Isotopic Evaluation on Nitrogen Reduction in Mangrove Ecosystems of Okinawa, Japan

概要

Anthropogenic loads of inorganic nitrogen (N) derived especially from fertilizer use in agriculture, livestock and domestic wastes and sewage are significant sources of excess N in the environment, which can finally cause severe damage to the natural coastal environment and the marine ecosystems. Mangrove, located at the ecotone of the land and sea, might act as a buffer zone by filtering excess N from the runoff waters and stabilize the coastal environment and protect the marine ecosystems. Despite providing valuable ecosystem services to nature and human beings, mangroves are combatting enormous threats all in the territory. In the tropical and sub-tropical maritime nations, a significant part of the national economy is based on marine resources. Therefore, conservation and management of mangroves are vital for healthy marine ecology, proper growth of coral lives, and ecosystem sustainability. The aims of this thesis are to gain better understanding of likely origin and relative availability of N inflowing into the mangrove ecosystems of Okinawa and the corresponding responses of mangroves under the influences of different magnitudes of anthropogenic activities in watersheds.

1. Foliar δ15N value and its relationship with land-use ratio in the mangrove watersheds
We collected foliar samples of mangrove and back-mangrove species from fifteen mangrove watersheds on Iriomote, Ishigaki, and Okinawa islands. Land-use ratio delineation through ArcGIS revealed that Urauchi watershed was the most pristine environment with 97 % of forest covers and was used as a reference site in this study. The comparison of foliarδ15N values among watersheds on the three islands revealed that all the watersheds on Iriomote Is. were almost in a natural condition having δ15N less than 3 ‰, whereas those ranged from 5 to 14 ‰ in watersheds on Okinawa and Ishigaki islands, which was an indication of heavier human disturbances and changes in the natural ecosystems. In addition, Todoroki watershed on Ishigaki Is. showed the highest value of δ15N (around 14 ‰), suggesting an intrusion of excess N in the environment through inland runoff from livestock farms. The results suggested that foliar δ15N values of mangroves were powerful ecological indicators for monitoring ecosystem conditions of Okinawa. Furthermore, the findings indicated that mangroves were potentially absorbing anthropogenic N from the ecosystems.

2. Adaptation of acetone methods forδ15N-NO - analysis of saline water
For identifying N sources in NO - in mangrove surface water samples, an adapted acetone method was developed for δ15N-NO3-analysis of saline water. Literature review on the pre-existing methodology for the determination of δ15N-NO - suggested that widely used protocols have several drawbacks, such as sensitivity to the sample type (i.e., freshwater or saline water), requirement of a large volume of sample containing high concentration of NO -, labor-intensive and time-consuming handling procedures, toxic substances, costly devices and especial bacterial culture, which is not available in many cases. Therefore, the adapted acetone method was developed for isolation of NO -from saline water containing a low concentration of NO - (4 µmol in an aliquot) forδ15N-NO -analysis. We elucidated the usability of the adapted acetone method in the determination of δ15N-NO3- in saline water (containing less than 6 g of Cl- in sample aliquot) with an acceptable range of precision and accuracy (< 0.1 ‰). The adapted acetone method was based on a modification of a solvent mixture (acetone/NaI/n-hexane as 21/0.35/10 mL) of the previous acetone method to a new mixing ratio as acetone/NaI/n-hexane as 30/0.25/5 mL and bringing a change in sample reduction and drying processes. Consequently, the adapted acetone method successfully removed the contaminants from the saline samples, such as organic matter and excess chlorides, and resulted in a satisfactory analysis ofδ15N-NO - in surface water samples collected from the mangrove watersheds.

3. Nitrogen availability, sources of surface water NO - and their relationships with foliar δ15N and land-use
A conventional approach for measuring concentration of dissolved N (DIN, DON) in surface water and soil pore water in six watersheds on Ishigaki and Iriomote islands, was carried out to investigate the relative availability of N. Besides, the adapted acetone method was used to investigate the source of NO3- in the surface water samples. A larger concentration of DIN, especially NO3-, along with higher values ofδ15N-NO3- of the surface water samples, ranging from 7 to 14 ‰ in the human affected watersheds compared to those of the reference site (less than 4 ‰), revealed that anthropogenically originated N were influencing the N dynamics in the systems. In addition, mangrove leafδ15N values were significantly correlated with the concentration of NO - and δ15N-NO3- values of the surface water, and the total forested areas in the watersheds, suggesting that the nutrient concentrations influenced by the land-use were responsible for the particular isotopic signature of leaf and water nitrates. In contrast, the DIN concentration of soil pore-water samples was not significantly correlated with δ15N-NO - and foliarδ15N values. Furthermore, along with DIN a considerable amount of DON was also observed in both surface and soil pore water in the mangrove ecosystems on Ishigaki Is., which was not shown in the case of mangrove watersheds on Okinawa Is. We assumed that higher concentration of DON in the study sites on Ishigaki Is. was due to livestock farms frequently found in the watersheds.

4. Soilδ15N and its relationships with foliar δ15N, surface water δ15N-NO - and land-use
Two sets of samples, the former collected from mangrove watersheds on Okinawa and Iriomote islands in 2017 and 2018, and the latter in 2019, were used for analytical experiments on DIN, foliarδ15N of mangroves and back-mangroves, and the floor soils, and δ15N-NO -of surface water. It was assumed that the results obtained from the two sets of samples would provide a better understanding of how mangroves responded to excess N, and whether the response would change in time. The concentration of DIN in the two sets of samples was distinct and relatively higher in the former set. Still, anthropogenic N inflowing into the mangrove ecosystems was evident from the elevated values of δ15N-NO - of surface water samples in the former set, the higher soilδ15N values in the latter set, and their significant correlation with land-use ratio in the watersheds. The δ15N values of the soils collected from both the mangrove and back-mangrove vegetative areas were compared among the study watersheds, and significantly higher values (from 4.7 to 7.3 ‰) were observed in all the human-influenced watersheds than those in the reference site (0.9 ‰). Additionally, significant positive correlations between the foliar mangrove δ15N and the floor soil δ15N, between the foliarδ15N and the surface water δ15N-NO -, and between the floor soilδ15N and the surface waterδ15N-NO3- indicated anthropogenically derived N uptake by the mangrove plants and reduction of excess N from the mangrove ecosystems.