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. A' and non-fertilizing point (B) of the breakwater (C) every months during the where the physical conditions (seawater tem- fertilization periods, at other points in February perature, salinity and flow, etc.) were similar. By every years. In addition, to understand the pulling up these ropes, blade length, blade wet nutrient status in the examination area before weight, maximum blade width in these rope fertilization, the bottom seawater sampling was were measured, and blade weight / blade length× also performed at Stn. A' and C on 10 Februa- blade width (substantiality value) were calcula- ry, 20 May, and 25 August 2009 by the same ted on 23 April, and 24 May 2010. Moreover, the method. Seawater samples were immediately levels of 15NinS. japonica var. religiosa sam- filtered, DIN (= NO3+NO2+NH4-N) concentration ples on 23 April and 24 May 2010 were deter- in the filtrate was determined using Auto Ana- mined by the above-mentioned method. The lyzer (QuAAtro2-HR: BL-TEC). investigation was conducted following the meth- od in Kuribayashi and Akaike (2014). C. 15N analysis of algal tissues b. Examination of 15N indication for detecting To compare 15N distribution of algal tissue nutrient supply effects to enhance algal bio- before and after fertilization, S. japonica var. mass religiosa and other species of seaweeds were The amount of algal biomass was investiga- collected randomly by SCUBA-diving from April ted by 0.25 m2 framed collection around Stn. A to June 2010, 2011, 2012, and 2013 when the blade before fertilization (on 27 September 2010) and length of S. japonica var. religiosa were maxi- after fertilization (on 24 May 2011). Moreover, 15N mum (Abe et al. 1983, 1984). For analysis of S. in algal samples were determined by the above- japonica var. religiosa and other species of mentioned method. The physical conditions seaweeds, samples with few cracks, tears, and (seawater temperature, salinity and flow, etc.) in attached organisms on the surface were select- biological investigation areas were similar. From ed. Any dirt attached to the sample surface was previous investigations, it has been clarified that carefully removed, and rinsed with filtered sea fertilization with removal of the influence of water, followed by washing with distilled water. grazing by herbivores (top down control) is
The samples of algal tissues and (NH 4)2SO4 were effective for forming seaweed beds (Kitching driedinanovenat60℃ and homogenized. and Ebling 1961; Miller 1985). Therefore, monthly Levels of 15N were determined using an ele- removals of sea urchins, Mesocentrotus nudus mental analyzer equipped with an isotope ratio (A. Agassiz) were continuously carried out. mass spectrometer (Fisons NA 1500-Finnigan
4 A new indicator of 15N signature for detecting nutrient supply effects to Saccharina japonica var. religiosa
Fig. 3. Seasonal variations of NO3-N, NO2-N and NH4-N concentration before and after fertilization at the fertilizing point (A, A') and the offshore side of the breakwater (C).
Fig. 4. DIN variations in February (2009-2013) in relation to distance from the fertilizing point (○: before fertilization, ●: after fertilization).
Results
A. DIN concentration Before fertilization, the DIN concentrations at Stn. A' and C were 5.4 Mand5.1Mwhich was the highest values in February, decreased in May, and were 0.7 Mand0.3MinAu- gust. Most of DIN in February was occurred as
NO3-N (Fig. 3). After fertilization, DIN concen- tration significantly increased and maintained more than 500 M from October 2009 to June 2010 and from October 2010 to June 2011, more than 200 M from November 2011 to June 2012 and from November 2012 to February 2013 for fertilization periods at the fertilizing point. Most of DIN for fertilization periods occurred as
NH4-N (Fig. 3). DIN concentration in February showed the distribution to approach to the levels before fertilization as the distance from fertilizing point all years (Fig. 4). The distance from fertilizing point to the point where the DIN
5 Takanori KURIBAYASHI, Shoichi AKAIKE and Shigeru MONTANI
Fig. 5. 15N changes in algal tissue in relation to distance from the fertilizing point (small symbols: before fertilization, large symbols: after fertilization).
6 A new indicator of 15N signature for detecting nutrient supply effects to Saccharina japonica var. religiosa concentrations became almost same as before fertilization differed among years: about 200 m fertilization in winter was different for among in 2010, 250 m in 2011, and 50 m in 2012 and 15 years: about 125 m in 2010 and 2011, 75 m in 2013. In addition, the Nof(NH4 )2 SO4 fertiliz- 2012 and 2013. On the other hand, the DIN er was -4.3 ± 2.0 ‰ (n= 10). concentration at Stn. C showed the same trend as before fertilization (Fig. 3). Most of the DIN C. Biological investigations 15 in February was also occurred as NO3-N. a. Examination of N indication for detecting the nutrient supply effects to S. japonica var. B. 15N values in algal tissue religiosa growth promotion Before the fertilization, the 15Nvaluesofalgal The algal body of S. japonica var. religiosa at tissues of S. japonica var. religiosa and other Stn. A' became significantly larger than that at species of seaweeds were in the range of 4.0-7.1 Stn. B. All of the items, i.e. blade length, blade ‰ in the examination area. After fertilization, wet weight, maximum blade width, and sub- the 15N in algal tissue was significantly deple- stantiality value of kelp individual at the ferti- ted at the fertilizing point in all years. The lizing point became remarkably larger (Fig. 6). levels of the 15N were different among years, Moreover, the 15N in algal tissue at Stn. A' was that is, from -15 ‰ to -20 ‰ in 2010, 2011, low (-15.7±1.1 ‰, n= 10) compared with that at from -7 ‰ to -15 ‰ in 2012, and from -2 ‰ to Stn. B (-4.7±1.0 ‰, n= 10). 3 ‰ in 2013 (Fig. 5). The 15Nvaluesshoweda b. Examination of 15N indication for detecting distribution to approach to the levels before nutrient supply effects to enhance algal bio- fertilization as the distance from the fertilizing mass point in all years. The 15N in algal tissues for Before fertilization (on 27 September 2010), distant locations from the fertilizing point were seaweed epiphytes occurred at 10 points in 28 hardly changed before and after the fertiliza- investigation points. Algal biomass was in the tion. The distance from the fertilizing point to range of 0-1,598 g m-2 except for 4,976 g m-2 at the point where the 15N in algal tissues be- one point. After fertilization (on 24 May 2011), came almostthesameasthelevelsbeforethe seaweed epiphytes occurred at all points. Algal
Fig. 6. Comparing algal growth, blade length (●, ○), blade wet weight (◆, ◇), maximam blade width (▲, △) and substantiality value (■, □)ofS. japonica var. religiosa at the fertilizing point (A') with the non-fertilizing point (B). The data on 29 March 2010 were referred from Kuribayashi and Akaike (2014).
7 Takanori KURIBAYASHI, Shoichi AKAIKE and Shigeru MONTANI
Fig. 7. Algal biomass and 15N distributions in algal tissue in the fertilizing areas before and after fertilization with density control of sea urchin.
-2 biomass was in the range of 12-2,762 g m ,with NH4-N accounted for more than 90% of DIN higher levels around Stn. A (Fig. 7). Moreover, there. By contrast, DIN concentration at Stn. C the 15N in algal tissue was low in the range was hardly changed before and after fertiliza- of -16 ‰ to -10 ‰ around Stn. A. tion. These results indicate that the high con-
centration of NH4-N was derived from fertilizer
Discussion (NH 4 )2SO4 and distributed around the fertili- zing point. The low 15N level of fertilizer means A. The 15N in algal tissue for detecting the DIN that the assimilation of fertilizer-derived DIN by sources assimilated by S. japonica var. S. japonica var. religiosa would be expected to religiosa and other species of seaweeds decrease the 15N in algal tissues. The 15Nin Seasonal changes of nutrient concentrations algal tissues around the fertilizing point showed on the west coast of Hokkaido, an area strongly significantly low in relation to high concentra- influenced by the Tsushima Warm Current tion for DIN. However, the 15Nofalgaltissues water, have been observed by monitoring the were low exceeding the 15N level of fertilizer nutrient status of the sea current (Dotsu et al. (NH 4)2SO4. 1999; Nakata et al. 2001; Kuribayashi et al. 2014). The 15N of primary producers contain both The DIN concentrations in the examination area information on the 15N-DIN sources and the before fertilization and on the west coast of influence of the isotopic fractionation, that is, on Hokkaido showed similar variations. Moreover, biological assimilation by primary producers, the 15N in algal tissues before fertilization were isotopically lower 14N-DIN is selectively used, in the range of 4.0-7.1 ‰, which was close to resulting in recording lower 15N in the tissue of 15 15 N-NO3 in subarctic sea areas (Miyake and primary producers than the NoftheDIN Wada 1967). These results indicated that S. source. If new 14N-DIN is not supplied, the 15N japonica var. religiosa in the examination area in the tissue elevates to close to the 15NofDIN grew by assimilated DIN mainly that origina- according to the Rayleigh distillation model. ted from the Tsushima Warm Current. When DIN is completely depleted, the 15Nin After fertilization, the DIN concentrations the tissue of primary producers are finally equal continuously showed the highest values at the to the 15N of DIN. In this study, high concen- fertilizing point. During fertilization periods, tration of DIN was continuously observed at the
8 A new indicator of 15N signature for detecting nutrient supply effects to Saccharina japonica var. religiosa fertilizing point. New 14N-DIN supply was con- In 2011-2012, the fertilization periods were for tinued during the fertilization period. There- about 8 months, the same as 2009-2010 and in fore, the 15NofS. japonica var. religiosa and 2010-2011. However, the fertilizing amount was other species of seaweeds reflected not only the less 1.9 t of nitrogen. Reducing the fertilizer 15N of the fertilizer DIN but also the influ- amount changed the 15N distribution of DIN ence of the isotopic fractionation, namely, isoto- resources in the examination area, would be pically lower 14N-DIN derived from fertilizer was expected to be reflected in the 15Ninalgal selectively continued to be used. tissues. Therefore, the 15N distribution in algal The 15N of algal tissue around the fertilizing tissues suggests that S. japonica var. religiosa and point had the lowest values, and approached to other species of seaweeds assimilated fertilizer- the 15N of algal tissue before the fertilization derived DIN for close distance of 75 m from the with distance from there in all years. Related to fertilizing point. the 15N distributions of algal tissue, DIN con- In 2012-2013, the fertilization periods were for centration in winter was the highest values about 4 months, the fertilizing amounts were around the fertilizing point, and approached to 0.7 t of nitrogen which was less than the val- the level before the fertilization with distance ues in the other years. Shortening the fertiliza- from there. These results indicate that DIN tion periods changed the 15N distribution of sources assimilated by S. japonica var. religiosa DIN sources in the examination area, and would and other species of seaweeds gradually changed be expected to reflect the 15N in the algal from being derived from fertilizer DIN to be tissues. Therefore, the 15N distribution in algal derived from Tsushima Warm Current water tissues suggest that S. japonica var. religiosa and with the distance from the fertilizing point as a other species of seaweeds assimilated fertilizer- reflection of 15N in algal tissue. The depleting derived DIN for closer distance of 50 m from the levels of 15N in algal tissue at the fertilizing fertilizing point. In other years, the nutrient point were different among years. Furthermore, supply was continued for the periods of S. the distance from the fertilizing point to the japonica var. religiosa gametophyte and sporo- point where the 15N of algal tissues became phyte growth season (Funano 1983), the fertili- almost the same level as before the fertiliza- zing point was covered with fertilizer-derived tion, were also different among years. These DIN. However, fertilization periods were from differences are discussed focusing on the ferti- 21 October 2012 to 28 February 2013. No fertili- lizing amounts and periods in each year below. zation was carried out in April-June when S. In 2009-2010 and 2010-2011, the fertilization japonica var. religiosa and other species of sea- periods and amounts were for about 8 months weeds were collected. Thus, the 15Ninalgal and 7.5-7.7 t of nitrogen. DIN concentration at tissue collected in 2013 reflected integrally the the fertilizing point was more than 500 M, the lower 15N of mainly fertilizer-derived DIN from distance from the fertilizing point to the point 21 October 2012 to 28 February 2013, and high- where the DIN concentration became almost the er 15N from natural seawater-derived DIN in the same as the value before fertilization was about period from March to April, May or June 2013. 125 m in February 2010 and 2011. The 15Nin As a result, the 15Ninalgaltissuesshowed algal tissue at the fertilizing point was from -15 values between the 15N from fertilizer-derived ‰ to -20 ‰, the distance from the fertilizing DIN and from natural seawater-derived DIN. point to the point where the 15N became al- The difference of nutrient supply related to most thesameasthevaluebeforefertilization, fertilizing amounts, periods, and the distance was about 200-250 m in April-June 2010 and from fertilizing point changed the 15N distribu- 2011. The 15N distribution in algal tissues sug- tion of DIN sources in the examination area, gests that S. japonica var. religiosa and other affected the biological uptake, and were reflec- species of seaweeds were able to assimilate the ted in the 15N levels of algal tissues. These fertilizer-derived DIN within a distance of 200- results suggest the possibility of 15Nasanin- 250 m from the fertilizing point. dicator in algal tissues for detecting of the DIN
9 Takanori KURIBAYASHI, Shoichi AKAIKE and Shigeru MONTANI sources assimilated by S. japonica var. religiosa. Asami, Abashiri Fisheries Research Institute, HRO for critical discussions and comments. B. 15N indication for detecting nutrient supply Thanks also due to Hokkaido fisheries techni- effects to enhance kelp production cal guidance office, Hiyama Subprefectural bu- Several researchers have reported that nutri- reau, Hokkaido Government. We thank the ent supply promotes the growth enhancement of members of Kaminokuni town government and saccharinan kelp (Akaike et al. 1998; Agatsuma fisheries co-operative association of Hiyama for et al. 2014). On the examination, it was observed helping. that the algal body of S. japonica var. religiosa at the fertilizing point became significantly larger References than that at the non-fertilizing point on 23 April and 24 May 2010. Similar tendency was shown Abe E, Kikuchi M, Matsuyama K, Kaneko T. on 29 March 2010 (Kuribayashi and Akaike Seasonal variations in growth and chemical 2014). The 15NinS. japonica var. religiosa of components in the blade of Laminaria which growth was promoted by DIN supply religiosa MIYABE in Oshoro Bay, Hokkaido. were lower. Therefore, the 15N can be used an Sci. Rep. Hokkaido Fish. Exp. Stn. 1983 ; 25 : indicator for detecting the nutrient supply ef- 47-60 (in Japanese with English Abstract). fects to promote S. japonica var. religiosa growth. Abe E, Kikuchi M, Matsuyama K, Kaneko T. Moreover, seaweeds epiphytes occurred on the On the estimating method of the blade area seabed of all points, algal biomass was en- in Laminaria religiosa MIYABE, Oshoro Bay, hanced around the fertilizing point with remo- Hokkaido. Sci. Rep. Hokkaido Fish. Exp. ving the influence of grazing by herbivores. The Stn. 1984 ; 26 :25-37 (in Japanese with Eng- average of quantity of sea urchin at the inves- lish Abstract). tigation area was 55.5 g m-2, which was less than Agatsuma Y, Endo H, Yoshida S, Ikemori C, 200 g m-2: the level to affect the vegetation on Takeuchi Y, Fujishima H, Nakajima K, the bottom of the sea. The 15Nvaluesinalgal Sano M, Kanezaki N, Imai H, Yamamoto N, tissues of seaweeds enhanced by DIN supply Kanahama H, Matsubara T, Takahashi S, were lower. Therefore, the 15N can be used as Isogai T, Taniguchi K. Enhancement of an indicator for detecting nutrient supply effects Saccharina kelp production by nutrient sup- to enhance algal biomass. S. japonica var. ply in the Sea of Japan off southwestern religiosa beds have been hardly formed. How- Hokkaido, Japan. J. Appl. Phycol. 2014 ; 26 : ever, DIN fertilization promotes S. japonica var. 1846-1852. religiosa growth and enhances algal biomass. If Akaike S, Kikuchi K, Monma H, Nozawa Y. S. japonica var. religiosa settled on the seabed, Effects of adding nitrogen and phosphorus the additional nutrient supply would be expec- fertilizer on the growth of sporophytes of ted to enhance S. japonica var. religiosa bio- Laminaria ochotensis, Phaeophyta in the mass. These results suggest the possibility of field. Aquaculture Sci. 1998 ; 46 :57-65 (in 15N signature in algal tissue as a new bioindi- Japanese with English abstract). cator for detecting nutrient supply effects to S. Atkinson MJ, Smith SV. C:N:P ratios of benthic japonica var. religiosa. marine plants. Limnol. Oceanogr. 1983 ; 28 : 568-574. Acknowledgments Costanzo D, O'Donohue MJ, Dennison WC, Loneragan NR, Thomas M. A new ap- This work was supported by the research proach for detecting and mapping sewage fund grants of Hokkaido Research Organiza- impacts. Mar. Pollut. Bull. 2001 ; 42 : 149-156. tion (HRO) and Hokkaido Government. We wish Dotsu K, Nomura H, Ohta M, lwakura Y. to gratefully thank A. Agui, Hokkaido Univer- Factors causing formation of Laminaria sity for technical assistance with 15N analysis, religiosa bedoncorallineflatsalongthe Drs. Y. Agatsuma, Tohoku University and H. southwest coast of Hokkaido. Nippon Su-
10 A new indicator of 15N signature for detecting nutrient supply effects to Saccharina japonica var. religiosa
isan Gakkaishi 1999 ; 65 : 216-222 (in Japa- stract). nese with English abstract). Kuribayashi T, Akaike S. Effects of nitrogen Fong P, Boyer KE, Zedler JB. Developing an fertilization with density control of sea ur- indicator of nutrient enrichment in coastal chins to reform seaweeds community, using estuaries and lagoons using tissue nitro- 15N signature in algal tissue of Saccharina gen content of the opportunistic alga, japonica var. religiosa (Miyabe). Fisheries Enteromorpha intestinalis (L.Link).J.Exp. Engineering 2014 ; 51 :47-54 (in Japanese with Mar. Biol. Ecol. 1998 ; 231 :63-79. English abstract). Fujiwara T. Oligotrophication of the Seto Inland Kuribayashi T, Abe T, Montani S. Nutritional Sea. Journal of Japan Society on Water status of seaweed communities along the Environment 2011 ; 34 :34-38 (in Japanese with west coast of the Japan Sea off Hokkaido, English abstract). Japan, from monitoring data and detecting Funano T. The ecology of Laminaria religiosa 15N records in Saccharina specimens. Bul- MIYABE I. The life history and the alter- letin on Coastal Oceanography 2014 ; 52 :75- nation of nuclear phases of Laminaria 81 (in Japanese with English abstract). religiosa, and the physiological ecology of McClelland JW, Valiela I, Michener RH. the gametophytes and the embryonal sporo- Nitrogen-stable isotope signatures in estua- phytes. Sci. Rep. Hokkaido Fish. Exp. Stn. rine food webs : a record of increasing ur- 1983 ; 25 :61-109 (in Japanese with English banization in coastal watersheds. Limnol. Abstract). Oceanogr. 1997 ; 42 :930-937. Harrold C, Reed DC. Food availability, sea Miller RJ. Succession in sea urchin and sea- urchin grazing, and kelp forest community weed abundance in Nova Scotia, Canada. structure. Ecology 1985 ; 66 : 1160-1169. Mar. Biol. 1985 ; 84 : 275-286. Hori Y, Mochizuki S, Shimamoto N. Relationship Miyake Y, Wada E. The abundance ratio of between the discoloration of cultivated 15N/14N in marine environments. Records of Porphyra thalli and long-term changes of Oceanographic Works in Japan 1967 ; 9 :37- the environmental factors in the northern 53. part of Harima-Nada, eastern Seto Inland Mizuta H, Hayasaki J, Yamamoto H. Relationship Sea, Japan. Bull. Jpn. Soc. Fish. Oceanogr. betweennitrogencontentandsorusforma- 2008 ; 72 : 107-112 (in Japanese with English tion in the brown alga Laminaria japonica abstract). cultivated in southern Hokkaido, Japan. Johnston HW. A detailed chemical analysis of Fisheries Science 1998 ; 64 : 909-913. some edible Japanese seaweeds. Proc. 7 th Mizuta H, Narumi H, Yamanoto H. Effect of Int. Seaweed Symp. 1971 : 429-435. nitrate and phosphate on the growth and Kawaguchi O, Takatsuzi H. Dynamics of dis- maturation of gametophytes of Laminaria solved inorganic nitrogen and its effect on religiosa Miyabe (Phaeophyceae). Aquacult. the discoloration of Porphyra yezoensis cul- Sci. 2001 ; 49 :175-180. tured along the eastern coast of Hiroshima Nakata A, Yagi H, Miyazono A, Yasunaga T, Prefecture, the Seto Inland Sea. Nippon Kawai T, Iizumi H. Relationships between Suisan Gakkaishi 2010 ; 76 : 849-854 (in Japa- sea surface temperature and nutrient con- nese with English abstract). centrations in Oshoro Bay, Hokkaido, Ja- Kitching JA, Ebling FJ. The ecology of Lough pan. Sci. Rep. Hokkaido Fish. Exp. Stn. Ine. . The control of algae by Paracentrotus 2001 ; 59 :31-41 (in Japanese with English lividus (Echinoidea). J. Anim. Ecol. 1961 ; 30 : Abstract). 373-383. North WJ, Gerard VA, Kuwabara JS. Farming KobayashiS,FujiwaraT.Analysisofnitrogen Macrocystis at coastal and oceanic sites. sources in Nori (Pyropia) cultivation area In:Srivastava LM (ed.). Synthetic Degrada- using stable isotope ratio. Aquabiology 2015; tive Processes in Marine Macrophytes. 37 : 269-273 (in Japanese with English ab- Walter de Gruyter, New York. 1982 ; 247-264.
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Nishikawa T, Hori Y, Nagai S, Miyahara K, the nori Pyropia cultivated in coastal sea Nakamura Y, Harada K, Tanda M, Manabe areas using nitrogen stable isotopes. T, Tada K. Nutrient and phytoplankton Nippon Suisan Gakkaishi 2013 ; 79 : 1002-1008 dymamics in Harima-Nada, eastern Seto (in Japanese with English abstract). Inland Sea, Japan during a 35 year period Tanda M, Akashige S, Ariyama H, Yamanori H, from 1973 to 2007. Estuaries and Coasts Kimura H, Dan A, Sakamoto H, Saiki Y, 2010 ; 33 : 417-427. Ishida Y, Kotobuki H, Yamada T. Nutri- Ogawa H, Fujita M. The effect of fertilizer ent environment and fisheries in the Seto application on farming of seaweed Undaria Inland Sea. Journal of Fisheries Technology pinnatifida (Laminariales, Phaeophyta). 2014 ; 7 :37-46 (in Japanese with English Phycol. Res. 1997 ; 45 : 113-116. abstract). Peterson BJ, Fry B. Stable isotopes in ecosys- Tarutani K. Long-term variations in water tem studies. Annu. Rev. Ecol. Syst. 1987 ; 18 : environments in the Seto Inland Sea of 293-320. Japan during 1973 to 2002 based on data Seto M, Kawai T, Makiguchi N. Study on the from the fisheries monitoring program. seaweed bed formation by water-drainage Japanese Journal of Benthology 2007 ; 62 :52- deep sea water. Proceedings of Civil Engi- 56 (in Japanese with English abstract). neering in the Ocean 2001 ; 17 :123-128 (in Umezawa Y, Miyajima T, Yamamuro M, Japanese with English abstract). Kayanne H, Koike I. Fine scale mapping of Tada K, Nishikawa T, Tarutani K, Yamamoto land-derived nitrogen in coral reefs by 15N K, Ichimi K, Yamaguchi K, Honjo T. values in macroalgae. Limnol. Oceanogr. Nutrient decrease in the eastern part of the 2002 ; 47 : 1405-1416. Seto Inland Sea and its influence on the Yamamoto T. The Seto Inland Sea-Eutrophic or ecosystem's lower trophic levels. Bulletin on oligotrophic ? Mar. Poll. Bull. 2003 ; 47 :37-42. Coastal Oceanography 2014 ; 52 :39-47 (in Zimmerman RC. Effects of El Nin oonlocal Japanese with English abstract). hydrography and growth of giant kelp, Takagi S, Nanba Y, Fujisawa T, Watanabe Y, Macrocystis pyrifera, at Santa Catalina Fujiwara T. River-water spread in Bisan Island, California. Limnol. Oceanogr. 1985 ; Strait with reference to nutrient supply to 30 :1298-1302. nori (Porphyra) farms. Bull. Jpn. Soc. Fish. Oceanogr. 2012; 76 : 197-204 (in Japanese with Received 12 April 2016 English abstract). Accepted 13 June 2016 Takagi S, Shimizu Y, Kusaka K, Kobayashi S, Fujiwara T. Evaluation of riverine DIN in
12 A new indicator of 15N signature for detecting nutrient supply effects to Saccharina japonica var. religiosa
ホソメコンブの 15Nを利用した栄養供給効果判定の可能性について
栗林貴範1,2・赤池章一3・門谷 茂2,4
要 旨
ホソメコンブ Saccharina japonica var. religiosa への栄養塩供給効果を検出するための藻 体の窒素安定同位体比（ 15N）の可能性について検証した。栄養塩供給は，2009年から2013年 にかけて，北海道南西部日本海沿岸海域において，海水中の硝酸塩より低い 15Nを示す硫酸ア ンモニウムを添加し，施肥量および施肥期間を毎年調整することで実施された。施肥開始後は， 施肥地点において溶存態無機窒素濃度の上昇，ホソメコンブの成長促進および海藻現存量の増 大が確認され，藻体の 15Nも対応して低下した。藻体の 15Nは，施肥地点からの離れるに従い上 昇し，施肥開始前の値に近づいた。藻体の 15Nが施肥開始前と同レベルになるまでの施肥地点 からの距離は，施肥量および施肥期間により異なった。これらの結果から，藻体の 15Nは，施 肥量，施肥期間および施肥地点からの距離を反映して変化し，ホソメコンブへの栄養塩供給効 果を検出する新たな指標としての可能性が示唆された。
1 北海道原子力環境センター，〒045-0123 北海道岩内郡共和町宮丘261-1 2 北海道大学大学院環境科学院，〒060-0810 北海道札幌市北区北10条西5丁目 3 北海道立総合研究機構網走水産試験場，〒099-3119 北海道網走市鱒浦1-1-1 4 北海道大学大学院水産科学研究院，〒041-8611 北海道函館市港町3-1-1
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