Stable isotopic evidence for the excess leaching of unprocessed atmospheric nitrate from forested catchments under high nitrogen saturation

. Owing to the elevated loading of nitrogen through atmospheric deposition, some forested ecosystems become nitrogen saturated, from which elevated levels of nitrate are exported. The average concentration of stream nitrate eluted from upstream and downstream of the Kasuya Research forested catchments (FK1 and FK2 catchments) in Japan were more than 90 µM, implying that these forested catch-ments were under nitrogen saturation. To verify that these forested catchments were under the nitrogen saturation, we determined the export ﬂux of unprocessed atmospheric nitrate relative to the entire deposition ﬂux ( M atm /D atm ratio) in these catchments; because the M atm /D atm ratio has recently been proposed as a reliable index to evaluate nitrogen saturation in forested catchments. Speciﬁcally, we determined the temporal variation in


Introduction
Nitrate is important as a nitrogenous nutrient in the biosphere. Traditionally, forested ecosystems have been considered as nitrogen limited (Vitousek and Howarth, 1991). However, owing to the elevated loading of nitrogen through atmospheric deposition, some forested ecosystems become nitrogen saturated (Aber et al., 1989), from which elevated levels of nitrate are exported Peterjohn et al., 1996). Such excessive leaching of nitrate from forested catchments degrades water quality and causes eutrophication in downstream areas (Galloway et al., 2003;Paerl and Huisman, 2009). Thus, evaluating the stage of nitrogen saturation in each forested catchment, including its temporal variation, is critical for sustainable forest management, especially for forested ecosystems under high nitrogen deposition.
Both concentration and seasonal variation of stream nitrate have been used as indexes to evaluate the nitrogen saturation of each forested catchment in past studies (Aber, 1992;Rose et al., 2015;Stoddard, 1994). A forested stream eluted from Fernow Experimental Forest USA, for instance, showed an elevated average nitrate concentration of 60 µM, along with the absence of a seasonal variation in the stream nitrate concentration, so the forest was classified into stage 3, the highest stage of nitrogen saturation (Rose et al., 2015).
However, using both the concentration level (high or low) and seasonal variation (clear or absent) of stream nitrate as indexes to evaluate nitrogen saturation has limitations, including the following: (1) seasonal variation of soil nitrate can be buffered by groundwater with long residence time, so that the seasonal variation is unclear in stream nitrate concentration in Japan, even in normal forests under the nitrogen saturation stage of 0 ; and (2) the stream nitrate concentration can be enriched or diluted depending on the volume of rainfall, so the concentration level can be high in low precipitation area irrespective of the stage of nitrogen saturation. Nakagawa et al. (2018) lately proposed that the M atm / D atm ratio, the export flux of unprocessed atmospheric nitrate (M atm ) relative to the deposition flux of NO − 3 atm (D atm ), can be an alternative, more robust index for evaluating nitrogen saturation in each forested catchment, because the M atm / D atm ratio directly reflects the demand for atmospheric nitrate deposited onto each forested catchment as a whole, and thus reflects the nitrogen saturation in each forested catchment. That is, we can expect high M atm / D atm ratios in forested catchments under nitrogen saturation and low M atm / D atm ratios in forested catchments with nitrogen deficiency.
To estimate the M atm / D atm ratio accurately and precisely in each forested catchment, the fraction of unprocessed atmospheric nitrate (NO − 3 atm ) in the stream needs to be estimated accurately and precisely. Triple oxygen isotopic compositions of nitrate ( 17 O) have recently been used as a conservative tracer of NO − 3 atm deposited onto each forested catchment (Inoue et al., 2021;Michalski et al., 2004;Nakagawa et al., 2018;Tsunogai et al., 2014;Ding et al., 2022), showing distinctively different 17 O from that of remineralized nitrate (NO − 3 re ) derived from organic nitrogen through general chemical reactions, including microbial N mineralization and microbial nitrification. While NO − 3 re , the oxygen atoms of which are derived from either terrestrial O 2 or H 2 O through microbial processing (i.e., nitrification), always shows the relation close to the "mass-dependent" relative relation between 17 (Alexander et al., 2009;Michalski et al., 2003;Morin et al., 2011;Nelson et al., 2018). As a result, the 17 O signature defined by the following equation (Kaiser et al., 2007) where the constant β is 0.5279 (Kaiser et al., 2007), 3 atm to reflect its accurate mole fraction within total NO − 3 , regardless of the progress of the partial metabolism (partial removal of nitrate through denitrification and assimilation) subsequent to deposition (Michalski et al., 2004;Nakagawa et al., 2013Nakagawa et al., , 2018Tsunogai et al., 2011Tsunogai et al., , 2014Tsunogai et al., , 2018.
Past studies reported that the maximum concentration of stream nitrate was 58.4 µM in the KJ forested catchment in Japan, with the maximum value of the M atm / D atm ratio as 9.4 % Sase et al., 2022). Whether the index of the M atm / D atm ratio can be applied to forested catchments, where the leaching of stream nitrate is much higher than the KJ forested catchment, remained unclarified. Besides, the advantages of the M atm / D atm ratio within the past indexes of nitrogen saturation have not been discussed.  has recently reported the enrichment of nitrate of more than 90 µM on the annual average in forested streams eluted from the catchments (FK1 and FK2) in the Kasuya Research Forest, Kyushu University, Japan ( Fig. 1a and b). The observed enrichment of stream nitrate implied that these forested catchments were under nitrogen saturation. Thus, in this study, we determined the M atm / D atm ratio in the FK1 and FK2 forested catchments by monitoring both the concentration and 17 O of stream nitrate for more than 2 years to verify that these forested catchments were under nitrogen saturation. For comparison, the catchment (MY catchment) in Shiiba Research Forest, Kyushu University, Japan ( Fig. 1a and c), was also monitored during the same period, where the average stream nitrate concentration was low (less than 10 µM). Furthermore, the M atm / D atm ratios in these forested catchments were compared with those reported in past studies to verify the reliability of the M atm / D atm ratio as an index of nitrogen saturation.  (FK and MY) in Japan (a), and the maps of FK1, FK2 (b), and MY catchments (c), shown by orange, yellow, and green areas, respectively, together with the sampling station A, B, and C, respectively, shown by orange, yellow, and green circles, respectively. The blue arrows indicate the flow direction of stream water.  (Chiwa, 2020.

Sampling
The stream water eluted from the FK1 (14 ha), FK2 (62 ha), and MY (43 ha) forested catchments was collected about once every month in principle from November 2019 to December 2021 ( Fig. 1). At the FK catchments, stream water was collected at upstream (station A) and downstream (station B) locations ( Fig. 1b). At the MY catchment, stream water was collected at station C (Fig. 1c). Samples of stream water to determine the concentration and stable isotopic com-positions (δ 15 N, δ 18 O, and 17 O) of stream nitrate were collected manually in bottles washed with deionized water before sampling and then rinsed at least twice with the sample before sampling at each sampling site.

Analysis
All the stream water samples were passed through a membrane filter (pore size 0.45 µm) within 2 days after sampling and stored in a refrigerator (4 • C) until analysis. The concentrations of nitrate were measured by ion chromatography (Prominence HIC-SP, Shimadzu, Japan). To determine the stable isotopic compositions of nitrate in the stream water samples, nitrate in each sample was chemically converted to N 2 O using a method originally developed to determine the 15 N/ 14 N and 18 O/ 16 O ratios of seawater and freshwater nitrate (McIlvin and Altabet, 2005) that was later modified (Konno et al., 2010;Tsunogai et al., 2011;Yamazaki et al., 2011). In brief, 11 mL of each sample solution was pipetted into a vial with a septum cap. Then, 0.5 g of spongy cadmium was added, followed by 150 µL of a 1 M NaHCO 3 solution. The sample was then shaken for 18-24 h at a rate of 2 cycles s −1 . Then, the sample solution (10 mL) was decanted into a different vial with a septum cap. After purging the solution using high-purity helium, 0.4 mL of an azideacetic acid buffer, which had also been purged using highpurity helium, was added. After 45 min, the solution was alkalinized by adding 0.2 mL of 6 M NaOH. Then, the stable isotopic compositions (δ 15 N, δ 18 O, and 17 O) of the N 2 O in each vial were determined using the continuousflow isotope ratio mass spectrometry (CF-IRMS) system at Nagoya University. The analytical procedures performed using the CF-IRMS system were the same as those detailed in previous studies (Hirota et al., 2010;D. D. Komatsu et al., 2008). The obtained values of δ 15 N, δ 18 O, and 17 O for the N 2 O derived from the nitrate in each sample were compared with those derived from our local laboratory nitrate standards to calibrate the values of the sample nitrate to an international scale and to correct for both isotope frac-tionation during the chemical conversion to N 2 O and the progress of oxygen isotope exchange between the nitratederived reaction intermediate and water (ca. 20 %). In this study, we adopted the internal standard method to calibrate the stable isotopic compositions of sample nitrate. Specifically, three kinds of the local laboratory nitrate standards were used in this study, which were named to be GG01 (δ 15 N = −3.07 ‰, δ 18 O = +1.10 ‰, and 17 O = 0 ‰), HDLW02 (δ 15 N = +8.94 ‰, δ 18 O = +24.07 ‰), and NF ( 17 O = +19.16 ‰), which the GG01 and the HDLW02 were used to determine the δ 15 N and δ 18 O of stream nitrate, and the GG01 and the NF was used to determine the 17 O of stream nitrate. The GG01, HDLW02, and NF had been calibrated using the internationally distributed isotope reference materials (USGS 34 and USGS 35). The oxygen exchange rate between nitrate and water during the chemical conversion was calculated through Eq. (2):  (Tsunogai et al., 2016;Nakagawa et al., 2013Nakagawa et al., , 2018Ding et al., 2022).
The δ 2 H and δ 18 O values of H 2 O of the stream water samples were analyzed using the cavity ring-down spectroscopy method by employing an L2120-i instrument (Picarro Inc., Santa Clara, CA, USA) equipped with an A0211 vaporizer and autosampler. The errors (standard errors of the mean) in this method were ±0.5 ‰ for δ 2 H and ±0.1 ‰ for δ 18 O. Both the VSMOW and standard light Antarctic precipitation (SLAP) were used to calibrate the values to the international scale. The δ 18 O values of H 2 O were used to calibrate the differences in δ 18 O of H 2 O between the samples and those our local laboratory nitrate standard samples (Tsunogai et al., , 2014. To determine whether the conversion rate from nitrate to N 2 O was sufficient, the concentration of nitrate in the samples was determined each time we analyzed the isotopic composition using CF-IRMS based on the N 2 O + or O + 2 outputs. We adopted the δ 15 N, δ 18 O, and 17 O values only when the concentration measured via CF-IRMS correlated with the concentration measured via ion chromatography prior to isotope analysis within a difference of 10 %. We repeated the analysis of δ 15 N, δ 18 O, and 17 O values for each sample at least three times to attain high precision. All samples had a nitrate concentration of greater than 3.5 µM, which corresponded to a nitrate quantity greater than 35 nmol in a 10 mL sample. Thus, all isotope values presented in this study have an error (standard error of the mean) better than ±0.2 ‰ for δ 15 N, ±0.3 ‰ for δ 18 O, and ±0.1 ‰ for 17 O.
Nitrite (NO − 2 ) in the samples interferes with the final N 2 O produced from nitrate, because the chemical method also converts NO − 2 to N 2 O (McIlvin and Altabet, 2005). There-fore, it is sometimes necessary to remove NO − 2 prior to converting nitrate to N 2 O. In this study, however, we skipped the processes for removing NO − 2 because all the stream samples analyzed for stable isotopic composition had NO − 2 concentrations lower than the detection limit (0.05 µM).

Deposition rate of atmospheric nitrate
The annual deposition rate of atmospheric nitrate (D atm ; total dry and wet deposition rate of atmospheric nitrate) in each catchment was estimated using the annual "bulk" deposition rate of atmospheric nitrate (D bulk ) calculated in Chiwa (2020) at each catchment by multiplying the volumeweighted mean concentration of nitrate in the bulk deposition samples collected every 2 weeks at each catchment for 10 years (from January 2009 to December 2018) by the annual amount of precipitation. The bulk deposition samples were those accumulated in a plastic bucket installed in an open site of each catchment 55 cm above the ground. The distances between the monitoring sites of bulk deposition in the FK1, FK2, and MY forested catchments and the stations of stream water sampling (stations A, B, and C) were 3.9, 2.9, and 4.5 km, respectively. The concentrations of nitrate in the bulk deposition samples were measured by ion chromatography.
The D bulk determined through this method, however, is less than D atm (Aikawa et al., 2003) because the dry deposition velocities of gases and particles on the water surface of the plastic bucket are smaller than those on the forest (Matsuda, 2008). Thus, we corrected the differences by using Eq. (3) to estimate D atm from D bulk : where D dry (W ) and D dry (F ) denote the annual dry deposition rates onto water and forest, respectively. The D dry (W ) and D dry (F ) at each catchment were determined using an inferential method (Endo et al., 2011) through Eqs. (4) and (5), respectively: where [NO − 3 atm ] gas denotes the concentration of gaseous nitrate in air; [NO − 3 atm ] p denotes the concentration of particle nitrate in air; V gas (W ) and V gas (F ) denote the deposition velocities of gaseous nitrate on the water surface and forest, respectively; and V p (W ) and V p (F ) denote the deposition velocities of particulate nitrate on the water surface and forest, respectively. Those determined by Chiwa (2010)

Flux of stream water
The flux of stream water (F stream ) in each catchment was not measured fully in this study. Instead, the water balance in each catchment was used to estimate F stream , assuming that the outflux of water from the study catchments to deep groundwater was negligible: where P denotes the annual average precipitation and E denotes the annual evapotranspiration flux of water in each catchment. In this paper, the equation obtained by H.  was used to estimate the E of the FK and MY catchments. Details on this equation are shown below. H.  compiled the annual flux of evapotranspiration determined in 43 forested catchments in Japan and found that E shows a positive correlation with the average temperature (T avg ) of each catchment. Thus, they proposed the modeled relation of E (mm) = 31.4 T avg ( • C) + 376 to estimate E in each forested catchment in Japan, where the standard error of 162.3 mm was included in the estimated evapotranspiration flux (E). They also confirmed that the estimated F stream using the model corresponded well with the observed F stream in three forested catchments, with estimated errors of less than 6 %. As a result, we utilized the water balance method proposed by H.  to quantify the F stream in each catchment.

Concentration of unprocessed NO − 3 atm in each water sample
The 17 O data of nitrate in each sample were used to estimate the concentration of NO − 3 atm ([NO − 3 atm ]) in each water sample by applying Eq. (7):  Tsunogai et al., 2016;Ding et al., 2022).
The annual export flux of unprocessed NO − 3 atm per unit area of the catchment (M atm ) was determined by applying Eq. (8):  (Fig. 1b). Assuming that the stream nitrate eluted from FK1 catchment was stable during the flow path from station A to station B, the concentration of stream nitrate eluted from the FK2 catchment was determined by applying Eq. (9):  (Sappa et al., 2015) were used for F FK1 (0.85 L s −1 ) and F FK1+FK2 (4.75 L s −1 ), respectively, and the measured [NO − 3 ] at stations A and B was used for [NO − 3 ] FK1 and [NO − 3 ] FK1+FK2 , respectively. Because the relation between the measured flow rates was comparable with the relation between the catchment area of FK1 (14 ha) and that of FK1 + FK2 (76 ha), we concluded that the measured flow rates of 0.85 L s −1 and 4.75 L s −1 were reasonable as for those representing the F FK1 and F FK1+FK2 , respectively. According to the mass balance of water, we can estimate the F FK2 eluted from the FK2 catchment only to be 3.90 L s −1 .
Assuming that the stream nitrate eluted from the FK1 catchment was stable during the flow path from station A to station B, the δ 15 N, δ 18 O, and 17 O values of stream nitrate eluted from the FK2 catchment only were determined by applying Eq. (10):

Deposition rate of atmospheric nitrate
The mean annual precipitation (P ) from 2009 to 2021 was 1777 and 3981 mm for FK and MY catchments, respectively (Chiwa, 2020;Masaaki Chiwa, personal communication, 21 September 2022). The mean annual temperature (T avg ) was reported to be 15.9 and 10.8 • C for FK and MY catchments, respectively (Chiwa, 2020). Based on these data, the annual flux of stream water (F stream ) was estimated to be 902.0 ± 162.3 mm at FK catchments and 3266.1 ± 162.3 mm at MY catchment, respectively, using Eq. (6). Chiwa (2020) reported the annual bulk deposition rates of atmospheric nitrate (D bulk ) to be 34.0 mmol m −2 yr −1 at the FK catchments and 24.2 mmol m −2 yr −1 at the MY catchment. On the other hand, the annual dry deposition rate of atmospheric nitrate (D dry ) deposited on the forest (D dry (F )) and on the water surface (D dry (W )) were estimated to be 39.9 and 4.1 mmol m −2 yr −1 , respectively, at FK catchments and 18.4 and 2.4 mmol m −2 yr −1 , respectively, at MY catchment. As a result, D atm was estimated to be 69.3 mmol m −2 yr −1 at FK catchments and 40.1 mmol m −2 yr −1 at MY catchment using Eq. (3).

Concentration and isotopic composition of stream nitrate
The concentrations of stream nitrate eluted from the FK1, FK2 (only), and MY catchments ranged from 97.5 to 121.3 µM, from 65.7 to 148.5 µM, and from 3.5 to 15.3 µM, respectively, with the average concentrations of 109.5, 90.9, and 7.3 µM, respectively, and the standard deviations (SD) of 6.3, 18.5, and 3.0 µM, respectively, which correspond to the coefficients of variation (CV) of 5.7 %, 20.4 %, and 40.7 %, respectively (Fig. 2a). All catchments showed no clear seasonal variation during the observation periods. The variation ranges and the average concentrations of stream nitrate eluted from the three catchments agreed well with the past observations performed in the same catchments .

Concentration of unprocessed atmospheric nitrate and the M atm / D atm ratio in each catchment
The concentration of unprocessed atmospheric nitrate ([NO − 3 atm ]) in the streams eluted from the FK1, FK2 (only), and MY catchments ranged from 8.64 to 14.30 µM, from 2.27 to 10.71 µM, and from 0.03 to 0.46 µM with the average concentration of 10.80 ± 1.30, 5.06 ± 0.67, and 0.16 ± 0.03 µM, respectively, even though these studied catchments showed little seasonal variations during the observation periods (Fig. 2e). The annual export flux of nitrate (M total ), the annual export flux of NO − 3 atm (M atm ), and the M atm / D atm ratio were 98.8 ± 17.8 mmol m −2 yr −1 , 9.7 ± 2.1 mmol m −2 yr −1 , and 14.1 ± 4.1 % at FK1 catchment, respectively, 82.0 ± 14.8 mmol m −2 yr −1 , 4.6 ± 1.0 mmol m −2 yr −1 , and 6.6 ± 2.0 % at FK2 catchment, respectively, 23.7 ± 1.2 mmol m −2 yr −1 , 0.5 ± 0.1 mmol m −2 yr −1 , and 1.3 ± 0.4 % at MY catchment, respectively (  Table S1, the D atm of the FK catchments exceeded the average level significantly. In addition, the D atm of the FK catchments corresponded to one of the highest among the Japanese forested areas (Table S1). All the catchments in Japan can be suffered from the long-range transport of air pollutants derived from megacities in the East Asian region Chiwa et al., 2012Chiwa et al., , 2013. In addition, the shorter transport distance from the Fukuoka metropolitan area (total population: 1.62 million people; population density: 4715 people km −2 ) may be mainly responsible for the D atm higher in FK than in MY, because the FK catchments are only 15 km west of the Fukuoka metropolitan area.

Excess leaching of unprocessed atmospheric nitrate from FK catchments
The isotopic compositions (δ 15 N, δ 18 O, and 17 O) of stream nitrate eluted from the FK and MY catchments were typical for those eluted from forested catchments (Hattori et al., 2019;Huang et al., 2020;Nakagawa et al., 2013Nakagawa et al., , 2018Riha et al., 2014;Sabo et al., 2016;Tsunogai et al., 2014Tsunogai et al., , 2016. The striking features found in the FK catchments were that, in addition to the high [NO − 3 ] and high M total that had been clarified in a past study , both [NO − 3 atm ] and M atm in FK were higher than those eluted from MY (Table 2). Especially, the average [NO − 3 atm ] in the stream eluted from the FK1 catchment was the highest ever reported in forested streams determined through continuous monitoring for more than 1 year (Bostic et al., 2021;Bourgeois et al., 2018a, b;Hattori et al., 2019;Huang et al., 2020;Nakagawa et al., 2018;Rose et al., 2015;Sabo et al., 2016;Tsunogai et al., 2014Tsunogai et al., , 2016.  Rose et al. (2015). d Tsunogai et al. (2014). e Huang et al. (2020). f N saturation stage estimated in past studies -means no data.
The observed high [NO − 3 atm ] in the stream eluted from the FK1 catchment could be caused just by the high [NO − 3 atm ] deposition in the catchment. Thus, we compiled all past data ever reported in forested streams through continuous monitoring in Table 3, where the data of average [NO − 3 ], average [NO − 3 atm ], M atm , M total , D atm , and M atm / D atm ratio were included for comparison. The result showed that the M atm / D atm ratio, along with M atm , was the highest as well in the FK1 catchment among the forested catchments (Table 3).
Elevated loading of nitrogen through atmospheric deposition was responsible for the occurrence of nitrogen saturation in forest ecosystems, from which elevated levels of nitrate are exported (Aber et al., 1989). Nakagawa et al. (2018) proposed that the M atm / D atm ratio can be an index for evaluating the nitrogen saturation in each forested catchment, because the M atm / D atm ratio directly reflects the present demand for atmospheric nitrate deposited in each forested catchment, and thus reflects the nitrogen saturation in each forested catchment. The high M atm / D atm ratios observed in the FK catchments implied that the demand for atmospheric nitrate was low in the FK catchments and that the stages of nitrogen saturation at the FK catchments were higher than those at other forested catchments. That is, the nitrogen saturation at the FK catchments was responsible for the observed high [NO − 3 ] and high M total at the FK catchments than at MY and any other catchment ever studied (Table 3).
The stand age of forests can affect the retention or loss of N (Fukushima et al., 2011;Ohrui and Mitchell, 1997). Fukushima et al. (2011) evaluated N uptake rates of Japanese cedars at different ages (5-89 years old) and demonstrated that the N uptake rates of Japanese cedars were higher in younger stands (53 kg N ha −1 yr −1 in 16 years old) than in older stands (29 kg N ha −1 yr −1 in 31 years old; 24 kg N ha −1 yr −1 in 42 years old; 34 kg N ha −1 yr −1 in 89 years old). In addition, Yang and Chiwa (2021) found that the nitrate concentration in the soil water taken beneath the rooting zone of matured artificial Japanese cedar plantations (607 ± 59 µM; 64-69 years old) was significantly higher than that of normal Japanese oak plantations (8.7 ± 8.1 µM; 24 years old). Moreover, by adding ammonium nitrate (50 kg N ha −1 yr −1 ) to the forest floor directly, Yang and Chiwa (2021) found that the nitrate concentration in the soil water of the matured artificial Japanese cedar plantations increased significantly faster than that of the normal Japanese oak plantations, probably because of the lower N uptake rates in the matured artificial Japanese cedar plantations. Because most of the artificial Japanese cedar/cypress plantations in the FK and MY catchments have reached their maturity (> 50 years; Yang and Chiwa, 2021), the higher proportion of matured artificial Japanese cedar/cypress plantations in the FK1 catchment (Table 1) was highly responsible for the observed elevated leaching of nitrate, caused by the reduction in N uptake rates.
As a result, we concluded that the FK forested catchments were under the high nitrogen saturation stage, FK1 catchment especially, and the nitrogen saturation in the FK1 catchment was responsible for the elevated M total , M atm , [NO − 3 ], [NO − 3 atm ] found in the stream eluted from the catchment (Fig. 3a, b, c and d).  Huang et al., 2020;KJ, IJ1, and IJ2: white squares, Nakagawa et al., 2018;Fernow 1, 2, and 3: white diamonds, Rose et al., 2015;Uryu: white triangle, Tsunogai et al., 2014). The data obtained in the Qingyuan forested catchment are shown in parentheses and excluded from the calculation to estimate correlation coefficients (see text for the reason).

The M atm / D atm ratio as an index of nitrogen saturation
Past studies have used the concentration of stream nitrate as one of the important indexes to evaluate the stage of nitrogen saturation in each forest (Aber, 1992;Huang et al., 2020;Rose et al., 2015;Stoddard, 1994). The strong linear relationship (R 2 = 0.76; P < 0.0001) between the stream ni-trate concentration and the M atm / D atm ratio, except for the Qingyuan forested catchment (Fig. 3d), further supported that the M atm / D atm ratio can be used as an alternative index of nitrogen saturation, as pointed out in . The differences in the number of storm and/or snowmelt events could affect the M atm / D atm ratio as well, because NO − 3 atm could be injected into the stream water directly, along with the storm/snowmelt water (Tsunogai et al., 2014;Ding et al., 2022;Inamdar and Mitchell, 2006). In a recent study, however, we found that storm events have little impact on the M atm / D atm ratio, based on monitoring temporal variation of [NO − 3 atm ] in stream water during storm events (Ding et al., 2022). In addition, the low M atm / D atm ratio found in Uryu forested catchment (0.7 %; Table 3) implied that the snowmelt has little impact on the M atm / D atm ratio as well, because 30 % of the annual mean precipitation was snow in Uryu forested catchment (Tsunogai et al., 2014).
The differences in the amount of precipitation, temperature, and the flux of stream water could affect the M atm / D atm ratio as well. As a result, the annual amount of precipitation, mean temperature, and the annual mean flux of stream water (F stream ) in the forested catchments were compiled in Table S2. While the stream nitrate concentration showed a strong linear relationship (R 2 = 0.76; P < 0.0001) with the M atm / D atm ratio (Fig. 3d), the precipitation, temperature, and F stream did not show a significant relationship with the M atm / D atm ratio (P > 0.14; Fig. 4). As a result, we concluded that the M atm / D atm ratio was mainly controlled by the progress of nitrogen saturation, rather than the differences in the number of storm and/or snowmelt events, the amount of precipitation, temperature, and the flux of stream water.
The differences in the residence time of water in each catchment could also impact the M atm / D atm ratio, as the residence time of water in forested catchments ranges from one month to more than one year (Asano et al., 2002;Farrick and Branfireun, 2015;Kabeya et al., 2008;Rodgers et al., 2005;Soulsby et al., 2006;Tetzlaff et al., 2007). While the M atm / D atm ratio could be higher in catchments with a shorter water residence time, it is difficult to explain high [NO − 3 ] and high M total eluted from the catchment by the residence time of water alone, as the majority of nitrate eluted from the catchment with a high M atm / D atm ratio was NO − 3 re produced by microbial nitrification. The significant correlation between M total and M atm / D atm ratios (P < 0.0001; Fig. 3a) supported nitrogen saturation as the leading cause of high M total in catchments with a high M atm / D atm ratio. Additionally, the high loading of atmospheric nitrogen, the type of plantation, and the old age of plantation in the FK1 catchment all supported the conclusion that the FK1 catchment was under nitrogen saturation.
The M atm / D atm ratio is a more reliable and robust index than the stream nitrate concentration, as explained below. The Qingyuan forested catchment can be classified into the highest nitrogen saturation stage based only on the highest stream nitrate concentration of 150 µM (Table 3). However, based on the leaching flux of nitrogen via stream water monitored by Huang et al. (2020) for 4 years in the Qingyuan forested catchment, along with the deposition flux of nitrogen, we can obtain the M atm / D atm ratio in the catchment to be a medium level of 5.8 ± 1.3 %, implying that the nitrogen saturation stage was not so high (Table 3). Huang et al. (2020) also concluded that the input of nitrogen exceeded the output in the catchment, and thus, the catchment was at stage 2 of nitrogen saturation. The M atm / D atm ratio in the Qingyuan forested catchment with a medium level among all forested catchments (Fig. 3d) should be a more reliable index of nitrogen saturation.
Compared with those in the other forested catchments in Table 3, the annual amount of precipitation (P ) has the lowest value of 709 mm in the Qingyuan forested catchment. The flux of stream water (F stream ) has the lowest value of 309 mm as well. Thus, we concluded that nitrate was relatively concentrated in the catchment because of the small precipitation, resulting in relative enrichment in the concentrations of both nitrate (150 µM) and unprocessed atmospheric nitrate (8.9 µM) in the stream.
While the concentration of stream nitrate, as an index of nitrogen saturation traditionally, can be influenced by the amount of precipitation, as demonstrated in the Qingyuan forested catchment, the M atm / D atm ratio is independent of the amount of precipitation (Fig. 5). Therefore, the M atm / D atm ratio can be used as a more robust index for evaluating nitrogen saturation in each forested catchment.

Conclusions
Both the concentrations and 17 O of stream nitrate were determined for more than 2 years in the forested catchments of FK (FK1 and FK2) and MY to determine the M atm / D atm ratio for each catchment. The FK catchments exhibited higher M atm / D atm ratio than the MY catchment and other forested catchments reported in past studies, implying that the progress of nitrogen saturation in the FK catchments was severe. Both age and proportion of artificial plantation in the FK catchments were responsible for the progress of nitrogen saturation. In addition, although past studies have commonly used the concentration of stream nitrate as an index to evaluate the progress of nitrogen saturation in forested catchments, it can be influenced by the amount of precipitation. As a result, we concluded that the M atm / D atm ratio should be used as a more reliable index for evaluating the progress of nitrogen saturation because the M atm / D atm ratio is independent from the amount of precipitation.
The uncertainty in M atm / D atm ratio was estimated from the uncertainties in M atm and in D atm according to the divisive equation of error propagation (Eq. A3): where σ M atm /D atm ratio , σ M atm , and σ D atm denote the uncertainty in M atm / D atm ratio, M atm , and D atm , respectively. Comparing the deposition rate of NO − 3 atm obtained at the other atmospheric monitoring stations nearby, the uncertainty of 20 % was adopted for those of D atm in each catchment, which corresponds to the uncertainty in D atm of ±13.9, ±13.9, ±8.0 mmol m −2 yr −1 at FK1, FK2, and MY catchments, respectively. As a result, the uncertainty in M atm / D atm ratio was ± 4.1 %, ±2.0 %, and ±0.4 % at FK1, FK2, and MY catchments, respectively. Data availability. All the primary data are presented in the Supplement. The other data are available upon request to the corresponding author (Weitian Ding).