 c Author(s) 2020. CC BY 4.0 License.