Interactive comment on “ Sources , fluxes , and behaviors of fluorescent dissolved organic matter ( FDOM ) in an estuarine mixing zone : Results from the Nakdong-River Estuary , Korea ” by Shin-Ah Lee and Guebuem

This manuscript by Lee and Kim set out to examine sources, fluxes, and mixing behavior of fluorescent dissolved organic matter in the Nakdong-River estuary in Korea. Time series samples were collected once a month during Sept 2014 – Aug 2015. In addition to humic-like and protein-like fluorescent DOM, the authors also measured bulk DOC concentration as well as its stable C isotopic composition to track DOM sources. They

Having said that some of the data need more discussion.For example, while both DOC and FDOM concentrations decreased linearly with increasing salinity showing an apparent conservative mixing behavior, stable carbon isotope signatures did not show a linear mixing between riverine and marine DOC, as also shown in other estuaries (e.g., Guo et al., 2009, Mar. Chem).The concave mixing curve indicated preferential removal of fluvial/terrestrial DOM during estuarine mixing (Zhou et al., 2016, GCA) and source of DOC had been changed to more marine or autochthonous to maintain the somewhat linear mixing of DOC and FDOM, as shown in Figure 2 in the manuscript.These are very interesting results and the authors could add a few sentences in their discussion.In addition, if DOM sources had shifted in the higher salinity regions, is the FDOM in the estuary terrestrial or marine?Or what portion is from terrestrial DOM? Also, if portions of DOC and FDOM in the estuary are from in situ produced DOM, then, terrestrial DOM is not conservative, at least in the middle and higher salinity regions?=> Thank you for your valuable suggestions.The stable carbon isotope mixing curve was constructed based on the two end-member mixing equation (Spiker, 1980;Peterson et al., 1994;Raymond and Bauer, 2001).The curved mixing line for the conservative mixing was clearly explained previously by Spiker (1980) and Peterson et al. (1994).In general, the conservative mixing line for δ 13 C-DOC is "not curved" for a constant DOC concentrations, however, it is "curved" when DOC concentrations decrease as salinities increase due to the weighting effect of the endmember isotopic compositions by the DOC concentrations (Spiker, 1980;Peterson et al., 1994).This is also shown in the equation.Since our DOC concentrations decrease as salinities increase, our data are generally fitted to the theoretical conservative mixing line.This suggests that DOC behaves conservatively in this system.The extrapolated endmember value of δ 13 C-DOC for each month ranged from -27.5 to -24.5, indicating that the main source of DOC is C3 plants.However, the value was heavier at lower salinity ranges (S<10) in March and April samples, perhaps in association with higher biological production in the river.This is mentioned in the revised version (lines 184-194).
As explained to Reviewer #1, it is very difficult to compare scatterings for different seasons in order to discuss the different behaviors of DOM since the scattering is generally larger for the narrow salinity ranges.This seems to be the limitation of our study.However, we added more discussion about the behavior of DOM based on correlation coefficients for the other seasons in the revised version (lines 221-230): "In March, during the highest biological production period in the river, the correlation coefficient was the highest for FDOMP and lowest for FDOMH against salinities.In contrast, in June, during the highest fluvial DOM discharge period, the correlation coefficient was the highest for FDOMH and lowest for FDOMP against salinities.This suggests that the biological production and removal, together with other generally known factors such as photo-degradation and sedimentary inputs, may affect the scattering of these FDOM components in the estuarine mixing zone".

Minor comments for the authors:
1) Title: could be shortened to read: "Sources, fluxes, and behaviors of fluorescent dissolved organic matter in the Nakdong-River Estuary, Korea".=> corrected as suggested.
2) Line-24: add p values if available; => In the revised version, p values were added.
3) Line-28/29: "…...due to higher fluvial production of humic-like FDOM"; This is not necessarily the case here.Data in Figure 3 show a constant slope for humic-like FDOM among different seasons although there is not a constant slope value for the protein-like FDOM.=> We rephrased this sentence to be read as "higher terrestrial input of humic-like FDOM".4) Line-31/32: Are there any difference in export fluxes between the bulk DOC and different FDOM?This could be an interesting issue.=> As shown in Figure 4, the fluxes of bulk DOC and FDOMH showed similar patterns over the seasons (the highest fluxes in July and lowest in January and February), while FDOMP showed the highest fluxes in April and lowest fluxes in January.This is mentioned in the revised version."The fluxes of FDOMP in December and March were twofold higher than those of FDOMH whereas the flux of FDOMH in July was 2-3 folds higher than that of FDOMP.This shows that the fluxes of both components of FDOM differ significantly by seasons owing to the different source inputs even though their magnitudes are controlled mainly by river discharges".5) Line-89: Here the sampling date is October 2014-Aug 2015, which is different from the date shown in the abstract: Sept 2014 to Aug 2015.This should be fixed.=> corrected.
6) Pg-7 on FDOM analysis: the extraction of FDOM-H and FDOM-P data from EEM-PARAFAC results should be mentioned briefly here.=> More details on PARAFAC analysis were added as suggested."Four components were identified in the water samples from the EEMs dataset.Based on the excitation-emission peak location, Component 1 (FDOMH, Ex/Em = 320/418 nm) is found to be a terrestrial humic-like component (C peak) shown by Coble (2007).Component 2 (FDOMP, Ex/Em = 280/328 nm) is found to be a tryptophan-like component (T peak), which is produced by microbial processes.Component 3 (Ex/Em = 300,325/364 nm) is found to be a marine humic-like component (M peak).Since Component 3 values were significantly correlated with Component 1 (r 2 =0.95) values, we simply focused on Component 1 (FDOMH) and Component 2 (FDOMP) for data interpretations" 7) Line-180/181: Both QSU and _M are used here for FDOM.How to convert QSU to _M should be mentioned a bit here or in M&M.=> corrected to QSU. 8) Pg-12: DOM fluxes are calculated from the integration of available time series data.Given the strong seasonality in DOC abundance and river discharge, how much difference will have in the fluxes if calculation is based on USGS LOADEST program although, in my opinion, showing the variability in DOM fluxes among sampling months, as provided in this manuscript, is very important.Maybe the authors can also plot data DOC vs discharge and show in Supplementary Materials.=> We attempted to calculate the fluxes using USGS LOADEST program as suggested.
However, we failed to obtain meaningful results since the river flow of this system was operated periodically by man and the concentrations were obtained from the slopes between DOC and salinity correlations at the downstream of the dam.Also, our sampling period (monthly average) does not meet the number of data needed for this program (i.e., 12 months are needed…but we have 11-month data).Although we see a quite significant correlation (r 2 =0.52) between DOC concentrations and discharge volumes each month, this can be simply due to higher discharge volume in summer monsoon season and higher temperatures in summer.In order to avoid this complication, we do not include this plot in supplementary.9) Line-251: add: "and specific river" after resolution.=> "and hydrogeologic settings of a specific river" is added.
12) Finally, are all the data, including DOC, FDOM, d13C, and discharge, documented in supplementary materials?=> added all data in supplementary materials as suggested. [END]
Generally, DOC includes fluorescent dissolved organic matter (FDOM), which emits fluorescent light due to its chemical characteristics.As FDOM accounts for 20 -70% of the DOC in coastal waters (Coble, 2007) and controls the penetration of harmful UV radiation in the euphotic zone, it plays a critical role in carbon cycles as well as biological production.In addition, FDOM is known as a powerful indicator of humic and protein-like substances (Coble, 2007) in coastal waters.River discharge is generally the main source of humic-like FDOM in coastal waters, although it is also produced through in situ microbial activity (Romera-Castillo et al., 2011).In contrast, protein-like FDOM is known to be from biological production as well as anthropogenic sources (Baker and Spencer, 2004).Terrestrial humic substances behave conservatively in coastal areas due to their refractory characteristics (Del Castillo et al., 2000), whereas protein substances behave non-conservatively in many estuaries due to their relatively rapid production and degradation (Vignudelli et al., 2004).
The magnitudes of DOC and FDOM fluxes from rivers are generally dependent on rainfall, discharge, and temperatures (Maie et al., 2006;Jaffé et al., 2004;Huang and Chen, 2009).In the estuarine mixing zone, intensive biogeochemical processes occur through photooxidation, microbial degradation, or physicochemical transformations (i.e., flocculation, sedimentation) (Bauer and Bianchi, 2011;Moran et al., 1991;Benner and Opsahl, 2001;Raymond and Bauer, 2001).Recent studies have demonstrated large seasonal variations as high as 40%, in DOC export from rivers to the ocean (Burns et al., 2008;Bianchi et al., 2004;Dai et al., 2012).However, the seasonal variations in sources, fluxes, and behaviors of DOC and FDOM in the estuarine mixing zone are still poorly understood.
In this study, we analyzed DOC, δ 13 C-DOC, and FDOM in estuarine water samples collected monthly from the Nakdong-River estuary.Sampling was conducted at a fixed platform, which has been utilized for monitoring various environmental parameters.This sampling station is advantageous because we can collect water samples for a wide range of salinities throughout tidal fluctuations.Using the data obtained from this unique station, we were able to determine (1) the behaviors of DOM in the estuarine mixing zone, (2) the fluxes of DOM from rivers based on the slopes between salinities and DOM components, and (3) the changes in DOM sources using δ 13 C-DOC in the estuarine samples.The slope measurement in the mixing zone may represent the endmember of DOM components in rivers better than sitespecific measurements in the river, by integrating larger spaces and times.

Study site
The Nakdong-River Estuary, which is the estuary of the longest river in Korea, is a major source of water supplying the needs for drinking, agriculture, and industry.The main channel of Nakdong River is approximately 510 km in length with a watershed area of approximately 23,380 km 2 .It faces the south-eastern coastal area of the Korean peninsula, passing through Busan which is the second largest city in Korea.The mean annual precipitation is 1150 mm, and most precipitation (60-70%) occurs during the summer monsoon and typhoon seasons (Jeong et al., 2007).To manage water supply and saltwater intrusion, estuary dams were constructed in the mouth of the river in 1987.

Sampling
Water samples were collected at the sampling site which is located 560 m downstream from the dam (Fig. 1).The sampling period was from October 2014 to August 2015.The 2-L water sampling was conducted every hour for 24 hours during spring tide using an auto-sampler (RoboChem TM Autosampler, Model S3-1224N, Centennial Technology, Korea), with a depth of the water intake 1 m below the surface.After samples were collected in acid-cleaned polyethylene bottles, they were moved to the laboratory within 24 hours.All water samples were filtered using pre-combusted GF/F filters.The FDOM samples were stored in precombusted amber glass vials and kept below 4°C in a refrigerator before analysis.The DOC and δ 13 C-DOC samples were acidified to pH ~2 using 6 M HCl to avoid bacterial activities and stored in pre-combusted glass ampoules.Ampoules were fire-sealed to prevent the samples from any contaminations.The samples were analyzed for DOC and CDOM within a week.
Salinity was measured using a YSI Pro Series conductivity probe sensor in the laboratory.The real-time and compulsory discharge volume data from the dam are available at http://www.water.or.kr, provided by K-Water.The monitoring program at this station is maintained by Korea Environment Management Corporation (KOEM).The water temperature data are recorded automatically at the site.The data are available at https://www.koem.or.kr.

Analytical methods
The concentrations of DOC were determined by a high-temperature catalytic oxidation (HTCO) method using a TOC-VCPH analyzer (Shimadzu, Japan).Standardization was performed based on the calibration curve of acetanilide in ultra-pure water.The acidified samples were purged with carbon dioxide (CO2) free carrier gas for 2 min to remove inorganic carbon.The samples were then injected into a combustion column packed with Pt-coated alumina beads and heated to 720°C.The CO2 evolving from combusted organic carbon was detected by a non-dispersive infrared detector (NDIR).Our DOC method was verified with Deep Seawater Reference (DSR) samples for DOC (44-46 μmol L -1 ) produced by the University of Miami, USA.
The values of δ 13 C-DOC were measured using a TOC-IR-MS instrument consisting of an IR-MS instrument coupled with a Vario TOC cube (Isoprime, Elementar, Germany).The TOC instrument uses a common high-temperature catalytic combustion method (Kirkels et al., 2014).The analytical method is fully described in Kim et al. (2015).Briefly, 10 mL of filtered samples was purged with O2 gas for 20-30 min to completely remove DIC after the samples were acidified to pH ~2.Then, 1 mL of the sample was injected into Pt-impregnated catalyst in a quartz tube.In this tube, the DOC was converted completely to CO2 at 750°C, which was then fed through a water trap followed by a halogen trap.After DOC was detected by an NDIR detector, the CO2 gas was entered the TOC-IR-MS interface by the O2 carrier gas.In the interface, the CO2 was transferred to the IR-MS instrument following the removal of any interfering gasses.The δ 13 C-DOC value of blank was measured using the Low Carbon Water (LCW) from Hansell lab (University of Miami), which contains less than 2 μM DOC.Certified IAEA-CH6 sucrose (International Atomic Energy Agency, −10.45 ± 0.03‰) prepared with the low carbon water was used as a standard solution.A standard sample was analyzed for every sample queue (once before or after ten samples) to check a drifting effect during the measurements.The blank correction was performed using a method previously described in De Troyer et al. ( 2010) and Panetta et al. (2008).Our measurement result of δ 13 C-DOC for the DSR (University of Miami) was −21.5 ± 0.1‰, which is consistent with the results reported by Panetta et al. (2008) and Lang et al. (2007).The reproducibility of TOC-IR-MS was ~0.3‰.
FDOM fluorescence was determined in a scan mode using a spectrofluorometer (SCINCO FluoroMate FS-2) within two days after sampling.Emission (Em) spectra were collected from 250 to 600 nm at 2 nm intervals at excitation (Ex) wavelengths from 250 nm to 500 nm at 5 nm intervals.Backgrounds were subtracted for fresh distilled water prepared daily from the sample data to eliminate Raman Scatter peaks (Zepp et al., 2004).All data were obtained in counts per second (cps) and converted to a ppb quinine sulfate standard solution in 0.1 N sulfuric acid at Ex/Em of 350/450 nm.The inner filter effect was negligible for these estuarine water samples using this instrument since the correlation between the uncorrected and corrected values for the inner filter effect was very significant for some samples (r 2 =1, n=5).EEMs-PARAFAC analysis was performed using a MATLAB R2013a program with a DOMFluor toolbox.

Results and Discussion
Salinities ranged from 0.1 to 28.5 over the sampling period of a year.Salinities in the sampling location were dependent primarily on the volume of river-water discharge from the dam.The volumes of river discharge were relatively larger in October, April, July, and May.
The mean annual surface water temperature was 16°C, with the lowest temperature (avg.8°C) in December and the highest temperature in August (avg.26°C).

Behaviors and sources of DOC in the estuarine mixing zone
The concentrations of DOC ranged from 100 to 300 μM, with the highest concentrations in July (avg.243 μM) and the lowest concentrations in February (avg.115 μM), consistent with the typical DOC concentration ranges in coastal waters (Wang et al., 2004;Raymond and Bauer, 2001).The concentrations of DOC correlated significantly with salinities (r 2 = 0.59-0.92,p < 0.0001), indicating that DOC behaves conservatively in the mixing zone of this estuary (Fig. 2A), which is commonly observed in estuarine mixing zones (Laane, 1980;Mantoura and Woodward, 1983;Del Castillo et al., 2000;Clark et al., 2002;Jaffé et al., 2004).
If the high salinity periods are excluded, both the slope and y-intercept of DOC concentrations versus salinities were highest in July (Fig. 1), which could be due to a higher terrestrial DOC loading in the summer period, as observed in Horsens Fjord, Denmark (Markager et al., 2011).For this comparison, we excluded the high-salinity periods (>20), including December, January, February, and June, since they showed a narrow and low DOC concentration range (103-163 μM), resulting in large uncertainties by extrapolating them to the fresh water.
The carbon isotope values in the Nakdong-River Estuary ranged from -28.2 ‰ to -17.6.In order to determine the source of DOC in fresh water, we plotted δ 13 C-DOC values against salinities (Fig. 2B).The conservative mixing curve of δ 13 C values can be obtained using the two endmember mixing equation (Spiker, 1980;Raymond and Bauer, 2001): where δ 13 Cs, δ 13 Cr and δ 13 Cm are the δ 13 C-DOC values at a given sample salinity, river endmember salinity, and marine endmember salinity, respectively; Fr is the riverine freshwater fraction calculated from the measured salinities; [DOC]s and [DOC]m are the DOC concentrations at a given salinity and marine endmember salinity, respectively; [DOC]r is the endmember DOC value for the river water (Fig. 1).
The riverine DOC endmember values (S=0‰) ranged from 174 to 284 μM.The marine endmember value (S=29‰) of DOC is 100 μM with the δ 13 C-DOC value of -19‰.If these values from each month are applied, the δ 13 C-DOC endmember values for the river water extrapolated to be from -27.5 to -24.5‰ (average: -26.2‰).Overall, the carbon isotope values of our samples are fitted well into the conservative mixing curve of the overall trend, with a slight change using different endmember values for different months (Fig. 2B).In general, δ 13 C-DOC values range from −22 to −18‰ for marine phytoplankton, from −34‰ to −23‰ for terrestrial C3 plants, and from −16‰ to −10‰ for terrestrial C4 plants (Gearing 1988;Clark and Fritz, 1997).Carbon isotope values in our study confirm that the main source of DOC in the estuarine mixing zone is dominantly from terrestrial C3 plants over all seasons.However, the value was heavier at lower salinity ranges (S<10) in March and April samples, perhaps in association with the higher biological production in the river.

Behaviors and sources of FDOM in the estuarine mixing zone
Four components were identified in the water samples from the EEMs dataset.Based on the excitation-emission peak location, Component 1 (FDOMH, Ex/Em = 320/418 nm) is found to be a terrestrial humic-like component (C peak) shown by Coble (2007).Component 2 (FDOMP, Ex/Em = 280/328 nm) is found to be a tryptophan-like component (T peak), which is produced by microbial processes.Component 3 (Ex/Em = 300,325/364 nm) is found to be a marine humic-like component (M peak).Since Component 3 values were significantly correlated with Component 1 (r 2 =0.95) values, we simply focused on Component 1 (FDOMH) and Component 2 (FDOMP) for data interpretations.
The concentrations of both FDOM components were significantly correlated with salinities (r 2 = 0.42-0.98,p<0.0001 for FDOMH and r 2 = 0.27-0.96,p<0.0001 for FDOMP), indicating that they are conservative in the mixing zone (Fig. 2).The slopes of FDOMH and FDOMP for each month ranged from -0.15 to -0.59 and -0.15 to -0.71, respectively.The higher FDOMH slopes in July and October were similar to the trend of DOC (Fig. 2C), which could be due to higher terrestrial FDOM production.However, the seasons (March and April) in which higher FDOMP slopes occurred differ from those of DOC and FDOMH, indicating that both FDOM components have different source inputs (Fig. 2D).
Although there are large differences in scattering of FDOM components against salinities, it is very difficult to compare scatterings for different seasons in order to discuss the different behaviors of DOM since the scattering is generally larger for the narrow salinity ranges.If the winter data are excluded, in March, during the highest biological production period in the river, the correlation coefficient against salinities was the highest for FDOMP and lowest for FDOMH.In contrast, in June, during the highest fluvial DOM discharge period, the correlation coefficient against salinities was the highest for FDOMH and lowest for FDOMP.
This suggests that the biological production and removal, together with other generally known factors such as photo-degradation and sedimentary inputs, may affect the scattering of these FDOM components in the estuarine mixing zone.
As such, there was a significant positive correlation between FDOMH and DOC concentrations throughout all sampling periods (r 2 = 0.93, p<0.0001) (Fig. 3A), suggesting that the main source of FDOMH and DOC is terrestrial based on δ 13 C-DOC values.Since FDOM does not usually contribute to a major portion of DOC, a positive correlation between FDOM and DOC has only been observed in specific areas, such as river-estuarine systems (Del Vecchio and Blough, 2004;Coble, 2007).Stedmon et al., (2006) demonstrated that stronger correlations were observed between DOC and FDOM as humic substances derived from terrestrial DOM are more colored than DOM produced in situ.In general, terrestrial DOM occurring in rivers originates mainly from plant decomposition and leaf litter in the form of humic substances (Huang and Chen, 2009).As such, Gueguen et al., (2006) showed that humic materials are more effectively extracted from soils during August and September under high temperatures.
Thus, higher FDOMH slopes in August, October, and November, relative to the other periods, could be associated with higher terrestrial inputs of degradation products of soil organic matter (Dowell, 1985;Qualls et al., 1991).
In the study region, FDOMP concentrations were poorly correlated with DOC concentrations (r 2 =0.11) (Fig. 3B).The slopes of FDOMP concentrations against DOC concentrations varied significantly over different seasons, with steeper gradients in the spring (March and April) and fall (October).In general, FDOMP is known to be produced efficiently by biological production in water (Coble, 1996;Belzile et al., 2002;Steinberg et al., 2004;Zhao et al., 2017).Thus, higher FDOMP concentrations, relative to DOC concentrations, in the spring and fall seems to be associated with the spring and fall phytoplankton blooms in river waters (Mayer et al., 1999;Zhang et al., 2009).

Fluxes of DOC and FDOM in the estuarine mixing zone
flux of FDOMH and FDOMP ranged from 1.4 × 10 9 QSU m 3 day -1 (December) to 23.1 × 10 9 QSU m 3 day -1 (July) and from 1.6 × 10 9 QSU m 3 day -1 (June) to 16.4 × 10 9 QSU m 3 day -1 (March), respectively.The seasonal variation trend of FDOMH was similar to that of DOC.The fluxes of FDOMP in December and March were twofold higher than those of FDOMH whereas the flux of FDOMH in July was 2-3 folds higher than that of FDOMP.This shows that the fluxes of both components of FDOM differ significantly by seasons owing to the different source inputs even though their magnitudes are controlled mainly by river discharges It is well known that the single sampling event is not enough to capture the full range of natural variability in DOM abundance over all seasons (Stedmon et al., 2006;Huang and Chen, 2009;Markager et al., 2011;Dai et al., 2012;Moyer et al., 2015).Overall, our results show that monthly variations are significant.This implies that our understanding of DOC fluxes from large rivers is largely biased, depending on sampling resolution, methods, and hydrogeological settings of a specific river.For example, if summer data are extrapolated to annual river water discharge, the DOC and FDOMH fluxes can be overestimated up to three times for the Nakdong River.

Conclusions
The concentrations of FDOMH and DOC showed significant negative correlations against salinities throughout all sampling periods, indicating that they behave conservatively in this estuarine mixing zone.The slopes of both DOC and FDOMH concentrations versus salinities were highest in July, due to the largest terrestrial DOC loadings.The carbon isotope values showed that the main source of DOC in the estuarine mixing zone is terrestrial C3 plants

Figure 1 .
Figure 1.Map of the Nakdong-River Estuary.The square indicates a fixed monitoring site,

Figure 3 .
Figure 3.The plots of the concentrations of DOC versus the concentrations of (A) FDOMH and