Suspended Particulate Matter drives the spatial segregation of nitrogen turnover along the hyper-turbid Ems estuary

12 Estuaries are nutrient filters and change riverine nutrient loads before they reach coastal oceans. Their morphology have been 13 extensively changed by anthropogenic activities like draining, deepening, and dredging to meet economic and social demand, 14 causing significant regime changes like tidal amplifications and in some cases to hyper-turbid conditions. Furthermore, 15 increased nutrient loads, especially nitrogen, mainly by agriculture cause coastal eutrophication. Estuaries can either act as a 16 sink or as a source of nitrate, depending on environmental and geomorphological conditions. These factors vary along an 17 estuary, and change nitrogen turnover in the system. Here, we investigate the factors controlling nitrogen turnover in the hyper- 18 turbid Ems estuary (Northern Germany), which has been strongly impacted by human activities. During two research cruises 19 in August 2014 and June 2020, we measured water column properties, dissolved inorganic nitrogen, dual stable isotopes of 20 nitrate and dissolved nitrous oxide concentration along the estuary. We found that three distinct biogeochemical zones exist 21 along the estuary. A strong fractionation (~ 26 ‰) of nitrate stable isotopes points towards nitrate removal via water column 22 denitrification in the hyper-turbid Tidal River, driven by anoxic conditions in deeper water layers. In the Middle Reaches of 23 the estuary nitrification gains importance, turning this section into


Equilibrator based nitrous oxide measurements and calculations 132
An nitrous oxide analyzer (Model 914-0022, Los Gatos Res. Inc.) coupled with a sea water/gas equilibrator measured the dry 133 mole fraction of nitrous oxide and water vapor in the water column using off-axis integrated cavity output spectroscopy. The 134 set-up and instrument precision is described in detail in Brase et al. (2017). The equilibration time of nitrous oxide of 135 approximately 7 min was taken into account for data processing. 136 For validation of the measurements, we measured two standard gas mixtures of nitrous oxide in synthetic air regularly (500.5 137 ppb ± 5 % and 321.2 ppb ± 3 %). No drift was detected. For further data processing, we calculated 1 min averages of nitrous 138 oxide detected dry mole fraction (ppm). We calculated the dissolved nitrous oxide concentration in water (N2Ocw) using the 139 Bunsen solubility function of Weiss and Price (1980) taking temperature differences between sample inlet and equilibrator 140 into account (Rhee et al., 2009). Nitrous oxide saturation (s) was calculated using Eq. (1), based on nitrous oxide concertation 141 in water (N2Ocw) and atmospheric nitrous oxide (N2Oair). 142 = 100 × 2 2 (1) 143 Atmospheric nitrous oxide was measured regularly during our cruise and was on average 0.33 ppm during our cruise in 2020. 144 The gas transfer coefficient (k) was calculated based on Borges et al. (2004), where u10 is wind speed 10 m above surface, and 145 Sc is the Schmidt number (Eq. (2)). Sea-to-air flux densities were calculated using Eq. (3). 146 = 0.24 × (4.045 + 2.58 10 ) × ( 600 ) −0.5 (2) 147 = × ( 2 − 2 ) (3) 148

Nitrate mixing calculations 149
Nitrate concentration from conservative mixing (CMix) between two endmembers was calculated for each sample using the 150 classical mixing model of Liss (1976). SMix, SM, SR denote the salinity of the sample, marine and riverine endmembers, respectively. We used the concentration-156 weighted mean of the isotopic values of the marine (ẟM) and riverine (ẟR) end-members to calculate the theoretical isotope 157 value of samples following conservative mixing (ẟMix) (Fry, 2002): 158 = × × +(1− )× × (6) 159

Isotope effect 160
During turnover processes, nitrogen isotopes ratios change along a specific isotope effect that helps to identify individual 161 process pathways (e.g. Kendall et al. 2007

Hydrographic properties and dissolved nutrients in surface water 177
To evaluate controls on nutrient cycling, we first look at the hydrochemical properties that were measured in 2014 and 2020 178 in surface waters, alongside with nutrient concentrations and nitrogen stable isotope composition (Fig. 2). 179

186
Discharge ranged from 59.7 m³ s -1 to 67.5 m 3 s -1 in 2014 and was ~ 30 m³ s -1 in 2020. The long-term average discharge is 187 30-40 m 3 s -1 in June and August (NLWKN Bst. Aurich and Engels, 2021). The mean water temperature was 23 °C in 2014 188 and 17 °C in 2020. Salinity ranged from ~ 0.5 to ~ 32 in both years. In 2014, the sampling section started with the onset of the 189 salinity gradient (km 20), whereas the most upstream sample in 2020 was taken near Herbrum (km -14) (Fig. 2g). This sample 190 and the sample at stream kilometer -9 were taken with a bucket from shore. The research vessel transect started in Papenburg 191 (km 0). 192 Nitrate was the major form of dissolved inorganic nitrogen (DIN) and decreased with increasing salinity. Nitrate concentration 193 decreased from 177 µmol L -1 to 3.9 µmol L -1 in 2014 and from 166 µmol L -1 to 4.9 µmol L -1 in 2020 (Fig. 2c). In 2020, we also measured dissolved nitrous oxide concentration. Measured values ranged between equilibrium concentrations 207 (~9 nmol L -1 ) and supersaturation of up to 40 nmol L -1 at km 0, which corresponded to a saturation of 400 %. Nitrous oxide 208 then decreased downstream to ~ 14 nmol L -1 (140 %) at km 30 and then increased to a local maximum of 21 µmol L -1 (210 %) 209 in the Tidal River/Dollard Reach transition at stream km 35. Further downstream, nitrous oxide decreased to near equilibrium 210 concentration towards the North Sea (Fig. 2f). 211

Suspended Particulate Matter properties 212
Near surface SPM concentration was highest in the Tidal River, reaching values of 2100 mg L -1 in 2014 and 1600 mg L -1 in 213 2020. SPM concentration decreased at the beginning of the Dollard Reach region (Fig. 2i). The δ 15 N-SPM values showed 214 considerable scatter (Fig. 2l): around 5 ‰ in the Tidal River/Dollard Reach, and 9 ‰ in the Middle Reaches. In the Outer 215 Reaches, δ 15 N-SPM dropped again to ~5 ‰. In 2014, δ 15 N-SPM were elevated (8 ‰), but the database during this cruise is 216 relatively sparse (Fig. 2l). 217 In 2020, C/N ratios of SPM (Fig. 2j) were relatively stable in the Tidal River (~11) and Dollard Reach, with a slightly lower 218 value of 9 in the most upstream sample. In the Middle Reaches, C/N ratios decreased, reaching the lowest value of 6.5 in the 219 most offshore sample. In 2014, C/N values were 11-15 in the Tidal River, increased to values as high as 20 in the Dollard 220 Reach and decreased to ~ 11 approaching the North Sea (Fig. 2j). 221 Particular organic carbon fraction (% POC) was high in the most upstream samples in 2020 (Fig. 2k), decreased to 4.5 % and 222 remained relatively stable in the Tidal River and Dollard before it increased in the Middle and Outer Reaches up to 11 %. In 223 2014, the values in Tidal River and Dollard were comparable, but we found a decreasing trend downstream, with a low POC 224 fraction of ~3 in the outermost sample (Fig. 2k). 225

Dissolved oxygen concentration in the Ems estuary 226
In surface water, oxygen concentrations in the Tidal River section were low during both cruises, and increased downstream 227 with rising salinity. The lowest values were measured in the Tidal River, where the minimum oxygen concentration was 228 ~72 µmol L -1 in 2014 and 76 µmol L -1 in 2020 (Fig. 2h), corresponding to a saturation of 27 % and 26 %, respectively. 229 Oxygen profiles showed strong vertical gradients with decreasing concentration in deeper water layers. The extent of hypoxia 230 in the water column depended on the tidal cycle and location, with lowest bottom water oxygen concentration measured at the 231 most upstream station at stream km 7.2 during low tide in 2020. Detailed profiles can be found in the supplementary material 232 (S1). 233 During the continuous near-bottom oxygen measurements, we found anoxic conditions during both of our cruises that lasted 234 for several hours over a tidal cycle (Fig. 3). Oxygen concentration was generally low at low tide, and elevated at high tide. In 235 2014, anoxia developed at stream km 11.8 and 18.5, and highest oxygen concentration in bottom water was only 60 µmol L -1 236 (km 24.5) and 70 µmol L -1 (km 11.8). At the beginning of August, oxygen concentration at kilometer 11.8 frequently exceeded 237 measured values at kilometer 24.5. 238 In 2020, oxygen concentration in bottom water was higher, and anoxia was only found at stream km 11.8. At all other stations, 239 oxygen concentration remained above 40 µmol L -1 even at low tide. 240 241 242

Nitrate mixing 250
We plotted nitrate vs salinity concentration and nitrate dual isotopes to evaluate mixing properties (Fry, 2002

Biogeochemical zones in the Ems Estuary 272
The first goal of this study was to identify distinct biogeochemical zones of nitrogen turnover within the Ems estuary to see if 273 changing environmental and geomorphological properties affect the occurring processes. The assessment of estuarine mixing 274 curves showed three zones of different nitrogen turnover along the salinity gradient (Fig. 4). 275 In both years, 2014 and 2020, nitrate concentration deviated clearly and in a similar manner from the conservative mixing line. 276 In the upper riverine part of the estuary, nitrate concentration fell below the conservative mixing line, indicating nitrate removal 277 (zone 1), followed by a zone with nitrate concentration slightly above the mixing line (zone 2) that acted as a net nitrate source. 278 In the third zone, nitrate mostly followed the conservative mixing line, with nitrate removal and isotopic enrichment near the 279 marine endmember in 2020, indicating nitrate uptake by phytoplankton. In 2014, the identification of the "Outer zone" / zone 280 3 is more difficult, as the outermost samples follow the conservative mixing line in Fig. 4. However, these outermost samples 281 are distinct from the prevailing processes in zone 2, because they do not show signs of nitrate production, a characteristic of 282 zone 2. 283 The PCA analysis showed that nitrogen turnover was comparable in both years. However, there are distinct differences 284 between the cruises. Seasonal and interannual variation may cause differences in dissolved inorganic nitrogen distribution and 285 nitrate stable isotope composition. The PCA independently confirms comparable zones of nitrogen turnover for both years. 286 The principle components loadings were also similar for both cruises. The PCA supports the suggested nitrate zonation taking 287 the other biogeochemical properties into account (Fig. 5). The three biogeochemical zones were mainly divided according to 288 PC1. Contributing parameters were oxygen, nitrate, C/N, SPM and silicate, which suggests a tight coupling of nitrate turnover 289 to suspended particulate matter. PC2 helped to differentiate zone 2. Contributing parameters (temperature, nitrite, and 290 phosphate) suggest a link to nutrient uptake processes. 291 Based on the location of the biogeochemical zones along the Ems (Fig. 1), we see a connection with the geomorphological 292 characteristics of the Ems estuary. In both years, zone 1 was located in the hyper-turbid Tidal River and the beginning of zone Overall, mixing properties as well as a PCA suggest that there are three distinct biogeochemical zones that act either as sinks 301 (zone 1 and 3) or sources (zone 2) of nitrate along the Ems. These ones are mainly defined by discharge and suspended 302 particulate matter (especially PC1). 303

Denitrification in the upper estuary 304
Zone 1, the most upstream region acted as a nitrate sink in both years, with nitrate concentrations below the conservative 305 mixing line and enriched ẟ 15 N-NO3and ẟ 18 O-NO3values ( Fig. 4d and 4e). Potential removal mechanisms are nitrate 306 respiration or nitrate assimilation. 307 High SPM values in the hyper-turbid Tidal River and Dollard Reach (Fig. 2i)  to little to no fractionation due to a diffusion limitation (Brandes and Devol, 1997;Lehmann et al., 2004;Sigman and Fripiat, 317 2018), water column denitrification has an isotope effect that fits our calculations (Kendall et al., 2007;Sigman and Fripiat, 318 2018), and can explain the observed patterns. 319 Water column denitrification occurs under anaerobic to low oxygen conditions in the water column (Tiedje, 1988). According 320 to Seitzinger (1988), denitrification occurs at oxygen concentration below 6.25 µM. We measured low oxygen concentration 321 in surface water during both years with lowest concentration of ~ 70 µmol L -1 (Fig. 2h), which is well above the threshold for 322 denitrification. However, vertical oxygen concentration profiles and continuous measurements in the estuary in near-bottom 323 water showed that deeper water became anoxic in both years. Even though these anoxic conditions only developed for a few 324 hours over a tidal cycle, we conclude that water column denitrification was the responsible nitrate sink mechanism in the Ems 325 in 2014 and 2020. 326 Overall, we find strong evidence for water column denitrification as in zone 1, likely in the anoxic bottom waters. Moreover, 334 coupled nitrification-denitrification can add to this nitrate sink in the hyper-turbid Tidal River. 335

Increasing importance of nitrification in the Middle Reaches 336
The mixing lines along the estuary displayed a significant shift of nitrogen turnover from the "Denitrification zone" / zone 1 337 to zone 2. Nitrate concentrations plotted above the mixing line, indicating a net nitrate source with lighter nitrate isotope values 338 (Fig. 4). 339 Nitrate is produced via nitrification, which was no longer oxygen limited in zone 2 due to increasing concentrations compared For denitrification, we calculated an isotope effect of 15 εDENIT = -26 ‰ in the "Denitrification zone" / zone 1. For nitrification, 362 the expression of the isotope effect depends on the abundance of ammonium. As long as ammonium is limiting, we assume 363 that any ammonium is converted to nitrite and nitrate, so that the apparent isotope effect is that of remineralisation, as long as 364 ammonium concentration is low. In most parts of zone 2, no ammonium was accumulated. A simultaneous increase of ẟ 15 N-365 SPM, ammonium and nitrite concentration at stream kilometer 50 in 2020 point towards remineralisation (Fig. 2a, 2b and 2l). concentrations, both processes influenced the fractionation caused by nitrification. Therefore, for total nitrification we assumed 373 a combined isotope effect of 15 εNITRI = -10 ‰, that we used to describe nitrification in samples with accumulated ammonium 374 and nitrite. This number is lower than previously measured for ammonium oxidation, and is based on nitrification rate from 375 incubations performed previously in the Elbe estuary (Sanders, unpublished data;Sanders and Dähnke, 2014). 376 Based on these input variables, the mapping approach can indeed explain the development of isotope effects and nitrate 377 concentration. In the most upstream samples, nitrate removal exceeded production: in 2014, denitrification removed 378 26 µmol L -1 , and nitrification added 10 µmol L -1 . In 2020, the mapping approach suggests an addition of 52 µmol L -1 and 379 simultaneous denitrification of 62 µmol L -1 . In the middle of zone 2, nitrification gained in relative importance with an 380 approximated production of 10 µmol L -1 in 2014 and 20 µmol L -1 in 2020, in contrast to denitrification of approximately 381 3 µmol L -1 and 10 µmol L -1 , respectively. In the most downstream samples, mixing was dominant, and we detected neither 382 nitrate production nor reduction. 383 Overall, nitrification and denitrification determined the evolution of nitrate isotopes and concentration in the estuary. Further 384 downstream of zone 2, nitrification becomes increasingly important, and the relevance of denitrification ceases. Both processes 385 lose in importance towards the North Sea, when mixing turns to be the most important process. 386

Mixing and nitrate uptake in the Outer Reaches 387
In the "Outer Zone " / zone 3 the mixing line shows divergent trends for our two cruises (Fig. 4) In the mixing plot (Fig. 4), the outermost isotope samples of our cruise in 2020 fall on the conservative mixing line. The good 397 fit is caused by the calculation with a marine endmember that has an isotopically enriched signature in comparison to average 398 global values (Sigman et al., 2000 and North Sea winter values of 5 ‰ (Dähnke et al., 2010). The increase of the isotope 399 signature shows that fractionation takes place, likely due to assimilation. 400 In contrast to the biogeochemical active inner zones, mixing dominated nitrate distribution in the Outer Reaches of the estuary 401 in 2014. In 2020 however, the Outer Reaches were a nitrate sink due to ongoing primary production in the coastal North Sea. 402

SPM as driving force of the spatial zonation 403
We identified three biogeochemical zones of nitrogen turnover along the estuary, which differ significantly in their coastal 404 filter function. The Tidal River was a nitrate sink with dominating water column denitrification. In the Middle Reaches, 405 nitrification gained in importance, turning this section in a net nitrate source. In the "Outer Zone" / zone 3, mixing gained in 406 importance but with a clear nutrient uptake in 2020. Other estuaries with high turbidity show denitrification zones as well 407 (Ogilvie et al., 1997;Middelburg and Nieuwenhuize, 2001). This finding and our analysis of the PCA and dominant nitrogen 408 turnover processes suggest that the overarching control on biogeochemical nitrogen cycling and zonation may be suspended 409 particulate matter. High C/N ratios (Fig. 2j), as well as a low and stable particular organic carbon (POC) fraction of the SPM in this region 415 (~ 4.5 %) in the Tidal River and Dollard Reach indicate low organic matter quality and a large contribution of mineral 416 associated organic matter of the present organic matter (Fig. 2k). In 2014, C/N ratios were extremely high, and uncharacteristic 417 for estuarine environments. We attribute this to a potential influence of peat soils or peat debris in sediments (Broder et  Nonetheless, and regardless of organic matter origin, degradation of organic carbon leads to anoxic conditions in the Tidal 422 River. Even though the low quality of organic matter fuels only low degradation rates with POC fractions of ~ 3 % (Fig. 2k) already showed that elevated discharge can relocate the ETM downstream. As we identified SPM concentrations as one of the 438 most important controls on nitrogen turnover in the Ems estuary, we assume that the described zones will move with shifting 439 SPM concentration along the estuary. 440 Overall, we find that the interplay of nitrification/denitrification and nitrogen assimilation is governed by SPM concentration 441 along the Ems estuary. We expect that changing discharge can lead to spatial offsets in SPM concentrations and thus influence 442 the spatial segregation of nitrogen turnover processes. 443 4.6 Nitrous oxide production and its controls in the Ems estuary 444 So far, we elucidated nitrogen turnover in the Ems Estuary. We found that nitrification and denitrification vary spatially in 445 importance. Both processes can produce nitrous oxide, and we accordingly found nitrous oxide peaks in the estuary in areas 446 with significant differences in their nitrogen turnover. Nitrous oxide was measured only in 2020, thus we will use the high-447 resolution data from this cruise to examine the importance of nitrification and denitrification for nitrous oxide production along 448 the estuary. We will also discuss controls that favor the emergence of nitrous oxide production areas. 449 The calculated average sea-to-air flux of 0.35 g-N2O m -2 a -1 results in a total nitrous oxide emission of 0.57 × 10 8 g-N2O a -1 450 along the Ems estuary. In June 1997, a significantly higher average sea-to-air flux density of 1.23 g-N2O m -2 a -1 was measured 451 (Barnes and Upstill-Goddard, 2011), which amounted to an annual nitrous oxide emission of 2.0 × 10 8 g-N2O a -1 over the 452 entire estuary. Upscaling from a single cruise to an entire year is somewhat questionable, but it is interesting to note that the 453 emissions may have halved since the 1990s. Furthermore, our results as well as those from 1997 were obtained from a single 454 survey in June, making the comparison intriguing. Since the 1990s, the DIN load of the Ems estuary was significantly reduced 455 due to management efforts (Bos et al., 2012). Phytoplankton biomass in the Outer Reaches (Station Huibertgat Oost, Van 456 Beusekom et al., 2018) decreased in response to decreasing nutrient loads, possibly contributing to the observed lower N2O 457 emissions. However, this hypothesis requires further verification in the future. 458 The nitrous oxide concentrations observed in 2020 can be linked to the prevailing biogeochemical conditions. The first nitrous 459 oxide maximum was located in the upstream region (stream kilometer 0). In this area, we identified water column 460 denitrification as the dominant nitrogen turnover process, and we found relatively low pH values and high nitrate concentration. 461 In their summary paper about nitrous oxide in streams and rivers, Quick et al. (2019) found that these factors are favorable for 462 nitrous oxide production via denitrification. Intermittent oxygen hypoxia and anoxia in the different water depths also enhance 463 nitrous oxide production in the Tidal River, which is in line with our tidal oxygen measurements in the Ems. Several studies 464 also showed a positive correlation between nitrous oxide concentration and SPM concentration (Tiedje, 1988;Liu et al., 2013;465 Zhou et al., 2019), and SPM concentration was also highest in this region of the Tidal River. Altogether, we suggest that the 466 Ems is well suited as a region with extremely high nitrous oxide production, triggered by high nutrient loss, intermittent anoxia, 467 and high SPM loads. 468 Further downstream, nitrous oxide concentrations decrease, along with oxygen concentrations, reaching a minimum around 469 km 22. The simultaneous reduction of nitrous oxide and oxygen concentration at first sight seems counterintuitive, but it may 470 be caused by complete denitrification that produces N2 instead of nitrous oxide (Knowles, 1982). 471 Based on our data, we cannot clearly say whether the source of nitrous oxide production was in the water column or in the 472 sediments. Other studies, e.g. in the muddy Colne estuary found high nitrous oxide production due to denitrification, but 473 assigned nitrous oxide production only to the sediments (Ogilvie et al., 1997;Robinson et al., 1998;Dong et al., 2002). 474 Sedimentary denitrification in our study may have contributed to this first nitrous oxide maximum. The beginning of ebb tide 475 during our campaign may have enhanced outgassing of nitrous oxide from the sediment, and low water levels may have caused 476 a mechanical release of nitrous oxide from the sediments caused by our research vessel. Thus, the "Denitrification zone" / zone 477 1 is an important nitrous oxide production zone, but the measured nitrous oxide concentration might in parts be affected by 478 sedimentary processes and might overestimate nitrous oxide production in the water column. 479 The second nitrous oxide maximum occurred around stream kilometer 35 at the transition between Tidal River and Dollard 480 Reach. In this area, our mapping approach indicates simultaneous denitrification and nitrification. The nitrous oxide peak 481 coincides with an increase of ammonium and nitrite concentration, as well as a slight rise in nitrate concentration, indicating 482 the onset of nitrification in the water column. 483 In contrast to condition leading to the first nitrous oxide peak, not enough fresh organic matter seems to be present in the 484 transition area to support nitrous oxide production. Lower SPM concentrations with comparable low POC fraction leads to 485 lower remineralisation rates and higher oxygen levels. Low organic matter availability and increasing oxygen concentration 486 favor nitrous oxide production via nitrification (Otte et al., 1999;Sutka et al., 2006). Similarly, Quick et al. (2019) summarized  487 aerobic or oxygen limited conditions with low organic carbon availability favorable for nitrous oxide production via 488 nitrification. As our data suggests additional denitrification, we speculate that in possible anoxic microsites on suspended 489 particles and anoxic deeper water layers, denitrification may have contributed to nitrous oxide production. Overall, we assume 490 that nitrification and denitrification jointly added to nitrous oxide production in this region. 491 In summary, we find that two nitrous oxide production hotspots exist in the Ems estuary. SPM plays a big role controlling the 492 nitrous oxide production along the Ems estuary. In the upstream region, where oxygen depletion occurs due to immense SPM 493 concentration, denitrification produces nitrous oxide. At the transition zone between Tidal River and Dollard Reach, SPM 494 concentration is lower, leading to higher oxygen concentration and nitrous oxide production via nitrification. Denitrification 495 prevails in deeper water layers where oxygen concentration is low, and possibly in anoxic microsites close to particles. 496

Conclusion 497
Overall, we find that three distinct biogeochemical zones exist along the Ems. Stable isotope changes point towards water 498 column denitrification in the turbid water column of the Tidal River. In the Dollard Reach/Middle Reaches nitrification gains 499 importance turning this section of the estuary into a net nitrate source. Nitrate uptake occurs in the Outer Reaches due to 500 primary production in the coastal North Sea, in August 2014 mixing dominated. Our analysis of the dominant nitrogen turnover 501 processes suggest that SPM concentration and the linked oxygen deficits exert the overarching control on biogeochemical 502 nitrogen cycling, zonation and nitrous oxide production in the Ems estuary. 503 Changing biogeochemical conditions can significantly alter estuarine nutrient processing. Deepening of river channels happens 504 not only in Germany (Kerner, 2007 2020), and this can change SPM loads and composition in estuaries. Increased SPM loads can enhance denitrification, but also 507 trigger nitrous oxide production and enhance oxygen-depleted zones, which is what we observe in the Ems estuary. Thus, the 508 interplay of SPM with riverine nutrient filter function and nitrous oxide emissions should be further evaluated. The common 509 practices of deepening and dredging affect SPM and this creates a direct link between pressing social and ecological problems 510 in coastal regions. 511

Data availability 512
The data sets are available under coastMap Geoportal (www.coastmap.org) connecting to PANGEA with DOI availabilty. The