Influence of nutrients pollution on the growth and organic matter output of Ulva prolifera in the southern Yellow Sea, China

Marine Pollution Bulletin xxx (2015) xxx–xxx

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Influence of nutrients pollution on the growth and organic matter output of Ulva prolifera in the southern Yellow Sea, China Zhou Yuping a,b, Tan Liju b,⇑, Pang Qiuting b, Li Feng a,b, Wang Jiangtao a,b,⇑ a b

Laboratory of Marine Chemistry Theory and Technology, Ministry of Education, Ocean University of China, 266100, PR China College of Chemistry and Chemical Engineering, Ocean University of China, 266100, PR China

a r t i c l e

i n f o

Article history: Available online xxxx Keywords: Ulva prolifera Field experiment Nutrient supply Organic matter output

a b s t r a c t The influence of nutrients on the growth of Ulva prolifera was studied in the SYS by field experiments. The wet weight of U. prolifera gradiently increased from 11.94% to 25.92% in proportion to contents of DIN supply, which indicated DIN content was essentially decisive for the output of U. prolifera blooms. Continuous nutrient supply could promote the growth of U. prolifera, indicated by the increase of growth rate from 10.46% of the batch culture to 42.17% of the in situ culture. The higher P utilized rate in all treatments showed P was the potential limited factor for the growth of U. prolifera. Moreover, it was calculated about 4.1  105 t organic matter was begot by U. prolifera in the whole Yellow Sea based on the statistical relationship between output of U. prolifera and DIN content. This work could be convenient to evaluate biomass and prepare enough tools to manage U. prolifera. Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction Blooms of green macroalgae belonging to Ulva spp. once restricted to several coastal areas have recently spread to more coastal and offshore areas including the Yellow Sea (Hu et al., 2010; Liu et al., 2010; Fan et al., 2012; Huo et al., in press), where Ulva prolifera (U. prolifera) caused serious damage to local tourism, aquaculture and the environment (Nelson et al., 2008; Choi et al., 2010; Huo et al., 2014). More researches on the geographical distribution, habit, origin, examination of U. prolifera have been carried out to investigate the consequence of U. prolifera blooms (Fortes and Lüning, 1980; Pihl et al., 1996; Guidone and Thornber, 2013; Zhang et al., 2014; Huo et al., in press). The tracing surveys suggested that the recurrence of U. prolifera blooms in the Yellow Sea was more likely to connect to coastal aquaculture of P. yezoensis and continuous expansion through floating in aquaculture (Hu et al., 2010; Keesing et al., 2011; Liu et al., 2013a). However, field surveys and laboratory works indicated that many environmental factors (temperature, salinity, irradiance and DO) arose U. prolifera blooms (Paerl, 1997; Valiela et al., 1997; Hauxwell et al., 1998, 2001; Charlier et al., 2007; Kim et al., 2011; Liu et al., 2013a,b; Sun et al., 2015). Laboratory studies showed that U. prolifera from Yellow Sea displayed growth temperature optima and salinity optima which parallel with the in situ temperature and salinity ⇑ Corresponding authors at: Chemistry and Chemical Engineering College, Ocean University of China, No. 238 Songling Road, Qiangdao 266100, PR China. E-mail addresses: [email protected] (L. Tan), [email protected] (J. Wang).

typical of the blooming period of U. prolifera (Fu et al., 2008). However, the highest U. prolifera growth rate in situ was not associated with the best temperature and salinity growth conditions (Xia et al., 2009). Furthermore, U. prolifera has a stronger capacity to adapt to varied environmental conditions (Vermaat and Sand-Jensen, 1987; Riccardi and Solidoro, 1996; Gao et al., 2012; Wu et al., 2013) via thalli exchanging nutrients with environment (Merceron et al., 2007; Liu et al., 2013a,b) and storing nutrients during pulses (Ramus and Venable, 1987). As general, environmental surveys carried out for monitoring U. prolifera reported high field nutrient concentrations in the nearby water column relative to other coastal areas, which implied U. prolifera bloom was the result of eutrophication (Smayda, 1989; Huang et al., 1997; Sun et al., 2008; Xia et al., 2009; Shi et al., 2015). Besides, laboratory studies indicated that the growth of U. prolifera was more sensitive to nitrate than phosphate (Li et al., 2010), and many field surveys confirmed that nitrate played the limiting role in the growth of U. prolifera (Hanisak, 1983; Sfriso et al., 1988; Fong et al., 1993; Ménesguen and Piriou, 1995; Pedersen, 1995; Merceron et al., 2007). Generally, nitrogen to phosphorous ratio (N/P) supply is a topic of particular concern for the development of nuisance algal blooms (Heisler et al., 2008; Shi et al., 2015). In many coastal areas, anthropogenic enrichment of N and P and the induced higher N/P ratios compared to the Redfield ratio have been shown to link with U. prolifera blooms (Redfield, 1963; Gao et al., 2012; Zhang et al., 2012).

http://dx.doi.org/10.1016/j.marpolbul.2015.04.034 0025-326X/Ó 2015 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Zhou, Y., et al. Influence of nutrients pollution on the growth and organic matter output of Ulva prolifera in the southern Yellow Sea, China. Mar. Pollut. Bull. (2015), http://dx.doi.org/10.1016/j.marpolbul.2015.04.034

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Y. Zhou et al. / Marine Pollution Bulletin xxx (2015) xxx–xxx

Although nutrient concentrations appear to correlate to U. prolifera growth, the previous investigations and laboratory studies were limited and the contribution of nutrient concentrations to the U. prolifera biomass was unknown (Fong et al., 1993; Fong et al., 1998; Barr et al., 2013; Wu et al., 2013). Therefore, we conducted mesocosm experiments to evaluate the effects of nutrients pollution, especially nitrogen, on U. prolifera blooms and contribution of nutrient contents to the U. prolifera biomass. These studies will be helpful to reveal the fundamental relationship between U. prolifera blooms and nutrients pollution and predict the green algae biomass derived from the nutrient contents. Moreover, the results from the experiments will give a glimpse to the influence of U. prolifera transition on the nutrient composition of different areas.

2. Materials and methods 2.1. Study site and field surveys The study area of the field experiment is located at 120–123°E, 32–35°N, covering most of the Southern Yellow Sea (SYS) (Fig. 1). The SYS is a semi-enclosed marginal sea, which locates between China and the Korean peninsula and bonds the North Yellow Sea to the north with the East China Sea. The SYS is a well-studied area in which annual blooms of U. prolifera caused a great economic losses to marine industries in May (e.g., fishery aquaculture and tourism) (Liu et al., 2009; Zhang et al., 2010, 2013; Wang et al., 2011a,b; Luo et al., 2012). The SYS has complex hydrographic conditions including Yellow Sea Warm Current (YSWC), Yellow Sea Coastal Current (YSCC), and Changjiang (Yangtze) Diluted Water (CDW), which result in variability of distribution of nutrients in the SYS. At all station that are marked in Fig. 1, Water samples were collected and filtered (Whatman glass fiber filters, pore size 0.7 lm) to measure nutrient concentrations (DIN, PO4–P) in the Southern Yellow Sea during 11 days between May 27 and June 6, 2012, which is the recurred season of mat-forming U. prolifera blooms (Lavery et al., 1991; Schramm and Nienhuis, 1996; Worm and Lotze, 2006; Nelson et al., 2008; Liu et al., 2009; Choi et al., 2001, 2010; Hu et al., 2010).

2.2. Field experiments 2.2.1. Acquisition of U. prolifera Thalli Free-floating green patches were found in the southern Yellow Sea by professional workers of ‘‘Runjiang 1’’ on May 27, 2012. These were recognized as U. prolifera by morphological description. The macroalgae U. prolifera used in the present study, which were visually healthy, were collected from the drift mats at the ge4 station (120°59.410 E, 34°29.840 N) and rinsed with field seawater to remove slime and epiphytes. 2.2.2. Field experiments The cultures were conducted aboard on cruise ‘‘Runjiang 1’’ during 11 days between May 27, 2012 and June 6, 2012. Experimental cultures were carried out in 25 L PE bottles containing 20 L autoclaved 0.45 lm filtered natural seawater collected from ge4 station. All culture bottles were placed in a container continuously exchanging seawater by cycling equipment to ensure the culture temperature the same with the field seawater and the temperature of field experiments ranged from 14.6 °C to 20.8 °C. Moreover, the container was placed in the main deck to make cultures under the natural light. In order to evaluate nutrients supply and N/P ratios effect on the growth of U. prolifera, the U. prolifera were inoculated to fresh media with the experimental nutrient conditions. Four different nutrient treatments were tested and control was only with the prepared seawater (1), and experimental cultures added stationary PO4–P concentration (0.5 lmol L1) with exponential DIN concentrations 10 lmol L1 (2), 20 lmol L1 (3), and 40 lmol L1 (4). For each nutrient treatment two series of cultures were set up. One series called the batch culture group (noted from M1 to M4) were supplied with nutrients only at the first day. The second series called the semi-continuous culture group (noted from M5 to M8) were daily replaced 15 L seawater with 15 L prepared natural seawater and supplied with nutrients following the treatment every day. Besides, the experiment added the third culture group (noted M9 to M10, called on-site culture) containing four enclosures made of tarlatan that assured the internal seawater agree with seawater around the enclosure. Moreover, the on-site culture group were put in the container and entirely submerged into the seawater to research the growth of U. prolifera in the field seawater. All treatments were conducted in triplicate in the same conditions as it shows. The initial nutrient concentrations in all groups were listed in Table 1. The inocula (2.0 g fresh U. prolifera thalli) were performed by using thalli collected from the ge4 station on May 27, 2012 at the three series of cultures described above. For all cultures, U. prolifera wet weight was measured every two days. Assuming that the changes in wet weight of U. prolifera were an indicator of U. prolifera growth, mean daily growth rates under different cultured conditions were calculated using the following modified formula (1) (Li et al., 2009):

  W iþ1  1  100% Wi

Ki ¼

Pn K¼

i¼1 K i

N

ð1Þ

where Ki is growth rate between i sample and (i + 1) sample, Wi+1 is wet weight of (i + 1) sample, Wi is wet weight of i sample, N is culture time, K is mean daily growth rate. Incremented weight percentage was calculated using the following equation (Li et al., 2010):

 Fig. 1. Location of the experiment monitoring site and sampling station in the southern Yellow Sea.

wi ¼

 W im  1  100% W om

ð2Þ

Please cite this article in press as: Zhou, Y., et al. Influence of nutrients pollution on the growth and organic matter output of Ulva prolifera in the southern Yellow Sea, China. Mar. Pollut. Bull. (2015), http://dx.doi.org/10.1016/j.marpolbul.2015.04.034

Y. Zhou et al. / Marine Pollution Bulletin xxx (2015) xxx–xxx Table 1 Addition of P–PO4, DIN and the initial nutrient concentrations after being added. No.

Initial nutrient concentration (lmol L1) P–PO4

DIN

Batch culture M1 M2 M3 M4

0.12 0.71 0.65 0.67

24.78 37.57 47.98 64.70

Semi-continuous culture M5 M6 M7 M8

0.16 0.67 0.67 0.64

23.67 35.46 47.94 58.51

On-site culture group M9 M10

0.26 0.26

41.10 41.10

Additions and the nutrient ratio were defined according to nutrient concentration measured in situ in the Yellow Sea when the U. prolifera erupted. The P–PO4 concentration was similar with that of the Yellow Sea, and the N/P ratio varied to test the influence of increasing DIN concentration. In batch culture M2 to M4, a gradient of DIN (NO3–N) addition was determined ranging from 10 lmol L1to a larger concentration. And the semi-continuous culture was dealt with in the same method and added nutrient every day. Besides, the nutrient condition of the third culture group agreed with the field seawater. The initial concentration was determined according to the sample analysis.

where wi is incremented weight percentage of i sample, W im is maximal wet weight of i enclosure, W om is maximal wet weight of control enclosure. 2.2.3. Nutrient analysis Water samples were daily decanted and filtered (0.7 lm Whatman glass fiber filters) from the raw water samples in the cultures and spectrophotometrically analyzed for the concentrations of DIN and PO4–P immediately according to Zhu (2006). Nutrient analysis in the second series was performed again after nutrient supply. The standard deviations from the spectrophotometric analysis (NO3–N, NO2–N, NH4–N, PO4–P, SiO3–Si) were ±0.05 mM, ±0.02 mM, ±0.03 mM, ±0.02 mM, and ±0.05 mM, respectively (Grasshoff et al., 1999). 3. Results 3.1. Nutrients distribution of the Southern Yellow Sea The surface distribution of nutrients in SYS were detected from May 27, 2012 to June 6, 2012. Due to the rapid economic development and population growth, the coastal waters have received increasing amounts of nutrients from rivers (Ma et al., 2013; Strokal et al., 2014). So the nutrients concentration of Yellow Sea peaked along the coastal sea due to human activities. The maximum concentration of DIN of 40 lM was observed near the coast of Yellow Sea (Fig. 3A), and it ranged from 10 lM to 40 lM. P concentration reached its highest values (0.9 lM) off the coast, while the P concentration showed a significant high value area near the coast (Fig. 3B). Algae eruption has consumed large of nutrients near the coast, and the Yellow Sea Warm Current supply lots of nutrients for the central area of Yellow Sea (Wei et al., 2013). Besides, the Yellow Sea Cold Water Mass (YSCWM) played an important role in the distribution of nutrient regimes of the SYS, and YSCWM could supplement nutrients to the upper layer (Su et al., 2013), so P concentration near the coast is lower than the central area of Yellow Sea. P concentration ranged from 0.3 lM to 0.6 lM, which indicated larger than Redfield ratio (N/P > 16). The higher N/P ratios indicated that phosphate is the limited factor for phytoplankton of Yellow Sea (Xie and Sun, 2012).

3

3.2. Evolution of nutrient concentrations in the culture groups The field experiment was conducted for 11 days and the nutrient changes during the culture period were shown in Fig. 2. Under the batch cultures M1 to M4, P dramatically decreased to the limiting levels (P < 0.1 lM) on day 2 (Fig. 2B). While DIN available were continuously accumulated till to low concentration of day 7 (Fig. 2A), although P concentration got close to the limiting levels. In the semi-continuous cultures M5 to M8, nutrient concentrations after nutrient supply increased to a higher value on day 4, and subsequently decreased to the end of experiment (Fig. 2C and D). Due the adaptation of U. prolifera to the new culture environment, the nutrient uptake content of U. prolifera is less than nutrient supply contents in the semi-continuous culture before day 4 and were converse after day 4, so they have higher nutrients concentration between day 2 and day 4. P uptake rates among semi-continuous cultures had no significant difference except for control culture M5 and higher P concentration promoted P uptake rate (Fig. 2D and Table 2). However, DIN uptake rates were complex. DIN uptake rates were similar among semi-continuous cultures before day 5, although they had different N/P ratios (Table 2). In contrast, after day 5 DIN concentration decided DIN uptake rates and ratios of the N uptake rate to the P uptake rate got close to N/P ratios of complementary nutrient (Fig. 2C and Table 2). Under the on-site culture conditions M9 to M10, nutrient concentrations varied following the sail (Fig. 2E). Consequently, nutrient concentrations in the third culture group decreased followed the ship sailing away from the coast. 3.3. Growth response of U. prolifera to different nutrient conditions The growth of U. prolifera was characterized by the wet weight and mean daily growth rate. Initial wet weight of U. prolifera were similarly on average, 2.0 g in all cultures. The obvious differences of U. prolifera wet weights were observed from day 6 (Fig. 4A). As shown in Fig. 4A, the wet weights of U. prolifera in on-site culture group were the highest and the highest increased wet weight was 20.6 g during exponential phase (from day 6 to day 11). The batch culture group was with the lowest increased wet weight of 2.4 g during exponential phase (Fig. 4A) and the N/P ratios did not have significant influence on wet weights of U. prolifera in the batch cultures (ANOVA, p > 0.05). While the wet weights of U. prolifera differed significantly among the semi-continuous cultures (p < 0.05) and the wet weight in the culture M8 with higher N/P ratio was higher than that in other cultures. The increased wet weight percentage in different groups on day 10 showed the similar trend with the wet weights (Table 3). Growth rates in all treatments, based on wet weights, are presented in Fig. 4B. Mean daily growth rates ranged from 10.46% to 42.17% during the culture time, consist with the laboratory works and in situ investigations (Liang et al., 2008). Similar with wet weight, the highest mean daily growth rate (42.17%) was obtained in on-site cultures, and it was 1.6- to 3.3-fold higher than the maximum values in the other culture groups. Besides, mean daily growth rates were not significantly different among the batch culture (M1M4) (ANOVA, p > 0.05), while the more DIN supply promoted the mean daily growth rates in the semi-continuous culture (ANOVA, p < 0.05). The maximum growth rates had a similar trend with mean daily growth rates in all culture groups (Fig. 4B). 3.4. The relationship between the increased mass of U. prolifera and nutrients uptake content Mean daily growth rates among semi-continuous culture were statistically significantly correlated with DIN concentrations (ANOVA, p < 0.05). To evaluate the relationship between production of U. prolifera and nutrients, the uptake content of DIN and

Please cite this article in press as: Zhou, Y., et al. Influence of nutrients pollution on the growth and organic matter output of Ulva prolifera in the southern Yellow Sea, China. Mar. Pollut. Bull. (2015), http://dx.doi.org/10.1016/j.marpolbul.2015.04.034

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Y. Zhou et al. / Marine Pollution Bulletin xxx (2015) xxx–xxx

70 60

DIN (μmol·L-1 )

B

M1 M2 M3 M4

50

1.0

M1 M2 M3 M4

0.8

PO 4 -P (μmol·L-1 )

A

40 30

0.6

0.4

20 0.2 10 0.0

0 0

2

4

6

8

10

0

12

2

4

Days

C

D

M5 M6 M7 M8

80 70

8

60

1.6

10

12

M5 M6 M7 M8

1.4 1.2

PO 4 -P (μmol·L-1)

50 40 30

1.0 0.8 0.6

20

0.4

10

0.2 0.0

0 0

2

4

6

8

10

12

0

2

4

PO 4 -P concentration (μmol·L - 1)

E

6

8

10

12

Days

Days 1.5

45

M9 PO 4 -P M10 PO4 -P M9 DIN M10 DIN

1.2

40 35 30

0.9

25 20

0.6

15 10

0.3

5

DIN concentration (μmol·L- 1)

DIN (μmol·L-1)

6

Days

0

0.0 0

2

4

6

8

10

12

Days Fig. 2. The variation of DIN (A) and PO4–P (B) concentrations in batch culture group, the variation of DIN (C) and PO4–P (D) concentrations after nutrient supply in semicontinuous culture group and the variation of DIN and PO4–P (E) concentrations in on-site culture group.

PO4–P was calculated in semis-continuous culture group for the early 10 days (Table 4). The result showed that the incremental wet weight of U. prolifera has a good linear correlation with DIN uptake content (Fig. 5, r2 = 0.903), but not with P uptake content (p > 0.05). The equations of the relationship between the incremental wet weight of U. prolifera and DIN uptake content were used as follow: DWet weight = 103.34NDIN + 1722. The strongest correlations found between increased wet weight and DIN uptake content indicate a determinant role of the limiting N over U. prolifera growth. 4. Discussion 4.1. Influence of nutrients on U. prolifera blooms Large scale eruption of green tides, especially U. prolifera, arose the question that why the rapid growth was caused. During the

cruise, it was found that U. prolifera was the predominant green tide. Previous works indicated that the optimum temperature from 15 °C to 25 °C was necessary for U. prolifera eruption (Wu et al., 2010; Xia et al., 2009). During the cruise, the temperature of field experiments was consistent with in situ temperature ranging from 14.6 °C to 20.8 °C, which was between the optimum temperature. Thus, sufficient nutrients concentration was the major factor of promoting the growth of U. prolifera in the field experiments. U. prolifera had strong ability of consuming nutrients (Raven and Taylor, 2003) so that U. prolifera showed high uptake rate of nutrients during the whole culture period in the field experiments. DIN and P had important roles of triggering U. prolifera blooms. P was a potential limited factor of U. prolifera blooms (Xia et al., 2009; Liu et al., 2013a,b; Wu et al., 2013; Shi et al., 2015) and the study found that U. prolifera had a higher utilized rate of phosphate before P concentration got to the limiting level. The P uptake content for a whole day was close to the P content in the media during the

Please cite this article in press as: Zhou, Y., et al. Influence of nutrients pollution on the growth and organic matter output of Ulva prolifera in the southern Yellow Sea, China. Mar. Pollut. Bull. (2015), http://dx.doi.org/10.1016/j.marpolbul.2015.04.034

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Y. Zhou et al. / Marine Pollution Bulletin xxx (2015) xxx–xxx

Fig. 3. DIN (A) and PO4–P (B) concentration in surface water during May 27, 2012 and June 6, 2012 at the sampling stations.

Table 2 Uptake rates of nitrogen and phosphorus (lM DIN or P day1) from the media and ratios of the N uptake rate to the P uptake rate for U. prolifera cultures grown under different nutrient treatments in semi-continuous culture group (values of uptake rates are not presented where nutrient concentrations increased). Time (days)

1–2 2–3 3–4 4–5 5–10

Control

N/P = 20

N/P = 40

N/P = 80

N

P

N/P

N

P

N/P

N

P

N/P

N

P

N/P

19.0 – 4.87 19.0 2.58

0.07 0.88 0.36 0.44 0.05

271 – 13.5 43.1 51.6

20.57 18.11 0.92 33.6 12.6

0.62 1.41 0.65 0.81 0.58

33.2 12.8 1.42 41.5 21.7

24.2 10.5 8.92 27.3 21.4

0.60 1.26 0.63 0.76 0.57

40.3 8.33 14.4 35.9 37.5

27.2 19.1 20.4 41.7 39.0

0.59 1.28 0.74 0.78 0.52

46.1 14.9 27.6 53.5 75.0

A

B M1 M2 M3 M4 M5 M6 M7 M8 M9 M10

25 20

80

Mean daily growth rate Maximal growth rate

70 60

Growth rate (%)

wet weight of U.prolifera (g)

30

15 10

50 40 30 20

5

10 0

0 0

2

4

6

8

10

12

Days

M1 M2 M3 M4 M5 M6 M7 M8 M9 M10

NO.

Fig. 4. The wet weight (A) and the mean daily growth rate (B) of U. prolifera in the experimental enclosures conducted under different nutrient conditions and different culture methods. The mean daily growth rate is calculated according to the wet weight of U. prolifera. In the experiment, the mean daily growth rate is an index that weighed the growth of U. prolifera. The maximal growth rate (B) represents the nutrient uptake ability of U. prolifera under different nutrient concentrations.

culture period (Fig. 2B and D), which was consist with the laboratory results of Hong (Hong et al., 2011). However, the growth of U. prolifera in the field experiment was more likely to be decided by DIN uptake content, as indicated by higher growth rate in the culture with higher N/P ratio. Moreover, the main role of DIN on growth of U. prolifera was reflected in exponential phase. In the semi-continuous culture, the uptake rate of DIN was significantly affected by N/P ratios in the exponential phase, while it did not happen before exponential phase (Fig. 4A). The lower P

concentration did not limit the DIN uptake of U. prolifera in the exponential period (Fig. 2C and Pedersen, 1995; Sfriso et al., 1988; Dailer et al., 2012). Besides, the difference of N/P did not work in the batch culture only with nutrient supply before exponential phase. These results clearly indicated DIN content was a major factor of U. prolifera blooms and DIN promotion primarily occurred during exponential phase. High growth rate of U. prolifera in the on-site culture group with more DIN uptake content further supported that DIN was a decisive factor of U. prolifera eruption.

Please cite this article in press as: Zhou, Y., et al. Influence of nutrients pollution on the growth and organic matter output of Ulva prolifera in the southern Yellow Sea, China. Mar. Pollut. Bull. (2015), http://dx.doi.org/10.1016/j.marpolbul.2015.04.034

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Table 3 Increased wet weight percentage of the first 10 days VS control and wet weight on day 10 in cultures with nutrients supply. No.

Wet weight on day 10 (g)

Increased wet weight percentage VS control

M2 M3 M4 M6 M7 M8

5.2 ± 0.1 5.1 ± 0.1 5.0 ± 0.1 7.5 ± 0.2 9.8 ± 0.3 11.7 ± 0.2

15.5% ± 2.2% 13.3% ± 2.2% 11.1% ± 2.2% 56.2% ± 4.2% 104.2% ± 6.2% 143.7% ± 4.2%

Si ¼

Table 4 The increased wet weight of U. prolifera, the quality of the absorbed nutrient in semiscontinuous culture group for the early 10 days. No.

M5

M6

M7

M8

Incremented wet weight Dm (g) Quality of absorbed DIN (mg) Quality of absorbed PO4–P (mg) Incremented organic matter (mg)

2.8 15.4 1.7 242

5.5 37.1 3.7 582

7.8 48.3 3.2 755

9.7 82.2 3.3 1283

10

Incremented wet weight Δm 䠄㼓䠅

9 8 7 6 5 4 3 2 10

20

30

40

50

60

2. The mean daily growth rate (k) assumed to be 35% according to the results of investigated sea (Liang et al., 2008) and the on-site culture enclosure M9–M10. 3. According to the mean daily growth rate and the entire floating area Sf of U. prolifera, the daily floating area Si of U. prolifera could be calculated as follows:

70

80

90

DIN uptake content (mg)

Sf ð1 þ kÞ

ni

ð4Þ

where n is the time of U. prolifera growth, i is the time of U. prolifera floating around the inshore, Sf is the entire floating area of U. prolifera, Si is the daily floating area of U. prolifera. According to the formula (3), (4) and the DIN concentration in the sea, the DIN amounts absorbed could be calculated. Then the weight of U. prolifera could be calculated with the DIN uptake content according to the linear relationship (Fig. 5). Using this method, we calculated the productivity of U. prolifera within a month in the Yellow Sea of 2008. The entire area Sf covered by U. prolifera patches was approximately 13,000 km2 (Sun et al., 2008) and mean DIN concentration w is 14.89 lmol L1 from June, 15th to July, 15th (Xia et al., 2009) in the sea where the U. prolifera patches accumulated. It was calculated that the mass of DIN uptake is 7.74  106 kg assuming h is 1 m, so the output of U. prolifera was calculated to be approximately 79.9  104 t according to the linear relation in Fig. 5, which came close to the practical weight of U. prolifera within a month (80  104 t). Different algae have different ability of absorbing nutrients and different C/N ratios. Kim et al. found that the C/N ratio of U. prolifera was about 17 when it floated away the coast of Yellow Sea (Kim et al., 2011). Choi et al. found that the C/N ratio is about 13–19 (Choi et al., 2010). Assuming C/N ratio of U. prolifera is about 17, incremental organic matters (OM) were evaluated in semi-continuous cultures (Table 4). Moreover, the incremental OM had a relative relationship with incremental wet weight of U. prolifera, OM = 0.141Dm  0.19 (p < 0.05), so the organic matters originated from output of U. prolifera could be calculated according to the relationship. Assuming floating areas of U. prolifera were limited between 32°N–36°N and 119°E–122.5°E of the Yellow Sea, the output of U. prolifera was about 4.28  106 t and the OM pollutant output derived U. prolifera was about 4.1  105 t.

Fig. 5. The relationship between incremental weight and DIN uptake content.

U. prolifera often firstly erupt in the southern Yellow Sea where the DIN concentration was higher. 4.2. OM output of U. prolifera evaluated from DIN content The wet weight output of U. prolifera was obtained based on the relationship between wet weight of U. prolifera and absorbed content of DIN (Fig. 5), which showed a method calculating the output of U. prolifera in the Yellow Sea. Using above assumption, the OM output of U. prolifera in the southern Yellow Sea was evaluated according to the uptake DIN content (Fig. 5). The calculation equation made following several hypotheses: 1. The depth h of seawater where U. prolifera absorbed nutrients was no more than 1 m and DIN was consumed completely in this depth by U. prolifera. The DIN amounts absorbed could be calculated as follows:

NDIN ¼ w  RSi  h

ð3Þ

where NDIN is the DIN content absorbed by floating U. prolifera, w is the mean concentration of DIN on the surface seawater, Si is the floating area of U. prolifera on day i, h is the depth of nutrients able to be absorbed.

4.3. The importance of field experiments on the management of U. prolifera Based on long-term analysis of nutrients in the Southern Yellow Sea, a clear increasing trend was observed in the horizontal distribution of NO3–N over the past three decades, especially since 2008 (Keesing et al., 2011). Besides, the nutrient composition also changed markedly since the occurrence of green tides in the SYS, marked by a sharp rise in the N/P ratio. The increase and change of nutrient composition may be one of the most critical causative factors contributing to green tides blooms in this area (Li et al., in press). The field experiments ensured conditions approaching the in situ environment and indicated that the DIN supply content of exponential phase controlled the output of U. prolifera eruption and P was the potential limited factor of U. prolifera blooms in the SYS. The field experiments made a better analysis of the effects of nutrients on U. prolifera, which supplied a comprehensive understanding of U. prolifera eruption. Besides, it implied that decreasing of nitrogen content was one of effective methods of controlling the output of U. prolifera eruption. The field experiments also showed semi-continuous culture was a better method to study the influence of different N substrates, such as urea and ammonium, on growth of U. prolifera (Wu et al., 2011). The large-scale eruption of U. prolifera required the government to adopt effective measures to deal with them and the common solution was physical gather

Please cite this article in press as: Zhou, Y., et al. Influence of nutrients pollution on the growth and organic matter output of Ulva prolifera in the southern Yellow Sea, China. Mar. Pollut. Bull. (2015), http://dx.doi.org/10.1016/j.marpolbul.2015.04.034

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that caused a large expense (Ye et al., 2011). The precise biomass data derived from evaluation by the DIN content will help workers to prepare enough tools for gathering U. prolifera. This method was more convenient than RS techniques and cruise observations, which had been used to investigate the biomass of U. prolifera (Qiao et al., 2009; Liu et al., in press). Moreover, the strong ability of storing DIN by U. prolifera derived from experiments indicated that the floating of U. prolifera would transit amounts of nitrogen from the Southern Yellow Sea to north with the push of wind, resulting in the change of nutrients composition. The tissue-N composition of U. prolifera was an indicator of sources of nitrogen (Barr et al., 2013).

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Please cite this article in press as: Zhou, Y., et al. Influence of nutrients pollution on the growth and organic matter output of Ulva prolifera in the southern Yellow Sea, China. Mar. Pollut. Bull. (2015), http://dx.doi.org/10.1016/j.marpolbul.2015.04.034