## 4 Discussion
In this study, similar values to the direct observation values were also obtained by the MPN method, indicating that diatom cells in the sediment collected at a depth of about 90 m were mostly viable (Figures 4a and 7). However, the species composition obtained with the MPN method showed a much lower ratio of _Chaetoceros_ compared to that with direct observation (Figure 7). In contrast, the ratio of pennate diatoms was confirmed to be high at all stations with the MPN method. For the direct microscopic observation, the sediment subsamples were consecutively sieved through 100, 20, and 10 \(\upmu\)m mesh and the remaining diatoms on the 20 and 10 \(\upmu\)m mesh were observed, while the sediment samples only diluted with the medium were used for the MPN method. The above-mentioneddifferences may be attributed to the compositions derived from different methods, notably the pennate diatoms smaller than 10 \(\upmu\)m and unidentified spherical cells (10 \(\upmu\)m in diameter) by the MPN method. [PERSON] et al. [37] calculated the concentration of diatom resting stages in sediment at a depth of 90 m in Funka Bay on the Pacific coast of Hokkaido, reporting that the concentration of _Chaetoceros_ (_C. sociale, C. compressus_, and _Chaetoceros_ spp.) was 18,000 cells g\({}^{-1}\) wet sediment in July 1995, corresponding to the timing of the present study (late July 2018). Although the concentration of _Chaetoceros_ at A2 in the present study was 8240 cells g\({}^{-1}\) wet sediment determined with direct microscopic observation, which was less than half of the value in Funka bay, it was more than fifteen times the _Chaetoceros_ concentration in Funka Bay in August 1995 (523 cells g\({}^{-1}\) wet sediment) [37]. However, diatom resting stages are generally more abundant in coastal than offshore regions [15] and the Sea of Japan off Hokkaido, where the present study was conducted, is also characterized as oligotrophic compared to other regions of Hokkaido [38]. Considering all the circumstances, the concentration of diatom resting stages at A2 was sufficiently high, subject to the effect of ARs. The resting stages of _Chaetoceros_ accounted for a large proportion (>50%) of the species composition at all of the stations. In the coastal waters of the Japan Sea and the Okhotsk Sea off Hokkaido, _Chaetoceros_ species are frequently observed and are dominant from April/May to November [39,40]. This is consistent with the results that _Chaetoceros_ are the most dominant in the sediment collected in the present study. Light is considered a critical factor for germination of diatom resting stages [13,15,41], and the minimum light intensity required for germination is reported to be 1.3 \(\upmu\)mol photons m\({}^{-2}\) s\({}^{-1}\) for resting spores of _Chaetoceros_ spp. [41]. Although the light intensity measured during the present study was mostly below 1.0 \(\upmu\)mol photons m\({}^{-2}\) s\({}^{-1}\), the light intensity monitored at the same location in September 2019 was often confirmed to be over 1.3 \(\upmu\)mol photons m\({}^{-2}\) s\({}^{-1}\) during the daytime, indicating that the minimum light intensity for germination reached the sampling stations (ca. 90 m) although not always. The germinated cells may not effectively enhance primary production on a regular basis if the cells are not carried to the euphotic zone by an upward flow such as upwelling and/or vertical mixing [16,17]. According to previous studies, the resting stages of _Chaetoceros_ spp. usually keep their viability for approximately 500 to 700 days [15,18,41], while a recent study succeeded with the germination of several millennia-old ancient _Chaetoceros_ resting spores from the Baltic Sea [42].