A New Indicator of 15N Signature for Detecting Nutrient Supply Effects To
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Algal Resources (2016) 9：1-13 A new indicator of 15N signature for detecting nutrient supply effects to Saccharina japonica var. religiosa Takanori KURIBAYASHI1,2*, Shoichi AKAIKE3 and Shigeru MONTANI2,4 Abstract : A possible new indicator of 15N for detecting nutrient supply effects to Saccharina japonica var. religiosa was investigated. Nutrient supply was examined by 15 15 adding (NH4)2SO4 fertilizer which shows lower Nthan N-NO3 of natural seawater on the southwest coast of Hokkaido, Japan, and the fertilizing amounts and periods were adjusted each year (2009-2013). The 15NinS. japonica var. religiosa and other species of 15 seaweeds before fertilization were close to N-NO3 in Tsushima Warm Current. After fertilization, S. japonica var. religiosa growth promotion and algal biomass enhancement were observed at the fertilizing point compared with the natural site. The 15Ninalgal tissue were significantly depleted at the fertilizing point, and approached levels before fertilization with distance from the fertilizing point. The distance from the fertilizing point at which the 15N in algal tissue were almost the same level as before fertilization dif- fered among years. These results indicate that the nutrient supply was related to fertilizing amounts, periods, and distance from the fertilizing point affected the algal uptake, and were reflected in the 15N values of algal tissue. We propose the applicability of 15N signature as a new indicator for detecting nutrient supply effects to S. japonica var. religiosa. Keywords : nutrient supply effects, indicator, oligotrophic sea, Saccharina japonica var. religiosa, 15N Introduction Fujiwara 2011; Tada et al. 2014), and this de- crease is causing damage such as the fishery Nutrients in seawater via“Bottom-up effects” production, the discoloration of the cultured often limit kelp production, which is funda- Porphyra (Hori et al. 2008; Kawaguchi and mental for coastal fisheries production (Harrold Takatsuzi 2010; Takagi et al. 2012; Tanda et al. and Reed 1985). Along the Californian coast of 2014). the United States, the productivity of giant kelp, It has been observed that the west coast of Macrocystis pyrifera (Linnaeus) C. Agardh de- Hokkaido has been characterized by oligotro- creased with the low nutrient concentrations phic conditions (Dotsu et al. 1999; Kuribayashi induced by the effects of the El Nin o(Zimmer- et al. 2014). In this region, low nutrient concen- man 1985). Moreover, the dissolved inorganic trations have a negative impact on growth and nitrogen (DIN) concentration has continued to maturation of the sporophytes and gameto- decrease in the Seto Inland Sea (Yamamoto phytes of the Saccharina japonica var. religiosa 2003; Tarutani 2007; Nishikawa et al. 2010; (Miyabe) Yotsukura et al. (Akaike et al. 1998; 1 Hokkaido Nuclear Energy Environmental Research Center, 261-1 Miyaoka, Kyowa, Iwanai, Hokkaido 045-0123, Japan 2 Graduate School of Environmental Science, Hokkaido University, North 10, West 5, Kita-ku, Sapporo, Hokkaido 060-0810, Japan 3Abashiri Fisheries Research Institute, Hokkaido Research Organization, 1-1-1 Masuura, Abashiri, Hokkaido 099-3119, Japan 4 Graduate School of Fisheries Sciences, Hokkaido University, 3-1-1 Minato-cho, Hakodate, Hokkaido 041-8611, Japan ＊Corresponding author : Tel: +81-135-74-3131, fax: +81-135-74-3135, e-mail: [email protected] 1 Takanori KURIBAYASHI, Shoichi AKAIKE and Shigeru MONTANI Mizuta et al. 1998). The female gametophytes of tions of the in situ water column DIN history, S. japonica var. religiosa matured and formed and provides integrated information on the bi- sporophytes at concentrations of more than 5.0 ologically available form of nitrogen in marine MNO3-N in laboratory experiments (Seto et al. ecosystems (Fong et al. 1998). Although water 15 2001; Mizuta et al. 2001). However, NO3-N con- quality parameters, N-DIN, DIN concentra- centrations have been often less than 5.0 Min tion etc., are useful for determining nutrient the field since 1989 (Kuribayashi et al. 2014). flows, pulses, and physical distributions of Moreover, NP ratio in the seawater is lower nutrients as non-continuous (snapshot) values, than that of algal tissues in S. japonica var. the 15N in algal tissue is expected to provide religiosa (Johnston 1971; Atkinson and Smith integrated information on biological uptake of 1983), DIN limits S. japonica var. religiosa pro- DIN (Costanzo et al. 2001). Previous studies have duction (Kuribayashi et al. 2014). reported that the 15NinPorphyra could be used It has been recognized that nutrient supply to estimate nutrient sources and nutrient supply in oligotrophic sea areas was an important effects (Takagi et al. 2013; Kobayashi and method for reconstruction of kelp forests for a Fujiwara 2015). long time (North et al. 1982; Ogawa and Fujita The purpose of this study is to propose a 1997). Nutrient supply enhances saccharinan kelp possibility of 15Ninalgaltissuesasanew growth with fertilizing in the west coast of indicator of the nutrient supply sources in Hokkaido, Japan (Akaike et al. 1998; Agatsuma order to detect nutrient supply effects to S. et al. 2014). For detecting nutrient supply ef- japonica var. religiosa. In this study, we carried fects, comparison of biological forms or assess- out nutrient supply by adding artificial fertilizer ment of algal biomass, body size were often which has a lower 15N than the ratio of natu- main methods. However, since biological infor- ral seawater, and compared the 15Ndistribu- mation on effects of fertilization is influenced by tion of algal tissue before and after fertilization other factors, e. g. changes in the light condi- on the southwest coast of Hokkaido, Japan. tions and the flow environment, influence of algal grazers (top down impacts). For this Materials and Methods reason, biochemical indicators which directly reflect nutrient supply effects are needed. A. Outline of nutrient supply To overcome this limitation, we have devel- Nutrient supply was carried out with DIN oped a technique involving analysis of the fertilizer by adding artificial NH4-N to the stable nitrogen isotope ratios (15N) signature in experimental area in Kaminokuni, Hiyama, algal tissues of S. japonica var. religiosa. Stable Hokkaido, located on the southwest coast of isotopes of elements, such as 14Nand15N, which Hokkaido, Japan, an area without major rivers exhibit different reactivities because of mass inflows or the impacts of anthropogenic resi- differences, result in isotopic fractionation dues (Fig. 1). The fertilizing point (A and A' at through a variety of physical and chemical pro- the depth of 0.5 m and 1.3 m) was set on the reef cesses. The various sources of nitrogen in the where seaweeds did not adhere except for ecosystems often have been characterized by coralline algae based on previous observations 15N:14N (expressed as 15N) values (Miyake and of the epiphytic situation of seaweeds on the Wada 1967; Peterson and Fry 1987). Since the bottom of the sea. The DIN fertilizer of NH4-N 15 late 1990s, N signatures in seaweeds have been was adjusted by dissolving (NH 4)2 SO4 in the often used as an indicator of the DIN source, mixing tanks where seawater is pumped up at such as anthropogenic inputs from sewage the quay. A rate of 4.2 t hour-1 (100.8 t day-1)of effluent (McClelland et al. 1997; Umezawa et al. liquid fertilizer was released to the fertilizing 2002). That is, algal species synthesize compo- point through an extended pipe from the mix- nents in the tissue by assimilating nutrients ing tanks continuously for 24 hours (Fig. 2). The from the surrounding water. This property fertilizing points, amounts and periods were indicates that the algal components are reflec- adjusted each year (2009-2013) as follows; 2 A new indicator of 15N signature for detecting nutrient supply effects to Saccharina japonica var. religiosa Fig. 1. Location of study area at Kaminokuni, Hiyama, Hokkaido, the southwest coast of Hokkaido, Japan. ●: Seawater sampling, and S. japonica var. religiosa and other species of seaweeds collection points; A': Fertilizing point from 24 October 2009 to 18 June 2010; A: Fertilizing point from 22 October 2010 to 12 June 2011, from 27 October 2011 to 21 June 2012, and from 21 October 2012 to 28 February 2013; B: Non-fertilizing point for comparison with Stn. A' to examine the 15N indication for detecting nutrient supply effects on S. japonica var. religiosa growth promotion; C: Offshore side of the breakwater compared with Stn. A' and A; □: Sea urchin removing areas. Fig. 2. Fertilization methods. 3 Takanori KURIBAYASHI, Shoichi AKAIKE and Shigeru MONTANI 15 A': 36.7 t (N: 7.7 t) from 24 October 2009 to 18 MAT 252). In addition, the Nof(NH4 )2 SO4 June 2010 fertilizer was also analyzed by the same meth- A: 35.8 t (N: 7.5 t) from 22 October 2010 to 12 od. Thestablenitrogenisotoperatio(15N) was June 2011 expressed as per mille (‰) deviation from the A: 9.0 t (N: 1.9 t) from 27 October 2011 to 21 standard (atmospheric N2) as defined by the June 2012 following equation: 15 15 14 15 14 A: 3.5 t (N: 0.7t) from 21 October 2012 to 28 N=[( N/ N) sample /( N/ N) standard－1]×1000 (‰) February 2013 The analytical error was within ± 0.2‰. B. Seawater sampling to determine DIN concen- D. Biological investigations tration a. Examination of 15N indication for detecting To understand the liquid fertilizer distribution nutrient supply effects to S. japonica var. in the examination area, bottom seawater sam- religiosa growth promotion pling at the depth in the range of 0.5 m to 14 On 26 November 2009, ropes of about 1.5m m was performed using a Van Dorn Water length with immature S. japonica var. religiosa Sampler (5026-A: Rigo) and syringes by SCUBA- seeded in the laboratory, were fixed on the diving at Stn. A (2010-2011, 2011-2012 and 2012- seabed with anchor bolts and were retrieved by 2013) and A' (2009-2010) and on the offshore side the float at Stn.