Chemosphere 121 (2015) 110–116

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Chemosphere

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Effect of linoleic acid sustained-release microspheres on Microcystisaeruginosa antioxidant enzymes activity and microcystins productionand release

http://dx.doi.org/10.1016/j.chemosphere.2014.11.0560045-6535/� 2014 Published by Elsevier Ltd.

⇑ Corresponding author. Tel.: +86 1 37 70993281; fax: +86 25 83786697.E-mail address: nilixiao@gmail.com (L. Ni).

Lixiao Ni a,⇑, Xiaoting Jie a, Peifang Wang a, Shiyin Li b, Guoxiang Wang b, Yiping Li a, Yong Li a,Kumud Acharya c

a Key Laboratory of Integrated Regulation and Resource Development on Shallow Lakes, MOE, School of Environment, Hohai University, 210098 Nanjing, Chinab Department of Environmental Science and Engineering, School of Geography Science, Nanjing Normal University, 210097 Nanjing, Chinac Desert Research Institute, Las Vegas, NV 89119, USA

h i g h l i g h t s

� LA sustained-release microspheres had the optimal dose with high inhibitory ratio.� LA sustained-release microspheres could affect algal antioxidant enzymes activity.� LA microspheres could decrease the production and release of microcystins.� LA sustained-release microspheres could represent a high degree of ecological safety.

a r t i c l e i n f o

Article history:Received 28 July 2014Received in revised form 18 September2014Accepted 19 November 2014

Handling Editor: Y. Liu

Keywords:Linoleic acid sustained-releasemicrospheresAntioxidant enzymes activityMicrocystis aeruginosaChlorophyll aMicrocystins

a b s t r a c t

The objective of this work was to identify the optimal dose range for good anti-algal effect of linoleic acid(LA) sustained-release microspheres and investigate their impact on the antioxidant enzymes (superoxide dismutase, Catalase and Peroxidase) activity changes of Microcystis aeruginosa, as well as the pro-duction and release of microcystins (MCs). Based on measured changes in algal cell density and inhibitoryratio (IR), the optimal dose of LA microspheres was 0.3 g L�1 with over 90% of IR in this study. The Chlo-rophyll a content and antioxidant enzymes activity in the LA microspheres group decreased markedlyuntil beyond the minimal detection limit after 16 d and 9 d, respectively. In addition, LA microspheresdemonstrated no significant impact on the extracellular release of MCs during the culturing period.The amount of intracellular microcystin-LR (MC-LR) per 106 algal cells in LA microspheres group washighest among all groups during the whole experimental process. Under the sustained stress of LAreleased from LA microspheres, the LA microspheres could decrease the production and release of algaltoxins. There was no increase in the total amount of MC-LR in the algal cell culture medium. These indi-cated that LA sustained-release microspheres represent a high degree of ecological safety and their prac-tical applications for the treatment of water undergoing algal blooms need further study.

� 2014 Published by Elsevier Ltd.

1. Introduction

In recent years, eutrophication has been an environmentalissue, which caused cyanobacteria blooms, especially Microcystisaeruginosa blooms, becoming a widespread problem in the aquaticenvironment (Codd et al., 2005; Chen et al., 2010). The addition ofalgae-killing chemicals (e.g. copper sulfate) to water is still thesimplest and most common method of inhibiting algal growth.

These algae killing chemicals rapidly cause algal death but are alsoseriously hazardous to the ecological environment and humanhealth (Zhang et al., 2001; Costas and Lopez-Rodas, 2006).

Over the past 20 years, there has been significant interest in thegrowth inhibition of cyanobacteria by allelochemicals released byaquatic macrophytes (Rice, 1984; Sun et al., 1989; Gross et al.,2003; Zhang et al., 2007). Allelochemicals extracted from variousaquatic plants have been confirmed to have certain inhibitoryeffects on the growth of algae involved in water blooms (Nakaiet al., 2000; Gross, 2003; Bauer et al., 2009; Gross and Gene,2009). Our previous research (Ni et al., 2011) showed that the med-

L. Ni et al. / Chemosphere 121 (2015) 110–116 111

ian effective concentration for the growth inhibition of M. aerugin-osa using linoleic acid (LA) was 3.67 mg L�1 and demonstrated apotent allelopathic inhibition on algal growth. However, theseallelochemical inhibitors were applied directly to the water body,which caused a quick loss without effective inhibitory concentra-tions. In order to solve this problem, a new algal inhibitor that tar-gets the harmful algae communities, was developed andcharacterized. Previous work found that this sustained-releasemicrospheres had a good thermal stability (decomposition temper-ature of 236 �C) and stable release property (release time of morethan 40 d) (Ni et al., 2015). However, the optimal dose range forgood anti-algal effect of LA sustained-release microspheres andtheir impact on the antioxidant enzymes activity changes of M.aeruginosa has not been studied.

Numerous types of water-bloom-causing algae can producetoxins, cyanobacterial microcystins (MCs) exhibit detrimentaleffects on organisms from zooplankton to humans including gas-troenteritis and liver damage, with reports linking MCs to humanhepatocellular carcinoma (Ueno et al., 1996; Chorus, 2001). Theimpact of allelochemicals on the production and release of algaltoxins is a strong determinant of the ecological safety of the appli-cation of allelochemicals for algal control. This has become a seri-ous concern of researchers (Boylan and Morris, 2003; Fistarol et al.,2003). Several researches showed that the application of allelo-chemical has certain influence on the MCs (Gross et al., 1996;Jang et al., 2003). Allelochemical extracted from Water lettuce caneffectively inhibit the proliferation of algal cells without increasingthe release of cyanotoxin (Wu et al., 2013). However, Boylan andMorris (2003) found that the application of anti-algal reagents incontrolling toxin-producing algae might cause the microcystin-LR(MC-LR) concentration in the water significantly increased. Onlya limited number of studies have focused on the impact of allelo-chemicals on the production and release of algal toxins frombloom-forming, toxin-producing algae. A systemic and compre-hensive research is greatly needed to clarify whether LA sus-tained-release microspheres with good algal inhibitory effects (Niet al., 2015) will cause the aforementioned problem.

The most common and most toxic cyanotoxin produced by M.aeruginosa were MC-LR, microcystin-RR (MC-RR) and microcy-stin-YR (MC-YR), which were used as representative algal toxins(Xian et al., 2006). Therefore, this research was carried out to deter-mine the optimal dose range for good anti-algal effect of LA sus-tained-release microspheres, and test production and release ofalgal toxins during the application of LA sustained-release micro-spheres for algal control.

2. Materials and methods

2.1. Algal cultivation

M. aeruginosa FACHB-905, purchased from Freshwater AlgaeCulture Collection of the institute of Hydrobiology (China), wascultured in the laboratory at Hohai University with a sterilizedBG11 medium (Hong et al., 2008). All cultures were grown in250 mL flasks with 50 mL of sterilized culture medium at 25 �Cunder 40–60 lmol photons m�2 s�1 (14 h light/10 h dark). Thealgae were cultivated in batch cultures (transferred and inoculatedonce every 5–6 d) to ensure that the experiments were always con-ducted with the algae in the exponential growth phase.

2.2. Algal inhibition test of LA sustained-release microspheres

Algal biomass (ISO, 8692-2004) was used to test the inhibi-tion effectiveness of the LA sustained-release microspheres. M.aeruginosa was inoculated into a culture medium in a 500 mL

flask using 2–3 � 106 cells mL�1 as the initial algal density. Theexperimental groups included a blank microspheres group, fourconcentrations of the pure LA group (0.05, 0.1, 0.2 and0.4 mg L�1), five concentrations of LA microspheres group (0.05,0.1, 0.3, 0.5 and 1 g L�1), and a control group. There were threereplicates for each group. All flasks were cultivated at 25 �Cunder 40–60 lmol photons m�2 s�1 (14 h light/10 h dark) condi-tions. The algal cell density was estimated daily in the first weekusing microscopic counting, and then estimated every 3 d fromday 7 to 30 of culturing to get the optimal dose range with goodalgal inhibitory effects.

2.3. Physiological assays

Effects of LA sustained-release microspheres on algae wereinvestigated. Specifically, changes in Chlorophyll a (Chl-a), superoxide dismutase (SOD) activity, Catalase (CAT) activity, and Perox-idase (POD) activity in M. aeruginosa were assessed using LA sus-tained-release microspheres, blank microspheres, pure LA andcontrol group. The doses of pure LA, LA microspheres were deter-mined to test the physiological assays and the production andrelease of MCs according to the experimental results of the optimaldose range with the good algal inhibitory effects.

The amount of Chl-a in M. aeruginosa was assayed according tothe ethanol extracting method (Eullaffroy and Vernet, 2003). 50 mLof algae mixture containing different samples was harvested bycentrifugation at 4000 rpm (Mini-10K) for 10 min, and the super-natant was discarded. The cell pellets were then washed with dis-tilled water and centrifuged twice to remove water. Harvested cellswere re-suspended to 5 mL ethanol (95%), and the extraction wascarried out at 4 �C without light for 24 h (Eullaffroy and Vernet,2003). 1 mL of extraction supernatant after centrifugation at8000 rpm for 10 min was collected to measure wavelengths (A)at 665 nm and 750 nm, and ethanol (95%) was used as a blank.The Chl-a content was calculated with the following formula(Yao, 1987):

CChl-a ðmg L�1Þ ¼ 11:93 ðA665 � A750Þ

Algal cells were harvested by centrifugation at 6000 rpm (Mini-10K) for 10 min. The cell pellets were then washed with distilledwater and centrifuged twice to remove any water. Harvested cellswere re-suspended in 5 mL distilled water to measure solublesugar content, and with 5 mL phosphate-buffered saline (PBS)solution (50 mM, pH 7.0) to measure SOD. The cells were homog-enized by an ultrasonic cell pulverizer (VCX130, US Sonics) for10 min. The homogenate was then centrifuged at 10000 rpm(Mini-10K) for 10 min at 4 �C. The supernatant, cell-free extractwas used for the following assays.

SOD activity was assayed according to the method ofBeauchamp and Fridovich (1971). The reaction mixture contained0.8 mL PBS solution (50 mM, pH 7.8), 0.3 mL methionine solution(130 mM), 0.3 mL Na2EDTA solution (100 lM), 0.3 mL riboflavinsolution (20 lM), 0.3 mL nitroblue tetrazolium (NBT) solution(750 lM), and 1 mL enzyme extract for a total volume of 3 mL.As SOD has the ability to inhibit the photochemical reduction ofNBT, this assay utilized negative controls (silver paper wrappedaround the test tube to mimic fully dark condition without anyphotochemical reduction of NBT), positive controls (deficiency ofSOD activity in light with full photochemical reduction of NBT),and treatment groups (in light with SOD inhibition on photochem-ical reduction of NBT). The absorbencies of all experimental tubeswere measured at 560 nm after a 20-min irradiance of 40–60 mmol photons m�2 s�1. One unit of SOD activity was definedas the amount of enzyme that inhibited 50% of photochemicalreduction of NBT.

112 L. Ni et al. / Chemosphere 121 (2015) 110–116

CAT activity was assayed according to the method ofGiannoplities and Ries (1977). The reaction mixture contained1 mL H2O2, 1.9 mL H2O and 1 mL crude enzyme, and then testthe reduce speed of absorbance at 240 nm.

POD activity was assayed according to the method of Evans(1965). The reaction mixture contained 1 mL H2O2, 1 mL 0.05 lMguaiacol and 1 mL crude enzyme. The reaction started when thecrude enzyme joined, and then test the reaction mixture at470 nm at 25 �C.

2.4. Determination of extracellular and intracellular MCs

The concentrations of MC-LR, MC-RR and MC-YR in cells andculture medium were detected. A solid-phase extraction methodcombined with high-performance liquid chromatography (HPLC)was applied to determine the amount of MCs (Song et al., 1999).The samples were first centrifuged at 9000 rpm (Mini-10K) for15 min at 4 �C. The supernatant after centrifugation was gatheredfor testing extracellular MCs. The remaining pellet was frozenand thawed three times, and suspended in 5% acetic solution forcentrifugation at 10000 rpm (Mini-10K) for 10 min to gather thesupernatant. Then the remaining pellet was resuspended in ultra-pure water for centrifugation at 10000 rpm (Mini-10K) for 10 min,and the mixed supernatant was gathered for testing intracellularMCs. The intracellular and extracellular MCs were extracted usingC18 solid phase extraction column and tested with HPLC. Thedetailed pretreatment method can refer to the research of Menand Hu (2007).

2.5. Statistical analysis

Statistical analysis was performed using SPSS for Windows Ver-sion 17.0 (SPSS, Chicago, IL, USA). All data were first log trans-formed so that the normality requirement for ANOVA was met,and then analyzed using one-way ANOVA followed by a test forsignificance at the p = 0.05 level.

3. Results and discussion

3.1. Inhibition effect of LA sustained-release microspheres on M.aeruginosa

The changes of algal density and inhibitory ratio (IR) with dif-ferent concentration groups of pure LA (0.05, 0.1, 0.2, 0.4 mg L�1)and LA sustained-release microspheres (0.05, 0.1, 0.2, 0.3,

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0.5 g L�1) were shown in Figs. 1 and 2. From Fig. 1, the algal densityof all groups increased with culture time exposed to pure LA(Fig. 1a), and IRs decreased with exposure concentration of LA(Fig. 1b). The low-concentration group (0.05 mg L�1 LA and 0.05and 0.1 g L�1 LA sustained-release microspheres) indicated a poorinhibitory effects and the algal cell growth was similar with thecontrol group (Figs. 1b and 2b). The maximum IRs value couldreach 90% in the high-concentration groups of pure LA (0.2 and0.4 mg L�1) at day 7, and then declined as algal growth recovered,but the decline trend was still much slower than that in other con-centration groups (Fig. 1b). The results of Fig. 1 was in agreementwith the research of Nakai et al. (1999) that algal cell growth beganto recover after approximately 8 d of culturing using a single doseof macrophyte extracts to inhibit the growth of M. aeruginosa,which might be due to the degradation or transformation of theeffective anti-algal components (Kong et al., 2006; Hong and Hu,2009). In the high-concentration groups of LA microspheres (0.3,0.5, and 1 g L�1), the inhibition of M. aeruginosa growth exhibiteda similar trend: the IR was about 90% during the whole experiment,which indicated a strong algal inhibitory effect of the LA micro-spheres. The algal cell growth of the blank microspheres groupwas similar with the control group which indicated that the blankmicrospheres had no impact on M. aeruginosa growth. LA micro-sphere could continually release LA to replenish the effectiveanti-algal component and inhibited M. aeruginosa to the non-growth state (Fig. 2). From Figs. 1 and 2, comparing the results ofhigher dose groups for pure LA (0.2 and 0.4 mg L�1) and LA micro-spheres (0.3, 0.5, and 1 g L�1), it was revealed that although ahigher dose yielded a stronger inhibitory effect on algal growth,the IR did not change significantly with an increase in dose oncethe dose exceeded a certain level, which indicated a thresholdeffect (Jin et al., 2003; Chang et al., 2012). Therefore, from an eco-nomic point of view, 0.2 mg L�1 and 0.3 g L�1 could be the optimaldoses with good anti-algal effect for pure LA and LA sustained-release microspheres, respectively.

3.2. Physiological effects of LA sustained-release microspheres on M.aeruginosa

3.2.1. Change of Chl-a of M. aeruginosaThe effects of LA sustained-release microspheres on Chl-a have

been investigated in Fig. 3a. Compared to the control test, the con-tents of Chl-a decreased during the first 7 d under the stress of pureLA (Fig. 3a), which indicated that LA can limit photosynthesis of M.aeruginosa (p < 0.05). As the exposure time increased, the content

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Fig. 3. Influence of different treated groups (0.2 mg L�1 LA, 0.3 g L�1 LA microspheres) on Chl-a content (a), activities of SOD (b), CAT (c) and POD (d) of M. aeruginosa.

L. Ni et al. / Chemosphere 121 (2015) 110–116 113

of Chl-a in the 0.2 mg L�1 pure LA group decreased then graduallyreturned to the control level. However, the content of Chl-a in theLA sustained-release microspheres group decreased markedly untilbeyond the minimal detection limit after 16 d (p < 0.05) whichindicated that photosynthesis of algae cells remained in an inhib-ited state, and the inhibition effect was enhanced progressivelydue to cumulative release of LA from the LA microspheres. The con-tent of Chl-a in the blank microspheres was similar to the controlgroup, which showed that the blank microspheres had not impacton the photosynthesis of M. aeruginosa.

The Chl-a in algae is the main photosynthetic pigment, and thecontent of the Chl-a was closely related to state of algal cell growthand photosynthesis, which can indicate the primary productivity ofwater bodies. The content of Chl-a can be a potential indicator pho-tosynthetic (Allen, 1995; Zhang et al., 2009). Chloroplasts can pro-duce reactive oxygen species (ROS) in the reductive side ofphotosystem I under illumination, and the ROS have a strongresponse capacity to intracellular components, and can directlydamage chlorophyll. In this study, LA stress may have resulted in

intracellular ROS increase, which reduced the chlorophyll content.Reduction of Chl-a may be due to the stress of LA decreased algalphoto-oxidation capacity (Eullaffroy and Vernet, 2003).

3.2.2. Changes of antioxidant enzymes activities of M. aeruginosaThe effects of LA sustained-release microspheres on SOD, CAT,

and POD activity of algae were observed in Fig. 3b–d. FromFig. 3b–d, the results showed that the activities of SOD, POD andCAT initially increased acutely when the algae were exposed toLA microspheres in the first 3 d, and then decreased markedly untilbelow the minimal detection limit after 9 d (p < 0.05). The threeantioxidant enzymes contents in the LA microspheres groups weremuch lower than other three groups. In the pure LA group, SOD,POD and CAT activities peaked on the 3rd d and then also beganto decrease until close to the level of the control group after 7 d(p < 0.05).

Several antioxidant enzymes and substances are involved in theprotective mechanisms adapted by plants to eliminate ROS andfree radicals (Allen, 1995). Environmental stresses can increase

114 L. Ni et al. / Chemosphere 121 (2015) 110–116

the generation of ROS, which can lead to severe cellular injury ordeath. It is therefore very important for cells to maintain appropri-ate levels of ROS (Blackhall et al., 2004). Specifically, SOD can con-vert O2� into H2O2 and O2, avoiding the cell damage (Wan et al.,2014). CAT can make the decomposition of H2O2 to molecular oxy-gen and water, to alleviate the oxidative damage from the poison ofH2O2. POD is an active enzyme, and can be used as a physiologicalindex of tissue aging (Blackhall et al., 2004). SOD, CAT and POD canmake up the oxidation and antioxidant defense system which isindispensable for organisms for defense against the toxic effect ofoxygen free radicals (Allen, 1995; Wan et al., 2014).

Our results showed that SOD, CAT and POD activities increasedsignificantly under LA stress in the first three days, but decreasedto the level of the control or even less (in LA microspheres group)during long-term exposure, which may suggest that the ROS wasincreased under LA stress and the collapse of defense system reg-istered as a decrease in SOD, POD and CAT activities (Hong et al.,2009; Zhao et al., 2012). In addition, the damage to active oxygenscavengers like POD, CAT and phycobiliproteins resulted in theantioxidant system not being able to thoroughly eliminate exces-sive active oxygen, which caused damage to photosynthetic pig-ments, bleaching of the algae, damage to algae cell membranesand inhibition of growth (Zhao et al., 2012).

3.3. Extracellular MCs release affected by LA microspheres

The MCs concentrations in culture medium of M. aeruginosawere detected when exposed to 0.2 mg L�1 LA and 0.3 g L�1 LAmicrospheres. The extracellular concentrations of MC-RR, MC-LR,and MC-YR were shown in Table 1. From Table 1, the release con-tent of MC-LR is the highest during the algal growth process, andextracellular MC-LR of the control group was at the same level asthe results reported by Robillot et al. (2000). The extracellularMC-LR concentrations of the control group were always stable dur-ing the experiment period (30 d), which suggested that few algaltoxins were released into the surrounding water in a normal envi-ronment. The release contents of extracellular MC-LR in blankmicrospheres group were similar with the control group, whichindicated that blank microspheres addition has no effects onrelease of MC-LR. With exposed to LA (including 0.2 mg L�1 pureLA group and 0.3 g L�1 LA sustained-release microspheres group),extracellular MC-LR contents in two groups were lower and similarwith control group under the first 9 d exposure, and then increasedwith the culture time. The content of MC-LR peaked at 1.532 lg L�1

in 28 d, more than 1 lg L�1 in 0.2 mg L�1 pure LA group, and MC-LRlevel in 0.3 g L�1 LA microspheres group was stable at about0.7 lg L�1.

MC-LR, as a predominantly algal toxin, is the most toxic andharmful of the known algae toxins (de Figueiredo et al., 2004).Without any external interference, the extracellular concentrationof algal toxins was very low and negligible during the early culturestage of algal cells and remained at a relatively low level prior tothe stable growth stage (Robillot et al., 2000), lower than the max-

Table 1Extracellular release of MC-LR, MC-YR and MC-RR of M. aeruginosa exposed to 0.2 mg L�1

Day Control Blank microspheres

MC-LR MC-RR MC-YR MC-LR MC-RR MC-YR

3 0.305 0.0652 0.0521 0.322 0.0598 0.01129 0.328 0.0644 ND⁄ 0.298 0.0612 ND

15 0.338 0.0633 ND 0.279 0.0682 ND21 0.342 0.0698 ND 0.331 0.0711 0.018828 0.357 0.0713 0.1233 0.276 0.0767 0.0906

Notes: ND⁄ means beyond the detection limit.

imum limit of 1 lg L�1 in drinking water suggested by the WorldHealth Organization (Codd et al., 2005; Umehara et al., 2012).However, the application of chemical algae-killing reagents causesthe death and disruption of algal cells, or the cells being inactiveand lysis, leading to a substantial release of intracellular algal tox-ins (Daly et al., 2007; Xiao et al., 2010). For example, Jones and Orr(1994) applied an algae-killing reagent (copper sulfate) to treatwater exhibiting algal blooms and found that amount of MC-LRdissolved in the water (i.e. extracellular MC-LR) increased signifi-cantly and harmed water quality. In contrast, under the conditionsused in this study, the utilization of pure LA and LA sustained-release microspheres to inhibit algal growth also effectively con-trolled the proliferation of algal cells but did not promote theextracellular release of algal toxins, and the MC-LR release contentin LA sustained-release microspheres group was lower than that inpure LA group. Wu et al. (2013) also found that allelochemicalextracted from water lettuce can effectively inhibit the prolifera-tion of algal cells without increasing the release of MCs. Our studyresults showed that the LA sustained-release microspheres repre-sent a higher degree of ecological safety and can therefore be usedin practical application for the water undergoing algal blooms.

3.4. Intracellular MCs production affected by LA microspheres

The concentrations of intracellular MC-RR and MC-YR were verylow and negligible. The production contents of intracellular MC-LR(the mass of MC-LR per 106 algal cells) in M. aeruginosa cells withthe different experimental groups (control group, the blank micro-spheres group, pure LA and LA microspheres) were shown in Fig. 4.From Fig. 4, in the control group, the amount of MC-LR was at thesame level as the results reported by Wiedner et al. (2003) andDowning et al. (2005). The amount of MC-LR per unit of algal cellsincreased in the LA and LA microspheres groups. At first 9 d cultur-ing, the intracellular MC-LR level increased significantly under LAstress, especially in LA microspheres group. 0.2 mg L�1 LA and0.3 g L�1 LA microspheres strongly inhibited M. aeruginosa growthwithin 7 days, and Chl-a content, SOD, POD and CAT activitiespeaked on the 3th d and then also began to decrease (p < 0.05).These results indicated that the surviving cells would produce agreater amount of toxins due to the external stress caused by thepresence of allelochemicals (LA), which is in agreement with theprevious research (Robillot et al., 2000; Jang et al., 2003; Wuet al., 2008). It is generally believed that algal intracellular energyis directed primarily towards two purposes: the synthesis of thenutrients required for growth and resistance against environmen-tal stress (Wang et al., 2011). As a type of environmental stress,allelochemicals can cause algal cells to consume more energy inthe synthesis of toxins, which is a possible reason for the slowgrowth of algal cells (Kearns and Hunter, 2000).

From the 7th d of culturing onwards, the inhibitory effect ofpure LA on the growth of M. aeruginosa decreased, and the algalcells in the culture medium exhibited restored growth. However,the intracellular MC-LR production in the pure LA group decreased

LA, 0.3 g L�1 LA microspheres (lg L�1).

LA LA microspheres

MC-LR MC-RR MC-YR MC-LR MC-RR MC-YR

0.312 0.0689 0.0182 0.311 0.0522 0.02820.347 0.0662 ND 0.256 0.0677 0.01920.983 0.0816 ND 0.662 0.0811 ND1.102 0.112 0.1028 0.722 0.0522 ND1.532 0.123 0.1228 0.698 0.0321 ND

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L. Ni et al. / Chemosphere 121 (2015) 110–116 115

with culturing time and was lower than the control level, indicat-ing that, with the consumption of LA and the environment stressreducing, the algae exhibited restored growth, and less intracellu-lar MC-LR produced. The intracellular MC-LR contents in LA micro-sphere group also began to decrease, but the intracellular MC-LRproduction in LA microspheres group were greatly higher thanthe other three groups. LA microspheres could continually releaseLA to replenish the effective anti-algal component and inhibited M.aeruginosa to the non-growth state (Fig. 2), which led to the higherlevel of intracellular MC-LR production per unit algal cell for resis-tance against LA stress.

The amount of extracellular MC-LR released was very small dur-ing culturing period (Table 1) and can be considered negligible. Asa result, the total amounts of MC-LR production can be calculatedbased on the intracellular MC-LR amount per 106 cells multipliedby the detected algal density (Men and Hu, 2007; Sager, 2009).From Fig. 5, the total MC-LR productions in the control and blankmicrosphere groups increased with the culturing time and weremuch higher than those in LA and LA microspheres groups, whichindicated that blank microspheres had no impact on the produc-tion and release of algal toxins. In the LA group, the total amountof MC-LR treated with 0.2 mg L�1 pure LA was lower than the con-

trol group after 21 d and then tended to be stable. Compared withalgal density results (Figs. 1 and 5), the algal cell growth decreasedgreatly first and then began to recover after 15 d with LA depletion,leading to the algal density being in change. The total amounts ofMC-LR production in LA group did not show significant differenceswith culturing time, which was consistent with results from Fig. 4.The total amount of MC-LR production in LA microspheres groupwas at the same level and lowest among all groups after 3 d. Thismight be explained by the fact that a stable LA stress from LAmicrospheres maintained continuously effective anti-algal compo-nent to ensure superior algal cell growth inhibition during cultur-ing periods. Consequently, compared to the other groups, the algaldensity was the lowest in LA microspheres group, leading to lowtotal MC-LR production even though the highest amount intracel-lular MC-LR production per 106 cells (Fig. 4). Therefore, the majorcalculation factor influencing the total amount of MC-LR produc-tion should be the detected algal density. Daly et al. (2007) foundthat largely numbers of intracellular MC-LR released into water asthe death of algal cells. In this study, M. aeruginosa has been inhib-ited to the non-growth state using LA sustained-release micro-sphere, but the total MC-LR production and extracellular MC-LRrelease in the cultures did not increase with culturing time. Themechanisms were being studied.

4. Conclusions

It was demonstrated that the optimal dose for the good anti-algal effect of LA microsphere was 0.3 g L�1, which resulted in anIR of 95%. LA microspheres had significant impact on the photosyn-thesis and antioxidant enzymes activity of M. aeruginosa. The con-tent of Chl-a and antioxidant enzymes (SOD, POD and CAT) activityin 0.3 g L�1 of LA sustained-release microspheres group decreasedmarkedly until beyond the minimal detection limit. The LA sus-tained-release microspheres demonstrated no significant impacton the extracellular release of MCs during the culturing period.The amount of intracellular MC-LR per 106 algal cells in LA micro-spheres group was highest among all groups during the wholeexperimental process. Under the sustained stress of LA releasedfrom LA microspheres, the LA microspheres could decrease theproduction and release of algal toxin. There was no increase inthe total amount of MC-LR in the algal cell culture medium. Theresults of this study suggested that the LA sustained-release micro-spheres may be a potential candidate for algal inhibition.

Acknowledgements

This work has been supported jointly by the National NaturalScience Foundation (Grant Nos. 51109061, 41373111, 51009049);the National Science Funds for Distinguished Young Scholars(Grant No. 51225901); the Research Fund for innovation team ofMinistry of education (IRT13061); the Major Project of the NationalWater Pollution Control (Grant No. 2012ZX07101-008); the JiangsuWater Resources Science and Technology Program (Grant No.201371).

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