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Perylene in Lake Biwa sediments originating from Cenococcum geophilum in its catchment area
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Geochimica et Cosmochimica Acta 95 (2012) 241–251 www.elsevier.com/locate/gca
Perylene in Lake Biwa sediments originating from Cenococcum geophilum in its catchment area Nobuyasu Itoh a,⇑, Nobuo Sakagami b, Masaki Torimura c, Makiko Watanabe d a
National Metrology Institute of Japan (NMIJ), National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1, Umezono, Tsukuba, Ibaraki 305-8563, Japan b College of Agriculture, Ibaraki University, 3-21-1, Chuuo, Ami, Inashiki, Ibaraki 300-0393, Japan c Research Institute for Environmental Management Technology, National Institute of Advanced, Industrial Science and Technology (AIST), 16-1 Onogawa, Tsukuba 305-8569, Japan d Graduate School of Urban Environmental Sciences, Tokyo Metropolitan University, 1-1, Minami-osawa, Hachioji, Tokyo 192-0397, Japan Received 21 February 2012; accepted in revised form 30 July 2012; available online 4 August 2012
Abstract Perylene, which is composed of five benzene rings, is commonly found in sediments throughout the world at concentrations and distributions that are different from those of other polycyclic aromatic hydrocarbons. The only information available on the origin of perylene comes from 4,9-dihydroxyperylene-3,10-quinone (DHPQ), which originates from fungal component symbiosis or from parasites on plants; however, there is no direct evidence of a mechanism of perylene formation. In this study, we examined the relationship between sedimentary perylene and Cenococcum geophilum (C. geophilum) in a catchment area at Lake Biwa. Sclerotium grains of C. geophilum containing DHPQ were found in this catchment area (approximately 40 balls kg1 dried soil for >1 mm-/), and small sclerotium grains were frequently found in the sediment. In the sediment sample, we also found broken particles containing perylene, and they had a porous structure characteristic of sclerotium grains. Furthermore, the particles contained DHPQ in different transformation stages to perylene via 3,10-perylenequinone (3,10-PQ). This finding was consistent with results from elemental analysis (oxygen/carbon). Because a remarkable amount of DHPQ originating from C. geophilum also exists in the humic acids of soils and because the inputs of compounds to the lake depend strongly on the rivers, perylene in the Lake Biwa sediment originates mainly from the DHPQ of C. geophilum in its catchment area. Ó 2012 Elsevier Ltd. All rights reserved.
1. INTRODUCTION Polycyclic aromatic hydrocarbons (PAHs), some of which are carcinogenic (Luch, 2004), are formed through incomplete combustion in anthropogenic processes, such as the burning of fossil fuels, and in natural processes such as forest fires (Finlayson-Pitts and Pitts Jr., 2000). Because they are well preserved for extended periods of time, their vertical distributions in sediment cores have been used to
⇑ Corresponding author. Tel.: +81 29 861 4318; fax: +81 29 861 6866. E-mail address: [email protected] (N. Itoh).
0016-7037/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.gca.2012.07.037
reconstruct human activities and natural events (Jiang et al., 2000; Grice et al., 2009; Itoh et al., 2010a). Perylene, which is composed of five benzene rings, is commonly found in sediments throughout the world at concentrations and distributions that are different from those of other polycyclic aromatic hydrocarbons (Venkatesan, 1988; Jiang et al., 2000; Grice et al., 2009; Itoh et al., 2010a). Since the discovery of perylene in aquatic sediments by Orr and Grady (1967: marine sediments) and Hodgson et al. (1968: fresh water lake), many researchers have reported the abundance of perylene in both marine and lacustrine sediments, and discussed on its source, route to sediments, reactions etc. One of the leading hypothesis is that the precursor material of perylene must be attributed
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N Japan Lake Biwa
Sampling point of soil
× 400 km
Ane River
90 70 40 20 90
The North Basin
The South Basin
Sampling point of sediment Seta River 10 km Fig. 1. Map of Lake Biwa and the sampling points. The dotted line is a catchment area of the lake.
to terrigenous materials, although there is another hypothesis that autochthonous organic matter from algal and crinoids’ productions are sources of precursor of perylene (Blumer, 1965; Wakeham et al., 1979; Venkatesan, 1988; De Riccardis et al., 1991). Recently, it has been suggested that perylene is originated from components of wooddegrading fungi (Jiang et al., 2000; Bechtel et al., 2007; Grice et al., 2009; Itoh and Hanari, 2010; Suzuki et al., 2010) and that the precursor compound of perylene may be 4,9-dihydroxyperylene-3,10-quinone (DHPQ) (Jiang et al., 2000; Grice et al., 2009; Itoh and Hanari, 2010) although perylene amount is also strongly dependent on their deposited environments (fungal spike; Nabbefeld et al., 2010). DHPQ from terrestrial organisms (e.g. fungi) has been suggested or proposed as an important candidate for a precursor of perylene by many authors since the discovery of perylene in sediments (e.g. Orr and Grady, 1967; Aizenshtat, 1973; Ishiwatari et al., 1980). Kumada found soil humic substance containing perylenequinone pigment (P-type humic acid), then he and his group found that Cenococcum graniforme (recently renamed to Cenococcum geophilum) is a precursor of a P-type humic acids containing the green fraction (Pg; Kumada and Hurst, 1967; Kumada, 1987). He suggests in his book a possibility of these perylenequinone pigments as a precursor of perylene in lake and marine sediments, writing “...the Pg in sclerotia as well as in soils may serve as one of the biogenic precursors of perylene in lake and sea sediments.” (Kumada, 1987). There is a published paper, reporting the presence of a large amount of precursor of perylene (DHPQ) in lake sediments including the Lake Biwa sediment, estimating its abundance to be
1.4–13 times larger than that of perylene (Ishiwatari and Matsushita, 1986). After sedimentation, DHPQ may be transformed to perylene over long periods of anoxic conditions (a precursor has a half-life of 58 years for the first-order rate and 110 years for the second-order rate; Gschwend et al., 1983). It is confirmed that perylene is transformed from pure DHPQ (Brown et al., 1954), DHPQ in fungal components (Sato, 1976b) and that in sediment samples (Ishiwatari and Matsushita, 1986) under extreme conditions (>290 °C). However, it is still unclear how the transformation from DHPQ to perylene occurs in environmental processes that require the removal of oxygen and aromatisation. Difficulties identifying the origin of perylene and the transformation process exist because transformation takes a long time, as mentioned above, and also because the DHPQ is not soluble in most organic solvents (Calderbank et al., 1954). Cenococcum geophilum (C. geophilum) is a type of rhizobia (Trappe, 1969; Massicotte et al., 1992; LoBuglio, 1999) that lives in catchment areas. Sclerotium grains are resting bodies of C. geophilum (Trappe, 1969; Massicotte et al., 1992; LoBuglio, 1999), and they are made of porous amorphous aluminium oxide (Watanabe et al., 2004, 2007; Sakagami, 2010). The sclerotium grains contain a remarkable amount of DHPQ (Kumada and Hurst, 1967; Sato, 1976b; Nakabayashi et al., 1982; Kumada, 1987), and DHPQ in the green fraction of humic acid (Pg) also originates from the C. geophilum sclerotium grains (Kumada and Hurst, 1967; Sato and Kumada, 1967; Sato, 1976b; Nakabayashi et al., 1982; Kumada, 1987). Moreover, both the sclerotium grains of C. geophilum (Trappe, 1969; Kumada, 1987; Massicotte et al., 1992; Watanabe et al., 2004, 2007; Sakagami, 2010) and Pg (Kumada and Hurst, 1967; Sato, 1976a; Kumada, 1987; Watanabe et al., 1996) are found throughout the world. Thus, there is a strong possibility that DHPQ from C. geophilum is a major precursor of sedimentary perylene around the world. Lake Biwa is one of the best locations for field studies to elucidate the origin of perylene because its sediments commonly contain very high amounts of perylene (lg g1-level; Ishiwatari and Hanya, 1975; Itoh et al., 2009, 2010a). These values are some of the highest concentrations of perylene found in sediment samples worldwide (Gschwend et al., 1983; Venkatesan, 1988; Jiang et al., 2000; Silliman et al., 2001; Grice et al., 2009; Suzuki et al., 2010). Recently, we reported that concentration of perylene increased with increase of terrestrial materials in the dated-sediment core collected from near the river’s mouth in Lake Biwa, and a transformation of perylene from its precursor compounds is ongoing within the deeper layers of sediments (Itoh et al., 2010a). Moreover, no remarkable concentration of perylene has been observed in the sinking particles collected from water at 5 m above the lake bottom (Itoh et al., 2010b). These results suggest that sedimentary perylene originates from outside the lake and that a transformation of perylene from its precursor compounds occurs mainly after deposition in Lake Biwa. Thus, in the Lake Biwa sediment samples, valuable information about not only the origin of perylene but also the transformation of perylene from its precursor compounds may be obtained.
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(a)
243
(b)
5 mm
20 µm
100 µm
(d)
(c)
200 µm
20 µm
Fig. 2. Photographs of the entire grains under white light (a) and SEM photographs of a cross section (b) of sclerotium grains of C. geophilum collected from soil and from sediment (c, d). The red arrows in (c) and (e) indicate septal pores.
In this study, we first attempted to find C. geophilum sclerotium grains in a soil sample collected from the Lake Biwa catchment area and in Lake Biwa sediments. Then, we observed each particle containing perylene using fluorescent microscopy and scanning electron microscopy (SEM). From spectrometric and elemental analyses, we found not only that perylene in the sediment of Lake Biwa originate from C. geophilum sclerotium grains from the catchment area but also that DHPQ transforms to perylene in sediment. 2. MATERIALS AND METHODS 2.1. Sampling location Lake Biwa is the largest lake in Japan (Fig. 1). The lake is surrounded by mountains and comprises a large northern basin (surface area, 614 km2; mean depth, 43 m; water volume, 27.3 km3) and a smaller southern basin (56 km2, 4 m, 0.2 km3). More than 100 rivers flow into the lake, and its catchment area is greater than 3000 km2. The input of compounds and sediment into the lake depends strongly on the rivers (Nakano et al., 2008). Most rivers flow into the north basin, whereas water drains from the lake via only one river (the Seta River) at the southern end of the south basin. Mt. Kanakuso is located at the riverhead of the Ane River (Fig. 1), which is one of the largest rivers flowing into the lake. 2.2. Samples and chemicals To represent the typical Lake Biwa sediment, NMIJ CRM 7307-a (National Metrology Institute of Japan, Ibaraki, Japan) was used because it not only contains perylene
at 2.1 lg g1 (Itoh et al., 2009), but it also is possible to obtain enough of this sample for analysis. It had been prepared from sediments collected from the south basin of Lake Biwa (35°40 1200 N, 135°550 1200 E) in 2001 (100 kg dry wt, surface sediment to a depth of 10 m from the lake bed) (Itoh et al., 2009). A brown earth soil sample of approximately 1 kg was collected from Mt. Kanakuso (35°320 1400 N, 136°200 3600 E), and C. geophilum sclerotium grains were collected from the soil. Chemical-analysis-grade perylene (used as the reagent perylene) and LC/MS-analysis-grade methanol (MeOH) as well as nitrobenzene, sodium dithionite, ethanol and chromium oxide (IV) were obtained from Wako Pure Chemical (Osaka, Japan). Aluminium oxide (approximately 150 mesh, surface area 155 m2 g1) was obtained from Sigma–Aldrich (MO, USA). Potassium hydroxide (KOH), concentrated sulphuric acid, hydrochloric acid, toluene, cyclohexane, anhydrous sodium sulphate (Na2SO4) and sodium chloride were obtained from Kanto Chemical (Tokyo, Japan). Crystalline [2H12]perylene was obtained from Cambridge Isotope Laboratories (Andover, MA, USA). The mixture of PAHs in toluene (SRM 2260a) used for GC/MS analysis was obtained from the US National Institute of Standards and Technology (NIST; Gaithersburg, MD, USA). 2.3. Sample preparation Sclerotium grains of C. geophilum collected from the soil were washed with Milli-Q water in quintuplicate using an ultrasonic bath. One gram of NMIJ CRM 7307-a was weighed in a 15 mL PP tube, and 12 mL of Milli-Q water was added, followed by
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Under white light
Under UV
(a)
(b)
(c)
(d)
(e)
(f)
(g)
Fig. 3. Photographs of particles from the top of the mesh obtained under white and UV light. Reagent perylene (a), 0.1% perylene doped on Al2O3 (b) and samples (c-g).
a 10 min wash in an ultrasonic bath. After 10 min, 10 mL of the supernatant was collected and filtered with a nylon mesh (40 lm) to separate the sclerotium grains from the mineral
solids and clays. The solution passing through the mesh (mesh-passed fraction) was collected and used for analyses of the carbon content and perylene content (see below).
N. Itoh et al. / Geochimica et Cosmochimica Acta 95 (2012) 241–251 120
Structural isomers
(a)
Relative intensity
20000
Intensity
15000
Perylene 10000
5000
30
35
40
45
50
(b)
252
80 60 40 20
126 112 100 120 140 160 180 200 220 240 260 280 300
55
120
20000
Relative intensity
(c) 15000
Intensity
100
0
0
10000
5000
100
(d)
252
80 60 40 125 112
20 0
0 30
35
40
45
50
100 120 140 160 180 200 220 240 260 280 300
55
1000
120
Relative intensity
(e)
800
Intensity
245
600 400 200 0
100
(f)
252
80 60 40 20
126 112
0 30
35
40
45
50
55
Time (min)
100 120 140 160 180 200 220 240 260 280 300
m/z
Fig. 4. Mass chromatogram of the reagent PAH mixture at m/z 252 (a) and mass spectrum of reagent perylene (b) and those of the fluorescent particles on the mesh (c and d) and the small yellow particles (e and f, Fig. 3g).
The sample collected on the mesh was washed again with 10 mL of Milli-Q water in an ultrasonic bath (5 min), and this procedure was repeated twice. The cleaned sample on the mesh was dried in the ambient air. In total, an entire bottle of NMIJ CRM 7307-a (60 g) was used. The small, black balls on the mesh were collected under white light. Each fluorescent particle was collected under UV light. Some fluorescent particles were used for perylene extraction, and the others were transferred to carbon tape for microscopic observation. 2.4. Absorption and fluorescence spectra of perylene The absorption (350–600 nm) and fluorescence spectra (Ex: 390 nm, Em: 400–600 nm) of the reagent perylene were obtained with the UV-2550 spectrophotometer (Shimadzu, Kyoto, Japan) and the RF-5300PC spectrofluorophotometer (Shimadzu, Kyoto, Japan), respectively. 2.5. Fluorescence microscopic observation The fluorescence microscope used was a BX51 (Olympus, Tokyo, Japan) equipped with a CCD camera (AxioCam MRc, Carl Zeiss, Oberkochen, Germany). A filter unit (excitation filter: 400–410 nm, absorption filter:
P455 nm, dichroic mirror: P450 nm) was selected for perylene fluorescence (Supplementary Fig. S1). A spectrometric system (Photonic Multichannel Analyzer, Hamamatsu Photonics, Shizuoka, Japan) was also used in combination with the fluorescence microscope for the spectral measurement of fluorescent particles (300–800 nm). 2.6. Identification and quantification of perylene The fluorescent particles collected under UV light were combined prior to extraction. Fluorescent particles too small to collect with micro-tweezers were flushed with cyclohexane and transferred to a back-deployed glass-fibre filter (only a few particles could be transferred due to the strong absorption on the mesh). The dried mesh-passed fraction and intact CRM7307-a were also used for perylene analysis; they were separately extracted using 30 mL of 1 M KOH/MeOH at 150 °C for 30 min using a MarsX MAE (microwave-assisted extraction) system (CEM, Matthews, NC, USA) and were analysed with a GC/MS as reported previously (Itoh et al., 2008), but with some modifications. In summary, the extract was filtered using a glass-fibre filter and shaken with 30 mL of Milli-Q water and 10 mL of toluene in a 250 mL separatory funnel. The lower layer was extracted again with 10 mL toluene, and this procedure
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White light
(a)
Fluoresence
(e)
SEM
(i)
Diagenetic Stage I
50 µm
(b)
(f)
(j)
Diagenetic Stage II
50 µm
(c)
(g)
(k)
Diagenetic Stage III
50 µm
(d)
(h)
(l)
Diagenetic Stage IV
50 µm
Fig. 5. Photographs of the fluorescent particles under white light (a–d), using a fluorescence microscope (e–h) and using SEM (i–l). The diagenetic stages correspond to those in Figs. 6 and 7 and Table 1.
was repeated twice. The combined toluene layers were washed with 30 mL of Milli-Q water and dried with anhydrous Na2SO4. The dried extracts were concentrated to 1 mL for the mesh-passed fraction and intact CRM7307-a (0.2 mL for fluorescent particles) using a rotary evaporator and a gentle stream of nitrogen after the addition of [2H12]perylene for quantification. The GC/MS analysis was carried out with a 6890/5975 system from Agilent Technologies (Palo Alto, CA, USA) equipped with a DB-17MS capillary column (30 m 0.25 mm, 0.25 lm film thickness) (J&W Scientific, Folsom, CA, USA) (Itoh et al., 2008, 2009). Data were obtained under the scan mode (m/z 100–300) for perylene identification and the selected ion mode (m/z 252.1 for perylene and m/z 264.2 for [2H12]perylene) for quantification. 2.7. Syntheses of 4,9-dihydroxy-3,10-perylenequinone and 3,10-perylenequinone The syntheses of 4,9-dihydroxy-3,10-perylenequinone (DHPQ) and 3,10-perylenequinone (3,10-PQ) are performed as described in the literature (Brass and Tengler, 1931; Calderbank et al., 1954), and the success of the syntheses was confirmed by comparison with absorption spectra from the literature (Calderbank et al., 1954; White et al., 1989) (Supplementary Fig. S2). 2.8. Scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDX) The SEM observations were performed with the TM1000 (Hitachi High-Tech, Tokyo, Japan), and the
EDX analyses were performed with the Genesis 4000 energy dispersive X-ray spectrometer (EDAX, Mahwah, NJ, USA) combined with the JSM-7400F scanning electron microscope (JEOL, Tokyo, Japan). 2.9. Total carbon, total organic carbon (TOC) and nitrogen contents of sediment 100-mg samples of sediment without and treated to remove carbonate (Itoh et al., 2003) were used to obtain the total carbon (organic + inorganic) and total organic carbon (TOC) contents with nitrogen content, respectively. They were analyzed with an elemental analyzer (Flash EA1112, Thermo Fisher Scientific). The total carbon, TOC and nitrogen contents of sediments (n = 4) were 1.11 ± 0.00 wt.%, 1.07 ± 0.01 wt.% and 0.070 ± 0.002 wt.%, respectively. 2.10. DHPQ in sclerotium grains DHPQ in sclerotium grains of C. geophilum was extracted with 1 M KOH/MeOH by ultrasonication in an ultrasonic water bath. The extract was centrifuged, and the supernatant was decanted. 1 M KOH/MeOH was then added to the precipitate, and the extraction and centrifugation were repeated twice. Although black colour of C. geophilum still remained, green colour of 1 M KOH/MeOH originating from DHPQ became scarce in the 3rd extract. The resulting supernatants were combined and compared with absorption spectrum of synthesized DHPQ for identification and quantification.
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3.1. Sclerotium grains of C. geophilum in soil and sediment Sclerotium grains of C. geophilum have been found throughout the world, including in central Japan (Trappe, 1969; Kumada, 1987; Massicotte et al., 1992; Watanabe et al., 2004, 2007; Sakagami, 2010). However, there are no reports of them in the Lake Biwa catchment area. Thus, we attempted to identify C. geophilum sclerotium grains from the catchment area of Lake Biwa. The location of soil sampling was chosen as Mt. Kanakuso because it is at the riverhead of the Ane River, which is one of the largest rivers flowing into the lake. In the soil sample, we found many gleaming black balls (approximately 40 balls kg1 dried soil for >1 mm-/, Fig. 2a). From their shape (with the bare eye and SEM), colour (gleaming black under white light) and elemental composition (mainly constituted from C, O and Al; see below), we identified these balls as sclerotium grains of C. geophilum (Fig. 2a and b). Presence of DHPQ in sclerotium grains of C. geophilum was also confirmed by absorption spectrum (Supplementary Fig. S3) and calculated to be more than 0.5% wt. From the sediment sample, we found many small black balls on the mesh. From their shape and their colour under white light (Fig. 2c) and from their porous structure with septal pores that were visible using SEM (Fig. 2d), we identified them as sclerotium grains. However, the elemental composition determined by EDX was different from that of the soil (see below). Because the sediment sample was collected near the downriver portion of the lake (Fig. 1), there is a strong possibility that the sclerotium grains had accumulated over the entire lake. 3.2. The characteristics of fluorescent particles on the mesh The particles collected on the mesh had various shapes and colours under white light. These included small black balls; small, clear minerals; pine pollen grains; filaments; and other coloured, rough particles. However, only some particles fluoresce under ultraviolet (UV) light (Fig. 3). Because perylene and solutions containing it fluoresce under UV light (Fig. 3 and Supplementary Fig. S1), we were able to pick these particles up with micro-tweezers and collect them. Fluorescent particles that were too small to collect with micro-tweezers (small yellow particles, Fig. 3g) were flushed with cyclohexane and transferred to a back-deployed glass-fibre filter. First, we confirmed the presence of perylene in the fluorescent particles by extraction and analysis of some of the fluorescent particles. Fig. 4 shows a chromatogram of the reagent PAH mixture (Fig. 4a) and a mass spectrum of reagent perylene (Fig. 4b), those of large fluorescent particles (Fig. 4c and d) and those of small yellow particles (Fig. 4e and f). From their retention time and mass spectra, the presence of perylene in both large fluorescent particles and small yellow particles can be confirmed. Then, each of the fluorescent particles on the mesh was transferred to carbon tape for microscopic observation. The
fluorescent particles commonly showed cream-to-white colours under white light (Fig. 5a–d) and blue fluorescence with UV light on the carbon tape. This blue fluorescence is characteristic of perylene; thus, these particles were observed using both fluorescence microscopy (Fig. 5e–h) and SEM (Fig. 5i–l). The different images provide different information (entire structures are visible with the fluorescence microscope and surface structures are observed with the SEM). The porous structures can be observed both in the bulk (Fig. 5e–h) and on the surface (Fig. 5i–l). Here, we categorised these particles into four diagenetic stages by their colour and surface structures. As the diagenetic
1.2
CO
1.0
Sclerotium grain (soil)
0.8
Al
0.6 0.4 0.2
Si
Fe
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1
2
C
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3
4
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7
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0.8 0.6
O
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Al Si
Fe
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C
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4
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Diagenetic Stage I
0.8 0.6
Relative intensity
3. RESULTS AND DISCUSSION
247
0.4
O
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Al Si
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C
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Diagenetic Stage II
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4
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Diagenetic Stage III
0.8 0.6 0.4
O
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1
2
3
C
1.0
4
5
6
7
Diagenetic Stage IV
0.8 0.6 0.4 0.2
O
Si
0.0 0
1
2
3
4
5
6
7
keV Fig. 6. EDX spectra of sclerotium grains and fluorescent particles. The elemental composition (C, O and Al) is reported in Table 1.
N. Itoh et al. / Geochimica et Cosmochimica Acta 95 (2012) 241–251
0.6
(a)
Reagent OH O
3.3. The process of transformation of DHPQ to perylene via 3,10-PQ
1.0
O
1.2
OH O
stage became more advanced, the colour observed with the fluorescence microscope changed from green to blue, and the surface lost its porosity.
O
248
0.8
Perylene 3,10-PQ (x200) DHPQ (x1000)
0.4 0.2
3.4. The transformation of the black balls in soil to fluorescent particles in sediment From the above results, transformation of DHPQ from perylene via 3,10-PQ (Fig. 7b–e) should be strongly associated with the loss of colour (Figs. 2a, c and 5a–d) and Al (Fig. 6) from sclerotium grains. Our results allowed us to
0.0 420 1.4 1.2
(b)
470
520
574 counts
570
620
670
Diagenetic Stage I
1.0 0.8 0.6 0.4 0.2 0.0 420 1.2 1.0
Relative intensity
From the photographs of the fluorescent microscope and SEM, it is apparent that shape and colour of fluorescent particles changed with advancing diagenetic stage. Because their constituents should change with advancing diagenesis, we examined them by EDX and fluorescent spectra. Fig. 6 shows the EDX spectra of sclerotium grains in soil and sediment as well as fluorescent particles at each diagenetic stage. The elemental compositions are reported in Table 1 with the results from fluorescent spectra (see below). In sclerotium grains collected from soil, C (62%), O (33%) and Al (4.5%) showed very high intensities. In contrast, sclerotium grains collected from sediment are dramatically lower in both O (21%) and Al (0.41%). Fluorescent particles contain approximately 10% O at any diagenetic stage, and Al decreased with increasing diagenetic stage (from 0.48% to 0.04%). Thus, the elemental compositions of fluorescent particles are quite different from those of the sclerotium grains collected from the soil. Prior to the collection of the fluorescent spectra, we synthesised DHPQ (the characteristic pigment of C. geophilum sclerotium grains; Kumada and Hurst, 1967; Sato, 1976b; Nakabayashi et al., 1982; Kumada, 1987) and 3,10-PQ (a possible intermediate compound in the transformation of DHPQ to perylene). Then, we attempted to explain each spectrum obtained from the particles in relation to these three compounds (Fig. 7a). Here, we did not consider dissociated DHPQ because the dissociation constant (pKa) of hypocrellin, containing DHPQ in its structure, is approximately 11 (He et al., 1999), and the pH of the sediments of Lake Biwa is approximately seven (Murphy et al., 2001). As the diagenetic stages increased (Fig. 7b–e), the fluorescent spectrum became that of perylene, and the relative amount of perylene increased (Table 1). Although both DHPQ (from 3438 to 98) and 3,10-PQ (from 554 to 70) decreased with increases diagenetic stage, the decrease was more dramatic for DHPQ than for 3,10-PQ. This result suggests that perylene was transformed from DHPQ via 3,10-PQ. Moreover, the calculated O/C ratios from the fluorescent spectra were 0.16–0.19 (as whole structures), which are consistent with those determined by EDX (0.11–0.15 at the surface; Table 1). Therefore, it can be concluded that perylene was transformed from DHPQ via 3,10PQ and that the organic content of these fluorescent particles is strongly dependent on DHPQ, 3,10-PQ and perylene.
(c)
470
520
1516 counts
570
620
670
Diagenetic Stage II
0.8 0.6 0.4 0.2 0.0 420 1.2 1.0
(d)
470
520
4281 counts
570
620
670
Diagenetic Stage III
0.8 0.6 0.4 0.2 0.0 420 1.2 1.0
(e)
470
520
5706 counts
570
620
670
Diagenetic Stage IV
0.8
Observed spectrum Calculated spectrum
0.6 0.4 0.2 0.0 420
470
520
570
620
670
Wavelength (nm) Fig. 7. The fluorescent spectra of perylene, 3,10-PQ and DHPQ with the filter unit (a) and the observed and calculated spectra of each fluorescent particle (b–e). The calculated spectra (b–e) were obtained from the spectra of perylene, 3,10-PQ and DHPQ (a).
elucidate the transformation of the black balls in soil to the fluorescent particles in sediment as follows. The sclerotium grains collected from the soil were gleaming black (Fig. 2a) and contained approximately 5% Al (Table 1), whereas the sclerotium grains in the sediment (Fig. 2c) were still black but had an Al content of less than 0.5%. During the transformation process, the colour became cream (Fig. 5a–d) and the grains had a comparable
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Table 1 Characteristics of samples, relative abundance of compounds calculated from fluorescence spectra and relative abundance of elements obtained by EDX. Sample
EDX
Fluoresence spectra
Relative abundance Name
Coloura
C
O
Al
Sclerotium grain (soil) Sclerotium grain (sediment) Diagenetic Stage I Diagenetic Stage II Diagenetic Stage III Diagenetic Stage IV
Gleaming Black Black Cream Cream Cream Cream
62 77 86 86 88 90
33 21 12 13 11 10
4.5 0.41 0.48 0.36 0.11 0.04
a b
O/Cb
O/C
Relative abundance Perylene
3,10-PQ
DHPQ
0.53 0.27 0.14 0.15 0.13 0.11
– – 1 1 1 1
– – 554 239 176 70
– – 3438 956 445 98
– – 0.19 0.18 0.17 0.16
observed colour under white light. calculated from the chemical compositions of perylene (C20H12), 3,10-PQ (C20H10O2), and DHPQ (C20H10O4).
Al content (0.11–0.48%) to that of black (Diagenetic stages IIII). Finally, the Al content decreased to 0.04%, and the fluorescence spectrum became similar to that of perylene (Fig. 7e, diagenetic stage IV). These results suggest that the loss of Al from the amorphous structure (Watanabe et al., 2004, 2007; Sakagami, 2010) should occur first, although DHPQ was not transformed to 3,10-PQ and perylene at this time. Then, the transformation of DHPQ to perylene via 3,10-PQ started increasing dramatically, and the particles lost their colour. Since remarkable amounts of 3,10-PQ exist, transformation of hydroxyl group to hydrogen should be occurred first followed by transformation of quinones to benzene (Blumer, 1965). Finally, Al was completely lost, and DHPQ became entirely perylene (Fig. 7e, Table 1). Although the transformation of DHPQ to perylene may begin in rivers and lakes, the last stage of the transformation to perylene likely occurs after deposition on the lake bottom (Gschwend et al., 1983; Silliman et al., 2001; Itoh et al., 2010a,b). Interestingly, the particles that had lost their colour were commonly part of large sclerotium grains, and black portions of large sclerotium grains were seldom observed, although the small sclerotium grains were nearly black (sometimes with white spots on their surfaces, Fig. 2c); no black balls lost their colour inside. Although some bacteria live inside sclerotium grains (Ohta et al., 2003; Nonoyama et al., 2009) and the microbiological transformation of DHPQ to perylene is suggested (Silliman et al., 2001), these bacteria should not be involved in DHPQ transformation. Thus, small sclerotium grains should be protected by the outer walls, and these walls should prevent the transformation of DHPQ to perylene. 3.5. The composition and state of perylene in the sediment samples We quantified perylene in the fluorescent particles by extraction, and the measured quantity only accounted for approximately 1/1000 of a given sediment sample (114 ng). However, this result is not surprising because not only we focused on collecting well-cleaned particles rather than on collecting as many particles as possible but also remarkable amount of DHPQ should be introduced
as Pg in humic acid (Kumada and Hurst, 1967; Kumada, 1987). In the mesh-passed fraction (approximately 1/6 of the total amount by weight), a higher perylene concentration and C content were observed and were responsible for approximately 1/3 of a given sample (150 lg perylene with 0.67 g carbon for a given sample and 42 lg perylene and 0.20 g carbon for the mesh-passed fraction). Since perylene relative to carbon in the mesh-passed fraction (210 lg g1) was comparable to that in bulk sediment (224 lg g1), the mesh-passed fraction represents the characteristics of a given sediment sample rather than the local presence of perylene and carbon in this fraction. On the other hand, perylene is also contained in small yellow particles adsorbed on the mesh (Figs. 3g, 4e and 4f). Thus, other parts of perylene and carbon (approximately 2/3 of a given sediment sample) could be significantly lost by both co-sinking with minerals (which likely have comparable characteristics to the mesh-passed fraction) and adsorption onto the mesh and PP tubes during our sample preparation. The hyphae of C. geophilum also contain DHPQ (Nakabayashi and Wada, 1991), and the DHPQ in the Pg is originated from sclerotium grains and hyphae of C. geophilum (Sato, 1976b; Nakabayashi et al., 1982; Kumada, 1987; Nakabayashi and Wada, 1991). Considering that the input of compounds to the lake depends strongly on the rivers (Nakano et al., 2008), the high (lg g1-level; Ishiwatari and Hanya, 1975; Itoh et al., 2009, 2010a) and continuous (over 200 m-core; Ishiwatari and Hanya, 1975) presence of perylene in the Lake Biwa sediments should have originated from the DHPQ of C. geophilum in its catchment area rather than from the insects and other fungi (Sato, 1976b; Jiang et al., 2000). 4. CONCLUSIONS We confirmed that the high and continuous presence of perylene in the Lake Biwa sediments originates mainly from C. geophilum in its catchment area. DHPQ in sclerotium grains of C. geophilum should be transformed to perylene via 3,10-PQ and loss of Al might be a trigger for this transformation. Although further investigation is required for other aquatic systems, there is a possibility that sedimentary
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perylene in other aquatic system is also contributed by C. geophilum attributed to global presence of C. geophilum and Pg. Furthermore, we anticipate that perylene in sediments will be a valuable indicator for the reconstruction of paleoenvironments through the growth characteristics of C. geophilum. ACKNOWLEDGEMENTS This work was supported by Grants-in-Aid for Young Scientists (B23710031 for N.I. and B23710014 for N.S.) from the Japan Society for the Promotion of Science (JSPS). The measurements of SEM/EDX were supported by the IBEC Center, AIST.
APPENDIX A. SUPPLEMENTARY DATA Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.gca.2012.07.037. REFERENCES Aizenshtat Z. (1973) Perylene and its geochemical significance. Geochim. Cosmochim. Acta 37, 559–567. Bechtel A., Widera M., Sachsenhofer R. F., Gratzer R., Lu¨cke A. and Woszczyk M. (2007) Biomarker and stable carbon isotope systematics of fossil wood from the second Lusatian lignite seam of the Lubsto´w deposit (Poland). Org. Geochem. 38, 1850– 1864. Blumer M. (1965) Organic pigments: their long-term fate. Science 149, 722–726. ¨ ber die Einwirkung von Brass K. and Tengler E. (1931) U Schwefelnatrium auf Dibrom-3,10-perylenechinon. Chem. Ber. 64, 1646–1649. Brown B. R., Johnson A. W., Quayle J. R. and Todd A. R. (1954) Colouring matters of the Aphididae Part VIII. Studies on the nature of the aromatic ring system in the Erythrophins. J. Chem. Soc. 1954, 107–112. Calderbank A., Johnson A. W. and Todd A. R. (1954) Colouring matters of the Aphidiae. Part X. Preparation and properties of 4:9-dihydroxyperylene-3:10-quinone. J. Chem. Soc. 1954, 1285– 1289. De Riccardis F., Iorizzi M., Minale L., Riccio R., de Forges B. R. and Debitus C. (1991) The gymnochromes: novel marine brominated phenanthroperylenequinone pigments from the stalked crinoid Gymnocrinus richeri. J. Org. Chem. 56, 6781– 6787. Finlayson-Pitts B. J. and Pitts, Jr., J. N. (2000) Chemistry of The Upper and Lower Atmosphere: Theory, Experiments, and Applications. Academic Press, London. Grice K., Lu H., Atahan P., Asif M., Hallmann C., Greenwood P., Maslen E., Tulipani S., Williford K. and Dodson J. (2009) New insights into the origin of perylene in geological samples. Geochim. Cosmochim. Acta 73, 6531–6543. Gschwend P. M., Chen P. H. and Hites R. A. (1983) On the formation of perylene in recent sediments: kinetic models. Geochim. Cosmochim. Acta 47, 2115–2119. He Y.-Y., An J.-Y. and Jiang L.-J. (1999) PH effect on the spectroscopic behavior and photoinduced generation of semiquinone anion radical of hypocrellin B. Dyes pigm. 41, 79–87. Hodgson G. W., Hitchon B., Taguchi K., Baker B. L. and Peake E. (1968) Geochemistry of porphyrins, chlorins and polycyclic
aromatics in soils, sediments and sedimentary rocks. Geochim. Cosmochim. Acta 32, 737–772. Ishiwatari R. and Hanya T. (1975) Organic geochemistry of a 200meter core sample from Lake Biwa II. Proc. Jpn. Acad. 51, 436– 441. Ishiwatari R. and Matsushita T. (1986) Determination of perylenequinones in sediments and soils. Proc. Jpn Acad. 62, 287–290. Ishiwatari R., Ogura K. and Horie S. (1980) Organic geochemistry of a lacustrine sediment (Lake Haruna, Japan). Chem. Geol. 29, 261–280. Itoh N. and Hanari N. (2010) Possible precursor of perylene in sediments of Lake Biwa elucidated by stable carbon isotope composition. Geochem. J. 44, 161–166. Itoh N., Numata M. and Yarita T. (2008) Alkaline extraction in combination with microwave-assisted extraction followed by solid-phase extraction treatment for polycyclic aromatic hydrocarbons in a sediment sample. Anal. Chim. Acta 615, 47–53. Itoh N., Aoyagi Y., Takatsu A. and Yarita T. (2009) Certified reference material for quantification of polycyclic aromatic hydrocarbons in sediment from the National Metrology Institute of Japan. Anal. Bioanal. Chem. 393, 2039–2049. Itoh N., Tamamura S. and Kumagai M. (2010a) Distributions of polycyclic aromatic hydrocarbons in a sediment core from the north basin of Lake Biwa, Japan. Org. Geochem. 41, 845–852. Itoh N., Tamamura S., Sato T. and Kumagai M. (2010b) Elucidation of polycyclic aromatic hydrocarbon sources in the sinking particles in Lake Biwa, Japan. Limnology 11, 241–250. Itoh N., Tani Y., Nagatani T. and Soma M. (2003) Phototrophic activity and redox condition in Lake Hamana, Japan, indicated by sedimentary photosynthetic pigments and molybdenum over the last 250 years. J. Paleolimnol. 29, 403–422. Jiang C., Alexander R., Kagi R. I. and Murray A. P. (2000) Origin of perylene in ancient sediments and its geological significance. Org. Geochem. 31, 1545–1559. Kumada K. (1987) Chemistry of Soil Organic Matter. Elsevier, Tokyo. Kumada K. and Hurst H. M. (1967) Green humic acid and its possible origin as a fungal metabolite. Nature 214, 631–633. LoBuglio K. F. (1999) Cenococcum. In Ectomycorrhizal Fungi. Key Genera in Profile (eds. J. W. G. Cairney and S. M. Chambers). Springer-Verlag, Berlin. pp. 287–309. Luch A. (2004) The Carcinogenetic Effects of Polycyclic Aromatic Hydrocarbons. Imperial College Press, London. Massicotte H. B., Trappe J. M., Peterson R. L. and Melville L. H. (1992) Studies on Cenococcum geophilum. II. Sclerotium morphology, germination, and formation in pure culture and growth puches. Can. J. Bot. 70, 125–132. Murphy T., Lawson A., Kumagai M. and Nalewajko C. (2001) Release of phosphorous from sediments in Lake Biwa. Limnology 2, 119–128. Nabbefeld B., Grice K., Summons R. E., Hays L. E. and Cao C. (2010) Significance of polycyclic aromatic hydrocarbons (PAHs) in Permian/Triassic boundary sections. Appl. Geochem. 25, 1374–1382. Nakabayashi K. and Wada H. (1991) Factors controlling Pg content in a dark brown forest soil. Soil Sci. Plant Nutr. 37, 93– 99. Nakabayashi K., Wada H. and Takai Y. (1982) Distribution patterns of Pg fraction of p-type humic acid and iron in a dark brown forest soil. Soil Sci. Plant Nutr. 28, 217–223. Nakano T., Tayasu I., Yamada Y., Hosono T., Igeta A., Hyodo F., Ando A., Saitoh Y., Tanaka T., Wada E. and Yachi S. (2008) Effect of agriculture on water quality of Lake Biwa tributaries, Japan. Sci. Total Environ. 389, 132–148. Nonoyama Y., Narisawa K., Ohta H. and Watanabe M. (2009) Bacterial community in sclerotia of Cenococcum species and soil
N. Itoh et al. / Geochimica et Cosmochimica Acta 95 (2012) 241–251 in sub-alpine forest, central Japan. Geophys. Res. Abstr. 11, EGU 2009–6512-1. Ohta H., Yagi M., Suzuki J., Fujitake N. and Watanabe M. (2003) Characterization of Sphingomonas species found as predominant members in the culturable bacterial community of a green pigment containing sclerotium grain from Mt. Myoko (Japan) volcanoic ash soil. Microbes Environ. 18, 126–132. Orr W. L. and Grady J. R. (1967) Perylene in basin sediments off southern California. Geochim. Cosmochim. Acta 31, 1201–1209. Sakagami N. (2010) Analysis on formation factor of sclerotia of Cenococcum geophilum in Picea abies forest, Harz Mts., Germany. Geogr. Rev. Jpn Ser. B 82, 184–195. Sato O. (1976a) The distribution of Pg (green fraction of P type humic acid) and the degree of humification of Pb (brown fraction of P type humic acid) in soils of central Japan. Soil Sci. Plant Nutr. 22, 159–167. Sato O. (1976b) A green pigment similar to the Pg fraction of P type humic acids and related compounds produced by litterdecomposing fungi. Soil Sci. Plant Nutr. 22, 269–275. Sato O. and Kumada K. (1967) The chemical nature of the green fraction of P type humic acid. Soil Sci. Plant Nutr. 13, 121–122. Silliman J. E., Meyers P. A., Eadie B. J. and Klump J. V. (2001) A hypothesis for the origin of perylene based on its low abundance in sediments of Green Bay, Wisconsin. Chem. Geol. 177, 309–322. Suzuki N., Yessalina S. and Kikuchi T. (2010) Possible fungal origin of perylene in Late Cretaceous to Paleogene terrestrial sedimentary rocks of northeastern Japan as indicated from stable carbon isotopes. Org. Geochem. 41, 234–241.
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Trappe J. M. (1969) Studies on Cenococcum graniforme. I. An efficient method for isolation from sclerotia. Can. J. Bot. 47, 1389–1390. Venkatesan M. I. (1988) Occurrence and possible sources of perylene in marine sediments – a review. Mar. Chem. 25, 1–27. Wakeham S. G., Schaffner C. and Giger W. (1979) Perylene in sediments from the Namibian Shelf. Geochim. Cosmochim. Acta 43, 1141–1144. Watanabe M., Tanaka H., Sakagami K., Aoki K. and Sugiyama S. (1996) Evaluation of Pg absorption strength of humic acids as a paleoenvironmental indicator in buried paleosols on tephra beds, Japan. Quat. Int. 34–36, 197–203. Watanabe M., Genseki A., Sakagami N., Inoue Y., Ohta H. and Fujitake N. (2004) Aluminium oxyhydroxide polymorphs and some micromorphological characteristics in sclerotium grains. Soil Sci. Plant Nutr. 50, 1205–1210. Watanabe M., Inoue Y., Sakagami N., Bolormaa O., Kawasaki K., Hiradate S., Fujitake N. and Ohta H. (2007) Characterization of major and trace elements in sclerotium grains. Eur. J. Soil Sci. 58, 786–793. White B., Nowakowska M. and Guillet J. E. (1989) Studies of the antenna effect in polymer molecules: photosensitized oxidation of perylene in an aqueous solution of poly(sodium styrenesulfonate-co-2-vynylnaphthalene. J. Photochem. Photobiol A 50, 147–156. Associate editor: Josef P. Werne
Geochimica et Cosmochimica Acta 95 (2012) 241–251 www.elsevier.com/locate/gca
Perylene in Lake Biwa sediments originating from Cenococcum geophilum in its catchment area Nobuyasu Itoh a,⇑, Nobuo Sakagami b, Masaki Torimura c, Makiko Watanabe d a
National Metrology Institute of Japan (NMIJ), National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1, Umezono, Tsukuba, Ibaraki 305-8563, Japan b College of Agriculture, Ibaraki University, 3-21-1, Chuuo, Ami, Inashiki, Ibaraki 300-0393, Japan c Research Institute for Environmental Management Technology, National Institute of Advanced, Industrial Science and Technology (AIST), 16-1 Onogawa, Tsukuba 305-8569, Japan d Graduate School of Urban Environmental Sciences, Tokyo Metropolitan University, 1-1, Minami-osawa, Hachioji, Tokyo 192-0397, Japan Received 21 February 2012; accepted in revised form 30 July 2012; available online 4 August 2012
Abstract Perylene, which is composed of five benzene rings, is commonly found in sediments throughout the world at concentrations and distributions that are different from those of other polycyclic aromatic hydrocarbons. The only information available on the origin of perylene comes from 4,9-dihydroxyperylene-3,10-quinone (DHPQ), which originates from fungal component symbiosis or from parasites on plants; however, there is no direct evidence of a mechanism of perylene formation. In this study, we examined the relationship between sedimentary perylene and Cenococcum geophilum (C. geophilum) in a catchment area at Lake Biwa. Sclerotium grains of C. geophilum containing DHPQ were found in this catchment area (approximately 40 balls kg1 dried soil for >1 mm-/), and small sclerotium grains were frequently found in the sediment. In the sediment sample, we also found broken particles containing perylene, and they had a porous structure characteristic of sclerotium grains. Furthermore, the particles contained DHPQ in different transformation stages to perylene via 3,10-perylenequinone (3,10-PQ). This finding was consistent with results from elemental analysis (oxygen/carbon). Because a remarkable amount of DHPQ originating from C. geophilum also exists in the humic acids of soils and because the inputs of compounds to the lake depend strongly on the rivers, perylene in the Lake Biwa sediment originates mainly from the DHPQ of C. geophilum in its catchment area. Ó 2012 Elsevier Ltd. All rights reserved.
1. INTRODUCTION Polycyclic aromatic hydrocarbons (PAHs), some of which are carcinogenic (Luch, 2004), are formed through incomplete combustion in anthropogenic processes, such as the burning of fossil fuels, and in natural processes such as forest fires (Finlayson-Pitts and Pitts Jr., 2000). Because they are well preserved for extended periods of time, their vertical distributions in sediment cores have been used to
⇑ Corresponding author. Tel.: +81 29 861 4318; fax: +81 29 861 6866. E-mail address: [email protected] (N. Itoh).
0016-7037/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.gca.2012.07.037
reconstruct human activities and natural events (Jiang et al., 2000; Grice et al., 2009; Itoh et al., 2010a). Perylene, which is composed of five benzene rings, is commonly found in sediments throughout the world at concentrations and distributions that are different from those of other polycyclic aromatic hydrocarbons (Venkatesan, 1988; Jiang et al., 2000; Grice et al., 2009; Itoh et al., 2010a). Since the discovery of perylene in aquatic sediments by Orr and Grady (1967: marine sediments) and Hodgson et al. (1968: fresh water lake), many researchers have reported the abundance of perylene in both marine and lacustrine sediments, and discussed on its source, route to sediments, reactions etc. One of the leading hypothesis is that the precursor material of perylene must be attributed
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N Japan Lake Biwa
Sampling point of soil
× 400 km
Ane River
90 70 40 20 90
The North Basin
The South Basin
Sampling point of sediment Seta River 10 km Fig. 1. Map of Lake Biwa and the sampling points. The dotted line is a catchment area of the lake.
to terrigenous materials, although there is another hypothesis that autochthonous organic matter from algal and crinoids’ productions are sources of precursor of perylene (Blumer, 1965; Wakeham et al., 1979; Venkatesan, 1988; De Riccardis et al., 1991). Recently, it has been suggested that perylene is originated from components of wooddegrading fungi (Jiang et al., 2000; Bechtel et al., 2007; Grice et al., 2009; Itoh and Hanari, 2010; Suzuki et al., 2010) and that the precursor compound of perylene may be 4,9-dihydroxyperylene-3,10-quinone (DHPQ) (Jiang et al., 2000; Grice et al., 2009; Itoh and Hanari, 2010) although perylene amount is also strongly dependent on their deposited environments (fungal spike; Nabbefeld et al., 2010). DHPQ from terrestrial organisms (e.g. fungi) has been suggested or proposed as an important candidate for a precursor of perylene by many authors since the discovery of perylene in sediments (e.g. Orr and Grady, 1967; Aizenshtat, 1973; Ishiwatari et al., 1980). Kumada found soil humic substance containing perylenequinone pigment (P-type humic acid), then he and his group found that Cenococcum graniforme (recently renamed to Cenococcum geophilum) is a precursor of a P-type humic acids containing the green fraction (Pg; Kumada and Hurst, 1967; Kumada, 1987). He suggests in his book a possibility of these perylenequinone pigments as a precursor of perylene in lake and marine sediments, writing “...the Pg in sclerotia as well as in soils may serve as one of the biogenic precursors of perylene in lake and sea sediments.” (Kumada, 1987). There is a published paper, reporting the presence of a large amount of precursor of perylene (DHPQ) in lake sediments including the Lake Biwa sediment, estimating its abundance to be
1.4–13 times larger than that of perylene (Ishiwatari and Matsushita, 1986). After sedimentation, DHPQ may be transformed to perylene over long periods of anoxic conditions (a precursor has a half-life of 58 years for the first-order rate and 110 years for the second-order rate; Gschwend et al., 1983). It is confirmed that perylene is transformed from pure DHPQ (Brown et al., 1954), DHPQ in fungal components (Sato, 1976b) and that in sediment samples (Ishiwatari and Matsushita, 1986) under extreme conditions (>290 °C). However, it is still unclear how the transformation from DHPQ to perylene occurs in environmental processes that require the removal of oxygen and aromatisation. Difficulties identifying the origin of perylene and the transformation process exist because transformation takes a long time, as mentioned above, and also because the DHPQ is not soluble in most organic solvents (Calderbank et al., 1954). Cenococcum geophilum (C. geophilum) is a type of rhizobia (Trappe, 1969; Massicotte et al., 1992; LoBuglio, 1999) that lives in catchment areas. Sclerotium grains are resting bodies of C. geophilum (Trappe, 1969; Massicotte et al., 1992; LoBuglio, 1999), and they are made of porous amorphous aluminium oxide (Watanabe et al., 2004, 2007; Sakagami, 2010). The sclerotium grains contain a remarkable amount of DHPQ (Kumada and Hurst, 1967; Sato, 1976b; Nakabayashi et al., 1982; Kumada, 1987), and DHPQ in the green fraction of humic acid (Pg) also originates from the C. geophilum sclerotium grains (Kumada and Hurst, 1967; Sato and Kumada, 1967; Sato, 1976b; Nakabayashi et al., 1982; Kumada, 1987). Moreover, both the sclerotium grains of C. geophilum (Trappe, 1969; Kumada, 1987; Massicotte et al., 1992; Watanabe et al., 2004, 2007; Sakagami, 2010) and Pg (Kumada and Hurst, 1967; Sato, 1976a; Kumada, 1987; Watanabe et al., 1996) are found throughout the world. Thus, there is a strong possibility that DHPQ from C. geophilum is a major precursor of sedimentary perylene around the world. Lake Biwa is one of the best locations for field studies to elucidate the origin of perylene because its sediments commonly contain very high amounts of perylene (lg g1-level; Ishiwatari and Hanya, 1975; Itoh et al., 2009, 2010a). These values are some of the highest concentrations of perylene found in sediment samples worldwide (Gschwend et al., 1983; Venkatesan, 1988; Jiang et al., 2000; Silliman et al., 2001; Grice et al., 2009; Suzuki et al., 2010). Recently, we reported that concentration of perylene increased with increase of terrestrial materials in the dated-sediment core collected from near the river’s mouth in Lake Biwa, and a transformation of perylene from its precursor compounds is ongoing within the deeper layers of sediments (Itoh et al., 2010a). Moreover, no remarkable concentration of perylene has been observed in the sinking particles collected from water at 5 m above the lake bottom (Itoh et al., 2010b). These results suggest that sedimentary perylene originates from outside the lake and that a transformation of perylene from its precursor compounds occurs mainly after deposition in Lake Biwa. Thus, in the Lake Biwa sediment samples, valuable information about not only the origin of perylene but also the transformation of perylene from its precursor compounds may be obtained.
N. Itoh et al. / Geochimica et Cosmochimica Acta 95 (2012) 241–251
(a)
243
(b)
5 mm
20 µm
100 µm
(d)
(c)
200 µm
20 µm
Fig. 2. Photographs of the entire grains under white light (a) and SEM photographs of a cross section (b) of sclerotium grains of C. geophilum collected from soil and from sediment (c, d). The red arrows in (c) and (e) indicate septal pores.
In this study, we first attempted to find C. geophilum sclerotium grains in a soil sample collected from the Lake Biwa catchment area and in Lake Biwa sediments. Then, we observed each particle containing perylene using fluorescent microscopy and scanning electron microscopy (SEM). From spectrometric and elemental analyses, we found not only that perylene in the sediment of Lake Biwa originate from C. geophilum sclerotium grains from the catchment area but also that DHPQ transforms to perylene in sediment. 2. MATERIALS AND METHODS 2.1. Sampling location Lake Biwa is the largest lake in Japan (Fig. 1). The lake is surrounded by mountains and comprises a large northern basin (surface area, 614 km2; mean depth, 43 m; water volume, 27.3 km3) and a smaller southern basin (56 km2, 4 m, 0.2 km3). More than 100 rivers flow into the lake, and its catchment area is greater than 3000 km2. The input of compounds and sediment into the lake depends strongly on the rivers (Nakano et al., 2008). Most rivers flow into the north basin, whereas water drains from the lake via only one river (the Seta River) at the southern end of the south basin. Mt. Kanakuso is located at the riverhead of the Ane River (Fig. 1), which is one of the largest rivers flowing into the lake. 2.2. Samples and chemicals To represent the typical Lake Biwa sediment, NMIJ CRM 7307-a (National Metrology Institute of Japan, Ibaraki, Japan) was used because it not only contains perylene
at 2.1 lg g1 (Itoh et al., 2009), but it also is possible to obtain enough of this sample for analysis. It had been prepared from sediments collected from the south basin of Lake Biwa (35°40 1200 N, 135°550 1200 E) in 2001 (100 kg dry wt, surface sediment to a depth of 10 m from the lake bed) (Itoh et al., 2009). A brown earth soil sample of approximately 1 kg was collected from Mt. Kanakuso (35°320 1400 N, 136°200 3600 E), and C. geophilum sclerotium grains were collected from the soil. Chemical-analysis-grade perylene (used as the reagent perylene) and LC/MS-analysis-grade methanol (MeOH) as well as nitrobenzene, sodium dithionite, ethanol and chromium oxide (IV) were obtained from Wako Pure Chemical (Osaka, Japan). Aluminium oxide (approximately 150 mesh, surface area 155 m2 g1) was obtained from Sigma–Aldrich (MO, USA). Potassium hydroxide (KOH), concentrated sulphuric acid, hydrochloric acid, toluene, cyclohexane, anhydrous sodium sulphate (Na2SO4) and sodium chloride were obtained from Kanto Chemical (Tokyo, Japan). Crystalline [2H12]perylene was obtained from Cambridge Isotope Laboratories (Andover, MA, USA). The mixture of PAHs in toluene (SRM 2260a) used for GC/MS analysis was obtained from the US National Institute of Standards and Technology (NIST; Gaithersburg, MD, USA). 2.3. Sample preparation Sclerotium grains of C. geophilum collected from the soil were washed with Milli-Q water in quintuplicate using an ultrasonic bath. One gram of NMIJ CRM 7307-a was weighed in a 15 mL PP tube, and 12 mL of Milli-Q water was added, followed by
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Under white light
Under UV
(a)
(b)
(c)
(d)
(e)
(f)
(g)
Fig. 3. Photographs of particles from the top of the mesh obtained under white and UV light. Reagent perylene (a), 0.1% perylene doped on Al2O3 (b) and samples (c-g).
a 10 min wash in an ultrasonic bath. After 10 min, 10 mL of the supernatant was collected and filtered with a nylon mesh (40 lm) to separate the sclerotium grains from the mineral
solids and clays. The solution passing through the mesh (mesh-passed fraction) was collected and used for analyses of the carbon content and perylene content (see below).
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m/z
Fig. 4. Mass chromatogram of the reagent PAH mixture at m/z 252 (a) and mass spectrum of reagent perylene (b) and those of the fluorescent particles on the mesh (c and d) and the small yellow particles (e and f, Fig. 3g).
The sample collected on the mesh was washed again with 10 mL of Milli-Q water in an ultrasonic bath (5 min), and this procedure was repeated twice. The cleaned sample on the mesh was dried in the ambient air. In total, an entire bottle of NMIJ CRM 7307-a (60 g) was used. The small, black balls on the mesh were collected under white light. Each fluorescent particle was collected under UV light. Some fluorescent particles were used for perylene extraction, and the others were transferred to carbon tape for microscopic observation. 2.4. Absorption and fluorescence spectra of perylene The absorption (350–600 nm) and fluorescence spectra (Ex: 390 nm, Em: 400–600 nm) of the reagent perylene were obtained with the UV-2550 spectrophotometer (Shimadzu, Kyoto, Japan) and the RF-5300PC spectrofluorophotometer (Shimadzu, Kyoto, Japan), respectively. 2.5. Fluorescence microscopic observation The fluorescence microscope used was a BX51 (Olympus, Tokyo, Japan) equipped with a CCD camera (AxioCam MRc, Carl Zeiss, Oberkochen, Germany). A filter unit (excitation filter: 400–410 nm, absorption filter:
P455 nm, dichroic mirror: P450 nm) was selected for perylene fluorescence (Supplementary Fig. S1). A spectrometric system (Photonic Multichannel Analyzer, Hamamatsu Photonics, Shizuoka, Japan) was also used in combination with the fluorescence microscope for the spectral measurement of fluorescent particles (300–800 nm). 2.6. Identification and quantification of perylene The fluorescent particles collected under UV light were combined prior to extraction. Fluorescent particles too small to collect with micro-tweezers were flushed with cyclohexane and transferred to a back-deployed glass-fibre filter (only a few particles could be transferred due to the strong absorption on the mesh). The dried mesh-passed fraction and intact CRM7307-a were also used for perylene analysis; they were separately extracted using 30 mL of 1 M KOH/MeOH at 150 °C for 30 min using a MarsX MAE (microwave-assisted extraction) system (CEM, Matthews, NC, USA) and were analysed with a GC/MS as reported previously (Itoh et al., 2008), but with some modifications. In summary, the extract was filtered using a glass-fibre filter and shaken with 30 mL of Milli-Q water and 10 mL of toluene in a 250 mL separatory funnel. The lower layer was extracted again with 10 mL toluene, and this procedure
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White light
(a)
Fluoresence
(e)
SEM
(i)
Diagenetic Stage I
50 µm
(b)
(f)
(j)
Diagenetic Stage II
50 µm
(c)
(g)
(k)
Diagenetic Stage III
50 µm
(d)
(h)
(l)
Diagenetic Stage IV
50 µm
Fig. 5. Photographs of the fluorescent particles under white light (a–d), using a fluorescence microscope (e–h) and using SEM (i–l). The diagenetic stages correspond to those in Figs. 6 and 7 and Table 1.
was repeated twice. The combined toluene layers were washed with 30 mL of Milli-Q water and dried with anhydrous Na2SO4. The dried extracts were concentrated to 1 mL for the mesh-passed fraction and intact CRM7307-a (0.2 mL for fluorescent particles) using a rotary evaporator and a gentle stream of nitrogen after the addition of [2H12]perylene for quantification. The GC/MS analysis was carried out with a 6890/5975 system from Agilent Technologies (Palo Alto, CA, USA) equipped with a DB-17MS capillary column (30 m 0.25 mm, 0.25 lm film thickness) (J&W Scientific, Folsom, CA, USA) (Itoh et al., 2008, 2009). Data were obtained under the scan mode (m/z 100–300) for perylene identification and the selected ion mode (m/z 252.1 for perylene and m/z 264.2 for [2H12]perylene) for quantification. 2.7. Syntheses of 4,9-dihydroxy-3,10-perylenequinone and 3,10-perylenequinone The syntheses of 4,9-dihydroxy-3,10-perylenequinone (DHPQ) and 3,10-perylenequinone (3,10-PQ) are performed as described in the literature (Brass and Tengler, 1931; Calderbank et al., 1954), and the success of the syntheses was confirmed by comparison with absorption spectra from the literature (Calderbank et al., 1954; White et al., 1989) (Supplementary Fig. S2). 2.8. Scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDX) The SEM observations were performed with the TM1000 (Hitachi High-Tech, Tokyo, Japan), and the
EDX analyses were performed with the Genesis 4000 energy dispersive X-ray spectrometer (EDAX, Mahwah, NJ, USA) combined with the JSM-7400F scanning electron microscope (JEOL, Tokyo, Japan). 2.9. Total carbon, total organic carbon (TOC) and nitrogen contents of sediment 100-mg samples of sediment without and treated to remove carbonate (Itoh et al., 2003) were used to obtain the total carbon (organic + inorganic) and total organic carbon (TOC) contents with nitrogen content, respectively. They were analyzed with an elemental analyzer (Flash EA1112, Thermo Fisher Scientific). The total carbon, TOC and nitrogen contents of sediments (n = 4) were 1.11 ± 0.00 wt.%, 1.07 ± 0.01 wt.% and 0.070 ± 0.002 wt.%, respectively. 2.10. DHPQ in sclerotium grains DHPQ in sclerotium grains of C. geophilum was extracted with 1 M KOH/MeOH by ultrasonication in an ultrasonic water bath. The extract was centrifuged, and the supernatant was decanted. 1 M KOH/MeOH was then added to the precipitate, and the extraction and centrifugation were repeated twice. Although black colour of C. geophilum still remained, green colour of 1 M KOH/MeOH originating from DHPQ became scarce in the 3rd extract. The resulting supernatants were combined and compared with absorption spectrum of synthesized DHPQ for identification and quantification.
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3.1. Sclerotium grains of C. geophilum in soil and sediment Sclerotium grains of C. geophilum have been found throughout the world, including in central Japan (Trappe, 1969; Kumada, 1987; Massicotte et al., 1992; Watanabe et al., 2004, 2007; Sakagami, 2010). However, there are no reports of them in the Lake Biwa catchment area. Thus, we attempted to identify C. geophilum sclerotium grains from the catchment area of Lake Biwa. The location of soil sampling was chosen as Mt. Kanakuso because it is at the riverhead of the Ane River, which is one of the largest rivers flowing into the lake. In the soil sample, we found many gleaming black balls (approximately 40 balls kg1 dried soil for >1 mm-/, Fig. 2a). From their shape (with the bare eye and SEM), colour (gleaming black under white light) and elemental composition (mainly constituted from C, O and Al; see below), we identified these balls as sclerotium grains of C. geophilum (Fig. 2a and b). Presence of DHPQ in sclerotium grains of C. geophilum was also confirmed by absorption spectrum (Supplementary Fig. S3) and calculated to be more than 0.5% wt. From the sediment sample, we found many small black balls on the mesh. From their shape and their colour under white light (Fig. 2c) and from their porous structure with septal pores that were visible using SEM (Fig. 2d), we identified them as sclerotium grains. However, the elemental composition determined by EDX was different from that of the soil (see below). Because the sediment sample was collected near the downriver portion of the lake (Fig. 1), there is a strong possibility that the sclerotium grains had accumulated over the entire lake. 3.2. The characteristics of fluorescent particles on the mesh The particles collected on the mesh had various shapes and colours under white light. These included small black balls; small, clear minerals; pine pollen grains; filaments; and other coloured, rough particles. However, only some particles fluoresce under ultraviolet (UV) light (Fig. 3). Because perylene and solutions containing it fluoresce under UV light (Fig. 3 and Supplementary Fig. S1), we were able to pick these particles up with micro-tweezers and collect them. Fluorescent particles that were too small to collect with micro-tweezers (small yellow particles, Fig. 3g) were flushed with cyclohexane and transferred to a back-deployed glass-fibre filter. First, we confirmed the presence of perylene in the fluorescent particles by extraction and analysis of some of the fluorescent particles. Fig. 4 shows a chromatogram of the reagent PAH mixture (Fig. 4a) and a mass spectrum of reagent perylene (Fig. 4b), those of large fluorescent particles (Fig. 4c and d) and those of small yellow particles (Fig. 4e and f). From their retention time and mass spectra, the presence of perylene in both large fluorescent particles and small yellow particles can be confirmed. Then, each of the fluorescent particles on the mesh was transferred to carbon tape for microscopic observation. The
fluorescent particles commonly showed cream-to-white colours under white light (Fig. 5a–d) and blue fluorescence with UV light on the carbon tape. This blue fluorescence is characteristic of perylene; thus, these particles were observed using both fluorescence microscopy (Fig. 5e–h) and SEM (Fig. 5i–l). The different images provide different information (entire structures are visible with the fluorescence microscope and surface structures are observed with the SEM). The porous structures can be observed both in the bulk (Fig. 5e–h) and on the surface (Fig. 5i–l). Here, we categorised these particles into four diagenetic stages by their colour and surface structures. As the diagenetic
1.2
CO
1.0
Sclerotium grain (soil)
0.8
Al
0.6 0.4 0.2
Si
Fe
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1
2
C
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3
4
5
6
7
Sclerotium grain (sediment)
0.8 0.6
O
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Al Si
Fe
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1.0
4
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7
Diagenetic Stage I
0.8 0.6
Relative intensity
3. RESULTS AND DISCUSSION
247
0.4
O
0.2
Al Si
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1
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3
C
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4
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7
Diagenetic Stage II
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Al Si
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1
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4
5
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Diagenetic Stage III
0.8 0.6 0.4
O
0.2
Si
0.0 0 1.2
1
2
3
C
1.0
4
5
6
7
Diagenetic Stage IV
0.8 0.6 0.4 0.2
O
Si
0.0 0
1
2
3
4
5
6
7
keV Fig. 6. EDX spectra of sclerotium grains and fluorescent particles. The elemental composition (C, O and Al) is reported in Table 1.
N. Itoh et al. / Geochimica et Cosmochimica Acta 95 (2012) 241–251
0.6
(a)
Reagent OH O
3.3. The process of transformation of DHPQ to perylene via 3,10-PQ
1.0
O
1.2
OH O
stage became more advanced, the colour observed with the fluorescence microscope changed from green to blue, and the surface lost its porosity.
O
248
0.8
Perylene 3,10-PQ (x200) DHPQ (x1000)
0.4 0.2
3.4. The transformation of the black balls in soil to fluorescent particles in sediment From the above results, transformation of DHPQ from perylene via 3,10-PQ (Fig. 7b–e) should be strongly associated with the loss of colour (Figs. 2a, c and 5a–d) and Al (Fig. 6) from sclerotium grains. Our results allowed us to
0.0 420 1.4 1.2
(b)
470
520
574 counts
570
620
670
Diagenetic Stage I
1.0 0.8 0.6 0.4 0.2 0.0 420 1.2 1.0
Relative intensity
From the photographs of the fluorescent microscope and SEM, it is apparent that shape and colour of fluorescent particles changed with advancing diagenetic stage. Because their constituents should change with advancing diagenesis, we examined them by EDX and fluorescent spectra. Fig. 6 shows the EDX spectra of sclerotium grains in soil and sediment as well as fluorescent particles at each diagenetic stage. The elemental compositions are reported in Table 1 with the results from fluorescent spectra (see below). In sclerotium grains collected from soil, C (62%), O (33%) and Al (4.5%) showed very high intensities. In contrast, sclerotium grains collected from sediment are dramatically lower in both O (21%) and Al (0.41%). Fluorescent particles contain approximately 10% O at any diagenetic stage, and Al decreased with increasing diagenetic stage (from 0.48% to 0.04%). Thus, the elemental compositions of fluorescent particles are quite different from those of the sclerotium grains collected from the soil. Prior to the collection of the fluorescent spectra, we synthesised DHPQ (the characteristic pigment of C. geophilum sclerotium grains; Kumada and Hurst, 1967; Sato, 1976b; Nakabayashi et al., 1982; Kumada, 1987) and 3,10-PQ (a possible intermediate compound in the transformation of DHPQ to perylene). Then, we attempted to explain each spectrum obtained from the particles in relation to these three compounds (Fig. 7a). Here, we did not consider dissociated DHPQ because the dissociation constant (pKa) of hypocrellin, containing DHPQ in its structure, is approximately 11 (He et al., 1999), and the pH of the sediments of Lake Biwa is approximately seven (Murphy et al., 2001). As the diagenetic stages increased (Fig. 7b–e), the fluorescent spectrum became that of perylene, and the relative amount of perylene increased (Table 1). Although both DHPQ (from 3438 to 98) and 3,10-PQ (from 554 to 70) decreased with increases diagenetic stage, the decrease was more dramatic for DHPQ than for 3,10-PQ. This result suggests that perylene was transformed from DHPQ via 3,10-PQ. Moreover, the calculated O/C ratios from the fluorescent spectra were 0.16–0.19 (as whole structures), which are consistent with those determined by EDX (0.11–0.15 at the surface; Table 1). Therefore, it can be concluded that perylene was transformed from DHPQ via 3,10PQ and that the organic content of these fluorescent particles is strongly dependent on DHPQ, 3,10-PQ and perylene.
(c)
470
520
1516 counts
570
620
670
Diagenetic Stage II
0.8 0.6 0.4 0.2 0.0 420 1.2 1.0
(d)
470
520
4281 counts
570
620
670
Diagenetic Stage III
0.8 0.6 0.4 0.2 0.0 420 1.2 1.0
(e)
470
520
5706 counts
570
620
670
Diagenetic Stage IV
0.8
Observed spectrum Calculated spectrum
0.6 0.4 0.2 0.0 420
470
520
570
620
670
Wavelength (nm) Fig. 7. The fluorescent spectra of perylene, 3,10-PQ and DHPQ with the filter unit (a) and the observed and calculated spectra of each fluorescent particle (b–e). The calculated spectra (b–e) were obtained from the spectra of perylene, 3,10-PQ and DHPQ (a).
elucidate the transformation of the black balls in soil to the fluorescent particles in sediment as follows. The sclerotium grains collected from the soil were gleaming black (Fig. 2a) and contained approximately 5% Al (Table 1), whereas the sclerotium grains in the sediment (Fig. 2c) were still black but had an Al content of less than 0.5%. During the transformation process, the colour became cream (Fig. 5a–d) and the grains had a comparable
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Table 1 Characteristics of samples, relative abundance of compounds calculated from fluorescence spectra and relative abundance of elements obtained by EDX. Sample
EDX
Fluoresence spectra
Relative abundance Name
Coloura
C
O
Al
Sclerotium grain (soil) Sclerotium grain (sediment) Diagenetic Stage I Diagenetic Stage II Diagenetic Stage III Diagenetic Stage IV
Gleaming Black Black Cream Cream Cream Cream
62 77 86 86 88 90
33 21 12 13 11 10
4.5 0.41 0.48 0.36 0.11 0.04
a b
O/Cb
O/C
Relative abundance Perylene
3,10-PQ
DHPQ
0.53 0.27 0.14 0.15 0.13 0.11
– – 1 1 1 1
– – 554 239 176 70
– – 3438 956 445 98
– – 0.19 0.18 0.17 0.16
observed colour under white light. calculated from the chemical compositions of perylene (C20H12), 3,10-PQ (C20H10O2), and DHPQ (C20H10O4).
Al content (0.11–0.48%) to that of black (Diagenetic stages IIII). Finally, the Al content decreased to 0.04%, and the fluorescence spectrum became similar to that of perylene (Fig. 7e, diagenetic stage IV). These results suggest that the loss of Al from the amorphous structure (Watanabe et al., 2004, 2007; Sakagami, 2010) should occur first, although DHPQ was not transformed to 3,10-PQ and perylene at this time. Then, the transformation of DHPQ to perylene via 3,10-PQ started increasing dramatically, and the particles lost their colour. Since remarkable amounts of 3,10-PQ exist, transformation of hydroxyl group to hydrogen should be occurred first followed by transformation of quinones to benzene (Blumer, 1965). Finally, Al was completely lost, and DHPQ became entirely perylene (Fig. 7e, Table 1). Although the transformation of DHPQ to perylene may begin in rivers and lakes, the last stage of the transformation to perylene likely occurs after deposition on the lake bottom (Gschwend et al., 1983; Silliman et al., 2001; Itoh et al., 2010a,b). Interestingly, the particles that had lost their colour were commonly part of large sclerotium grains, and black portions of large sclerotium grains were seldom observed, although the small sclerotium grains were nearly black (sometimes with white spots on their surfaces, Fig. 2c); no black balls lost their colour inside. Although some bacteria live inside sclerotium grains (Ohta et al., 2003; Nonoyama et al., 2009) and the microbiological transformation of DHPQ to perylene is suggested (Silliman et al., 2001), these bacteria should not be involved in DHPQ transformation. Thus, small sclerotium grains should be protected by the outer walls, and these walls should prevent the transformation of DHPQ to perylene. 3.5. The composition and state of perylene in the sediment samples We quantified perylene in the fluorescent particles by extraction, and the measured quantity only accounted for approximately 1/1000 of a given sediment sample (114 ng). However, this result is not surprising because not only we focused on collecting well-cleaned particles rather than on collecting as many particles as possible but also remarkable amount of DHPQ should be introduced
as Pg in humic acid (Kumada and Hurst, 1967; Kumada, 1987). In the mesh-passed fraction (approximately 1/6 of the total amount by weight), a higher perylene concentration and C content were observed and were responsible for approximately 1/3 of a given sample (150 lg perylene with 0.67 g carbon for a given sample and 42 lg perylene and 0.20 g carbon for the mesh-passed fraction). Since perylene relative to carbon in the mesh-passed fraction (210 lg g1) was comparable to that in bulk sediment (224 lg g1), the mesh-passed fraction represents the characteristics of a given sediment sample rather than the local presence of perylene and carbon in this fraction. On the other hand, perylene is also contained in small yellow particles adsorbed on the mesh (Figs. 3g, 4e and 4f). Thus, other parts of perylene and carbon (approximately 2/3 of a given sediment sample) could be significantly lost by both co-sinking with minerals (which likely have comparable characteristics to the mesh-passed fraction) and adsorption onto the mesh and PP tubes during our sample preparation. The hyphae of C. geophilum also contain DHPQ (Nakabayashi and Wada, 1991), and the DHPQ in the Pg is originated from sclerotium grains and hyphae of C. geophilum (Sato, 1976b; Nakabayashi et al., 1982; Kumada, 1987; Nakabayashi and Wada, 1991). Considering that the input of compounds to the lake depends strongly on the rivers (Nakano et al., 2008), the high (lg g1-level; Ishiwatari and Hanya, 1975; Itoh et al., 2009, 2010a) and continuous (over 200 m-core; Ishiwatari and Hanya, 1975) presence of perylene in the Lake Biwa sediments should have originated from the DHPQ of C. geophilum in its catchment area rather than from the insects and other fungi (Sato, 1976b; Jiang et al., 2000). 4. CONCLUSIONS We confirmed that the high and continuous presence of perylene in the Lake Biwa sediments originates mainly from C. geophilum in its catchment area. DHPQ in sclerotium grains of C. geophilum should be transformed to perylene via 3,10-PQ and loss of Al might be a trigger for this transformation. Although further investigation is required for other aquatic systems, there is a possibility that sedimentary
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perylene in other aquatic system is also contributed by C. geophilum attributed to global presence of C. geophilum and Pg. Furthermore, we anticipate that perylene in sediments will be a valuable indicator for the reconstruction of paleoenvironments through the growth characteristics of C. geophilum. ACKNOWLEDGEMENTS This work was supported by Grants-in-Aid for Young Scientists (B23710031 for N.I. and B23710014 for N.S.) from the Japan Society for the Promotion of Science (JSPS). The measurements of SEM/EDX were supported by the IBEC Center, AIST.
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