Journal of Oceanology and Limnology   2019, Vol. 37 issue(5): 1555-1565     PDF       
http://dx.doi.org/10.1007/s00343-019-8348-x
Institute of Oceanology, Chinese Academy of Sciences
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Article Information

GENG Huixia, YU Rencheng, ZHANG Qingchun, YAN Tian, KONG Fanzhou, ZHOU Mingjiang
Tracing the settlement region of massive floating green algae in the Yellow Sea
Journal of Oceanology and Limnology, 37(5): 1555-1565
http://dx.doi.org/10.1007/s00343-019-8348-x

Article History

Received Dec. 6, 2018
accepted in principle Jan. 6, 2019
accepted for publication Feb. 18, 2019
Tracing the settlement region of massive floating green algae in the Yellow Sea
GENG Huixia1,2,4, YU Rencheng1,2,3,4, ZHANG Qingchun1,2,4, YAN Tian1,2,4, KONG Fanzhou1,2,4, ZHOU Mingjiang1     
1 Key Laboratory of Marine Ecology and Environmental Sciences, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China;
2 Laboratory for Marine Ecology and Environmental Science, Pilot National Laboratory for Marine Science and Technology(Qingdao), Qingdao 266237, China;
3 University of Chinese Academy of Sciences, Beijing 100049, China;
4 Center for Ocean Mega-Science, Chinese Academy of Sciences, Qingdao 266071, China
Abstract: Outbreaks of large-scale green tides formed by Ulva prolifera in the Yellow Sea (YS) cause heavy ecological damages and huge economic losses. However, the ecological consequences of green tides remain poorly understood due to the lack of knowledge on the settlement region of massive green algae floating in the YS. In this study, we established a method to trace the settlement region of floating green algae, using 28-isofucosterol preserved in sediment as the specific biomarker for green algae. Sterols including 28-isofucosterol in surface sediment samples collected during an investigation in the YS and the Bohai Sea (BS) in August 2015 were analyzed using gas chromatography coupled with a mass spectrometer (GC-MS). Based on the content of 28-isofucosterol in sediment samples, the potential settlement region of floating green algae was identified in the sea area southeast to the Shandong Peninsula around the sampling site H06 (122.66°E, 36.00°N). This paper presents a first result on the settlement region of floating green algae in the YS for providing a solid basis to elucidate the ecological consequences of green tides in the area.
Keywords: green tide    biomarker    sterol    Ulva prolifera    
1 INTRODUCTION

Green tide, as a unique type of harmful algal bloom, is mainly caused by the rapid growth and accumulation of green algae. In the southern Yellow Sea (YS), large-scale green tides have been reported for twelve years since 2007 (Zhou et al., 2015). The major bloom-forming species of green tides was identified as Ulva prolifera (Leliaert et al., 2009). During the occurrence of green tides from May to August, a huge amount of floating green algae could affect an extensive sea area in the southern YS (Liu et al., 2015). In 2015, for example, the maximum sea area affected by the green tide was about 52 700 km2. Outbreaks of large-scale green tides have seriously affected the coastal region of the YS and aroused substantial scientific concerns (Liu et al., 2016).

Green tides in the YS of China are known of several features, such as huge biomass of green algae, extensive sea area affected, and long-distance transportation. In fact, green tides in the YS have been known originated from Subei Shoal along the coast of Jiangsu Province (Liu et al., 2010, 2013). Under the influence of monsoons and currents, the massive floating green algae moved northward from the Subei Shoal (Bao et al., 2015), during which they kept growing and the total biomass of floating green algae could reach several million tons (Liu et al., 2015; Zhou et al., 2015). Parts of the floating green algae could pile up on the shore, while the floating green algae remaining in the sea could settle to the bottom in late July and early August when environmental conditions were not favorable for the growth of green algae. According to a simulated experiment in the laboratory, settled green algae can undergo a rapid decomposition process and release nutrients including ammonia and phosphate (Wang et al., 2012). This process can lead to changes in nutrient concentration and composition in seawater, and inevitably affect the biomass and composition of phytoplankton communities. However, knowledge of the region where massive floating green algae settle and decompose in the YS is scarce. Lü and Qiao (2008) predicted the distribution of settled green algae in the coastal waters of Qingdao after the green tide in 2008 using a numerical model based on the Princeton Ocean Model (POM). However, the study area was confined in the coastal waters of Qingdao, and there was no result on the settlement region of floating green algae in the YS. It is almost impossible to track the settled green algae through remote sensing, and difficult to find them using an approach like bottom trawling during field investigations. Furthermore, the settled green algae will undergo a rapid decomposition process, which further increases the difficulty in tracking the settled algae. Therefore, an effective means or indicator is urgently needed to find out the settlement region of green algae.

Biomarkers conserved in the sediment are widely used in biogeochemical studies, including biogenic silica (Nelson et al., 1995), phytoplankton pigments (Harris et al., 1996), sterols (Volkman et al., 1998; Menzel et al., 2003), and other organic compounds (Canuel et al., 1997; Balakrishna and Probst, 2005). Among those biomarkers, sterols are often used to indicate the composition of phytoplankton communities (Tian et al., 1992; Fattore et al., 1996). Sterols are important components of biological cells and play an important role in their life activities (Hartmann, 1998). Some sterols are widely spread among different algal groups, while others are specific to certain classes (Volkman, 1986). For instances, brassicasterol is specific for diatoms (Werne et al., 2000), dinosterol for dinoflagellates (Volkman, 2003), and (24Z)-24-propylidenecholesterol (24Z) for pelagophyte Aureococcus anophagefferens (Giner et al., 2001, 2009). Some sterols, like β-sitosterol and stigmasterol, have been suggested as indicators of terrestrial higher plants (Volkman, 1986). Through the analysis of these sterols in surface sediment or sediment cores, the structure of the phytoplankton community in a specific time or the long-term changes of algal groups can be deduced.

Sterol has a great potential to be used as an indicator for green algae. In many previous studies, 28-isofucosterol has been found as a major sterol in green algae Ulva spp. and Cladophora spp. (Geng et al., 2017). It was also detected in a few brown algal species like Ascophyllum nodosum and Cladophora flexuosa but the content was extremely low (Bolger et al., 1969; Knights, 1970; Itoh et al., 1980). Its isomer, fucosterol, is mainly found in brown algae (Ikekawa et al., 1968). 28-isofucosterol and cholesterol are major sterols in green-tide forming algae U. prolifera. Okano et al. (1983) found that 28-isofucosterol always dominated the sterol components in U. prolifera, although its content showed apparent seasonal fluctuation. In the previous studies, a method for detection of 28-isofucosterol was established using gas chromatography coupled with a mass spectrometer (GC-MS), and the feasibility of using 28-isofucosterol to represent U. prolifera was examined (Geng et al., 2017, 2018). Therefore, 28-isofucosterol could serve as a promising biomarker to trace the settlement region of green algae in the southern YS.

The intensive green tides in the YS may lead to significant impacts on marine ecosystems. It was estimated that green algae in the YS could transport 360 000 t of carbon, 23 000 t of nitrogen, 400 t of phosphorus and 16 000 t of sulfur from south to north during a single green tide event (Ding, 2014). During the development stage of green tides, floating green algae will absorb and assimilate inorganic nitrogen and phosphorus from seawater. After the settlement of the floating green algae, the decomposition takes place and releases nitrogen and phosphorus, partially in organic forms, into seawater. Therefore, it would lead to the changes in the distribution and composition, and the concentration of nitrogen and phosphorus, and in turn affect the phytoplankton communities in the southern YS. In addition, the decomposition of green algae could produce toxic chemicals like hydrogen sulfide and ammonium, which have lethal effects on some marine animals (Wang et al., 2011). Due to the lack of knowledge on the final settlement region of floating green algae, potential ecological consequences of green tides are still poorly understood.

In this study, surface sediment samples collected during an investigation in the YS and the Bohai Sea (BS) in August 2015 were analyzed with GC-MS, and the content of 28-isofucosterol, as the biomarker for green algae, was analyzed to trace the potential settlement region of floating green algae.

2 MATERIAL AND METHOD 2.1 Cruise and sample collection

Sediment samples analyzed in this study were collected during a field investigation organized by National Natural Science Foundation of China (NSFC) in August 2015. The sea area investigated (longitude 118.90°–124.00°E, latitude 32.50°– 39.60°N) covered both the BS and the YS (Fig. 1). All together 80 sediment samples were obtained using a stainless-steel box corer, and the surface sediment samples (0–5 cm) were collected and stored at -20℃ until analysis.

Fig.1 Investigation area and sampling sites in the Yellow Sea and the Bohai Sea in August 2015
2.2 Chemical reagents and standards

All the reagents (n-hexane, methanol, and dichloromethane) used in this study were purchased from Merck (Germany). Hexamethylbenzene, 5α-cholestane, bis (trimethylsilyl) trifluoroacetamide and trimethylchlorosilane (99% BSTFA + 1% TMCS), sterol standards (stigmasterol, cholesterol, brassicasterol, β-sitosterol, campesterol and fucosterol), and phenylalanine standard (99%, C: 65.44%, N: 8.48%) were purchased from SigmaAldrich (Steinheim, Germany). 28-isofucosterol and dinosterol standards are not commercially available.High purity helium and oxygen gas (volume fraction >99.99%) were used for GC-MS analysis and element analysis.

2.3 Sample analysis 2.3.1 Sterol analysis 2.3.1.1 Sample preparation

Sediment samples were lyophilized using an Alpha 1-2 LDplus freeze dryer (Christ, Osterode am Harz, Germany), then ground with a ceramic mortar. After sieved through a 150-μm mesh, 10 g of sediment was weighed into 250 mL Erlenmeyer flasks. The internal standard, 5α-cholestane, was added. Samples were extracted with dichloromethane and methanol (v/v= 2/1) in an ultrasonic bath (SB25-12DT, China). The supernatant was transferred to a flask after centrifugation (3-16K centrifuge, Sigma, Germany), and evaporated into dryness at 40℃ using a rotary evaporator (RV 10 basic, IKA, Germany). Potassium hydroxide in methanol solution (6%) was then added into the flask, and the extract was saponified at 80℃ for 1 h. The mixture was extracted with n-hexane, and derivatized with 500 μL of 99% BSTFA + 1% TMCS before analysis. A known amount of hexamethylbenzene was added prior to injection to test the instrument stability. The final extract was analyzed using GC-MS.

2.3.1.2 Sterol analysis in gas chromatography-mass spectrometry

Sterols extracted from surface sediment were analyzed with gas chromatography (Agilent 7890A, with an autosampler, USA) coupled with a mass spectrometer (GC-5975C MS, USA) using a silica capillary column (30 m×0.25 mm i.d., 0.25 μm film thickness SPE-50 column, SUPELCO, USA), using helium as carrier gas. Injection volume was 1 μL, with a split ratio of 10:1. Instrument setting of the gas flow rate and inlet temperature has been published previously (Geng et al., 2018). The gas flow rate was set at 1.0 mL/min. The inlet temperature was 300℃. The oven program was as follows: 80℃ for 1 min, increased to 250℃ at a rate of 15℃/min, then to 280℃ at a rate of 1℃/min, 300℃ at a rate of 5℃/ min, and remained at 300℃ for 10 min. Electron impact ion source was set at 70 eV. The ions for identification and quantitative analysis in the selected ion scan mode (SIM) were listed in Table 1.

Table 1 Retention time, qualitative ions and quantitative ions of internal standard and various sterols (m/z indicates the ratio of mass to charge)
2.3.1.3 Test of matrix effects

In the analysis of sterols with GC-MS, matrix effects are mainly caused by interferences of components other than the target compounds in the sample, which may affect the accuracy of analytical results. In this study, matrix effects are evaluated by comparing signals of target compounds in n-hexane (the theoretical value AT) and in sediment extract (the measured value AM), respectively. Three samples collected from the BS, the northern YS, and the southern YS are selected to examine their matrix effects (Table 2). The sediment samples were extracted according to the procedure described in Section 2.3.1.1, and divided into an experimental group (A1) and a control group (A2). Known amounts of sterol standards were spiked into the experimental group, while an equal volume of n-hexane was added into the control. Peak areas of A1 and A2 were calculated for different sterols, and the measured value for each sterol standard was calculated as AM=A1-A2. In addition, the same amount of sterol standards dissolved in n-hexane was analyzed directly to get the theoretical value of each sterol's peak area AT.

Table 2 Sampling sites of sediment used for matrix effect testing
2.3.2 Analyses of total organic carbon and total nitrogen 2.3.2.1 Sample preparation

To analyze the total organic carbon (TOC) and total nitrogen (TN), a portion of surface sediment sample is lyophilized using an Alpha 1-2 LD plus freeze dryer (Christ, Osterode am Harz, Germany), and ground with a ceramic mortar and pestle. Then a sample of approximately 20 mg sediment is put into a silver boat (6 mm×6 mm×12 mm) and weighed accurately with an electronic balance (MS 105DU, METTLER TOLEDO, Switzerland). The content of TOC and TN are analyzed with an elemental analyzer (Vario MACRO, Elemental Analyzer, Germany). Before the determination of TOC, samples were acidified with hydrochloric acid for more than 48 h to remove inorganic carbon. TN was measured directly.

2.3.2.2 Total organic carbon and total nitrogen analysis

TOC and TN were analyzed with an element analyzer (Vario MACRO cube, Elementar, Germany). Instrument conditions were as follows: helium pressure was 0.12 MPa, oxygen pressure was 0.22 MPa. The temperature of the primary combustion tube was 960℃, the secondary combustion tube was 900℃ and the reducing tube was 830℃. O2 was burned at a flow rate of 25 mL/min for 70 s.

The instrument was calibrated in advance, and different amount of phenylalanine (0.57, 0.62, 0.90, 1.02, 1.59, 1.65, 1.96, 2.15, 2.55, 2.78, 3.39, 3.61 and 4.29 mg) were analyzed to obtain the calibration curve. The detection limits of organic carbon and nitrogen were 0.42% and 0.042% respectively.

2.3.3 Grain size analysis

A portion of sediment sample (0.5 g) was placed in a 20-mL beaker and sonicated for 1 min, then analyzed with a laser particle size analyzer (1 190 L, Cilas, France). The measuring range was 0.04-2 500 μm. The relative error in particle size of duplicate samples was less than 3% (n=6).

2.4 Data analysis

Matrix effects of samples collected from different locations were tested by one-way analysis of variance and a multiple-comparison test (Tukey HSD). Variance assumptions were analyzed by ShapiroWilks' test and Levene's test, respectively. In this study, P < 0.05 indicated significant correlation, while P < 0.01 indicated extremely significant.

3 RESULT 3.1 Matrix effects of sediment samples

Notable matrix effects were observed in all the three sediment samples collected from the BS, the northern YS and the southern YS, but no significant difference (P>0.05) was found among the three samples (Fig. 2). The measured values of sterols in sediment extracts were around 2.1±0.3 times as high as the corresponding theoretical values of sterols dissolved in n-hexane.

Fig.2 Matrix effects of three sediment samples (B13, H13, and B69) based on GC-MS analysis of sterols as indicated by the difference between signals of sterols in sediment extract (AM) and in n-hexane (AT)
3.2 Establishment of the calibration curve

To eliminate the interference caused by matrix effects in the analysis of sterols, 10 sediment samples from the YS and the BS were selected randomly and a part of these samples was mixed and extracted according to the procedure in 2.3.1.1. Different concentrations of sterol standards were then added into the extract, and the final concentrations were set at 0, 1.0, 2.0, 4.0, 6.0, 8.0, and 10 μg/mL, respectively. The calibration curve was established using the standards spiked into the raw extract. Based on the recovery rate and calibration curve, sterol content in sediment samples was quantified. The content of 28-isofucosterol was determined using the calibration curve of its isomer fucosterol, due to the lack of a 28-isofucosterol standard. The two isomers had similar mass spectra and fragment ions, but different retention time (28-isofucosterol, 42.4 min; fucosterol, 42.2 min) (Geng et al., 2017). Dinosterol was quantified using the calibration curve of cholesterol, supposing that they have similar responses. The recovery of this method was 87%-124%.

3.3 Sterol content in surface sediment 3.3.1 Content of 28-isofucosterol in sediment

The results showed that 28-isofucosterol content in the investigation area was in a range of 0.03-0.36 μg/g dry weight (Fig. 3). For most of the sampling stations, the content of 28-isofucosterol was less than 0.09 μg/g dry weight. The maximum value was recorded at the station H06 (122.66°E, 36.00°N) in the southern YS. In the sea area north to the coastline of Shandong Peninsula, relatively high content of 28-isofucosterol was also detected near the station B26 (121.99°E, 37.70°N).

Fig.3 Distribution and content of 28-isofucosterol (μg/g dry weight) in surface sediment of the Yellow Sea and the Bohai Sea Black dots in the figure indicating sampling sites.
3.3.2 Content of other sterols in sediment

The content of total sterols (cholesterol, coprosterol, brassicasterol, campesterol, stigmasterol, β-sitosterol, fucosterol, 28-isofucosterol, and dinosterol) in the investigation area was 0.34-7.56 μg/g dry weight and the highest value was recorded at the H05 station (122.33°E, 36.00°N) in the southern YS. The main component was cholesterol. For most of the sterols, the distribution patterns were similar, with the highlevel region in the sea area southeast to the Shandong Peninsula. Cholesterol was widely distributed in the study area, and the range of content was 0.07−5.24 μg/g dry weight. The maximum content was recorded in the H05 station. It was the major sterol component in the sediment sample collected from H05 station. The content of coprosterol was 0.34-7.56 μg/g dry weight. Nearshore waters and the sea area around H06 station (122.66°E, 36.00°N) had a relatively high content of coprosterol. Contents of dinosterol, brassicasterol, fucosterol, stigmasterol, β-sitosterol, and campesterol were similar, which were relatively high in the sea areas around H06 station and north to the coastline of Shandong Peninsula. Besides, high level of campesterol, brassicasterol, stigmasterol, and β-sitosterol were also found in the sea area south to the Liaodong Peninsula in the northern YS (Fig. 4).

Fig.4 Distribution and content of total sterols (μg/g dry weight) and each corresponding sterol including cholesterol, campesterol, dinosterol, brassicasterol, fucosterol, stigmasterol, β-sitosterol, and coprosterol, in the Yellow Sea and the Bohai Sea Black dots in the figure indicating sampling sites.
3.4 TOC and TN content in surface sediment

Distribution patterns of TOC and TN in surface sediment are consistent with that of total sterols, and the highest values of TOC and TN appeared in the sea area around H06 station (122.66°E, 36.00°N) southeast to the Shandong Peninsula. Another sea area around the sampling station B26 (121.99°E, 37.70°N) north to the Shandong Peninsula also had a relatively high level of TOC and TN. For most of the sampling stations, the molar ratio of TOC/TN (C/N) was less than 10, while C/N ratio (molar ratio of TOC content to TN content) in the H06 station had the highest value of 37. Besides, another area with a relatively high value of C/N ratio was observed near the Liaodong Peninsula (Fig. 5).

Fig.5 Distribution and content of total organic carbon (TOC, %), total nitrogen (TN, %), and molar ratio of TOC content to TN content (C/N ratio) in the Yellow Sea and the Bohai Sea Black dots in the figure indicating sampling sites.
3.5 Grain size of surface sediment

Most of the surface sediment samples collected from the YS and BS were composed of silt and clay, with the medium diameter less than 63 μm. The two areas with much lower values of medium diameter were found in the southern YS (122.0°‒124.0°E, 34.0°‒36.0°N) and the northern YS (121.5°‒123.0°E, 37.5°‒38.5°N), respectively. These two regions correspond to the sea areas with high level of sterols, TOC, and TN (Fig. 6).

Fig.6 Medium diameter values (μm) of surface sediment in the Yellow Sea and the Bohai Sea Black dots in the figure indicating sampling sites.
4 DISCUSSION 4.1 Feasibility of using 28-isofucosterol preserved in surface sediment as a biomarker for green algae

Many studies found that green algae in genus Ulva, such as U. prolifera and U. lactuca, had 28-isofucosterol as the major sterol component (Gibbons et al., 1967, 1968; Patterson, 1974; Okano and Aratani, 1979; Okano et al., 1983; Ji, 1997). Although 28-isofucosterol has been detected in other algae besides those in genus Ulva, the content is extremely low (Bolger et al., 1969; Knights, 1970; Itoh et al., 1980). Geng et al. (2017) analyzed sterol composition in 13 major bloom-forming species of algae in China, which belong to Dinophyceae, Bacillariophyceae, Ulvophyceae, and Pelagophyceae, and found that 28-isofucosterol was produced by U. prolifera only. This further confirms its specificity for green algal species. For the green-tide forming green alga U. prolifera in the southern YS, 28-isofucosterol and cholesterol are major sterol components. Therefore, 28-isofucosterol, as the specific and major sterol of green algae in genus Ulva, can serve as a reliable indicator to trace the settled green algae in the southern YS.

Previous studies showed that green alga U. prolifera could totally decompose within 1-2 months under the environmental conditions at 20℃ in darkness, and there was a positive correlation between decomposed biomass of U. prolifera and 28-isofucosterol content conserved in sediment. Both laboratory simulation experiments and field investigations demonstrated that 28-isofucosterol released from decomposed U. prolifera could remain stable in surface sediment for at least 1 month (Geng et al., 2018). Therefore, the distribution of settled green algae could be well reflected by 28-isofucosterol conserved in surface sediment shortly after the decline of green tides.

4.2 Settlement region of massive floating green algae in the Yellow Sea

According to the analytical results of 28-isofucosterol in surface sediment, it is suggested that massive floating green algae mainly settled in the sea area around the sampling site H06 at 122.66°E, 36.00°N southeast to the Shandong Peninsula in the southern YS. The highest value of 28-isofucosterol content at H06 station (0.36 μg/g dry weight) is even higher than that in surface sediment of Tuandao Bay (0.14 μg/g dry weight) and Jiaozhou Bay (0.11 μg/g dry weight) (Geng et al., 2018), where huge amounts of green algae often pile up after green tides. A field survey before the settlement of green algae was conducted in June 2016. The content of 28-isofucosterol at station H06 in June was 0.05 μg/g dry weight (unpublished data), which was much lower than that in August 2015. Meanwhile, the average content of 28-isofucosterol in the YS was 0.05 μg/g dry weight in June, also lower than its average content in August (0.09 μg/g dry weight). These results indicated that 28-isofucosterol content has apparent change before and after the settlement of floating green algae, and annual surveys should be carried out to further demonstrate the settlement region.

In addition, the distribution of 28-isofucosterol in surface sediment is in consistence with both TOC and TN. The highest value of C/N ratio was found at station H06, where the maximum content of 28-isofucosterol was recorded. It is generally believed that organic matter synthesized by marine organism has a lower C/N ratio less than 8, while the terrestrial organic matter has a much higher C/N ratio above 12 (Prahl et al., 1980; Meyers and Ishiwatari, 1993; Meyers, 2003). The exceptionally high C/N ratio at the H06 station is likely to be affected by the settlement of green algae. In fact, the C/N ratio of U. prolifera varies significantly in different growth stages. At the late stage of green tides when floating green algae arrived in the coastal waters of Shandong Peninsula, the nutritional status of U. prolifera decreases significantly with an extremely low level of crude protein content (Ding, 2014). This would lead to an elevated level of C/N ratio compared to the green algae floating in Subei Shoal. In a previous study, we analyzed the TOC and TN content in samples of U. prolifera collected from Subei Shoal and coastal waters of the Shandong Peninsula. The results showed that those samples had similar content of TOC, while TN content was significantly different. TN content of U. prolifera samples collected from the coastal waters of Shandong Peninsula was significantly lower than that from Subei Shoal, and the C/N ratio could reach 37 (Table 3), similar to the C/N ratio in the sediment of the station H06. This result further confirms the settlement region of floating green algae as indicated by 28-isofucosterol.

Table 3 The content of TOC (total organic carbon), TN (total nitrogen), and molar ratio of TOC content to TN content (C/N ratio) of U. prolifera samples collected from different sea areas

In addition, most sterols detected in the YS, including dinosterol, brassicasterol, coprosterol, stigmasterol, and β-sitosterol, have similar distribution patterns, and the sea area around the sampling site H06 southeast to the Shandong Peninsula always has a relatively high level of sterols. It is suggested that the sea area southeast to the Shandong Peninsula around the sampling site H06 is a major settlement region for most of the biogenic organic matter. The distribution pattern of biogenic silica in the YS is also in consistence with the findings of this study (Ye et al., 2004). Besides, Cho and Matsuoka (2001) and Shin et al. (2013) investigated the distribution of dinoflagellate cysts in the YS, which had a similar pattern to dinosterol in this study.

In addition to the sea area southeast to the Shandong Peninsula, relatively high content of 28-isofucosterol was also found in the sea area around station B26 north to the coastline of Shandong Peninsula. This, however, could be related to the settlement of floating green algae in the BS and the northern YS. Floating green algae were observed occasionally in the coastal waters of Yantai and Qinhuangdao in last several years. The blooming species, however, is different from U. prolifera in the Southern YS (Zhang Qingchun, unpublished data).

Combined the distribution pattern of both 28-isofucosterol, TOC, and TN, it can be deduced that the settlement region of floating green algae in the YS should be around the sampling site H06 southeast to the Shandong Peninsula. The identification of settlement region of green algae in the YS is of great importance to understand in depth the ecological consequences of green tides. Decomposed green algae will release a huge amount of reduced nitrogen like ammonium and phosphate into seawater, which could change nutrient structure and composition of phytoplankton communities in the sea area around the settlement region of floating green algae. Based on a laboratory experiment, it was found that effluent of decomposed green algae could promote the growth of raphidophyte Heterosigma akashiwo, while dramatically inhibit the growth of diatom Skeletonema costatum. Low-concentration of the decomposing green algal effluent promoted the growth of dinoflagellates Alexandrium tamarense and Prorocentrum donghaiense, but high-concentration effluent inhibited the growth of dinoflagellates (Wang et al., 2012). The selective promoting effect of decomposed green algal effluent on different microalgae is likely to trigger the formation of specific red tides. In addition, the sea area is close to the YS Cold Water Mass, which is the over-summering site for copepods Calanus sinicus (Copepoda, Crustacea), a keystone species of zooplankton in the YS (Wang et al., 2003; Pu et al., 2004). Copepods C. sinicus dominates the mesozooplankton in the BS and YS, and provide important food for most of the commercially important fish stocks (e.g. sardine, anchovy etc.). However, settlement and decomposition of green algae may lead to decreased dissolved oxygen and the production of toxic chemicals like hydrogen sulfide and ammonium, which pose potent threats to copepods C. sinicus and benthic communities in the YS.

We identified the settlement region of floating green algae in the southern YS, and provide an important basis for future studies. Future field investigations in the settlement region with high spatial and temporal resolutions are still needed to examine the settlement and decomposition processes of green algae and to have a better understanding on the ecological consequences of green tides.

5 CONCLUSION

In this study, sterol content in surface sediment samples collected during an investigation in the YS and BS in August 2015 was analyzed with GC-MS. According to the distribution pattern of 28-isofucosterol content, a reliable biomarker for bloom-forming green algae in the YS, the potential settlement region of massive floating green algae is primarily identified in the sea area around 122.66°E, 36.00°N southeast to the Shandong Peninsula. The high value of C/N ratio in this region further supports this conclusion. The identification of settlement region of floating green algae will significantly improve current understandings on the ecological consequences of green tides in the YS.

6 DADA AVAILABILITY STATEMENT

The datasets analyzed during the current study are available from the corresponding author on reasonable request.

7 ACKNOWLEDGEMENT

The sampling for this study is supported by the open cruise organized by NSFC. We sincerely thank Dr. PENG Quancai and LI Chen for the help in sample analysis and Dr. CHEN Xue for collecting sediment samples.

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