Ecosystem Organic Carbon Stock Estimations in the Sile River, North Eastern Italy

: River ecosystems are one of the dynamic components of the terrestrial carbon cycle that provide a crucial function in ecosystem processes and high value to ecosystem services. A large amount of carbon is transported from terrestrial to the ocean through river ﬂows. In order to evaluate the contribution of Sile River ecosystem to the global carbon stock, the river ecosystem Organic Carbon (OC) stock was quantiﬁed for sediments and dominant submerged aquatic macrophytes (SAMs) during the two sampling periods at three different stations along the Sile River (North Eastern Italy). The total mean ecosystem OC stock was 95.2 ± 13.8 Mg C ha − 1 while those of SAMs ranged from 7.0 to 10.9 Mg C ha − 1 which accounted for approx. 10% of the total OC stock. The total aboveground biomass retains approx. 90% of the SAM carbon stock, with a mean of 8.9 ± 1.6 Mg C ha − 1 . The mean sediment OC stock was 86.6 ± 14.5 Mg C ha − 1 with low seasonal variations among the sites. Indeed, various environmental parameters and hydrodynamics appear to affect the accumulation of OC within the river ecosystem. The results highlight the role that freshwater river ecosystems play in the global carbon cycle, which consequently provide a baseline for future river ecosystem monitoring programs. Furthermore, future studies with additional sites and seasonal surveys of the river will enhance our understanding of the effects of global climate change on the river ecosystem and improve the ecosystem services.


Introduction
Rivers represent the most dynamic components of the terrestrial carbon cycle and provide important functions in ecosystem processes. Significant amounts of organic and inorganic carbon typically deliver rivers from the surrounding landscape or originate from the photosynthesis of algae and plants in the water [1]. Despite their dynamic role in the terrestrial carbon stock cycle, river systems and the potential mechanistic controls of OC storage are among the least investigated [2], in comparison to other ecosystems. To date, the limited conducted research suggests that river networks might store a significant proportion of terrestrial carbon [3,4]. Research in ecosystem processing underlines the importance of Organic Carbon (OC) in river systems. Indeed, the fluxes in OC in terrestrial freshwater systems indicate that a marked portion of terrestrial carbon is stored within river networks [2]. River OC fluxes exert an important control of freshwater ecosystems and their ecosystem services. Therefore, understanding physical and biological processes in rivers is crucial for determining the potential impacts of global climate change, land-use, and land-cover changes on OC dynamics within river systems [5].
Different studies highlight the difficulty in identifying the major sources of carbon to freshwater ecosystems, which is critical to properly link the terrestrial and aquatic carbon cycles [6].
Globally, rivers receive about 2.9 Pg (one billion tonnes) C each year, a quantity that represents the differences between global annual terrestrial production and respiration [5,7].

•
Storga: a resurgent station with spring characteristics and clear shallow water (ca. 7.0 m transept and 0.5 m depth). Storga is located inside the park "Parco dello Storga" of about 58 ha. The park is characterized by riparian formations of willows (Salix sp.), poplars (Populus sp.) and alder (Alnus glutinosa L.) and by the presence of fountains, streams, and flooded lowlands and hygrophilous woods [30]. The mean water column temperature is 13.7 • C [20]; • Morgano: station before Treviso city centres and located inside the oasis of Santa Cristina. The oasis represents a great marsh of Sile River and a biotope of high naturalistic value with numerous springs. The reserve is part of the Natura 2000 Network as a Site of Community Interest (SCI) and a Special Protection Area (SPA). The station is characterized by ca. 10.0 m transept with maximum 2.5 m water depth. The mean water column temperature is 13.7 • C [20]; • Treviso: is located within the town of Treviso (ca. 25.0 m transepts and ca. 2.5 m depth) with anthropogenic impacts from densely populated neighbourhoods and commercial activities and near a hydroelectric plant. The mean water column temperature is 13.9 • C [20].

Study Area
Submerged aquatic macrophytes (SAMs) and sediments were collected from Sile River in two different sampling campaigns: January (winter) and late July (summer) 2019 in order to estimate the variations in OC stock during spring, the season with the highest primary productivity [28,29]. Three stations were selected along the Sile River according to their characteristics ( Figure 1):

SAM Classification and Carbon Quantification
Samples of SAMs were collected in 5 m transept at each station along Sile River in order to calculate the biomass (kg/m 2 wet weight) for each species according to Madsen and Wersal [31]. SAMs were collected using a rake within a square of 50 × 50 cm 2 and kept

SAM Classification and Carbon Quantification
Samples of SAMs were collected in 5 m transept at each station along Sile River in order to calculate the biomass (kg/m 2 wet weight) for each species according to Madsen and Wersal [31]. SAMs were collected using a rake within a square of 50 × 50 cm 2 and kept separately in plastic bags [32]. In laboratory, SAMs were identified according to Pignatti et al. [33]. After separating roots and rhizomes (below-ground part) from leaves (aboveground part), organic carbon (OC) contents were measured using a CHNS elemental analyser (Elementar Vario MICRO Tube, precision <0.1% abs.) after sample parts' pulverizing [34,35]. All analyses were performed in duplicate on different days to obtain a Water 2021, 13, 80 4 of 12 coefficient of variation <5%. The concentrations of OC are reported in Mg C ha −1 , after considering the cover areas of each SAM species.
Organic carbon (OC) was determined indirectly as differences in Total Carbon (TC) and Inorganic Carbon (IC). Total carbon was determined directly by CHNS Elemental Analyser after sediment pulverization with a sediment mill. Inorganic carbon concentration was determined after sample combustion for two hours at 440 • C [36]. All analyses were performed in duplicate in different days to obtain coefficient of variation (CV) ≤5%. The concentrations of sediment OC were reported in Mg C ha −1 . The percentage of Fines (fraction ≤ 63 µm) was obtained by wet sieving approx. 50 g of dried sediment throughout Endecotts sieves (ENCO Scientific Equipment, Spinea, Italy), after removing shell fragments [37]. Sediment dry density (g/cm −3 ) was determined by gravimetric method weighting the sediment before and after sediment desiccation at 110 • C [38].

Water Environmental Parameters
Temperature ( • C), redox potential (Eh) (accuracy ± 0.15%) and pH (accuracy ±0.015 units) were determined using a portable pH meter (pHenomenal, pH 1100 H, VWR,). Dissolved oxygen was determined using an oxygen portable meter (WTW ProfiLine™ Oxi 3310, accuracy ±0.01 mg/L, White Plains, NY, USA). Three surface water samples were collected. Subsamples were filtered through GF/F Whatman glass microfiber filters (porosity 0.7 µm). Filtered waters were analyzed for nutrient (ammonium, nitrite, nitrate, reactive phosphorus, silicate) determination according to the spectrophotometric analysis procedures reported by Strickland and Parson [39]. The quantification at different wavelengths of phosphates, silicates and inorganic nitrogen compounds occurred by comparison with calibration curves made with sea water. All colorimetric analyses were performed in triplicate. Chlorophyll-a (Chl-a) concentrations were measured by spectrophotometric analysis [40] using the acetone (90%) extraction protocol. Two additional 500 mL water sub-samples were filtered in situ through GF/F Whatman glass microfiber filters (porosity 0.7 µm), previously dried at 110 • C for two hours, in order to obtain the concentration (mg L −1 ) of the total suspended sediment (TSS), as described previously by Sfriso et al. [41].
Visualization of data was performed using R software (version 3.5.3) and R studio [RStudio, PBC, Austria] (version 1.2.5033) using the packages: ggplot2; ggpubr; RColor-Brewer. The study stations map was created using software QGIS (version 2.18.28).

SAMs Community Composition and Biomass
The in-field sampling revealed that eight species of SAMs were present along the Sile River during the two monitoring periods (summer and winter) in the sampling stations (Table 1). In total, two alien species were identified: Elodea canadensis and Ceratophyllum demersum L. The station at Treviso was dominated by all the listed species, except Myosotis scorpioides L. and Callitriche stagnalis Scop. Instead, the latter were present in the SAM community at Storga. Myriophyllum spicatum L. was found only at Treviso, with an annual average biomass of approx. 1.15 kg/m 2 whereas Ceratophyllum demersum was collected from all stations, with the highest biomass (3.95 kg/m 2 ) recorded at Storga during the summer period. The biomass of this species decreased during winter and showed the lowest biomass (2.58 kg/m 2 ) at Treviso.
The lowest vegetation biomass was represented from Elodea canadensis, with values <0.6 kg/m 2 both at Morgano and Treviso. The dominant SAM recorded at Treviso during summer was Vallisneria spiralis L. (5.26 kg/m 2 ), while in winter Stuckenia pectinata (L.) Börner showed the highest biomass (4.91 kg/m 2 ). Conversely, at Morgano during the summer, the SAM with the highest biomass was Stuckenia pectinata (5.11 kg/m 2 ) while in winter the most species abundant was Callitriche brutia Petagna (4.86 kg/m 2 ). At Storga Callitriche stagnalis, Myosotis scorpioides and Ceratophyllum demersum represented ca. 95% of the total biomass. Indeed, the biomass of these species did not show significant seasonal variations. For instance, the biomass of Myosotis scorpioides and Ceratophyllum demersum decreased by 4% and 1%, respectively, from summer to winter. Regarding the biomass variability, the species that had the greatest loss (>70%) during the winter were: Stuckenia pectinata at Morgano and Vallisneria spiralis at Morgano and Treviso.

Water and Sediment Parameter Variations
The variations in the water column parameters of the three stations are shown in Table  S1. Regarding the sediment (Table S1), pH ranged between 7.04 and 7.40 in all stations with a mean value of 7.22. The redox potential (Eh) ranged from 82.3 to 228 mV with the lowest values at Treviso. The highest percentage of Fines (Table S2) was measured at Storga (87% for the top 35 cm), while at Treviso the percentage of Fines decreased to 67%. In contrast, Morgano showed the sediment with the lowest percentage of Fines (44%). Moreover, the highest Fine percentage was found at 25-35 cm depth at all stations. Regarding the total nitrogen (TN) concentration (Table S3) in the top 35 cm sediment (4.18-5.87 Mg TN ha −1 ), higher values were observed during summer with little variability between stations.

Sediment Carbon Determination
The distribution of OC in the sediment profile of Sile River sampling sites is shown in Figure 2. The average OC concentration (0-35 cm) was 86.6 Mg C ha −1 . The highest OC content (0-35 cm) was recorded at Storga during the two sampling periods (104.7 and 104.2 Mg C ha −1 in summer and winter, respectively), whereas the lowest content was recorded at Treviso (71.8 Mg C ha −1 ) in winter. Morgano showed an increasing trend from 75. 8

Sediment Carbon Determination
The distribution of OC in the sediment profile of Sile River sampling sites is shown in Figure 2

SAMs Carbon Determination
The total aboveground biomass stored ca. 90% of the SAM carbon stock (Figure 3)

SAMs Carbon Determination
The total aboveground biomass stored ca. 90% of the SAM carbon stock (Figure 3)

Ecosystem OC Stock
The distribution of the OC stock of the Sile river ecosystem (both sediment and SAMs) is shown in Figure 4. The mean ecosystem OC was 96.2 ± 14.3 Mg C ha −1 in summer and slightly lower in winter (94.2 ± 16.4 Mg C ha −1 ). The sediment contributed with 86.6 Mg C ha −1 , while SAM biomass with 8.66 Mg C ha −1 accounting for 9.9% of the total ecosystem OC stock. Storga showed the highest level of OC (112.2 Mg C ha −1 , i.e., 104.5 and 7.7 Mg C ha −1 in sediments and SAMs, respectively), without seasonal differences. In the other two stations (Morgano and Treviso) OC stocks were 88.4 and 85.0 Mg C ha −1 , respectively, with slight decreases in winter (6.3% and 12.2%, respectively). In these stations the sediments fraction was approx. 90% of the total.

Ecosystem OC Stock
The distribution of the OC stock of the Sile river ecosystem (both sediment and SAMs) is shown in Figure 4. The mean ecosystem OC was 96.2 ± 14.3 Mg C ha −1 in summer and slightly lower in winter (94.2 ± 16.4 Mg C ha −1 ). The sediment contributed with 86.6 Mg C ha −1 , while SAM biomass with 8.66 Mg C ha −1 accounting for 9.9% of the total ecosystem OC stock. Storga showed the highest level of OC (112.2 Mg C ha −1 , i.e., 104.5 and 7.7 Mg C ha −1 in sediments and SAMs, respectively), without seasonal differences. In the other two stations (Morgano and Treviso) OC stocks were 88.4 and 85.0 Mg C ha −1 , respectively, with slight decreases in winter (6.3% and 12.2%, respectively). In these stations the sediments fraction was approx. 90% of the total. Water 2021, 13, 80 8 of 12

SAM Carbon Estimation and Environmental Considerations
This study highlights the capacity of the Sile River ecosystem to store OC in sediments and aquatic plants. The mean ecosystem OC stock ranged from 79.4 to 112.5 Mg C ha −1 with no significant seasonal variations among stations. This can be related to the low variability of vegetation biomass among seasons. The SAM accounted for ca. 10% of the ecosystems OC with a mean value of 8.6 ± 1.7 Mg C ha −1 . Furthermore, the total aboveground biomass accounted for 90% (8.9 ± 1.6 Mg C ha −1 ) of the vegetation OC stock, contrary to the seagrass meadows [42] and mangroves [43], which had the largest proportion of OC stock in belowground parts.
The highest OC amount in the Sile River was stored in the sediment; this could be due to the high sediment capacity for C sequestration [44]. In addition, the highest OC stock was recorded at Storga, which is a resurgent zone of shallow water with spring characteristics influenced by groundwater discharges developed both by springs feeding local sinkholes and waterways sourced inland [30]. As a result, the site is characterized by the finest sediment grain-size and relatively high sediment C:N ratio. The increased storage of carbon in the sediment at Storga could be related to the high affinity of carbon for fine sediments [45]. On the contrary, the sediment OC stock at Morgano and Treviso was lower because sediments were coarser, as previously described by Kauffman et al. [43].
The vegetation composition and structure of river ecosystems is determined by a combination of environmental parameters, including topography, water depth, salinity, nutrient concentration, sediment type, and inter-vegetation competition [46]. Indeed, the seasonal variations in the DO and DIN of water at Morgano and Treviso which were dominated by S. pectinata and V. spiralis could result in higher OC storage variability among the sampling periods, with a 25% reduction in OC during winter. Moreover, the SAM biomass in these stations increased from winter to summer in agreement with the increasing OC stock of SAMs. Similarly, the size and morphometry of water bodies influenced the SAM distribution, which was affected by the concentrations of DO and pH in water column [47]. There was no clear relationship between OC stock in SAMs and C and N concentrations in sediments. Equally important, several other unmeasured factors were assumed to influence variations in the river carbon stock. For instance, phytoplankton and epiphytes probably had an influence on the water quality and may have been responsible for the SAM biomass changes as recorded from Frodge et al. [47]. These authors showed that growing photosynthesizing plants within the water column contributed to higher mid-water column DO concentrations within patches of E. canadensis and C. demersum.

SAM Carbon Estimation and Environmental Considerations
This study highlights the capacity of the Sile River ecosystem to store OC in sediments and aquatic plants. The mean ecosystem OC stock ranged from 79.4 to 112.5 Mg C ha −1 with no significant seasonal variations among stations. This can be related to the low variability of vegetation biomass among seasons. The SAM accounted for ca. 10% of the ecosystems OC with a mean value of 8.6 ± 1.7 Mg C ha −1 . Furthermore, the total aboveground biomass accounted for 90% (8.9 ± 1.6 Mg C ha −1 ) of the vegetation OC stock, contrary to the seagrass meadows [42] and mangroves [43], which had the largest proportion of OC stock in belowground parts.
The highest OC amount in the Sile River was stored in the sediment; this could be due to the high sediment capacity for C sequestration [44]. In addition, the highest OC stock was recorded at Storga, which is a resurgent zone of shallow water with spring characteristics influenced by groundwater discharges developed both by springs feeding local sinkholes and waterways sourced inland [30]. As a result, the site is characterized by the finest sediment grain-size and relatively high sediment C:N ratio. The increased storage of carbon in the sediment at Storga could be related to the high affinity of carbon for fine sediments [45]. On the contrary, the sediment OC stock at Morgano and Treviso was lower because sediments were coarser, as previously described by Kauffman et al. [43].
The vegetation composition and structure of river ecosystems is determined by a combination of environmental parameters, including topography, water depth, salinity, nutrient concentration, sediment type, and inter-vegetation competition [46]. Indeed, the seasonal variations in the DO and DIN of water at Morgano and Treviso which were dominated by S. pectinata and V. spiralis could result in higher OC storage variability among the sampling periods, with a 25% reduction in OC during winter. Moreover, the SAM biomass in these stations increased from winter to summer in agreement with the increasing OC stock of SAMs. Similarly, the size and morphometry of water bodies influenced the SAM distribution, which was affected by the concentrations of DO and pH in water column [47]. There was no clear relationship between OC stock in SAMs and C and N concentrations in sediments. Equally important, several other unmeasured factors were assumed to influence variations in the river carbon stock. For instance, phytoplankton and epiphytes probably had an influence on the water quality and may have been responsible for the SAM biomass changes as recorded from Frodge et al. [47]. These authors showed that growing photosynthesizing plants within the water column contributed to higher mid-water column DO concentrations within patches of E. canadensis and C. demersum. The low water flow and lower TSS at Storga may favor the development of SAMs of a distinctive vegetation assemblage, like Myosotis scorpioides and Callitriche stagnalis. In this context, Chambers et al. [48] showed that the biomass and shoot density of SAMs significantly decreased with the increasing current velocity within the weed beds. Indeed, the lowest SAM diversity and highest biomass were observed at Storga which was characterized by low water flow. It is not surprising that M. scorpioides, which was only recorded at Storga, is commonly present in freshwater marshes and slow-moving water. Furthermore, this species grows best in saturated soils with high carbon content and is usually found growing with many obligate plant species such as water parsley (Oenanthe sarmentosa C. Presl ex DC, [49]).
The highest OC accumulation at Treviso and Morgano was recorded in Callitriche brutia Petagna and Stuckenia pectinata. In contrast, V. spiralis accumulated lower OC during summer despite the highest biomass obtained for this species in the same period. In fact, the experimental evidence of a study by Coa and Ruan, [50] to investigate how V. natans (Hydrocharitaceae) responded to high temperature and CO 2, showed that the increase in these parameters significantly enhanced the photosynthetic performance, growth and clonal propagation of V. natans.
Moreover, it is important to state that the ecological status of the study stations was in accordance with the Water Framework Directive (2000/60/EC), as previously described by ARPAV, 2019 [20]. The station of Storga was classified as an area with "Good" ecological status, whereas the stations of Treviso and Morgano were assessed as "moderate". Indeed, this highlighted that the ecological status of the aquatic ecosystems might be related with the ecosystem carbon stock capacity, as previously described by Buosi et al. [51] for the sediment Carbon concentration in the seagrass meadows as bioindicator for the ecological status of the Lagoon of Venice [52].

Ecosystems Carbon Stock Comparison
River ecosystems stored a significant proportion of terrestrial carbon stock [2]. The studies that quantify the carbon stored in the River ecosystems are scarce [2,7,45]. The capacity of ecosystems to store carbon is widely variable and related to the primary productivity of ecosystem components [45]. Interestingly, the estimated carbon stock of the Sile river ecosystem was comparable with the other ecosystems' capacity to store carbon. In particular, the seagrass ecosystem had a globally carbon storage capacity ca. 200 Mg C ha −1 [42]. This was related to the high primary production of seagrass meadows and their capacity to filter out particles from the water column and store them in sediments [53]. However, the mean carbon stock of seagrass biomass was 7.29 ± 1.52 Mg C ha −1 , which is low when compared with that in forests, which ranges from 30 Mg C ha −1 , for boreal tundra woodlands, to 300 Mg C ha −1 for tropical rainforests [54]. Similar values were obtained in the wetlands in South Eastern Australia [45]. Shallow and deep freshwater marshes showed a mean carbon stock of 200 ± 20 and 230 ± 190 Mg C ha −1 , respectively. The permanent open freshwater areas, however, exhibited a mean OC of 110 ± 12 Mg C ha −1 for the same study [45]. Carbon is accumulated mainly in mangrove and salt marsh ecosystems, in comparison to Sile river ecosystem. Kauffman et al. [43] showed that the mean ecosystem carbon stock in salt marshes east of the mouth of Amazon river (Brazil) was 257 Mg C ha −1 , whereas the mean mangrove carbon stock was 361-746 Mg C ha −1 . Similarly, the estimated carbon stock of the mangrove ecosystem within the delta of the Zambezi River (Mozambique) ranged between 373 and 620 Mg C ha −1 [55]. In the tropical riverine wetlands of the southern pacific coast of Mexico, Adame et al. [56] estimated that the carbon stock of the mangrove ecosystem was 4-fold higher than the average carbon stock of the Sile river, whereas salt marshes had a mean carbon stock of 336 Mg C ha −1 .

Conclusions
This study highlights the contribution of the river ecosystems in the global carbon stock. The quantification of stored OC within sediment and SAMs from the Sile River implied that the storage capacity for Carbon of the river ecosystem was varied, depending on the differences of vegetation biomass and SAMs biodiversity. Furthermore, the variability of water and sediment environmental parameters were found to affect the ecosystem OC storage capabilities. These findings shed light on the role freshwater rivers play in the global carbon cycle. Indeed, the estimated OC storage capacity from this preliminary research showed that river ecosystems, like Sile River, can significantly contribute to the global assimilation and storage of carbon dioxide. The conservation and protection of river ecosystems have the capacity to reduce the emissions of greenhouse gas and increase the storage of carbon stock while delivering key ecosystem services to river communities. For this reason, the study and protection of river ecosystems and the conservation of SAMs, threatened by anthropogenic pressures and climate change, is fundamental for environmental improvement and ecosystem services. Furthermore, future studies with additional sites and seasonal surveys of the river will enhance our understanding of the effects of global climate changes on the river ecosystem and improve the ecosystem services.
Supplementary Materials: The following are available online at https://www.mdpi.com/2073-444 1/13/1/80/s1, Table S1: Water and sediment environmental parameters; Table S2: Sediment grain size distribution with depth, Table S3: Sediment total nitrogen (TN) concentrations with depth. Funding: This research received department grant from the department of environmental sciences, informatics and statistics, Ca' Foscari University of Venice, Italy.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author. The data are not publicly available due to privacy.