ANALYTICAL SCIENCES JANUARY 2004, VOL. 20 159 2004 © The Society for Analytical Chemistry

Determinations of Humic Substances and Other Dissolved Organic Matter and Their Effects on the Increase of COD in

Shinichi AOKI,* Yasuro FUSE,** and Etsu YAMADA*,**†

*Department of Chemistry and Material Technology, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606Ð8585, Japan **Center for Environmental Science, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606Ð8585, Japan

Humic substances and other dissolved organic matter (DOM) in Lake Biwa and the surrounding rivers were investigated to elucidate their origins and behavior. An annual increase in chemical oxygen demand (COD) has been observed in the northern basin of Lake Biwa since 1985. The concentrations of dissolved organic carbon (DOC) in the northern and southern basins of Lake Biwa were 1.7 Ð 2.4 mgC/l and 1.9 Ð 2.6 mgC/l, respectively. The DOC concentrations tended to be high in summer and low in winter, and the seasonal changes in the concentrations of humic substances were small. The humic substances content of DOM was considered to be comparatively small because the ratio of the concentration of humic substances to DOC was in the range of 0.14 Ð 0.32. From the results of the fractionation of DOM in lake waters, it was estimated that hydrophobic acids, such as humic substances and hydrophilic acids, were about 25% and 45%, respectively. The main origin of hydrophobic acids in Lake Biwa may be humic substances from soils around the rivers that flow into Lake Biwa, while hydrophilic acids may be due to the inner production by phytoplankton. Therefore, the increase of COD in the northern basin of Lake Biwa may be attributed to the contributions of not only humic substances but also hydrophilic acids.

(Received September 25, 2003; Accepted November 5, 2003)

hydrous iron oxide (HIO) and their reactivity with Introduction permanganate oxidant. They found that the fraction with adsorptivity to HIO consisted primarily of refractory organic Chemical oxygen demand (COD) and biochemical oxygen carbon.4Ð6 However, the dynamics of DOC could not be demand (BOD) have been used as the main indexes of water quantitatively explained by these data alone. pollution in lakes and rivers. Recently, total organic carbon In our previous study, humic and fulvic acids in environmental (TOC) and dissolved organic carbon (DOC) are also being used as another indexes of water quality. In Lake Biwa, the largest lake in Japan, the annual increase of COD has been observed since 1985, although the values of BOD and chlorophyll-a have been almost constant. Moreover, the values of COD in the rivers that flow into Lake Biwa have not increased (Fig. 1).1 These results suggest that refractory organic matter may have accumulated in Lake Biwa. To clarify the reason for the increase in COD, it is important to analyze the dynamics of both pedogenic and autochthonous DOC separately. In several lakes, chemical fractionation methods have been applied to analyze the temporal changes of DOC.2 Imai et al. divided DOC occurring in Lake Biwa into six fractions: hydrophobic acid, hydrophobic base, hydrophobic neutral, hydrophilic acid, hydrophilic base, and hydrophilic neutral. As a result, they reported that the hydrophilic acid fraction contained more than 40% of the total DOC, whereas the hydrophobic acid fraction contained less than 30%.3 Hori et al. attempted to characterize organic carbon samples in Lake Biwa and the surrounding rivers according to their adsorptivity to Fig. 1 Annual changes in chemical oxygen demand (COD) and † To whom correspondence should be addressed. biochemical oxygen demand (BOD) in the northern basin of Lake E-mail: [email protected] Biwa.1 , COD; , BOD; , CODÐBOD. 160 ANALYTICAL SCIENCES JANUARY 2004, VOL. 20 water were determined by UV spectrophotometry after preconcentration with diethylaminoethyl(DEAE)-cellulose and separation at pH 1 by centrifugation, where the relationship between aquatic humic substances and trihalomethane (THM) formation was further examined.7 Furthermore, simultaneous determinations of the concentration and molecular weights of humic substances in environmental water were developed by gel chromatography with a fluorescence detector. Such methods were applied to the direct determination of humic substances in the Katsura, Uji, Kidzu, and Yodo Rivers.8 In the present study, our method was applied to the determination of humic substances in Lake Biwa and the surrounding rivers to clarify their contributions to the increase of COD in Lake Biwa. Moreover, other dissolved organic matter (DOM) in Lake Biwa and the rivers were investigated to elucidate their origins and behavior.

Experimental

Reagents and apparatus Humic acid (extracted from peat soil; Aldrich Chemicals) was purified by repeated dissolution in 0.1 M sodium hydroxide and precipitation in 0.1 M hydrochloric acid until the color of the Fig. 2 Sampling stations on Lake Biwa and in the surrounding supernatant solution was eliminated. Fulvic acids obtained rivers. Lake Biwa: L1 Hikone, L2 Biwako-ohashi, L3 Akanoi. from A-horizons of brown forest soil (Dystric Cambisol, Dando, Rivers: R1 Ane River, R2 Amano River, R3 Yagura River, R4 Aichi, Japan) and ando soil (Humic Andozol, Inogashira, , R5 Uso River, R6 , R7 , R8 Shizuoka, Japan) were used. Fulvic acids prepared according to Uji River, R9 Kidzu River, R10 . the methods developed by the International Humic Substances Society (IHSS) were supplied by the Japan Humic Substances Society and used as a standard without purification. Procedure for the determination of humic and fulvic acids in Diethylaminoethyl(DEAE)-cellulose (Wako Pure Chemicals, environmental water samples after preconcentration Osaka, Japan) was purified according to the previous study.7 A The environmental water samples were collected from Lake weakly basic anion exchanger with a DEAE group (Toyopak Biwa and the rivers (Fig. 2). The lake water samples were DEAE; Tosoh) was used instead of DEAE-cellulose for the collected at two sites in the northern basin (Hikone and Biwako- direct determination of humic substances. A macroporous resin ohashi) and one site in the southern basin (Akanoi). The river (Amberlite XAD-7HP, 28 Ð 73 mesh), a strongly acidic cation water samples were collected from the Yasu, Uso, Inukami, exchanger (Bio-Rad AG-MP-50, 100 Ð 200 mesh), and a Yagura, and Ane Rivers, which flow into Lake Biwa. The river strongly basic anion exchanger (Bio-Rad AG-MP-1M, 50 Ð 100 water samples were also collected from the Katsura, Uji, Kidzu, mesh) were used for the fractionation of humic substances and and Yodo Rivers.7,8 At the stations, water samples were taken at other DOM. The XAD-7HP resin was soaked in 0.1 M sodium the depth of 0.5 m. All water samples were filtered through a hydroxide, washed with pure water, and sequentially purified by membrane filter (0.45 µm, Millipore) as soon as possible to Soxlet extraction with methanol, diethyleter, acetonitrile, and avoid biodegradation. The membrane filters were used after methanol. The ionic exchangers were washed by Soxlet extraction rinsing with 1 M hydrochloric acid and distilled water. with methanol and further washed with pure water. All other Preconcentration and determination of humic and fulvic acids chemicals were of the best commercial grade. Pure water was in river water samples were carried out according to the prepared by a Millipore Milli-Q water purification system. methods used in the previous study.7 The apparatus used for HPLC was a Shimadzu LC-10AD chromatography pump equipped with a Shimadzu RF-535 Procedure for the direct determination of humic substances in fluorescence detector. The excitation and emission wavelengths environmental water samples8 were 340 nm and 435 nm, respectively. A Shimadzu C-R7A A 100 µl aliquot of filtrated water samples without chromatopac was used for data analysis. The test samples were preconcentration after adjusting to 0.01 M sodium hydroxide applied to a gel filtration column, Superose12 HR10/30 (300 × solution was measured by gel chromatography with a 10 mm i.d.); Pharmacia Biotech, using a sample injector fluorescence detector (chromatogram A). Further, a filtered (Rheodyne 7125) with a 100 µl loop. A 0.01 M sodium water sample passed through an anion exchanger with DEAE was hydroxide solution was used as an eluent at a flow rate of 0.4 ml measured similarly (chromatogram B). The concentration and minÐ1. Two TOC meters, a Shimadzu TOC-5000 and a Uniflex molecular weights of humic substances in river water samples TOC-1010, were used for the determination of DOC. A were calculated by means of the differential chromatogram (C) Shimadzu UV-160A spectrophotometer was used for the obtained as the difference of the chromatograms (A) and (B). measurement of UV-absorbame (260 nm) of water samples. A The concentration of humic substances was calculated with Kubota KN-70 centrifuge was used for the separation of humic reference to the standard of fulvic acid because fulvic acid is substances. A Horiba F-15 pH meter, a Denki-Kagaku Keiki predominant in environmental waters. AOL-40 EC meter, and a Mitsubishi-Kagaku TN100 TN meter were used for the measurement of pH, electric conductivity, and Procedure for the fractionation of DOM in environmental water trace total nitrogen in environmental water samples, samples respectively. The fractionations of DOM in Lake Biwa and the rivers were ANALYTICAL SCIENCES JANUARY 2004, VOL. 20 161

Table 1 Analytical results for humic substances (HS) and dissolved organic carbon (DOC) in Lake Biwa and the surrounding rivers

HS DOC HS/DOC Sampling stationa Dateb mg lÐ1 MeanmgC lÐ1 Mean Mean Lake Biwa Hikone (L1) A 0.44 2.8 0.16 B 0.39 1.8 0.22 C 0.33 1.8 0.18

Biwako-ohashi (L2) A 0.18 1.9 0.09 B 0.34 1.7 0.20 C 0.31 1.9 0.16

Akanoi (L3) A 1.13 3.0 0.38 B 0.60 1.9 0.32 C 1.27 2.5 0.51

Rivers flowing into Lake Biwa (6 rivers) (R1 Ð R6) A 0.28 Ð 1.04 0.61 0.7 Ð 2.2 1.4 0.28 Ð 0.53 0.42 B 0.34 Ð 1.09 0.56 1.0 Ð 1.7 1.5 0.28 Ð 0.64 0.38 C 0.23 Ð 1.76 0.66 0.6 Ð 3.1 1.3 0.39 Ð 0.62 0.48

Yodo rivers (4 rivers) (R7 Ð R10) B 0.50 Ð 1.53 0.96 1.9 Ð 2.7 2.2 0.26 Ð 0.61 0.43 a. See Fig. 2. b. Sampling on A, July 2001; B, January 2002; C, August 2002.

(HiA), hydrophilic base (HiB), and hydrophilic neutral (HiN) were calculated using the following equations:

HoB = (DOC2 Ð B1)V2/V1 (1)

HoA = (DOC3 Ð B2)V2/V1 (2)

HoN = DOC1 Ð (DOC4 Ð B3) Ð HoB Ð HoA (3)

HiB = (DOC4 Ð B3) Ð (DOC5 Ð B4) (4)

HiA = (DOC5 Ð B4) Ð (DOC6 Ð B5) (5)

HiN = DOC6 Ð B5 (6)

Here, DOC1 Ð DOC6 are the concentrations of DOCs obtained

in the procedures shown in Fig. 3, and V1 and V2 are sample volume and eluent volume, respectively. The concentrations of DOC were measured by the high-temperature catalytic oxidation method by using a TOC meter. B1 and B2 are blank DOC values of 0.1 M HCl and 0.1 M NaOH passed through a XAD-7HP resin column. B3, B4 and B5 are blank DOC values of Milli-Q water (pH 2) passed through a XAD-7HP resin column, an AG-MP-50 resin column, (AG-MP-50 resin + AG- MP-resin) columns, respectively. The average values of the blank samples B1, B2 and B3, were 0.073 ± 0.011 mgC/l (n = 3), 0.74 ± 0.11 mgC/l (n = 12) and 0.13 ± 0.02 mgC/l (n = 6), respectively. The values of B4 and B5 were almost zero. The relative standard deviation values of DOC concentrations in Fig. 3 Flowchart for fractionation of hydrophobic and hydrophilic Lake Biwa (Biwako-ohashi) collected on October 2002 were dissolved organic matter (DOM) fractions from water samples of Lake Biwa and the surrounding rivers. 17.6% (n = 3) for DOC1 and less than 8% (n = 5) for DOC2 Ð DOC6, respectively. carried out according to the method of Imai et al.3 The hydrophobic and hydrophilic fractions of DOM were isolated Results and Discussion using the XAD-7HP resin, AG-MP-50 resin, and AG-MP-1M resin. Figure 3 shows the procedure for the fractionation of Behavior of humic substances and DOC in Lake Biwa and DOM. surrounding rivers The concentrations of hydrophobic acid (HoA), hydrophobic The concentrations of humic substances in Lake Biwa and the base (HoB), hydrophobic neutral (HoN), hydrophilic acid surrounding rivers determined by the present experiment are 162 ANALYTICAL SCIENCES JANUARY 2004, VOL. 20

Fig. 4 Relationships between the concentration of humic substances and DOC at Biwako-ohashi ( ) and Akanoi ( ) in Lake Biwa. Fig. 6 Gel chromatograms for humic substances in water samples of the Lake Biwa without preconcentration (July 2001). (A) Direct analysis of lake water after filtration; (B) analysis of lake water through a DEAE-cellulose column; (C) the difference of the chromatograms (A) and (B).

northern basin of Lake Biwa. The average concentrations of humic substances and DOC in the Yodo River were about 1.5 times higher than those in the rivers flowing into Lake Biwa. The relationships between the concentrations of humic substances and DOC at Biwako-ohashi and Akanoi are shown in Fig. 4. A positive correlation was observed between humic substances and DOC at Akanoi (r2 = 0.425), but not at Biwako- ohashi (r2 = 0.012). The humic substances content of DOM in the northern basin of Lake Biwa was considered to be comparatively small because the ratio of the concentration of humic substances to DOC was in the range of 0.14 Ð 0.32, while that at Akanoi was relatively large. The seasonal changes in the concentrations of humic substances and DOC at Biwako-ohashi and Akanoi in Lake Biwa are shown in Fig. 5. At Akanoi, the concentrations of humic substances and DOC were higher in warmer seasons and Fig. 5 Seasonal changes in the concentrations of humic substances ( , ) and DOC ( , ) at Biwako-ohashi and Akanoi in Lake Biwa. lower in cooler seasons. At Biwako-ohashi, the DOC concentrations tended to be high in summer and low in winter, while seasonal changes in the concentrations of humic substances were small. listed in Table 1. The analytical results for DOC and the ratios of the concentrations of humic substances to DOC are also Characteristics of humic substances and DOC in Lake Biwa listed in Table 1. The concentrations of humic substances in the and surrounding rivers northern and southern basins of Lake Biwa were 0.18 Ð 0.44 Figure 6 shows the gel chromatograms of humic substances in mg/l and 0.6 Ð 1.27 mg/l, respectively. The mean values of water samples of Lake Biwa collected in July 23, 2001. The gel humic substances in water samples from the northern basin of chromatograms (A), (B) and (C) at Akanoi and chromatogram Lake Biwa (Hikone and Biwako-ohashi) and the rivers flowing (C) at Hikone are shown. The chromatogram (C) was obtained into Lake Biwa collected in July 2001 were 0.44 mg/l, 0.18 from the difference of the chromatograms (A) and (B) as mg/l, and 0.61 mg/l, respectively. The DOC concentrations in previously described. Three peaks, peak 1 at Rt (retention time) the northern and southern basins of Lake Biwa were 1.7 Ð 2.8 = 30 min, peak 2 at Rt = 32 min, and peak 3 at Rt = 35 min, mgC/l and 1.9 Ð 3.0 mgC/l, respectively. The mean values of were detected in water samples from Lake Biwa and the rivers DOC in water samples from the northern basin of Lake Biwa flowing into Lake Biwa, which were similar to those in the (Hikone and Biwako-ohashi) and the rivers flowing into Lake Katsura, Uji, Kidzu, and Yodo Rivers.8 The seasonal changes Biwa collected in July 2001 were 2.8 mgC/l, 1.9 mgC/l, and 1.4 in the concentrations of humic substances and the ratio of peak mgC/l, respectively. The concentrations of humic substances in 1/peak 2 calculated by chromatograms of humic substances in the rivers flowing into Lake Biwa were higher than those in the water samples from Lake Biwa and the rivers are listed in Table northern basin of Lake Biwa, while the DOC concentrations in 2. The molecular weights of peaks 1 and 2 were estimated to be the rivers flowing into Lake Biwa were lower than those in the 5000 Ð 10000 Da and 3000 Ð 5000 Da, respectively.8 The ANALYTICAL SCIENCES JANUARY 2004, VOL. 20 163

Table 2 Seasonal changes in the concentrations of humic substances (HS) and intensity ratios of peak 1/peak 2 (P1/P2) observed in the gel chromatograms for humic substances in lake and river waters Jul. 2001 Jan. 2002 Aug. 2002 Sampling station HS/ HS/ HS/ P1/P2a P1/P2a P1/P2a mg lÐ1 mg lÐ1 mg lÐ1 Lake Biwa Hikone (L1) 0.44 0.67 0.39 0.77 0.33 0.67 Biwako-ohashi (L2) 0.18 0.74 0.34 0.76 0.31 0.53 Akanoi (L3) 1.13 0.55 0.60 0.53 1.27 0.58 Rivers flowing into Lake Biwa Ane River (R1) 0.28 0.48 0.34 1.02 0.29 0.64 Amano River (R2) 0.42 0.62 0.57 0.74 0.44 0.46 Yagura River (R3) 1.04 0.41 1.09 0.56 1.76 0.51 Inukami River (R4) 0.34 0.74 0.36 0.72 0.23 0.44 Uso River (R5) 0.88 0.78 0.60 0.46 0.68 0.66 Fig. 7 Relationship between the concentrations of humic Yasu River (R6) 0.69 0.48 0.42 0.65 0.54 0.61 substances and UV-absorbance at 260 nm (E260) in Lake Biwa. , Hikone; , Biwako-ohashi; , Akanoi. a. The peak intensity ratios in the gel chromatograms (see Fig. 6). The molecular weights of peaks 1 and 2 are 5000 Ð 10000 Da and 3000 Ð 5000 Da, respectively.

seasonal changes in the ratios of peak 1/peak 2 of humic substances in the Katsura, Uji, Kidzu, and Yodo Rivers collected from Aug. 1995 to Dec. 1996 were 0.58 Ð 1.37, while those in the northern and southern basins of Lake Biwa and the rivers flowing into it collected from Jul. 2001 to Aug. 2002 were 0.53 Ð 0.77, 0.53 Ð 0.58, and 0.41 Ð 1.02, respectively. The ratios of peak 1/peak 2 of humic substances in water samples from Lake Biwa and the rivers were lower than those in samples from the Katsura, Uji, Kidzu, and Yodo Rivers. From these results, the molecular weights of most humic substances in Lake Biwa and the rivers were estimated to be lower than 5000 Da, which is lower than that of the humic substances in the Katsura, Uji, Kidzu, and Yodo Rivers, about 3000 Ð 10000 Da.8 Most humic substances in the Katsura, Uji, Kidzu, and Yodo Rivers are assumed to originate from soils around the rivers.7 As for DOC in the open sea, it is now thought that the higher- Fig. 8 Seasonal changes in the ratios of E260/DOC in Lake Biwa. molecular-weight DOC is degradable and the lower-molecular- , Hikone; , Biwako-ohashi; , Akanoi. weight DOC is refractory.9 These results indicate that the higher-molecular-weight DOC and humic substances in Lake Biwa and the rivers may decompose easier with biodegradation than the lower-molecular-weight DOC or that the aquatic humic were reported to be 23 Ð 58 and 8 Ð 16 cmÐ1 (g C)Ð1 l, substances with the lower molecular weights in them may have respectively.11,12 Seasonal changes in the ratios of E260/DOC in different origins, such as autochthonous DOC. Lake Biwa are shown in Fig. 8. The maximum, minimum, and The relationship between humic substances and UV- average values of the ratios of E260/DOC were 19, 9 and 13 absorbance at 260 nm (E260) is shown in Fig. 7. The cmÐ1 (g C)Ð1 l, respectively, in the northern basin of Lake Biwa, absorbance of lake and river waters was measured at 260 nm and 23, 11, and 18 cmÐ1 (g C)Ð1 l, at Akanoi. On the other hand, because humic substances that contain aromatic compounds the maximum, minimum, and average values of the ratios of substituted with COOH and OH groups have absorbances at 200 E260/DOC in the rivers flowing into Lake Biwa were 26, 10, Ð 290 nm and the effects of nitrate and bromide ions on the and 16 cmÐ1 (g C)Ð1 l, respectively. The ratios of E260/DOC in absorbance of 260 nm are neglected.10 At Akanoi, a fairly the northern basin of Lake Biwa tended to be low with the positive correlation between humic substances and UV- increase of DOC in summer, while those at Akanoi tended to be absorbance (r2 = 0.739) was observed, while lower positive higher as DOC increased. These results suggest that the correlations were observed at Hikone (r2 = 0.553) and Biwako- decrease in the ratios of E260/DOC in the northern basin of ohashi (r2 = 0.281). Specific ultraviolet absorbance (SUVA) is Lake Biwa in summer may be due to the inner production by defined as the UV-absorbance of a water sample at a given phytoplankton, whereas their increase at Akanoi in summer may wavelength normalized for DOC concentration. It is expressed be due to humic substances from soils around the rivers or Ð1 Ð1 in units of cm (g C) l. SUVA260 (E260/DOC) was indicated sediments of Akanoi. to be strongly correlated with aromatic compounds. The ratios of E260/DOC for pedogenic DOC and autochthonous DOC 164 ANALYTICAL SCIENCES JANUARY 2004, VOL. 20

Table 3 Fractionation of dissolved organic matter (DOM) in hydrophobic acids in Lake Biwa may be humic substances from Lake Biwa soils around the rivers, while hydrophilic acids may be due to Humic the inner production by phytoplankton. Therefore, the increase DOC/ Ratio, E260/ Fraction Datea substances/ of COD in the northern basin of Lake Biwa may be attributed to mgC lÐ1b % DOC mgC lÐ1 the contribution of not only humic substances but also hydrophilic acids. Hydrophobic acid A 0.32 24.6 26 0.27 B 0.32 22.4 — 0.18 Hydrophobic base A 0.05 3.8 6 — Acknowledgements B 0.06 4.2 —— Hydrophobic neutral A 0.26 20.0 —— B 0.28 19.6 ——The authors thank their students for their assistance in the Hydrophilic acid A 0.59 45.4=< 10 — collection of environmental water samples and in the B 0.55 38.5 ——measurements of humic substances. Hydrophilic base A 0.07 5.4 —— B 0.16 11.2 —— Hydrophilic neutral A 0.01 0.8 14 — References B 0.06 4.2 —— Total A 1.30 100.0 16 1. , Kankyohakusyo, 2002, 110. B 1.43 100.0 — 2. H. Tatsumoto, T. Hattori, T. Furukawa, I. Ikushima, M. Kurihara, and I. Abe, Nippon Kagaku Kaishi, 1991, 852. a. Sampling on A, October 11, 2002; B, April 14, 2003. b. DOC: dissolved organic carbon. 3. A. Imai, T. Fukushima, K. Matsushige, T. Inoue, and T. Ishibashi, Jpn. J. Limnol., 1998, 59, 53. 4. T. Hori, Y. Sugiyama, and M. Sugiyama, Jpn. J. Limnol., 1998, 59, 39. 5. Y. Sugiyama, M. Sugiyama, and T. Hori, Limnology, 2000, Fractionation of DOC in Lake Biwa and the surrounding rivers 1, 171. The fractionations of DOM in Lake Biwa and the rivers were 6. Y. Sugiyama and T. Kumagai, Anal. Sci., 2001, 17, 77. carried out according to the method of Imai et al.3 The 7. E. Yamada, T. Ozaki, and M. Kimura, Anal. Sci., 1998, 14, hydrophobic and hydrophilic fractions of DOM were isolated 327. using XAD-7HP resin, AG-MP-50 resin, and AG-MP-1M resin. 8. E. Yamada, K. Doi, K. Okano, and Y. Fuse, Anal. Sci., The fraction species of DOC in Lake Biwa (Biwako-ohashi) 2000, 16, 125. collected in October 2002 and April 2003 are listed in Table 3. 9. R. M. W. Amon and R. Benner, Nature, 1994, 369, 549. From the results of the fractionation of DOM in lake waters, it 10. N. Tanbo and T. Kamei, “Aquatic Humic Substances”, was estimated that hydrophobic acids, such as humic substances 1989, American Chemical Society, Washington D.C., 453. and hydrophilic acids, were about 20 Ð 25% and 40 Ð 45%, 11. T. Fukushima, J. Park, A. Imai, and K. Matsushige, Aquatic respectively. The present results in Lake Biwa were in good Sci., 1996, 58, 139. agreement with the results reported by Imai et al. from spring in 12. J. Buffle, P. Delandoety, J. Zumstein, W. Haerdi, and Z. 1995 to winter 1996. Thurman et al. reported that dissolved Schweiz, Hydrol., 1982, 44, 325. humic substances are 40 Ð 60% of DOC in natural waters and 13. E. M. Thurman, “Organic Geochemistry of Natural about 40% of DOC in lake waters. Mcknight et al. have Waters”, 1985, Martinus Nijhoff/Dr. W. Junk Publishers, reported that dissolved humic substances were 13 Ð 20% of Dordrecht. DOC in lake waters in which the main origin of DOC is 14. D. M. Mcknight, G. R. Aiken, and R. L. Smith, Limnol. autochthonous.14,15 It was then considered that the ratio of Oceanogr., 1991, 36, 998. humic substances is relatively low in waters in which the main 15. D. M. Mcknight, E. D. Andrews, S. A. Spaulding, and G. origin of DOC is autochthonous. The main origin of R. Aiken, Limnol. Oceanogr., 1994, 39, 1972.