Introduction

Eutrophication is generally considered as the principal cause of red tides and the deterioration of marine coastal environments (Schramm et al. 1996). The coastal area is closely associated with human economic activities and has become an important region for human society. Over the last few decades, eutrophication of coastal seas has been aggravated by human activities (Schramm 1999; Capriulo et al. 2002). In coastal areas of China, rapid population increases and human activities such as agricultural production, discharge of industrial wastewater, urban runoff, and large-scale finfish and shrimp aquaculture have caused large inputs of nutrients (He et al. 2008), many times greater than those generated by natural processes (Victor et al. 2002), and many areas exhibit typical symptoms of eutrophication (Xu and He 2006). The detrimental effects of eutrophication include harmful phytoplankton and unwanted macroalgae blooms (Cuomo et al. 1993; Naylor et al. 2000), as well as development of hypoxia and anoxia (Bonsdorff et al. 2002; Sfriso et al. 1992), which ultimately result in the breakdown of continuous economic development (Fei 2004).

Among different measures to control eutrophication, seaweed cultivation is receiving more attention, because of the low cost of cultivation and possible pollutant removal by the seaweeds (Xu and He 2006). Until now, most bioremediation studies have focused on the green seaweed Ulva, the red seaweeds Gracilaria (Anderson et al. 1999; Mariachiara and Pierluigi 2002; Suzuki et al. 2005; Hernández et al. 2006; Yang et al. 2006), Porphyra (Chopin and Yarish 1999; Chopin et al. 1999; Chung et al. 2002; McVey et al. 2002; Kraemer et al. 2004; Carmona et al. 2006; Pereira et al. 2006) and the kelp, Laminaria (Chopin et al. 2003; Fei 2004; Yang et al. 2004). Many studies have shown that large-scale cultivation of macroalgae may be a feasible approach to reduce eutrophication by reducing the concentration of nutrients in the sea water. However, to our knowledge, few studies are available on the utilization of macroalgae for the bioremediation of low salinity sea areas close to estuaries.

Hangzhou Bay is located in the northern part of East China Sea. Water from Yangtze River, Qiantang River, Yong River, and Caoe River containing large amount of inland pollutants converge in Hangzhou Bay (Chen and Chen 2003). The Bulletin of Marine Environmental Quality of China issued by the State Bureau of Oceanic Administration from 2004 to 2008 showed that the concentration of dissolved inorganic nitrogen (DIN) in all the Hangzhou Bay sea area and the PO4-P concentration in some sea areas of the Hangzhou Bay are worse than the water quality of grade IV (S.O.A. 2004–2008). In addition, these impacts tend to be most severe in Hangzhou Bay with poor water exchange, leading to severe eutrophication and frequent red tides. The purpose of this study was to cultivate Gracilaria verrucosa in a low salinity enclosed sea area of Hangzhou Bay close to the entrance of Yangtze River and evaluate its role in the bioremediation of the eutrophicated area. At the same time, the matched pattern of bioremediation for the utilization of G. verrucosa in an enclosed sea area was evaluated. The results of the present paper could provide references for the effective control of eutrophication and the consequent red tides.

Materials and methods

The Jinshan enclosed sea and Fengxian enclosed sea are located in the coastal region of northern Hangzhou Bay in the East China Sea (Fig. 1). This sea area has a subtropical monsoon climate with an annual average temperature of 15.7–15.9°C and annual average precipitation of 988.1–1197.2 mm (Ji et al. 2004). The average salinity of Hangzhou Bay is only 11–15 psu due to the influence of fresh water from the Yangtze, Qiantang, Yong, and Caoe rivers. The length of the coastline and dam for the Jinshan enclosed sea is 3.16 km and 3.63 km, respectively. The Jinshan enclosed sea has an average water depth of 5 m and is divided into two parts by a ramp bridge (Fig. 1a). The east part of Jinshan enclosed sea was selected to cultivate G. verrucosa (dashed square in Fig. 1a) because the outside sea water containing high nutrients was introduced into this region. The length of the dam for the Fengxian enclosed sea was 3.83 km with a total area of 2.30 km2 (Fig. 1b), which was used as control. A total of six monitoring sites (A, B, C, D, E and F) were established in the both of Jinshan and Fengxian enclosed sea. The monitoring sites D, E and F of the Jinshan enclosed sea were located in the area where G. verrucosa was cultivated.

Fig. 1
figure 1

The location of the Jinshan and Fengxian enclosed sea in northern part of the Hangzhou Bay. a and b show the schematic diagram of the Jinshan and Fengxian enclosed sea, respectively. The thick dashed line around the enclosure was dam in a and b. The dashed square in the east part of a was selected to cultivate G. verrucosa. The letters indicate the location of monitoring sites

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Cultivation of G. verrucosa and the growth rate determination

The equipment for cultivating G. verrucosa was composed of 63 cables (each cable 250 m long). The distance between the cables was 3 m. The ends of each cable were fixed by bamboo stakes. The middle part of the cable was supported by five to six floats, forming floating ropes with a total area of 75,000 m2. A 3.5-t fresh weight of G. verrucosa was transferred from a seaweed culture factory in the Xiangshan Harbor, Zhejiang province and placed in polyethylene bags with a mesh size of 0.5 cm, a length of 10 m, a width of 0.5 m and a height of 0.5 m. The polyethylene bags were hung on the cables with five to six bags per cable, giving a total of 350 bags initially containing an average 10 kg G. verrucosa per bag. The bags on adjacent cables were interlaced. By hanging heavy objects, bags were kept 1–2 m below the surface of water in order to avoid damage by strong light intensity. Cultivation of G. verrucosa started in August 2006 and finished in July 2007. Every 14 days, three to five bags of G. verrucosa were removed from the cables and spun for 3 min in a laundry-drier and weighed. The growth rate was calculated according to the following equation: \( {\hbox{SGR}} = \frac{{100 \times \left( {\ln {W_i} - \ln {W_{i - 1}}} \right)}}{t} \) where SGR represents specific growth rate (% day−1), W i represents the fresh weight of the G. verrucosa collected at time i, W i − 1 represents the fresh weight of the G. verrucosa collected at time i −1 and t represents days (d) between i and i − 1. To prevent reduced growth rate of G. verrucosa in bags due to the crowding effect resulting from fast growth, the additional production was harvested periodically to keep 10–15 kg of G. verrucosa in one bag.

Water sample collection and the water quality

Water samples were collected in November 2005 and in February, May, August 2006 prior to the cultivation of G. verrucosa and once every month during the G. verrucosa cultivation period. Temperature, pH, salinity, and dissolved oxygen (DO) were measured in the field by using a YSI multi-parameter water quality meter. Chemical oxygen demand (COD) and biological oxygen demand (BOD) were determined by using an AWA analyzer. Three water samples were collected at 0.5 m below the sea surface at every monitoring site for the determination of chlorophyll a (Chl a) and inorganic nutrients. The water sample (500 mL) used for Chl a determination was immediately filtered onto Whatman GF/F filters and then stored frozen. For the determination, the filter was extracted with 90% acetone and a Turner Designs fluorescent analyzer was used (Parsons et al. 1984). The water sample (250 mL) used for dissolved inorganic nutrient determination was filtered through cellulose membranes (Millipore® HAWP 0.45 μm) and a small amount of HgCl2 was added. The samples were transported to the laboratory in cold condition and nutrients were determined using SKALAR flow analyzer.

Collection and determination of phytoplankton

Phytoplankton were sampled with a conical plankton net (0.37 m mouth diameter, 77 mm mesh size), which was towed vertically from near the bottom to the surface. Simultaneously, 1,000 mL sea water was also sampled at different depths of water at every site. The samples were immediately fixed with Lugol’s solution at a final concentration of 1%. After settling over 24 h and concentration, the phytoplankton species were determined and counted. The dominance index (Y) of the phytoplankton species, species diversity index (H′), richness index (D) and evenness index (J) were calculated according the Wang et al. (2005), Shannon and Weaver (1963), Margalef (1968) and Pielou (1975). A species with a Y > 0.02 was considered as a dominant phytoplankton species.

Data analysis

Average values obtained from the each monitoring site from the Jinshan and Fengxian enclosed seas were used for water quality and phytoplankton comparison. ANOVA was used to compare the phytoplankton index and water quality. Differences were considered significant when p < 0.05 and all the data are displayed as mean ± standard deviation.

Results

Eutrophication of Hangzhou Bay

The concentration of NH4-N, NO3-N, NO2-N, and PO4-P in the both Jinshan and Fengxian enclosed seas was similar during the four seasons prior to the cultivation of G. verrucosa (Table 1), with a range of 0.22–0.38 mg L−1, 1.05–1.45 mg L−1, 0.020–0.042 mg L−1, and 0.018–0.037 mg L−1, respectively. NH4-N and NO3-N were the main forms of dissolved inorganic nitrogen, accounting for 14.55–23.26% and 74.29–84.12%, respectively. DO, COD, BOD, and Chl a were 5.92–6.96 mg L−1, 4.17–5.15 mg L−1, 4.21–5.13 mg L−1 and 9.25–16.14 µg L−1, respectively, in the both enclosed seas. These results suggested that both Jinshan and Fengxian enclosed seas of Hangzhou Bay were severely eutrophicated.

Table 1 Concentrations of DIN, PO4-P, DO, BOD, COD and Chl a in both Jinshan and Fengxian enclosed seas before the cultivation of G. verrucosa
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Growth rate of G. verrucosa

The water temperature varied between 28.93 and 31.80°C in summer, and 5.20 to 11.80°C in winter (Fig. 2). The growth rate of G. verrucosa in the Jinshan enclosed sea from September to November of 2006 and from April to June of 2007 was significantly higher than in other months (p < 0.05). The highest SGR of 15.88%·day−1 and 15.16%·day−1 occurred in September and May. At the end of December 2006, most of the G. verrucosa was harvested and nearly 1 t fresh weight of G. verrucosa was left in order to observe the growth during the winter. In January, the sea water temperature could drop to 5°C, and the growth of G. verrucosa stopped or was negative. However, when the water temperature increased in February, G. verrucosa growth increased and the biomass was doubled at the end of February. The additional production of G. verrucosa was used as seedlings for extensive cultivation. And at the end of the experiment, a cumulative 15 t dry weight of G. verrucosa was harvested from the Jinshan enclosed sea. Our results suggested that G. verrucosa could survive in both winter and summer in Hangzhou Bay.

Fig. 2
figure 2

Variations of the specific growth rate of G. verrucosa in the Jinshan enclosed sea

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Concentrations of dissolved inorganic nutrients

The monthly changes of NH4-N, NO3-N, NO2-N and PO4-P concentrations in the water column of the Jinshan and Fengxian enclosed seas are shown in Fig. 3a–d. Before G. verrucosa cultivation (prior to August 2006), there was no significant difference in the concentration of these nutrients between the two seas, with the exception of PO4-P in the Jinshan enclosed sea which was significantly higher than in the Fengxian enclosed sea (p < 0.01). During the period of G. verrucosa cultivation from September 2006 to July 2007, the concentrations of dissolved inorganic nutrients in the Jinshan enclosed sea were significantly lower than those in the Fengxian enclosed sea (p < 0.05) with the concentrations of NH4-N, NO3-N, NO2-N, and PO4-P decreased by 54.12%, 75.54%, 49.81%, and 49.00%, respectively.

Fig. 3
figure 3

Concentration of NH4-N (a), NO3-N (b), NO2-N (c), and PO4-P (d) of the sea water in the Jinshan and Fengxian enclosed sea in difference months

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Biomass and species diversity of phytoplankton

The highest phytoplankton density in the Fengxian enclosed sea occurred from June to August with an average density of 8584.91 × 104 ind. m−3, while the lowest densities occurred from January to February with an average density of 9.52 × 104 ind. m−3 (Fig. 4). During the period of G. verrucosa cultivation in the Jinshan enclosed sea, the average density of phytoplankton was 126.53 × 104 ind. m−3 in summer/fall and 6.90 × 104 ind. m−3 in winter/spring, which was significantly lower than that in the Fengxian enclosed sea (p < 0.05).

Fig. 4
figure 4

Monthly changes of phytoplankton density in the Jinshan and Fengxian enclosed sea

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The dominant species in the Jinshan enclosed sea were Cerataulina bergonii, Chaetoceros subtilis, Chaetoceros subsecundus, Melosira moniiformis, Coscinodiscus oculus-iridis, and Pleurosigma formosum. The most abundant species was C. bergonii with an average density of 2850.35 × 104 ind. m−3. While the dominant species in the Fengxian enclosed sea was Skeletonema costatum, C. subsecundus, M. moniiformis, Prorocentrum micans and Prorocentrum donghaiense. In spring, the average density in the Fengxian Sea was 3050.82 × 104 ind. m−3. In addition, the abundance of dinoflagellates was much higher than that of diatoms, and P. micans accounted for 72.74% of the total phytoplankton. In summer, the average abundance of phytoplankton was 8584.91 × 104 ind. m−3 and the dominant species was S. costatum, accounting for 70.30% (Table 2). The annual diversity index (H′) and richness index (D) in the Jinshan enclosed sea were significantly higher than in the Fengxian enclosed sea (p < 0.05; Table 3). However, there was no significant difference in the evenness index (J; p > 0.05; Table 3).

Table 2 Dominant species as well as values of dominance index (Y) of phytoplankton in both Jinshan and Fengxian enclosed seas in different seasons
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Table 3 Seasonal changes in H′, D and J of phytoplankton in both Jinshan Jinshan and Fengxian enclosed seas
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Discussion

Eutrophication of Hangzhou Bay

From November 2005 to August 2006, the sea water of Hangzhou Bay was considered as severely eutrophicated. The COD had reached to the level of grade IV or worse than grade IV level. Although DIN (29.56–45.36 μmol L−1) was lower than in other coastal areas in China (57.1–108.0 μmol L−1), e.g., coastal areas of Lvsi harbor in Jiangsu province, Xiangshan Harbor in Zhejiang province and Yaling Bay in Guangdong province (Ye et al. 2002; Jia et al. 2005; He et al. 2008), it also reached the level of grade IV or worse than grade IV level. The high concentration of DIN in Hangzhou Bay was mainly caused by inland activities and nutrient discharge or runoff to the coastal area. According to the Bulletin of Marine Environmental Quality of China issued by State Bureau of Oceanic Administration (S.O.A. 2006–2007), in 2006 and 2007, COD introduced into the sea water through Yangtze River was 5.0 × 106 and 4.9 × 106 t year−1 and nutrients were 1.2 × 106 and 1.4 × 106 t year−1, respectively. In addition, nutrients introduced by mariculture are also an important factor causing high concentrations of inorganic nutrients in Hangzhou Bay. Until the end of 2004, the total yield of aquatic products in Zhejiang province where the Hangzhou Bay is located reached 4.9 × 106 t, among which shellfish was 0.75 × 106 t, accounting for 80.75% of the total yield (Xu et al. 2007). Cultivation of shellfish produces large amount of biological deposition, which easily results in eutrophication. In addition, industrial and domestic sewage also plays a role in the introduction of nutrients. Although the nutrients introduced through wet precipitation accounted for a small percentage of the total nutrients in the entrance of Yangtze River, sudden precipitation can cause eutrophication on the surface of the seawater (Zhang et al. 2003). Our results showed that the concentration of PO4-P was relatively low (0.19–0.39 μmol L−1). The overall N:P indicated that P was the main limiting nutrient in the coastal waters of Hangzhou Bay. Even the lowest N:P ratio (44.75:1) surpassed the threshold at which phosphorus becomes limiting (about 25:1; Guildford and Hecky 2000). These results indicate that any efforts to reduce eutrophication in the coastal waters of Hangzhou Bay must focus on nitrogen removal.

Growth of G. verrucosa in Hangzhou Bay

The growth of G. verrucosa was significantly affected by the water temperature. When the water temperature ranged from 16 to 29°C, SGR could reach 15.88% day−1. In winter, the low water temperature inhibited the growth of G. verrucosa. Therefore high temperatures in summer and low temperature in winter are not appropriate for the growth and survival of G. verrucosa. We adopted a series of measures including utilization of polyethylene bags and cultivation of G. verrucosa in a lower depth of water. The results showed that G. verrucosa could survive the low temperatures in winter and once the temperature increased in spring, the seedlings of G. verrucosa can be used for extensive cultivation. The annual average growth rate of G. verrucosa in Hangzhou Bay was 9.42% day−1, which was lower than that in Xiangshan Harbor cage culture sea area (11.52% day−1) and lower than the growth rate of G. lemaneiformis (11∼13% day−1) (Zhou et al. 2006; Yang et al. 2006). However, the growth rate of G. verrucosa was higher than that of G. chilensis (5.6–6.7% day−1; Troell et al. 1997) and G. longissima (6% day−1) (Hernández et al. 2006) in the cage culture sea areas. Therefore, G. verrucosa can be successfully cultivated and used for bioremediation of eutrophication in Hangzhou Bay.

Bioremediation by G. verrucosa

The high value of Gracilaria in human food, abalone feed, and agar production has led to the development of farming systems in the sea and in ponds in Brazil, Chile, China, Israel, Mexico, South Africa, Taiwan, and the United States (Mclachlan and Bird 1986; Neto 1987; Fei et al. 1999; Buschmann et al. 1995, 2001; Wakibia et al. 2001). In China, Gracilaria has been cultivated on a large scale in Guangdong, Shandong, Hainan, Guangxi, Fujian, Zhejiang, Jiangsu, and Liaoning provinces, etc. (Fei et al. 1998; Tseng 2001; Yang and Fei 2003; Yang et al. 2004, 2006). In addition to the production of agar, many studies have shown that Gracilaria can effectively remove nutrients (e.g., N and P) in the polyculture system of fish, or scallop, or shrimp with algae (Buschmann et al. 1996; Troell et al. 1997; Neori et al. 1998; Jones et al. 2001; Yang et al. 2005, 2006; Hernández et al. 2006; Zhou et al. 2006; Xu et al. 2008a, b; Mao et al. 2009; Jiang et al. 2010). In the cage culture sea area of Dongshan Island in Zhejiang Province, G. lemaneaformis could remove more than 80% of DIN and PO4-P (Tang et al. 2005). Yang et al. (2006) found that after 40 days of G. lemaneiformis cultivation, the removal rate of NH4-N and PO4-P reached 69.45% and 26.74%, respectively. Xu et al. (2007) reported that the concentrations of inorganic nitrogen and inorganic phosphate in the polyculture ponds of Gracilaria lichenoides with Litopenaeus vannamei, and with Epinephelus awoara were lower and more stable than those in the ponds without Gracilaria. Mao et al. (2009) reported that the maximum reduction efficiency of ammonium and phosphorus was 83.7% and 70.4%, respectively, in one polyculture system of G. lemaneiformis and scallop Chlamys farreri.

The present study also demonstrates that cultivation of G. verrucosa played a significant role in the bioremediation of eutrophicated water in the Jinshan enclosed sea of Hangzhou Bay. Bioremediation effects of G. verrucosa cultivated in eutrophicated seas were achieved through absorption and utilization of excessive nutrients (Xu and He 2006). Over the period of G. verrucosa cultivation water quality in the Jinshan enclosed sea was transformed from grade IV (from August to October in 2006) to grades II–III (from November 2006 to July 2007). From August to December in 2006, the dissolved inorganic nutrients decreased to the lowest level because of absorption by G. verrucosa during the period of fast growth. During winter from December 2006 to February 2007, even though the most of G. verrucosa biomass was harvested, the dissolved inorganic nutrients still remained at the low level due to less rainfall and no water exchange with the external sea water. From March to July 2007, although the external sea water containing relatively high concentrations of nutrients the water quality still remained good because of the fast growth of G. verrucosa. The nutrient removal efficiency achieved by G. verrucosa cultivation in the Jinshan enclosed sea was higher than that achieved by G. lemaneaformis cultivation (Tang et al. 2005) or that achieved by G. verrucosa cultivated in the cage culture sea area of Dongshan Island (Tang et al. 2005).

The dam of the Jinshan enclosed sea prevented the effect of Hangzhou Bay tides on internal seawater of enclosure. The current of the water in the enclosed sea was slow and mainly initiated by the wind. Exchange of water was artificially controlled and the enclosed sea was regularly replenished with external sea water to maintain stable water level. These provided relatively stable conditions to investigate the bioremediation efficiencies of G. verrucosa cultivated in one eutrophicated sea. In addition, these stable conditions increased the efficiency of nutrient uptake and growth rate of G. verrucosa. The results of the present paper also show that G. verrucosa could inhibit the growth of phytoplankton but increased phytoplankton species diversity. Cultivation of G. verrucosa could not only control the eutrophication, but also prevent and reduce the occurrence of red tides.

To maintain the low level of DIN in the Jinshan enclosed sea, the matched pattern of G. verrucosa cultivated could be evaluated based on the results of present study. The volume of the seawater in the Jinshan enclosed sea is adjusted by periodically artificial sluicing, so according to the method of calculating sea water exchange rate (Makoto 1984), the horizontal distribution of the nutrients inside and outside of the enclosed sea, and the assuming that the concentration of nutrients in the enclosed sea did not change once the nutrients and water exchange are balanced, 0.78 mg·L−1 average DIN could be calculated in the Jinshan enclosed sea. The annual average growth rate of G. verrucosa was 9.42% day−1, the average nitrogen content in the tissue was 4.80%, and dry/wet ratio was 0.169 (Xu et al. 2008a, b). In addition, on the basis of the enclosure area of 1.72 km2 and average water depth of 5 m, it was evaluated that 21.8 t of G. verrucosa need to be cultivated to achieve a grade I water quality (DIN ≤0.20 mg·L−1) and 18.0 t of G. verrucosa need to be cultivated to achieve a grade II water quality (DIN ≤0.30 mg·L−1). However, this calculation is only based on average growth rate, average DIN concentration and average nitrogen content of G. verrucosa. The precise requirement of G. verrucosa cultivation in different months in the Jinshan enclosed sea needs to be further studied.