Chemosphere 127 (2015) 188–194
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Environmental effects of modified clay flocculation on Alexandrium tamarense and paralytic shellfish poisoning toxins (PSTs) Guangyuan Lu a,b,c, Xiuxian Song a,⇑, Zhiming Yu a,⇑, Xihua Cao a, Yongquan Yuan a a
Key Laboratory of Marine Ecology and Environmental Sciences, Institute of Oceanology, Chinese Academy of Sciences, IOCAS, Qingdao 266071, PR China University of Chinese Academy of Sciences, Beijing 100049, PR China c School of Environmental Science and Engineering, Tianjin University, Tianjin 300072, PR China b
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
1 of MC could efficiently remove >90% cells of A. tamarense within 3.5 h. MC quickly eliminated both inorganic and organic macronutrients from seawater. Firstly demonstrated the potential detoxification of PSTs by using MC treatment. The high toxicity GTX1 and GTX4 transformed into the lower toxicity GTX2 and dcGTX3.
0.25 g L
a r t i c l e
i n f o
Article history: Received 12 March 2014 Received in revised form 13 January 2015 Accepted 25 January 2015
Handling Editor: X. Cao Keywords: Modified clay HABs mitigation Paralytic shellfish poisoning toxin (PSTs) Alexandrium tamarense Environmental effect
a b s t r a c t Among various mitigation strategies for harmful algal blooms (HABs), the flocculation of algal cells by using modified clay (MC) has been widely applied in the field, particularly in Japan, Korea and China. However, to examine the long-term effects and the environmental safety of this method, we investigated alterations in macronutrients and paralytic shellfish poisoning toxins (PSTs) induced by the application of MC treatment to a toxic bloom, Alexandrium tamarense. The control, algal cells grew in nature condition (A1), was compared to the only MC flocculation (A2) and the MC-sediment co-matrix systems of A. tamarense (A3). The low-dosage of 0.25 g L1 MC could efficiently remove >90% of the A. tamarense cells within 3.5 h. The mechanisms underlying the effects elicited by MC flocculation on nutrient cycling, PSTs and Chl-a degradation were also discussed. This study demonstrated that MC treatment was able to significantly remove the macronutrients (43–60% TP removal and 17–30% TN removal) and scavenge most of the PSTs from seawater, thereby speeding up the nutrient settling and the transformation and degradation of PSTs (83% decreasing in A2). Simultaneously, the study firstly demonstrated the potential detoxification of PSTs by using MC treatment, from the high toxicity of gonyautoxin 1 and 4 (GTX1 and GTX4) to the lower toxicity decarbamoyl gonyautoxins (dcGTX3) and gonyautoxin 2 (GTX2), particularly within the water-sediment environment during the two month incubation. Ó 2015 Elsevier Ltd. All rights reserved.
⇑ Corresponding authors at: Biological Building 417, Institute of Oceanology, Chinese Academy of Sciences, IOCAS, Nanhai Road 7#, Shinan District, Qingdao 266071, PR China. Tel.: +86 532 82898587; fax: +86 532 82898566 (X. Song). Tel.: +86 532 82898581; fax: +86 532 82898581 (Z. Yu). E-mail addresses: [email protected] (G. Lu), [email protected] (X. Song), [email protected] (Z. Yu), [email protected] (X. Cao), [email protected] (Y. Yuan). http://dx.doi.org/10.1016/j.chemosphere.2015.01.039 0045-6535/Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction The frequency and scales of harmful algal blooms (HABs) have increased over the past three decades (Anderson et al., 2012). The coagulation–flocculation of algae cells using modified clay
G. Lu et al. / Chemosphere 127 (2015) 188–194
(MC) is a promising method among the HAB-control treatments when applied in several coastal waters, particularly in Korea, Japan and China (Pierce et al., 2004; Yu et al., 2004; Hagström et al., 2010; Anderson et al., 2012). Paralytic shellfish poisoning toxins (PSTs) are a broad group of highly potent neurotoxins and gonyautoxins produced by the harmful phytoplankton Alexandrium, Gymonodinium and Pyrodinium. Moreover, the accumulation of PSTs by marine organisms, such as mussels, clams, and fishes, through the filter-food web induces a disease that is lethal to marine mammals and humans (Hall et al., 1990) and is currently without antidotes or detoxification pathway (Faber, 2012). Every year, the amount of paralytic shellfish poisoning (PSP) event is reported around most coasts of the world (Burkholder et al., 1992; Jiang et al., 2013). A PSP event is not a recent problem, having been described in the exploration record of Captain George Vancouver in 1793 as an illness caused by eating mussels (NOAA, marine biotoxins). In 1909, Field found that most poisonings attributed to mussels and other shellfish are due to ptomaines, which are poisonous substances resulting from the action of micro-organisms upon the animal tissue. In 1948, the pure PST was obtained directly from Gonyaulax catenella (renamed Alexandrium catenella) (Sommer et al., 1948). In 1957–1962, the properties and structure of saxitoxin (STX) were described (Schantz et al., 1957; Schuett and Rapoport, 1962). Today, PSTs, identified as one of the four recognized syndromes of shellfish poisoning, are approximately 57 hydrophobic analogues of SXT. Of particular concern for the control of toxic algal blooms, only a handful of studies were based on the removal of toxic algae and toxins. Most studies focused on the removal of Pseudo-nitzschia pungens (Yu and Subba, 1998), Prymnesium parvum (Hagström and Granéli, 2005; Sengco et al., 2005; Hagström et al., 2010), Karenia brevis (Pierce et al., 2004), Microcystis aeruginosa (Chang et al., } 2014) and cyanobacterium Anabaena sp. (Engström-Ost et al., 2013). The toxins produced by the phytoplankton could also bind to clay, thereby enabling the use of clay to remove and/or neutralize the toxins; however, there was a concern that the clay might have a negative effect on sessile organisms (Hagström et al., 2010). Thus, particular consideration should be taken regarding the potential long-term effects of MC treatment of toxic HABs. This study used Alexandrium tamarense as a model toxic algal bloom species that produced PSTs and blooms in the coastal waters of China, particularly blooms in the East China Sea every year since 2000 (Jiang et al., 2013). The aim of the present study was to elucidate how MC flocculation of A. tamarense influences the variability of nitrogen, phosphorus, and PSTs’ roles in the aquatic environment. This approach enabled us to test whether the potential environmental effects of the MC strategy was attributed to the adsorptive preservation of toxins.
189
approximately 60–65 lmol photons m2 S1 and a 12:12 h light/ dark cycle at a temperature of 20 ± 1 °C throughout the experiments (Lu et al., 2014). The MC slurry used in this study was based on polyaluminum chloride (PACl, analytical reagent) to modify Kaolin (1:5) in a hyperpure water of 25.0 g L1 as stock solution (Yu et al., 1999; Yu et al., 2004). The slurry was not mixed until required in the algae removal experiments. Freeze-dried sediment samples were obtained from the East China Sea Coast, China, where A. tamarense blooms often occur (Chen et al., 2013). The TN and TOC concentrations of the freezedried samples (site ZA3 in the Zhejiang coast, 29°310 N, 122°360 4800 E, where A. tamarense blooms often occur) were 0.0608% and 0.52%, respectively. 2.2. Cell removal experiment Prior to the incubation contrast experiments, 500 mL of algal culture, at exponential state of 3.1 107 cells L1 was thoroughly stirred and divided equally into 50 mL-tubes. After culturing the tubes of algae, 0-, 0.05-, 0.25-, 0.50-, 1.0-, 2.08- and 4.35-mL aliquots of the MC mixture were added into the 50-mL algal cultures to final concentrations of 0, 0.025, 0.125, 0.25, 0.50, 1.00 and 2.00 g L1 MC, respectively. The tube contents were thoroughly mixed and then allowed to settle for 3.5 h. The samples were collected from 3 cm below the seawater surface. Simultaneously, we calculated the removal efficiency (RE) and selected the specific dosage for the subsequent incubation experiments. 2.3. Incubation experiments After culturing for 21 d, the stock cultures were divided equally into 75 test tubes to establish three treatments in triplicate at day 22, with each tube containing 100 mL of culture (Fig. S1). Treatment A1 was used to determine the normal degradation of A. tamarense aggregates; treatment A2 was used to investigate the effects of MC flocculation from day 25 to day 80; and treatment A3 was used to distinguish the effects of MC flocculation and sediments on the depositional environment from day 25 to day 80. Additionally, the filtrate of the algal culture at day 22 was divided into treatments B1, B2, and B3, which were then treated at day 25 to estimate the effects of light, MC particle, and sediment, respectively, on dissolved water. Before the MC removal experiment, 2.0 g of sediment was added into A3 and B3 and then algal culture slowly flowed into the tube without disturbance. At day 25, 1.0-mL aliquots of the selected MC mixture were carefully added to the 100-mL algal cultures of A2, A3, B2, and B3. During these incubation periods, the samples were carefully collected to minimize contamination at days 22, 25, 28, 32, 40, 66 and 80 (in triplicate), respectively (Fig. S1).
2. Materials and methods 2.4. Sampling and parameter analysis 2.1. Algal culture and preparation A. tamarense (CCMM1005, strain AT5-3) was isolated from Dapeng Bay and maintained by the Institute of Hydrobiology, Jinan University, Guangzhou, China. Prior to the incubation experiments, all of the glassware was pre-rinsed with 5% v/v HCl to remove possible contaminants for 24 h and then thoroughly washed with Milli-Q water. The 0.45-lm membrane-filtered seawater and glassware were sterilized at 121 °C for 20 min. Batch cultures at the exponential phase were inoculated in 2 L of sterilized seawater supplemented with a modified f/4 medium in 5-L flasks in quadruplicate (Guillard and Ryther, 1962; Guillard et al., 1973). The initial cellular concentration of the A. tamarense cultures was 1 104 cells L1. The stock culture was cultivated under light intensity of
The cell concentrations, pH and in vivo chlorophyll fluorescence Fa (Fluorescence Turner Designs, TD700; fsu) of the algal cultures were simultaneously monitored during the cultivation and flocculation period. The cellular concentrations were counted in triplicate under a microscope (OLYMPUS IX71, Tokyo, Japan) after fixation with a 5% glutaraldehyde solution (Churro et al., 2010; Maruyama and Kim, 2013). All of the nutrient samples were stored and frozen at 20 °C for further analysis by using a nutrients continuous flow analyser (Skalar San++, Breda, the Netherlands, see in Supplementary Data). The sediment samples were filtered through 0.22-lm glass–fibre membranes and then frozen until analysis. The Chl-a samples were treated for 24 h in 10 mL of 100% N,
G. Lu et al. / Chemosphere 127 (2015) 188–194
2.5. Data analysis The growth rates were calculated as the differences between two sampling intervals of the cell numbers (Guillard et al., 1973; Yu and Subba, 1998), l = [ln (Nt) ln (N0)]/(t t0), units of d1, where t0 and t represent the initial and incubation times, respectively; where No and Nt represent the cell numbers at days t0 and t, respectively. Conversion factors were used to express the toxicity of the sum of the variants as saxitoxin toxicity equivalents (STXeq., Table S1) due to the differing toxicities and concentrations of the individual saxitoxin variants (Hall et al., 1990; Kayal et al., 2008). The values were all averaged from triplicate treatments to produce the mean concentrations/values ± one standard deviation. Quantitative data analysis was based on SPSS 16.0 (multiple comparisons using Fisher’s LSD and Dunnett’s T3 method). The significant data was considered as p < 0.05.
3. Results 3.1. Algae removal and the basic parameters change in incubation experiment The RE value of A. tamarense cells increased with the pre-set MC concentrations, and the highest RE was found to be up to 99% at a concentration of 2.00 g L1 of MC (Fig. S2). According to the previous treatment, we used the concentration of 90% cell removal in the field application (unpublished data). Thus, we selected 0.25 g L1 as the representative concentration for use in comparison test (Fig. S2). A. tamarense cells in A1 were maintained in an exponential phase for the first 20 d, with a maximum cellular concentration of (19.00 ± 1.12) 106 cells L1 (Fig. 1) at day 32. When MC was added in A2 and A3 at day 25, the cellular concentrations in seawater dropped sharply from (17.44 ± 1.01) 106 cells L1 to (0.14 ± 0.04) 106 cells L1 (p 0.000 < 0.05, n = 9) and (0.11 ± 0.02) 106 cells L1, respectively (p 0.000 < 0.05, n = 9). Subsequently, the cellular concentration of A2 re-bloomed at a low density of (6.44 ± 1.01) 106 cells L1; however, the cellular concentration of A3 reached undetectable levels at day 80 (Fig. 1). After the addition of MC, the Fa of A2 and A3 suddenly decreased from 22.20 ± 1.95 fsu to 2.07 ± 0.32 fsu (p = 0.005 < 0.5, n = 3) and 1.95 ± 0.05 fsu (p = 0.006 < 0.05, n = 3), respectively, at day 25, which coincided with the cell density during the subsequent period (Fig. 1).
A1 cells A2 cells A3 cells
A1 Fa A2 Fa A3 Fa
40
20 Modified Clay Addition 15 20
6
-1
(*10 cells L )
25
10 5
A. tamarense 0
0 0
10
20
30
40
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in vivo Chlorophyll Fluorescence Fa
N-dimethylformamide (DMF) for efficient extraction (Inskeep and Bloom, 1985; Qin et al., 2013). For toxins analysis in the phytoplankton detritus and sediments, glass–fibre membranes were cut into small pieces and extracted with 5.0 mL of 80% v/v acetonitrile solution (including 0.1% v/v formic acid). PST extraction was conducted based on previous PST determination studies (Sayfritz et al., 2008; van de Riet et al., 2009; DeGrasse et al., 2011). The PSTs analysis used a postcolumn oxidation HPLC-FD (Water Corporation e2695 separation and 2475 detector, Milford, MA, USA), based closely on the AOAC Official Method 2011.02 (Turner et al., 2013, details in Supplementary data). All of the chemical reagents and procedures used for the carbamate gonyautoxins (GTX1, GTX2, GTX3, GTX4, STX and neoSTX), decarbamoyl toxins (dcGTX2, dcGTX3 and dcSTX), N-sulfocarbamoyl toxin GTX5, and N-sulfocarbamoyl toxins (C1 and C2) analysis were based on Chen et al. (2013) study.
Cell density of A. tamarense
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80
Time (d) Fig. 1. Growth curve of the cell density (left y-axis) and the in vivo chlorophyll fluorescence Fa (right y-axis) of A. tamarense using different comparative tests (here and in the following figures, solid and empty squares represent the cells and Fa in A1 natural growth; solid and empty cycles represent the cells and Fa in A2 only modified clay applied in the algal culture from day 25; solid and empty triangles represent the cells and Fa in A3 modified clay flocculation with algal cells under the water-sediment systems; the arrows indicate the modified clay addition time at day 25).
3.2. Characteristics of the nutrients in seawater The ammonium concentration in A1 was 8.42 ± 0.65 lM at day 80 (Table S2; Fig. S4). The nitrite concentrations increased to 13.78 ± 2.85 lM at day 80. In contrast, the nitrate and phosphate concentrations exhibited an obvious decrease to the level of 96.79 ± 6.06 lM and 1.06 ± 0.09 lM, respectively, at day 80. After the addition of MC, the ammonium concentrations in A2 exhibited no differences (p = 0.296 > 0.05, n = 3). However, the performance of phosphate in A2 first decreased significantly to 0.87 ± 0.05 lM at day 25 (p = 0.001 < 0.05, n = 3). The nitrite concentrations in A2 increased to >40 lM, whereas the nitrite level of A1 was less than 16 lM (p 0.000 < 0.05, n = 3). Within the sediment environment, the ammonium in A3 significantly increased to 34.58 ± 6.63 lM at day 25 (p = 0.001 < 0.05, n = 3) and to 103.23 ± 8.52 lM at day 80 (p 0.000 < 0.05, n = 3), which was much higher than that observed in A1 and A2. Simultaneously, the ammonium in B3 was about 60 lM (Fig. S4). The nitrate concentration in A3 decreased to 70.47 ± 11.94 lM at day 40 (Fig. S4). At day 80, there was significant differences of DIP concentration between A1 and A2 (p 0.000 < 0.05, n = 3), A1 and A3 (p 0.000 < 0.05, n = 3). 3.3. Changes in the chlorophyll a content of the sediments The Chl-a trend of the bottom water in A1 gradually decreased from 0.202 ± 0.012 lg mL1 at day 22 to 0.156 ± 0.003 lg mL1 at day 80 (Fig. 2). Upon the addition of MC, the Chl-a concentration in A2 sediment changed from 0.456 ± 0.010 lg mL1 at day 25 to 0.053 ± 0.001 lg mL1 at day 80. The Chl-a concentration in A3 sediment sharply decreased to 0.154 ± 0.004 lg mL1 at day 25, whereas the Chl-a concentration in the original sediment was 0.150 ± 0.006 lg mL1 in B3 (this part was deducted in Fig. 2). The q value in A1 ranged from 0.07 to 0.03 d1 with synchronous changes of the cell density in the upper waters. However, the q value in A2 gradually decreased to 0.04 d1. Simultaneously, the q values of A3 were approximately zero. 3.4. Changes of PSTs To detect the changes of total PSTs, we measured 12 main toxin concentrations of the bottom 50 mL-volume water and sediments.
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Chl-a in bottom water / sediment (mg L-1)
A1 Chl-a A2 Chl-a A3 Chl-a
0.6
0.4
0.2 Modified Clay Addition
0.0 20
30
40
50
60
70
80
Time (d)
the total PSTs concentration (STX-eq. nM)
b
a
A1 total PSTs A2 total PSTs A3 total PSTs
100
50 Modified Clay Addition
0 20
30
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50
60
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80
Time (d)
Fig. 2. The chlorophyll-a (mg L1; (a)) and the total PSTs (STX-eq. nM; (b)) changes of A. tamarense cells of bottom water and sediments in three compared treatments.
The total PSTs in A1 exhibited the auto-deposition of algal toxins, and the concentration gradually ranged from 36.75 STX-eq. nM to 78.76 STX-eq. nM (Fig. 2). Upon the addition of MC, the total PSTs in A2 sediments initially increased to 106.02 ± 0.19 STX-eq. nM at day 25 and gradually decreased to a minimum concentration of 17.75 STX-eq. nM at day 80 (Fig. 2). In contrast, the total PSTs concentration in A3 sediments initially decreased to 15.02 STXeq. nM at day 25 and remained
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Chemosphere journal homepage: www.elsevier.com/locate/chemosphere
Environmental effects of modified clay flocculation on Alexandrium tamarense and paralytic shellfish poisoning toxins (PSTs) Guangyuan Lu a,b,c, Xiuxian Song a,⇑, Zhiming Yu a,⇑, Xihua Cao a, Yongquan Yuan a a
Key Laboratory of Marine Ecology and Environmental Sciences, Institute of Oceanology, Chinese Academy of Sciences, IOCAS, Qingdao 266071, PR China University of Chinese Academy of Sciences, Beijing 100049, PR China c School of Environmental Science and Engineering, Tianjin University, Tianjin 300072, PR China b
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
1 of MC could efficiently remove >90% cells of A. tamarense within 3.5 h. MC quickly eliminated both inorganic and organic macronutrients from seawater. Firstly demonstrated the potential detoxification of PSTs by using MC treatment. The high toxicity GTX1 and GTX4 transformed into the lower toxicity GTX2 and dcGTX3.
0.25 g L
a r t i c l e
i n f o
Article history: Received 12 March 2014 Received in revised form 13 January 2015 Accepted 25 January 2015
Handling Editor: X. Cao Keywords: Modified clay HABs mitigation Paralytic shellfish poisoning toxin (PSTs) Alexandrium tamarense Environmental effect
a b s t r a c t Among various mitigation strategies for harmful algal blooms (HABs), the flocculation of algal cells by using modified clay (MC) has been widely applied in the field, particularly in Japan, Korea and China. However, to examine the long-term effects and the environmental safety of this method, we investigated alterations in macronutrients and paralytic shellfish poisoning toxins (PSTs) induced by the application of MC treatment to a toxic bloom, Alexandrium tamarense. The control, algal cells grew in nature condition (A1), was compared to the only MC flocculation (A2) and the MC-sediment co-matrix systems of A. tamarense (A3). The low-dosage of 0.25 g L1 MC could efficiently remove >90% of the A. tamarense cells within 3.5 h. The mechanisms underlying the effects elicited by MC flocculation on nutrient cycling, PSTs and Chl-a degradation were also discussed. This study demonstrated that MC treatment was able to significantly remove the macronutrients (43–60% TP removal and 17–30% TN removal) and scavenge most of the PSTs from seawater, thereby speeding up the nutrient settling and the transformation and degradation of PSTs (83% decreasing in A2). Simultaneously, the study firstly demonstrated the potential detoxification of PSTs by using MC treatment, from the high toxicity of gonyautoxin 1 and 4 (GTX1 and GTX4) to the lower toxicity decarbamoyl gonyautoxins (dcGTX3) and gonyautoxin 2 (GTX2), particularly within the water-sediment environment during the two month incubation. Ó 2015 Elsevier Ltd. All rights reserved.
⇑ Corresponding authors at: Biological Building 417, Institute of Oceanology, Chinese Academy of Sciences, IOCAS, Nanhai Road 7#, Shinan District, Qingdao 266071, PR China. Tel.: +86 532 82898587; fax: +86 532 82898566 (X. Song). Tel.: +86 532 82898581; fax: +86 532 82898581 (Z. Yu). E-mail addresses: [email protected] (G. Lu), [email protected] (X. Song), [email protected] (Z. Yu), [email protected] (X. Cao), [email protected] (Y. Yuan). http://dx.doi.org/10.1016/j.chemosphere.2015.01.039 0045-6535/Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction The frequency and scales of harmful algal blooms (HABs) have increased over the past three decades (Anderson et al., 2012). The coagulation–flocculation of algae cells using modified clay
G. Lu et al. / Chemosphere 127 (2015) 188–194
(MC) is a promising method among the HAB-control treatments when applied in several coastal waters, particularly in Korea, Japan and China (Pierce et al., 2004; Yu et al., 2004; Hagström et al., 2010; Anderson et al., 2012). Paralytic shellfish poisoning toxins (PSTs) are a broad group of highly potent neurotoxins and gonyautoxins produced by the harmful phytoplankton Alexandrium, Gymonodinium and Pyrodinium. Moreover, the accumulation of PSTs by marine organisms, such as mussels, clams, and fishes, through the filter-food web induces a disease that is lethal to marine mammals and humans (Hall et al., 1990) and is currently without antidotes or detoxification pathway (Faber, 2012). Every year, the amount of paralytic shellfish poisoning (PSP) event is reported around most coasts of the world (Burkholder et al., 1992; Jiang et al., 2013). A PSP event is not a recent problem, having been described in the exploration record of Captain George Vancouver in 1793 as an illness caused by eating mussels (NOAA, marine biotoxins). In 1909, Field found that most poisonings attributed to mussels and other shellfish are due to ptomaines, which are poisonous substances resulting from the action of micro-organisms upon the animal tissue. In 1948, the pure PST was obtained directly from Gonyaulax catenella (renamed Alexandrium catenella) (Sommer et al., 1948). In 1957–1962, the properties and structure of saxitoxin (STX) were described (Schantz et al., 1957; Schuett and Rapoport, 1962). Today, PSTs, identified as one of the four recognized syndromes of shellfish poisoning, are approximately 57 hydrophobic analogues of SXT. Of particular concern for the control of toxic algal blooms, only a handful of studies were based on the removal of toxic algae and toxins. Most studies focused on the removal of Pseudo-nitzschia pungens (Yu and Subba, 1998), Prymnesium parvum (Hagström and Granéli, 2005; Sengco et al., 2005; Hagström et al., 2010), Karenia brevis (Pierce et al., 2004), Microcystis aeruginosa (Chang et al., } 2014) and cyanobacterium Anabaena sp. (Engström-Ost et al., 2013). The toxins produced by the phytoplankton could also bind to clay, thereby enabling the use of clay to remove and/or neutralize the toxins; however, there was a concern that the clay might have a negative effect on sessile organisms (Hagström et al., 2010). Thus, particular consideration should be taken regarding the potential long-term effects of MC treatment of toxic HABs. This study used Alexandrium tamarense as a model toxic algal bloom species that produced PSTs and blooms in the coastal waters of China, particularly blooms in the East China Sea every year since 2000 (Jiang et al., 2013). The aim of the present study was to elucidate how MC flocculation of A. tamarense influences the variability of nitrogen, phosphorus, and PSTs’ roles in the aquatic environment. This approach enabled us to test whether the potential environmental effects of the MC strategy was attributed to the adsorptive preservation of toxins.
189
approximately 60–65 lmol photons m2 S1 and a 12:12 h light/ dark cycle at a temperature of 20 ± 1 °C throughout the experiments (Lu et al., 2014). The MC slurry used in this study was based on polyaluminum chloride (PACl, analytical reagent) to modify Kaolin (1:5) in a hyperpure water of 25.0 g L1 as stock solution (Yu et al., 1999; Yu et al., 2004). The slurry was not mixed until required in the algae removal experiments. Freeze-dried sediment samples were obtained from the East China Sea Coast, China, where A. tamarense blooms often occur (Chen et al., 2013). The TN and TOC concentrations of the freezedried samples (site ZA3 in the Zhejiang coast, 29°310 N, 122°360 4800 E, where A. tamarense blooms often occur) were 0.0608% and 0.52%, respectively. 2.2. Cell removal experiment Prior to the incubation contrast experiments, 500 mL of algal culture, at exponential state of 3.1 107 cells L1 was thoroughly stirred and divided equally into 50 mL-tubes. After culturing the tubes of algae, 0-, 0.05-, 0.25-, 0.50-, 1.0-, 2.08- and 4.35-mL aliquots of the MC mixture were added into the 50-mL algal cultures to final concentrations of 0, 0.025, 0.125, 0.25, 0.50, 1.00 and 2.00 g L1 MC, respectively. The tube contents were thoroughly mixed and then allowed to settle for 3.5 h. The samples were collected from 3 cm below the seawater surface. Simultaneously, we calculated the removal efficiency (RE) and selected the specific dosage for the subsequent incubation experiments. 2.3. Incubation experiments After culturing for 21 d, the stock cultures were divided equally into 75 test tubes to establish three treatments in triplicate at day 22, with each tube containing 100 mL of culture (Fig. S1). Treatment A1 was used to determine the normal degradation of A. tamarense aggregates; treatment A2 was used to investigate the effects of MC flocculation from day 25 to day 80; and treatment A3 was used to distinguish the effects of MC flocculation and sediments on the depositional environment from day 25 to day 80. Additionally, the filtrate of the algal culture at day 22 was divided into treatments B1, B2, and B3, which were then treated at day 25 to estimate the effects of light, MC particle, and sediment, respectively, on dissolved water. Before the MC removal experiment, 2.0 g of sediment was added into A3 and B3 and then algal culture slowly flowed into the tube without disturbance. At day 25, 1.0-mL aliquots of the selected MC mixture were carefully added to the 100-mL algal cultures of A2, A3, B2, and B3. During these incubation periods, the samples were carefully collected to minimize contamination at days 22, 25, 28, 32, 40, 66 and 80 (in triplicate), respectively (Fig. S1).
2. Materials and methods 2.4. Sampling and parameter analysis 2.1. Algal culture and preparation A. tamarense (CCMM1005, strain AT5-3) was isolated from Dapeng Bay and maintained by the Institute of Hydrobiology, Jinan University, Guangzhou, China. Prior to the incubation experiments, all of the glassware was pre-rinsed with 5% v/v HCl to remove possible contaminants for 24 h and then thoroughly washed with Milli-Q water. The 0.45-lm membrane-filtered seawater and glassware were sterilized at 121 °C for 20 min. Batch cultures at the exponential phase were inoculated in 2 L of sterilized seawater supplemented with a modified f/4 medium in 5-L flasks in quadruplicate (Guillard and Ryther, 1962; Guillard et al., 1973). The initial cellular concentration of the A. tamarense cultures was 1 104 cells L1. The stock culture was cultivated under light intensity of
The cell concentrations, pH and in vivo chlorophyll fluorescence Fa (Fluorescence Turner Designs, TD700; fsu) of the algal cultures were simultaneously monitored during the cultivation and flocculation period. The cellular concentrations were counted in triplicate under a microscope (OLYMPUS IX71, Tokyo, Japan) after fixation with a 5% glutaraldehyde solution (Churro et al., 2010; Maruyama and Kim, 2013). All of the nutrient samples were stored and frozen at 20 °C for further analysis by using a nutrients continuous flow analyser (Skalar San++, Breda, the Netherlands, see in Supplementary Data). The sediment samples were filtered through 0.22-lm glass–fibre membranes and then frozen until analysis. The Chl-a samples were treated for 24 h in 10 mL of 100% N,
G. Lu et al. / Chemosphere 127 (2015) 188–194
2.5. Data analysis The growth rates were calculated as the differences between two sampling intervals of the cell numbers (Guillard et al., 1973; Yu and Subba, 1998), l = [ln (Nt) ln (N0)]/(t t0), units of d1, where t0 and t represent the initial and incubation times, respectively; where No and Nt represent the cell numbers at days t0 and t, respectively. Conversion factors were used to express the toxicity of the sum of the variants as saxitoxin toxicity equivalents (STXeq., Table S1) due to the differing toxicities and concentrations of the individual saxitoxin variants (Hall et al., 1990; Kayal et al., 2008). The values were all averaged from triplicate treatments to produce the mean concentrations/values ± one standard deviation. Quantitative data analysis was based on SPSS 16.0 (multiple comparisons using Fisher’s LSD and Dunnett’s T3 method). The significant data was considered as p < 0.05.
3. Results 3.1. Algae removal and the basic parameters change in incubation experiment The RE value of A. tamarense cells increased with the pre-set MC concentrations, and the highest RE was found to be up to 99% at a concentration of 2.00 g L1 of MC (Fig. S2). According to the previous treatment, we used the concentration of 90% cell removal in the field application (unpublished data). Thus, we selected 0.25 g L1 as the representative concentration for use in comparison test (Fig. S2). A. tamarense cells in A1 were maintained in an exponential phase for the first 20 d, with a maximum cellular concentration of (19.00 ± 1.12) 106 cells L1 (Fig. 1) at day 32. When MC was added in A2 and A3 at day 25, the cellular concentrations in seawater dropped sharply from (17.44 ± 1.01) 106 cells L1 to (0.14 ± 0.04) 106 cells L1 (p 0.000 < 0.05, n = 9) and (0.11 ± 0.02) 106 cells L1, respectively (p 0.000 < 0.05, n = 9). Subsequently, the cellular concentration of A2 re-bloomed at a low density of (6.44 ± 1.01) 106 cells L1; however, the cellular concentration of A3 reached undetectable levels at day 80 (Fig. 1). After the addition of MC, the Fa of A2 and A3 suddenly decreased from 22.20 ± 1.95 fsu to 2.07 ± 0.32 fsu (p = 0.005 < 0.5, n = 3) and 1.95 ± 0.05 fsu (p = 0.006 < 0.05, n = 3), respectively, at day 25, which coincided with the cell density during the subsequent period (Fig. 1).
A1 cells A2 cells A3 cells
A1 Fa A2 Fa A3 Fa
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20 Modified Clay Addition 15 20
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-1
(*10 cells L )
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A. tamarense 0
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in vivo Chlorophyll Fluorescence Fa
N-dimethylformamide (DMF) for efficient extraction (Inskeep and Bloom, 1985; Qin et al., 2013). For toxins analysis in the phytoplankton detritus and sediments, glass–fibre membranes were cut into small pieces and extracted with 5.0 mL of 80% v/v acetonitrile solution (including 0.1% v/v formic acid). PST extraction was conducted based on previous PST determination studies (Sayfritz et al., 2008; van de Riet et al., 2009; DeGrasse et al., 2011). The PSTs analysis used a postcolumn oxidation HPLC-FD (Water Corporation e2695 separation and 2475 detector, Milford, MA, USA), based closely on the AOAC Official Method 2011.02 (Turner et al., 2013, details in Supplementary data). All of the chemical reagents and procedures used for the carbamate gonyautoxins (GTX1, GTX2, GTX3, GTX4, STX and neoSTX), decarbamoyl toxins (dcGTX2, dcGTX3 and dcSTX), N-sulfocarbamoyl toxin GTX5, and N-sulfocarbamoyl toxins (C1 and C2) analysis were based on Chen et al. (2013) study.
Cell density of A. tamarense
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80
Time (d) Fig. 1. Growth curve of the cell density (left y-axis) and the in vivo chlorophyll fluorescence Fa (right y-axis) of A. tamarense using different comparative tests (here and in the following figures, solid and empty squares represent the cells and Fa in A1 natural growth; solid and empty cycles represent the cells and Fa in A2 only modified clay applied in the algal culture from day 25; solid and empty triangles represent the cells and Fa in A3 modified clay flocculation with algal cells under the water-sediment systems; the arrows indicate the modified clay addition time at day 25).
3.2. Characteristics of the nutrients in seawater The ammonium concentration in A1 was 8.42 ± 0.65 lM at day 80 (Table S2; Fig. S4). The nitrite concentrations increased to 13.78 ± 2.85 lM at day 80. In contrast, the nitrate and phosphate concentrations exhibited an obvious decrease to the level of 96.79 ± 6.06 lM and 1.06 ± 0.09 lM, respectively, at day 80. After the addition of MC, the ammonium concentrations in A2 exhibited no differences (p = 0.296 > 0.05, n = 3). However, the performance of phosphate in A2 first decreased significantly to 0.87 ± 0.05 lM at day 25 (p = 0.001 < 0.05, n = 3). The nitrite concentrations in A2 increased to >40 lM, whereas the nitrite level of A1 was less than 16 lM (p 0.000 < 0.05, n = 3). Within the sediment environment, the ammonium in A3 significantly increased to 34.58 ± 6.63 lM at day 25 (p = 0.001 < 0.05, n = 3) and to 103.23 ± 8.52 lM at day 80 (p 0.000 < 0.05, n = 3), which was much higher than that observed in A1 and A2. Simultaneously, the ammonium in B3 was about 60 lM (Fig. S4). The nitrate concentration in A3 decreased to 70.47 ± 11.94 lM at day 40 (Fig. S4). At day 80, there was significant differences of DIP concentration between A1 and A2 (p 0.000 < 0.05, n = 3), A1 and A3 (p 0.000 < 0.05, n = 3). 3.3. Changes in the chlorophyll a content of the sediments The Chl-a trend of the bottom water in A1 gradually decreased from 0.202 ± 0.012 lg mL1 at day 22 to 0.156 ± 0.003 lg mL1 at day 80 (Fig. 2). Upon the addition of MC, the Chl-a concentration in A2 sediment changed from 0.456 ± 0.010 lg mL1 at day 25 to 0.053 ± 0.001 lg mL1 at day 80. The Chl-a concentration in A3 sediment sharply decreased to 0.154 ± 0.004 lg mL1 at day 25, whereas the Chl-a concentration in the original sediment was 0.150 ± 0.006 lg mL1 in B3 (this part was deducted in Fig. 2). The q value in A1 ranged from 0.07 to 0.03 d1 with synchronous changes of the cell density in the upper waters. However, the q value in A2 gradually decreased to 0.04 d1. Simultaneously, the q values of A3 were approximately zero. 3.4. Changes of PSTs To detect the changes of total PSTs, we measured 12 main toxin concentrations of the bottom 50 mL-volume water and sediments.
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Chl-a in bottom water / sediment (mg L-1)
A1 Chl-a A2 Chl-a A3 Chl-a
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Time (d)
the total PSTs concentration (STX-eq. nM)
b
a
A1 total PSTs A2 total PSTs A3 total PSTs
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50 Modified Clay Addition
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Time (d)
Fig. 2. The chlorophyll-a (mg L1; (a)) and the total PSTs (STX-eq. nM; (b)) changes of A. tamarense cells of bottom water and sediments in three compared treatments.
The total PSTs in A1 exhibited the auto-deposition of algal toxins, and the concentration gradually ranged from 36.75 STX-eq. nM to 78.76 STX-eq. nM (Fig. 2). Upon the addition of MC, the total PSTs in A2 sediments initially increased to 106.02 ± 0.19 STX-eq. nM at day 25 and gradually decreased to a minimum concentration of 17.75 STX-eq. nM at day 80 (Fig. 2). In contrast, the total PSTs concentration in A3 sediments initially decreased to 15.02 STXeq. nM at day 25 and remained
