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To investigate the mass mortality of the macrobenthos community, primarily asari clams, triggered by upwelling-driven hypoxia, we conducted continuous observations of temperature, salinity, and DO, and monthly macrobenthos monitoring on the Rokujo tidal flat in Mikawa Bay, central Japan, from 2014 to 2016. Additionally, laboratory experiments were conducted using sediments on a tidal flat containing macrobenthos to examine the possibility of hydrogen sulfide formation in tidal flats. The bottom layer at the offshore station was intermittently hypoxic, and the station of the tidal flat was occasionally hypoxic in August and September for three years. Hypoxia was mostly observed on the tidal flat when constant easterly winds were recorded offshore. The biomass of asari clams decreased considerably from September to October 2016 when hypoxia was intermittent. Hypoxia persisted for approximately one week from 20 September, which was associated with the calm weather and stagnation of tidal currents owing to the neap tide. Conversely, the hydrogen sulfide concentration in the water directly above the sediment exceeded 30 mg L−1 after 3 days of incubation in the laboratory experiment. Therefore, the possibility of oxygen consumption on tidal flats due to hydrogen sulfide formed by biological die-offs was considered in the long-term persistence of hypoxia.