要
JR

Abstract

1. Introduction
Many hot springs containing humic substances such as humic and fulvic acids, so-called Kuroyu hot springs (black springs) and/or Moal hot springs are widely distributed throughout Japan.  Especially, they are found in certain plain areas of Hokkaido, Aomori, Akita and Yamagata Prefectures of Tohoku region, Saitama, Chiba and Kanagawa Prefectures and Tokyo of the south Kanto region, Niigata and Gifu Prefectures of Chubu region, Osaka Prefecture of Kinki region, Miyazaki and Kagoshima Prefectures of Kyushu region (Kanroji, 2010).  These colored hot springs containing humic substances are associated with the area of water-soluble natural gas deposit (Fig. 1, Petroleum Technology Association,1983 ; Kanroji, 2010).  Humic substances, formed by diagenesis of biological materials, are chemically unidentified complex mixture of polymers (Ishiwatari, 2008 ; Kanroji, 2010). Very little is known on organic components in Kuroyu and Mohl hot springs, and thus these studies are strongly required in hot spring science and geochemistry.  We studied fatty acids in humic acid by pyrolysis-tetramethylammoniumhydroxide methylation gas chromatography/mass spectrometry (Py-TMAH-Me-GC/MS) in Kuroyu hot springs from Kamata area in Tokyo (Matsumoto and Ohmiya, 2019).  Pyrolysis cut chemical bonds of ethers, esters and amides, and/ or cause decarboxylation and dehydration reactions.  TMAH forms methyl esters and/or methyl ethers of pyrolyzed products, and GC/MS determines esters and/or ethers with molecule levels. Py-TMAH-Me-GC/MS needs very small amounts of samples without any pretreatment, and very useful for the study of organic components in humic substances (Yamamoto et al ,, 2007 ; Matsumoto and Ohmiya, 2019). Here we studied geochemical characteristics, especially biomarkers such as fatty acids and sterols in ethyl acetate extracts and in humic acids by Py-TMAH-Me-GC/MS in Kuroyu hot springs (Sentou) from the Ohta-ku area in Tokyo to elucidate their geochemical features and sources as well as sedimentary environments.
Materials and Methods
2.
2.1  Kuroyu hot spring samples
Tea brown and black brown with transparent groundwaters are widely distributed in Koto- ku, Edogawa-ku and the south region of Tokyo wards area (Fig. 2, Tokyo Metropolitan Bureau of Economics, 1955 ; Kawashima et al , 1996).  In August 25 and 26, 2016, Kuroyu hot springs (OT01-OT10) were sampled from hot water supply port of Kuroyu hot springs in 2L polyethylene bottles from the Ohta-ku area in Tokyo (Fig. 2, Table 1).  The Kuroyu hot springs were kept in a refrigerator until analyses.
2.2  Analytical methods
1)   Transparency and humic acid
Transparency was measured with a 30cm transparency meter (Shibata Science Co. Ltd.). Humic acid contents were determined by the method of Mineral Spring Analysis Guidelines (Natural Environment Bureau, Ministry of the Environment, 2014).  Kuroyu hot springs were fractionated in 100mL glass bottles with screw caps, and added 2mL concentrated hydrochloric acid to precipitate humic acid, and then to stand for overnight at room temperatures.  Humic
*Hot spring analytical data sheet and personal communication. acid was precipitated completely and transparent water were obtained (Fig. 3).  Humic acid was collected by filtration through glass fiber paper (Whatman, GF/C pore size 0.12 μ m, pretreated 500℃ for 2h in an electric furnace, weight measured) attached in all glass filtering apparatus under reduced pressure.  No pure water wash was done to avoid redissolution of humic acid.  Water and hydrochloric acid on glass fiber paper was evaporated under reduced pressure with an aspirator in a screw-in desiccator at 40℃ in a water bath.  After confirmation of no hydro- chloric acid odor of glass fiber filters, these filters were kept in a desiccator with drying agent of silica gel for overnight, and weighed humic acid contents.  The filters packed in aluminum foil were kept in a freezer at  – 28℃ until Py-TMAH-Me-GC/MS. 2)    Lipid biomarker in ethyl acetate extract Analytical methods of lipid biomarkers (hydrocarbon, fatty acids and sterols) are shown elsewhere (Matsumoto et al. 1979, 1982, 2003 : Matsumoto and Watanuki, 1992).  Briefly, selected five Kuroyu hot springs (OT01-OT05, A 300mL) were acidified with hydrochloric acid (pH<2), and extracted 3 times with ethyl ace¬ tate (50 mLx3).  The ethyl acetate extracts were concentrated to dryness and saponified with 0.5 mol/L potassium hydroxide/methanol B 0T10 (2 mL, 80℃, 2 h).  The ethyl acetate extracts were fractionated by column chromatography on a silica gel column (160mm×6mm i.d., 100 mesh, 5% water).  Hydrocarbon and fatty acid- Kuroyu hot springs (OT01-OT10) from the Ohta-ku area in Tokyo. sterol fractions were obtained by the elution A : Before addition of hydrochloric acid.  B : with hexane and ethyl acetate, respectively. Precipitation of humic acid after addition of hydrochloric acid A half volume subsample of the fatty acid- sterol fraction was methylated with diazo¬ methane.  The other half volume of the fatty acid-sterol fraction was trimethylsilylated (TMS) with 25% N, O -bis(trimethylsilyl acetamide) acetonitrile solution, to obtain sterol TMS derivatives. Hydrocarbons, fatty acid methyl esters and sterol-TMS derivatives were analyzed by a JEOL Q1000 gas chromatograph-mass spectrometer (GC/MS) equipped with a fused silica capillary column (J&W DB5ms, 30m, 0.25mm i.d., film thickness 0.1 μ m).  Splitless mode was employed. Column oven temperature was programmed from 70 to 120℃ at 30℃/min, from 120 to 320℃ at 8℃/min and kept at 320℃ for 3.5 min.  The flow rate of helium carrier gas was 1.2mL/min.  The temperatures of injector, interface and ion source were maintained at 300, 300 and 250℃, respec- tively.  Ionization energy, filament current and detector voltage were 70 eV, 200 μ A and -1,000 V, respectively.
3)    Py-TMAH-Me-GC/MS of humic acid
Glass fiber filter retained humic acid was cut (ca. 10×2mm) with scissors. Py-TMAH-Me- GC/MS was carried out by the method reported elsewhere (Matsumoto and Ohmiya, 2019).  Cut glass fiber paper was wrapped in pyrofoil (curie point at 445℃), together with TMAH reagent (25% tetramethylammoniumhydroxide methanol 10 μ L) and nonadecanoic acid (10 ng) as internal standard.  The solvent was removed by retained heat of the GC/MS column oven.  Py-TMAH- Me-GC/MS was carried out by a JAI-5 Curie point pyrolyzer at 445℃ connected directly with a JEOL Q1000GC/MS.  Oven and needle temperatures of the pyrolyzer were kept at 398 and 250℃, respectively.  Column oven temperatures were programmed from 60 to 320℃ at 8℃/min and kept at 320℃ for 2.5 min. Split mode with split ratio of 1/50 was employed. Other GC/MS conditions were same as the lipid biomarker analysis stated above.
3.
Results and Discussion
3.1 Sedimentary environment in the Ohta-ku area
Tokyo Metropolitan Bureau of Port and Harbor (1993) surveyed basic geology of the port of Tokyo, and taken a number of sediment cores including 400m AU2-1 core in the northwest of Tokyo Bay (35°36′19″N, 139°47′02″E) approximately 7.8 km of the Ohta Ward Office (Fig. 2).  The AU2-1 sediment core consists of Kazusa Group (Kiwada Formation, Ohtadai F., Umegase F. and Kokumoto F.), Shimousa Group (Edogawa F., Tokyo F.), Yurakucho Formation and reclaimed land layer (Fig. 4, Ishiwata, 2004 ; Sekimoto et al , 2009 ; Akiyama et al , 2012). Kazusa Group in AU2-1 core from the bottom to the upward is formed in the lower, middle and upper marine land shelf slopes, respectively (Sekimoto et al ., 2009).  Shimousa Group is formed in Paleo Tokyo Bay of shallow marine environments (Kikuchi, 1980).  The sea levels are largely fluctuated with glacial and inter glacial cycles.  The boundary of Kazusa and Shimousa Group is
Fig. 4 Sedimentary sequence and sedimentary paleoenvironments (reorganized from Sekimoto et al. , 2009).  * Arakawa Peil (Tokyo Peil-1.134m).  * Kikuchi (1980).  * Tokyo Metropolitan Bureau of Port and Harbor (1993).  9 Okada and Bukry (1980). assumed to be marine oxygen isotope stage 16 (ca. 0.65 Ma, Kikuchi, 1997, 2004 ; Fig. 4).  Matuyama- Brunhes transition (0.774Ma) was found at 91.2m of AU2-1 core (Sekimoto et al , 2009).  Extinction of Helicosphaera sellii was found at 340m (1.2Ma).  Well depths of Kuroyu hot springs ranged from 30 to 120m, and thus the sediment ages are expected to be ca. 0.50-1 Ma.
3.2  Hot spring quality
Table 1 shows physico-chemical properties of Kuroyu hot springs from the Ohta-ku area in Tokyo.  Water temperature, pH, electric conductivity, chemical components, total dissolved matter and well depth were used with the hot spring analysis data sheet of each Kuroyu hot spring and personal communication of the staff.  Water temperatures ranging from 16.1 to 18.7℃ with an average of 17.5±0.6 (standard deviation) showed all cold mineral springs.  pH ranging from 7.6 to 8.5 with an average of 8.2±0.5 showed all weak alkaline reflecting major hot spring quality of sodium-hydrogen carbonate and sodium-hydrogen carbonate/chloride (Table 1).  Well depths ranged from 30 to 120m correspond to the Shimousa Group formed in shallow marine environment of Paleo Tokyo Bay (Kikuchi, 1980) or upper Kazusa Group formed in marine land shelf slope (Sekimoto et al., 2009, Figs. 4). Major cations and anions in the Kuroyu hot springs are shown in Fig. 5.  Most predominant cation was Na * in all Kuroyu hot springs, especially OT01-OT07 samples Na * exceed greater than 80%.  OT08-OT10 samples contain considerable amounts of Ca ²* (17.2-33.9%).  OT10 sample con- tains Mg 2+ greater than 33%.  Most predominant anion of HCO 3  is found in OT01-OT06, OT08 and OT09 samples, while Cl − is the most predominant anion of OT10 and OT07 samples. It is well known that alkaline sodium-hydrogen carbonate hot springs with low concentration and low temperatures are widely distributed in granite and/or rhyolite zones, etc. (Mashiko et al., 1959 : Suzuki, 1979 ; Takamatsu et al ., 1981 ; Seki et al ., 2004 ; Muramatsu et al ., 2008).  The for- mation processes of major cations and anions in Kuroyu hot springs in the Ohta-ku area can be
explained as follows.
Usually, origin of water in alkaline hot springs with low dissolved matter is ascribed to meteoric water based on δD and δ 88 O isotope ratios (Seki et al ., 2004).  For pH increase, water reacts with granitic rocks (Takamatsu et al ., 1981) and felsic rocks (Ichikuni et al ., 1982) under limited carbon dioxide supply. In addition, Iwatsuki and Yoshida (1999) reported alteration of plagioclase in deeper groundwater system.  They contribute, probably, to the formation of alkaline pH of Kuroyu hot springs in the Ohta-ku area (Table 1). Takamatsu et al . (1981) conducted dissolution experiments of granitic rocks in distilled water, and showed that K/Na, Ca/Na and Mg/Na weight ratios quickly decreased with decrease of distilled water/granite powder weight ratios (g/g) in 260 hours experiment (Fig. 6).  This result suggests that low contribution of granitic rocks provides a mixture of Na + , K + , Ca 2+ and Mg 2+ cation Kuroyu hot springs such as OT08-OT10 samples, while high contribution of granitic rocks forms Na + cation dominant Kuroyu hot springs such as OT01-OT07 samples (Fig. 5). Seki et al. (2004) reported on the predominance of Na t cation that there are two processes 1) exchange reac- tion of kaolinite to Ca-montmorillonite, and 2) cation exchange reaction of montmorillonite.  Cl − may be derived from sedimentary environments.  NaHCO 3 -type Kuroyu hot springs of the Tokyo Bay area are formed from CaHCO z -type hot springs by ion exchange reaction of Na-montmorillonite (Muramatsu, 2011).  Sodium-hydrogen carbonate and sodium-hydrogen carbonate/chloride type Kuroyu hot springs may, therefore, be predominant in the Ohta-ku area.  Further study on the formation mechanisms of Kuroyu hot spring quality will be required.
3.3 Transparency and humic acid contents
Very little is known on transparency of Kuroyu hot springs.  Transparency of Kuroyu hot springs varied widely from 1.8cm to >30cm (Table 1).  OT01-OT05 samples have very low transparency (1.8-7.5cm) which is similar to the previous study of Kuroyu hot springs from Kamata area ranging from 3.0 to 9.3cm (Matsumoto and Ohmiya, 2019).  Humic acid contents showed wide range of variation ranging from 5.8 to 325mg/L (Table 1) which is comparable to those of Kamata hot spring samples ranging from 85 to 217 mg/L (Matsumoto and Ohmiya, 2019). Kanroji (1999a, 1999b, 2010) reported humic acid contents in Tokyo since 1950th and obtained less than 1 mg/L-490mg/L. Humic acid contents of Mohl hot springs in Hokkaido and Kuroyu hot springs in Tokyo Bay area were 0.2-70mg/kg (Aoyanagi et al , 2005 ; Takano et al , 2016) and 0.34-194 mg/L (Imahashi, 2015), respectively. The Log-transparency and humic acid contents in Kuroyu hot springs in the Ohta-ku area are inversely correlated with high correlation coefficient of r ² = 0.965 (Fig. 7).  This result revealed that humic acid contents reflect color level of Kuroyu hot springs.
3.4    Features of fatty acids and sterols
3.4.1 Hydrocarbons were very low concentrations and not detected in all the Kuroyu hot springs. This is the first report of fatty acids in organic solvent extracts in Kuroyu hot springs.  Normal- alkanoic acids ranging in carbon-chain length from n-C 12 to n-C 22 with a predominance of even- carbon numbers were found in OT01 sample, together with branched ( iso- and anteiso-C 13-17 ), and monounsaturated fatty acids (C 16:1 and C 18:1 , carbon chain length : number of unsaturation, Fig. 8). Four C 16:1 peaks and two C 18:1 peaks were found in the gas chromatogram, although no polyun- saturated fatty acids were detected in OT01 sample (Fig. 9).  To determine double bond position of these peaks, we calculated equivalent carbon chain length (ECL) of the methyl esters of fatty acids (Table 2, Christe, 1989).  The peaks C 16:1 r , C 16:1c, C 16:14, C 18:14 and C 18:15 coincided with and tentatively identified to n-C 16:1 (9, double bond position), n-C 16:1 (8?), n-C 16:1 (7), n-C 16:1 (5), n-C 18:1 (11) and n-C 18:1 (7), respectively (Table 2).  Normal-C 16:1 (7), n-C 16:1 (9), n-C 18:1 (9) and n-C 18:1 (11)
*Christe (1989). have been detected in Lake Yugama of Kusatsu-shirane Volcano, but no polyunsaturated fatty acids are found in the lake (Matsumoto and Watanuki, 1992). The major fatty acids ( > 10%) were short-chain n -alkanoic acids ( n -C 12 , n -C 14 and/or n -C 16 ) and n-alkenoic acids (n-C 16:1 (9), n-C 16:1 (8?) and/or n-C 18:1 (7), Table 3). The most predominant fatty acid was n-C 16 alkanoic acid in all Kuroyu hot springs. Fatty acid compositions revealed that short- chain n -alkanoic acids, long-chain n -alkanoic acids, branched acids and n -alkenoic acids were 47.97-77.51%, 2.69-5.49%, 2.69-3.68% and 15.89-46.32%, respectively (Table 3).  Short-chain n-alkanoic acids were most dominant fatty acids in all Kuroyu hot springs.  Unexpectedly, n-alkenoic acids are abundant in OT04, OT02 and OT01 samples.  These fatty acid compositions are similar to those in Toyotomi hot springs (Matsumoto et al. (2019), and inland waters from Ogasawara Islands (Matsumoto, 1981) and hydrothermal sediments of Tateyama (Matsumoto and Watanuki, 1990).  Total fatty acid concentration ranged from 17.0 to 77.1 μg/L (Table 3).  They are similar to those in some unpolluted inland waters from Ogasawara Islands (Matsumoto, 1981). This is the first report of sterols of organic solvent extracts in Kuroyu hot springs.  Cholesta-5, 22-dien-3 β -ol, cholesterol, cholestanol, brassicasterol, 24-methyl-cholesterol, 24-methylcholestanol, 24-ethylcholesterol and 24-ethylcholestanol were found in OT01 sample from the Ohta-ku area (Fig. 10).  The most abundant sterol was cholesterol in all Kuroyu hot springs (40.9–77.5%) and thus C n sterol was most abundant in all Kuroyu hot springs (53.1–85.6%, Table 4).  Total sterol concentrations ranged from 1.74 to 8.41 μ g/L (Table 4).  These concentrations are very low as compared with polluted Tamagawa River waters (20–29 μg/L, Ogura et al ., 1975). 3.4.2    Fatty acids in Py-TMAH-Me products of humic acid Normal-alkanoic acids ranging in carbon-chain length from C 7 to C 31 with a predominance of even-carbon numbers were found in Py-TMAH-Me products of humic acid, together with branched ( iso- and anteiso-C 13-17 ), and alkenoic acids (four peaks C 16:1 and two peaks C 18:1 ), although no polyunsaturated fatty acids were detected in OT01 sample as in the case of ethyl acetate extract, Fig. 11).  Interestingly, the pattern of alkenoic acids in Py-TMAH-Me product of humic acid is similar to that of ethyl acetate extract (OT01, Fig. 8).  Four C 16:1 peaks and two C 18:1 peaks in the gas chromatogram (Fig. 9) were tentatively identified as in the case of ethyl acetate extract, as
n-C 16:1(9), n-C 16:1(8?), n-C 16:1(7), n-C 16:1(5), n-C 18:1(11) and n-C 18:1(7) (Table 2). The major fatty acids ( > 10%) were n-C 14 , n-C 16 , n-C 16:1 (9), n-C 16:1 (8?) and/or n-C 16:1 (5) (Table 5). The most predominant fatty acid was n-C 16 , n-C 16:1 (9) or n-C 16:1 (8?).  As compared with fatty acids of ethyl acetate extracts, abundance of n-C 16:1 (9) or n-C 16:1 (8?) is originated from degradation products of humic acid.  Fatty acid compositions revealed that short-chain n -alknoic acids ( n -C 12 − n-C 3 t ), long-chain n -alkanoic acids ( n-C 20-n-C 3 t ), branched acids ( n-C 13-n-C 1; ) and n -alkenoic acids (n-C 16:1, n-C 18:1 ) were 30.78-47.31, 2.05-16.19, 1.34-4.86 and 17.03-64.05%, respectively (Table 5). Very short-chain n -alkanoic acids ( n -C 7 –n -C 11 ) were present in all Kuroyu hot springs.  Short-chain n-alkanoic acids were most dominant in OT01, OT03, OT05, OT06 and OT07 samples, while interestingly n -alkenoic acids were most abundant in OT02, OT08, OT09 and OT10 samples (Table 5).  This result suggests strongly that n -alkenoic acids are major structural components of humic acid.
3.5 Sources of organic components
Fatty acids are widely distributed in every organism except for Archaea.  Short-chain n-alkanoic acids ( n-C 10 –n-C 19 ) are major components of algae, and are biomarker of algae including plankton, while long-chain n -alkanoic acids ( n -C 29 –n-C 24 ) are abundant in the waxes of vascular plants, and are biomarker of vascular plants (Matsumoto and Watanuki, 1992 ; Matsumoto, 2014). Branched acids ( iso and anteiso-C 12 –C 17 ) are biomarkers of bacteria (O'Leary, 1982 ; Reddy et al,, 2002, 2003a, 2003b).  Polyunsaturated fatty acids are generally not produced by bacteria (O'Learly, 1982 ; Reddy et al , 2002, 2003a, 2003b).  Very short-chain n-alkanoic acids ( n-C 7-n-C s ) are not common in natural products except for the milk of various mammals (Curro et al , 2019). The abundance of short-chain n -alkanoic acids with small amounts of long-chain n -alkanoic acids and branched acids revealed that the major sources of fatty acids are algae with small contribution of vascular plants and bacteria.  It is similar to those of fatty acids of Py-TMAH-Me products of humic acid (Table 5).  Humic acid is, therefore, mainly derived from algae.  Unusual abundance of n -monousaturated fatty acid in ethyl acetate extracts and Py-TMAH-Me products of humic acid can be explained by the degradation loss of polyunsaturated fatty acids in the sedimentary environments, since mono- and polyunsaturated fatty acids (C 16:1 , C 16:2 , C 18:1 , C 18:2 , C 18:3 C 20:5, C 22:6, etc., Weete, 1976 ; Kawamura et al , 1980 ; Kawamura and Ishiwatari, 1981) are
widely distributed in algae including phytoplankton and zooplankton in lacustrine and marine environments (Weete, 1976 ; Kawamura et al., 1980 ; Kawamura and Ishiwatari, 1981). Wakeham (2000) studied particulate organic carbon and lipid fluxes in the water column and surface sediments in 3 sites of the equatorial Pacific Ocean (9°N, 5°N and equator along 140°W). Estimates of lipid production by plankton in the euphotic zone range from 30-290mg lipid/m²d. Fluxes of lipids decreased quickly in the water column, resulting in delivery rates of lipids to sediments that were 5–6 orders of magnitude reduced (0.00004–0.032mg lipid/m²d, Wakeham, 2000). A diagenesis proceeded down the water column that polyunsaturated fatty acids decreased and disappeared in the water column and surface sediments, but monounsaturated fatty acids and branched acids increased (Wakeham, 2000).  Thus, absence of polyunsaturated acids and abundance of n-mono-alkenoic acids in Kuroyu hot springs are attributed to the preferential degradation loss of polyunsaturated acids.  Very little is known on the double bond position of n -alkenoic acids in natural environments.  Further geochemical studies on double bond position of n-alkenoic acids in relation to their characteristics and source organisms are strongly required. Sterols (stenols and stanols) are widely distributed in lacustrine and marine environments. Chlolesterol is a typical sterol of algae including phytoplankton and is biomarker of their organisms (Matsumoto et al ., 1982, 2006 ; Volkman et al ., 1998).  24-Methylcholesterol is often abundant in certain diatoms and is a biomarker of diatoms (Rampen et al , 2010).  C 29 sterols (24-ethylcholesterol and 24-ethylcholesta-5,22-dien-3 β -ol) are predominant in vascular plants, and generally accepted as their biomarker (Matsumoto et al. 1982, 2006). Sterol composition showed that cholesterol is most predominant sterol in all Kuroyu hot springs (Table 3).  This is derived from algae including plankton with some contribution of diatom and vascular plants. Stanols are found in certain microalgae such as dinoflagellates, diatoms and raphidphyte (Volkman et al. 1998) and are also formed by bacterial reduction of stenols (Nishimura 1982). Stanols are increased in the water column and sediments in the Pacific Ocean (Wakeham, 2000). Stanols are, therefore, derived from microalgae in the sedimentary environments in addition to microbial reduction of stenols.
4.
Conclusion
Geochemical studies on Kuroyu hot springs in the Ohta-ku area of Tokyo were carried out to elucidate their characteristics, especially focused on organic components including humic acid, together with source organisms and sedimentary environments. (1) Kuroyu hot spring qualities are mainly sodium-hydrogen carbonate or sodium-hydrogen carbonate/chloride.  These hot springs may be formed by contribution levels of granitic rocks and ion exchange reaction of montmorillonite. The inverse correlation between Log-transparency and humic acid contents showed (2) that color levels of Kuroyu hot springs are attributed to humic acid contents. Saturated fatty acids are mainly composed of short-chain n -alkanoic acid (C 12 -C 18 ) with (3) small amounts of long-chain n -alkanoic acids (C 20 –C 31 ) and branched acids ( iso- and anteiso-C 12 –C 17 ) in ethyl acetate extracts and Py-TMAH-Me products of humic acid. Unusually, n-alkenoic acids (n-C 16:1 (9), n-C 16:1 (8?), n-C 16:1 (7), n-C 16:1 (5), n-C 18:1 (11) and (4) n-C 18:1 (7)) are abundant in ethyl acetate extracts and Py-TMAH-Me products of humic acids. Major sterols in ethyl acetate extracts were all cholesterol with small amounts of (5) 24-methylcholesterol and 24-ethylcholesterol and others. Organic components in Kuroyu hot springs are mainly originated from algae with some (6) contribution of vascular plants and bacteria. Unusual abundance of n -mono-unsaturated fatty acids can be explained by the degradation loss of polyunsaturated fatty acids in the sedimentary environments. (7) Well depths (30–120m) imply that sedimentary environments are shallow Paleo Tokyo Bay of Shimousa Group and marine land shelf slope of Kazusa Group in ages 0.5-1 Ma.
Acknowledgments
The authors are greatly appreciated to the owners of Kuroyu hot springs (Sentous) for their kind supports of the sampling and offering hot spring analysis data sheet and well depth infor- mation.  We thank two anonymous reviewers for their useful comments.