1. The introduction of the denitrifier method for the analysis of nitrate  15 N and  18 O and their variation in the surface water of Lake Kasumigaura from 2000 to 2005 A. Kohzu*, A. Imai*, Y. Nakajima**, K. Osaka***, K. Komatsu*, N. Kawasaki*, S. Miura*, T. Satou* * Center for Regional Environmental Research Division, National Institute for Environmental Studies ** Carbon and Nutrient Cycles, National Institute for Agro-Environmental Sciences ***Environmental Science Graduate School, The University of Shiga prefecture 2011.10.13 福島 FS 発表資料

2. 14 NO 3 - N 16 O 3 - 15 NO 3 - N 18 O 3 -  15 N  18 O Nitrogen isotope ratioOxygen isotope ratio 15 N 18 O High Low High Low Sewage Animal waste Rain N 2 -fixed N Rain Surface soil Groundwater Sewage, waste Human impact Atmospheric effect High traceability of N and O isotope ratios in nitrate

3. NO 3 - ➞ NO 2 ➞ N 2 O The advantage of the denitrifier method for  15 N and  18 O measurement of nitrate Quick measurement 30-40 min/sample Dual isotope measurement  15 N,  18 O Lower detection limit 0.3  g-N Good traceability by triple parameters [NO 3 - ],  15 N,  18 O by the dinitrifier bacteria (Pseudomonas aureofaciens) lack of N 2 O reductase Suitable for synoptic survey Small amount of sample requirement (< 10 ml in most of samples)

4. Inexpensive auto-injection system of N 2 O to IRMS that was self made to measure  15 N and  18 O of nitrate Kohzu et al.(2011) RADIOISOTOPES, 60, 231-240 Analytical precision SD (n = 12) ± 0.19 ‰ in  15 N ± 0.59 ‰ in  18 O at 30 nmol N 2 O injection Good precision and high throughput relative to those in the commercial expensive systems

5. What is the isotope fractionation? Phyto- plankton 14 N selectively assimilated NH 4 + -N diffused from sediment First stage f ≒ 1 Middle stage 0 < f < 1 Last stage f ≒ 0 15 N 14 N 15 N 14 N 15 N 14 N 15 N 14 N 15 N 14 N 15 N ≒ ≒ Approxi- mately equal Fractionation large ↑ Fractionation large ↓ Roughly isotope ratios of the matter A are determined by those of the origin of A However, different due to isotope fractionation

6. Sediment NH 4 + -N diffused from sediment Phyto- plankton NH 4 + -N from SPOM NO 3 - -N NH 3 gas volatilization, anamox Denitrification SedimentationAssimilation Nitrification Processes with large 15 N fractionation Processes with minor to negligible 15 N fractionation The schematic figure describing primary N processes within water column in Lake Kasumigaura. Mineralization

7. Monthly sampling sites at the center of Lake Kasumigaura Study Periods: from Apr. 2000 to Mar. 2005 Way of water sampling: 0-2m column sampling

8. 0 50 100 150 0100200300400500600 NH 4 + -N (  g/L) NO 3 - -N (  g/L) Phase 1 with high [NH 4 + ] Phase 3 with low [DIN] Phase X The N status characterized by three kinds of phases that were different in DIN concentration Phase 2 with high [NO 3 - ]

9. Phase 1 with high [NH 4 + ] Phase 3 with low [DIN] Phase X Phase 2 with high [NO 3 - ] Different  15 N nitrate and  18 O nitrate characteristics between phases -10 -5 0 5 10 15 -505101520  18 O nitrate (‰)  15 N nitrate (‰)  18 O water  15 N of surface (0 to 8 cm) sediment

10. -10 -5 0 5 10 15 -505101520  18 O nitrate (‰)  15 N nitrate (‰)  18 O water  15 N of surface sediment In phase 1, why  15 N nitrate was varied and often enriched, while  18 O nitrate kept low ? Because water column in this lake was almost always oxic, nitrification would be enhanced within ammonium rich water column. NH 4 + NH 2 OH NO 2 - NO 3 - H2OH2OH2OH2OO2O2  18 O(NO 3 - ) = 2/3  18 O(H 2 O) + 1/3  18 O(O 2 ) ≒  18 O(H 2 O) in autotrophic lake ≳  18 O(H 2 O) in heterotrophic lake Nitrate produced by nitrification would be low in  18 O nitrate

11. Meanwhile, why only  15 N nitrate was varied and sometimes higher than that of surface sediment in phase 1 ? The N status in phase 1 should be... Occasional nitrification from large NH 4 + pool under phytoplanktpon uptake (Case B) Sediment Phyto- plankton Minerali- zation NH 4 + -N In water column Nitrification NO 3 - -N Phyto- plankton Case A: Remained nitrate enriched both in  18 O and  15 N Case B: Remained nitrate enriched only in  15 N Case A Case B

12. -10 -5 0 5 10 15 -505101520  18 O nitrate (‰)  15 N nitrate (‰)  18 O water  15 N of surface sediment In phase 2, both  15 N nitrate and  18 O nitrate varied and their lowest end was close to that of surface sediment and water, respectively. Phase 1 with high [NH 4 + ] Phase 2 with high [NO 3 - ]

13. The N status in phase 2 should be... NO 3 - pool under phytoplanktpon uptake (Case A) Sediment Phyto- plankton Minerali- zation NH 4 + -N In water column Nitrification NO 3 - -N Phyto- plankton Case A: Remained nitrate enriched both in  18 O and  15 N Case B: Remained nitrate enriched only in  15 N Case A Case B Which kind of N status predominated in phase 2 ?

14. Another evidence that algal uptake of nitrate enriched remained nitrate in 15 N and 18 O r = 0.84, n = 27 p < 0.001 r = 0.78, n = 27 p < 0.001 4 8 12 16 20406080 [Chl-a] (  g/L)  18 O nitrate (‰) -4 0 4 8 12 16  15 N nitrate (‰) Strong positive relationships were found in phase 2 between  15 N nitrate and [Chla] and between  18 O nitrate and [Chla]

15. Phase 1 [NH 4 + -N]/[NO 3 - -N] > 0.4 [NH 4 + -N] > 33  g/L Phase 2 [NH 4 + -N]/[NO 3 - -N] < 0.4 [NO 3 - -N] > 85  g/L Phase 3 [NH 4 + -N] < 25  g/L [NO 3 - -N] < 25  g/L Enhanced phyto- plankton uptake of DIN Enhanced NH 4 + supply from sediment Enhanced nitrification The environmental factors that caused phase 1, 2, 3

16. The occurrence of phase 1 to 3 during 2000-2005

17. Phase 1Phase 3 Phase 2 Apr. 2000 to Aug. 2002 Aug. 2002 to Mar. 2005 PON NH 4 -N NH 4 diffused from sediment Nitrification Nitrate uptake Ammonium uptake Sedimentation to sediment NO 3 -N N cyclings were different before and after Aug. 2002 In summer Mineralization NH 4 -N NO 3 -NPON Three kinds of primary N pools

18. Kohzu et al. G-cubed, in press Possible mechanisms that brought the different N cyclings before and after Aug. 2002 0.1 1 0.3 10 3 3030 0 1 2 3 4 5 6 Conc. of SO 4 2- in pore water (mg/l) Conc. of ammonium in pore water (NH 4 + -N mg/l) Depth: 7 cm [NH 4 + -N] [SO 4 2- ] 19982000200220042006 2008 Oxic environment in sediment was found in 2002-2004. Highly oxic environment in surface sediment enhanced nitrification that brought N cycling of phase 1 → 2 → 3 → 1… after Aug. 2003. Phase 1 ↔ 3 Phase 1 → 2 → 3

19. Conclusion 1.Selfmade auto-injection system with denitrifier method enabled good precision and high throughput in the measurement of  15 N nitrate and  18 O nitrate 2.Three kinds of different N status were found within water column 3.Phase 1 with high [NH 4 + ] was found in summer, that suggested accumulation of ammonium diffused from sediment 4.Accumulated ammonium in water column was exhausted by phytoplankton before Aug. 2002 5.Accumulated ammonium in water column was first nitrified and then uptaken by phytoplankton after Aug. 2002 6.The timing of N status shift should be associated with oxic/anoxic shift in surface sediment

20. Acknowledgeme nt Thank you for your attention! Cooperation (Water and sediment sampling ): Members in GEMS/Water Trend Monitoring Project at Lake Kasumigaura. Grant-in-Aid for Scientific Research: No. 21241008 from the Japan Society for the Promotion of Science.