Chemosphere 174 (2017) 732e738

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Enhanced toxicity to the cyanobacterium aeruginosa by low-dosage repeated exposure to the allelochemical N-phenyl-1- naphthylamine

* Y.N. Gao b, 1, F.J. Ge a, 1, L.P. Zhang a,Y.Hea, Z.Y. Lu a, Y.Y. Zhang a, B.Y. Liu a, Q.H. Zhou a, , Z.B. Wu a a State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei, 430072, China b College of Fisheries, Engineering Technology Research Center of Henan Province for Aquatic Animal Cultivation, Henan Normal University, Xinxiang, Henan, 453007, China highlights

Severe damage on M. aeruginosa exposed repeatedly to low-dosage N-phenyl-1-naphthylamine (NPN) was observed. Continual stress of low-dosage allelochemicals was proved to be a potent mode for allelopathy of aquatic plants. It is essential to introduce low-dosage continual exposure pattern of allelochemicals into further research. article info abstract

Article history: It has been puzzling whether and how a plant could exert a strong allelopathic inhibition to the target Received 22 August 2016 organisms by releasing low concentrations of allelochemicals. Plant allelochemicals have been proposed Received in revised form to be released continuously, however, direct evidence from specific allelochemicals is urgently required. 7 December 2016 In the present study, the toxicity of allelochemical N-phenyl-1-naphthylamine (NPN) towards the Accepted 20 January 2017 cyanobacterium Microcystis aeruginosa by two different exposure patterns was compared. One was low- Available online 21 January 2017 dosage repeated exposure (LRE), in which 50 mgL 1 NPN was repeatedly dosed to simulate the continual Handling Editor: Caroline Gaus release of allelochemicals, and the other one was high-dosage single exposure (HSE) as per the routine toxicity assay. The results showed a significant growth inhibition to M. aeruginosa in the LRE group, Keywords: where the inhibition rate reached above 90% from day 6 to day 9. The cell-membrane damage ratio N-phenyl-1-naphthylamine increased from 64.05% on day 5 up to 96.60% on day 9. PSII photosynthesis activity expressed as Fv/Fm, Repeated exposure FPSII, NPQ and ETRmax was also thoroughly inhibited in this group. Whereas the growth and PSII Microcystis aeruginosa photosynthesis activity of M. aeruginosa in the HSE group were inhibited initially, but recovered grad- Photosynthesis inhibition ually from day 4 or 5, which was accompanied by a continuous reduction of NPN content in culture Cell membrane damage solutions. Although NPN content in the LRE group was relatively lower, it remained at a more stable level Residual dynamic throughout the experiment. These results indicate that continual release of low-dosage allelochemicals by aquatic plants plays crucial roles in their potent inhibition against . Low-dosage continual exposure pattern needs to be investigated further. © 2017 Elsevier Ltd. All rights reserved.

1. Introduction problems and enormous economic losses (Anderson et al., 2012). Suppression of cyanobacterial development through the exudation The frequent occurrences of harmful cyanobacterial blooms in of allelochemicals is supposed as one effective strategy for mac- eutrophic water bodies worldwide cause serious environment rophytes in aquatic ecosystems (Hilt and Gross, 2008). As compared to the traditional chemical algicides, plant allelochemicals attract much attention because they are supposed to be more selective, * Corresponding author. easily degradable and environmentally safe as natural secondary E-mail address: [email protected] (Q.H. Zhou). metabolites (Narwal, 2006). Various aquatic macrophytes have 1 These authors contributed equally to this study and share first authorship. http://dx.doi.org/10.1016/j.chemosphere.2017.01.102 0045-6535/© 2017 Elsevier Ltd. All rights reserved. Y.N. Gao et al. / Chemosphere 174 (2017) 732e738 733 been confirmed to be able to allelopathically inhibit the growth and the growth and physiological activity of M. aeruginosa between propagation of cyanobacteria. Emergent plants such as Phragmites low-dosage repeated exposure (LRE) pattern simulating the communis and Thalia dealbata (Li and Hu, 2005a; Zhang et al., 2011), continual release of allelochemicals by plants, and high-dosage floating plants Eichhornia crassipes (Sun et al., 1990), submerged single exposure (HSE) pattern as per conventional toxicity assay. macrophytes and Stratiotes aloides (Nakai During a 9-day exposure duration, in addition to daily cell density et al., 1999; Mulderij et al., 2009) are some of the typical exam- changes, flow cytometry technology and chlorophyll fluorescence ples. Reestablishment of aquatic vegetation including these plants technique were used to determine time variation of cellular has been applied successfully to the restoration of eutrophic membrane integrity and photosynthesis activity of M. aeruginosa shallow lakes, and it is supposed that plant allelopathy is involved cells, respectively. in suppression of cyanobacteria and maintenance of clear-water states (Deng et al., 2007; Zuo et al., 2014). The allelochemicals, 2. Materials and methods isolated and identified from allelopathic plants, mainly include phenolic acids (Gross et al., 1996), fatty acids (Nakai et al., 2005; Hu 2.1. Test organisms and chemicals et al., 2010), alkaloids (Semenov and Granlk, 2004; Wang et al., 2010), esters (Li and Hu, 2005b), flavonoids (Les and Sheridan, The axenic unicellular strain of M. aeruginosa (FACHB 905), ob- 1990) and aromatic amine (Yang et al., 1992), etc. The aromatic tained from the Culture Collection of the Freshwater Algae, the amine N-phenyl-1-naphthylamine (NPN), as a potent antialgal Institute of Hydrobiology, the Chinese Academy of Sciences allelochemical, was firstly identified and isolated from root exu- (Wuhan, China), was used as the test organism. The cyanobacteria dates of floating plant Eichhornia crassipes in laboratory and field was cultured in autoclaved BG11 medium (Rippka et al., 1979) conditions (Sun et al., 1993). It was also detected in the enriched under a 12:12 h light/dark cycle with a light intensity of 2000 lux at exudates of three submerged macrophytes, Elodea nuttallii, Hydrilla 25 C and were manually shaken twice a day during the experi- verticillata and Vallisneria spiralis, by our recent GC/MS analysis ment. The cells in the exponential phase were prepared for the (unpublished data). exposure experiment. However, the effective inhibition concentrations of aquatic plant NPN was purchased from Sigma-Aldrich, and its stock solutions allelochemicals, e.g. the EC50s measured by conventional toxicity were prepared in dimethyl sulfoxide (DMSO, >98%). Fluorescence assay under laboratory conditions, were much higher than their probe propidium iodide (PI, Cat No. F4170-10 MG) for cell mem- releasing concentrations in the ambient water environment. For brane integrity analysis as well as 3-(3, 4-dichlorophenyl)-1, 1- example, it was reported that the EC50 of pyrogallol on the growth dimethylurea (DCMU, CatNo. D2425-100G) for fluorimetric of Microcystis aeruginosa in laboratory toxicity tests was 0.7 mg L 1, parameter analysis was also purchased from Sigma -Aldrich. whereas its release content from M. spicatum was only 5.2 mgL 1 (Nakai et al., 2000). The detected concentrations of allelochemicals 2.2. Experimental design released by aquatic plants didn’t exceed 80 mgL 1 according to available literature (Hilt et al., 2006; Gao et al., 2011). However, the M. aeruginosa cells in exponential growth phase were trans- lowest EC50 values of the most potent allelochemicals on the ferred to 100 mL fresh BG11 medium in 250 mL glass flasks with an growth of M. aeruginosa were above 0.5 mg L 1 (Nakai et al., 2005; initial density of 1.0 106 cells mL 1. The exposure protocols are Li and Hu, 2005b). Similar phenomenon has also been reported in presented in Fig. 1. For LRE group, NPN was added to the culture terraneous plant allelopathy. Centaurea maculosa (spotted knap- with 50 mgL 1 each dose, which was repeated 10 times at 1-h in- weed), an invasive species in the western United States, displaced terval per day for continuing 5 days with total nominal concen- native plant species by exuding the phytotoxin ()- from tration of 2.5 mg L 1. HSE group was performed as per conventional its roots. However, the releasing amount of ()-catechin was toxicity tests (Nakai et al., 2005; Li and Hu, 2005b), in which significantly lower than its effective inhibition dose on the growth 2.5 mg L 1 NPN was dosed to the culture immediately following of Arabidopsis thaliana and Idaho fescue (Bais et al., 2003, 2010; Blair inoculation on day 1. The stock solutions of NPN were prepared et al., 2005). How low-dose allelochemicals released by plants exert with DMSO, whose final concentration in culture medium was the effective inhibition on target organisms remains a crucial 0.02% (v/v). The solvent (DMSO) control for the respective exposure puzzle. group and the blank using the culture without any treatment were It has been demonstrated that aquatic macrophytes release prepared simultaneously. All treatments and controls were pre- allelochemicals continually. Nakai et al. (1999) found that the pared in triplicate, and the culture conditions were the same as that allelopathic effects of M. spicatum on M. aeruginosa were the mentioned above. Cyanobacterial growth was determined daily by strongest in plant-cyanobacteria co-cultured systems, but weaker microscope counting over a 9-day period. Physiological parameters by semi-continuous addition and weakest by initial addition of were determined on day 1, 2, 3, 4, 5 and 9, respectively. The plant culture solutions. The growth of Scenedesmus obliquus was exposure experiment was repeated once. significantly inhibited by Potamogeton malaianus in the co-culture experiment but not by P. malaianus filtrates with initial addition 2.3. Measurement of cellular membrane integrity (Wu et al., 2007). Nakai et al. (2012) determined the release rate of fatty acids and found that their concentrations increased linearly The cyanobacterial cells sampled from the treatments and over time in the M. spicatum culture medium. Tharayil and controls were stained with propidium iodide (PI) at final concen- Triebwasser (2010) found that catechin exudation exhibited a tration of 7.5 mM. PI is a fluorescent dye of the nucleic acid, and possible diurnal rhythm with respect to light intensity, with the could only enter into damaged or dead cells, whose membranes are highest concentration at 6 h after exposure to sunlight. These no longer intact, to form orange fluorescence when staining the studies suggested that the release of allelochemicals to surrounding nucleic acid (Xiao et al., 2011). After 5 min of incubation in the dark, water should be in a continual way and give a consistent stress to the cells were subjected to Cytomics FC 500 Flow cytometry the target organism. However, it is not clear whether specific plant (Beckman Coulter, Inc., Fullerton, CA, USA) with a 488 nm laser. allelochemicals at releasing levels could exert strong inhibitory Negative controls were prepared in each determination including effects to the target organisms with continual exposure. heat-treated cells (100 C for 5 min) and unstained live cells (Wang The present study aimed to compare inhibitory effects of NPN on et al., 2013). 734 Y.N. Gao et al. / Chemosphere 174 (2017) 732e738

Fig. 1. Two exposure patterns (A, B) of N-phenyl-1-naphthylamine (NPN) in the present study.

2.4. Determination of chlorophyll fluorescence parameters in the splitless mode. Helium carrier gas was maintained at a constant flow rate of 1 mL/min. The ionization source temperature The chlorophyll fluorescence parameters were determined with and the MS quadrupole temperature were set at 230 C and 150 C, a pulse amplitude modulated fluorometer (Phyto-PAM Walz, respectively. For quantitative analysis, the MS was operated in Effeltrich, Germany). The measurements were performed at room selected ion monitoring (SIM) mode. The quantitative ion (m/z*) temperature. The cyanobacterial cells sampled from the treatments monitored for NPN was 219, and the confirmation ions (m/z)were and controls were dark-adapted for 15 min before analysis. Fo 109 and 218. The pretreated standard solutions dissolved in ethyl (original fluorescence) was measured under a low measuring light acetate with different known concentrations were determined to immediately after dark adaption. F (steady state fluorescence) and plot the standard curves, based on which the NPN contents were 0 s Fm (maximum fluorescence in the light adapted state) were ach- calculated. ieved by illuminating for 3 min with actinic light at 256 mmol photons m 2 s 1, and a subsequent saturating light pulse (about 2.6. Data analysis 3000 mmol photons m 2 s 1). Rapid light curves were plotted including 10 steps of actinic irradiance: 1, 32, 64, 96, 192, 320, 448, Data were presented as mean ± standard error of the mean 576, 707, 1216 mmol photons m 2 s 1, and a saturation light pulse (SEM). Cyanobacterial growth and physiology inhibition ratio for (about 3000 mmol photons m 2 s 1) with a 10s interval between each treatment was expressed as their percent change based on any two adjacent steps. F was determined in the presence of m those of the corresponding controls on the same day. The signifi- 10 mM DCMU with a saturating light pulse to avoid the influence of cant differences of cyanobacterial growth and physiology inhibition state transitions (Campbell et al., 1998). The maximum PSII quan- ratio between two exposure groups were tested with one-way tum yield (Fv/F ), operational PSII quantum yield (F ), non- m PSII ANOVA. Values were considered significantly different when the photochemical quenching (NPQ) and electron transport rate (ETR) probability (p) was less than 0.05 or 0.01. Cell density and physio- were calculated as: Fv/Fm ¼ (FmFo)/Fm (Ting and Owens, 1992); 0 0 0 0 logical parameters in the blank culture without any treatment were FPSII ¼ (FmFs)/Fm (Genty et al., 1989); NPQ ¼ (FmFm)/Fm (Krause 0 0 used as the control in the figures, since these parameters in two et al.,1982); ETR ¼ (F F )/F 0.42 PPFD (Schreiber et al.,1998) m s m solvent controls were not significantly different from those in the (PPFD: photosynthetic photo flux density). Maximum electron blank culture (p > 0.05). To model NPN content changes in culture transport rate (ETRmax) was derived from rapid light curves when solutions, exponential decay equation was used to fit the changes in the curve reached plateau phase. the HSE group, and nonlinear curve fit was for the LRE group in OriginPro8. 2.5. Residual dynamic analysis of NPN in culture medium 3. Results and discussion Liquid-liquid extraction was used to extract NPN in culture so- lutions (Gao et al., 2011), specifically, 2 mL of cyanobacteria me- 3.1. Growth inhibition of M. aeruginosa under different exposure dium was sampled every other day and centrifuged at 3000 rpm for patterns 10min, and then 1 mL supernatant was extracted with ethyl ace- tate. The extracts were diluted and analyzed by an Agilent 6890 N Effects of the allelochemical NPN on M. aeruginosa growth under GC with an Agilent 5973Inert MS operated in electron impact different exposure patterns are shown in Fig. 2, in which the mean mode. A HP-5 ms (30 m 0.25 mm 0.25 mm) capillary column value and standard deviation of daily cell density in blank culture was used. The initial oven temperature was 40 C, held for 2 min, representing the control are shown, since growth curves in blank then raised to 280 Cat20C/min with a final isotherm of 10 min. culture and two solvent controls were similar. However, there were The injection port and interface temperature were 250 C and significant differences amongst the control and two exposure pat- 280 C, respectively. One microliter of diluted extract was injected terns. Cyanobacterial growth in the HSE group was retarded Y.N. Gao et al. / Chemosphere 174 (2017) 732e738 735

Fig. 2. Effects of N-phenyl-1-naphthylamine (NPN) on the growth of M. aeruginosa under different exposure patterns, where * and ** indicate significant differences between two groups (p < 0.05 and p < 0.01). obviously until day 5, when the growth inhibition ratio reached up continual stress to target organisms, which might be an effective to 74.27%. However, from day 7 to day 9, rapid growth appeared and mode for aquatic plants to inhibit cyanobacterial growth and inhibition ratio decreased from 57.53% to below zero. M. aeruginosa physiological activity through allelopathy in natural water bodies. growth in the LRE group did not rise during the whole experiment, and inhibition ratio was higher than that in the HSE group from day 3.2. Cellular membrane damage 4 and significant difference occurred from day 5 (p < 0.01). Inhi- bition ratio in the LRE group remained above 90% during the later Effects of NPN on cellular membrane integrity of M. aeruginosa four days. under different exposure patterns were measured, and the results fi It was found for the rst time that the allelochemical NPN in the are demonstrated in Fig. 3. In the HSE group, slightly stronger LRE pattern exhibited much more detrimental effects on cyano- membrane damage was observed during the first five days, and the than that in the HSE pattern during a 9-day experiment. At highest damage ratio of 9.84% was found on day 2. On day 9, it was the same total nominal concentration of NPN, the M. aeruginosa only 3.23%, showing no statistical difference from the control growth was inhibited thoroughly in the LRE group whereas tran- (p > 0.01). However, cell membrane damage ratio of M. aeruginosa sitory growth inhibition was observed in the HSE group. It was once was obviously increased over time in the LRE group. On day 5, reported that growth of M. aeruginosa was recovered from day 5 membrane-damaged cell percentage reached up to 64.05%, signif- 1 after exposure to nonanoic acid at 4 mg L of nominal initial icantly higher than that of the HSE group (p < 0.01). On day 9, the fi concentration, though signi cant inhibition effect was observed ratio increased to 96.60%. fi during the rst 3 days (Shao et al., 2008). Our results showed that In the investigation on antialgal effects of salcolin A and B, iso- growth inhibition activity of NPN in the LRE group evidently lated from barley straw, Xiao et al. (2014) observed an increasing exceeded that in the HSE group on day 4, when nominal NPN broken-membrane cells during first 5 days, but a significant content added in cyanobacterial cultures for the LRE group was 1.5 mg L 1 in total, which is much lower than that added in the HSE group, 2.5 mg L 1. It indicated that repeated exposure was an efficient way for low-dosage allelochemicals to exert strong anti- cyanobacterial effects. Daily exposure with a concentration close to EC50 values of allelochemicals was reported to exert effective inhibition on M. aeruginosa, i.e. daily exposure of pyrogallol significantly inhibi- ted growth and physiology of M. aeruginosa at a single dose above 0.5 mg L 1 (Lu et al., 2014). The content of the polyphenols and fatty acids mixtures added every time to cyanobacteria cultures was higher than 1.0 mg L 1, which significantly reduced their maximum growth (Nakai et al., 2012). Here the hourly dose of NPN in the LRE treatment was as low as 50 mgL1, which was among 0.9e76.6 mgL 1, the releasing levels of allelochemical from aquatic plants such as M. spicatum, H. verticillata and V. spiralis (Nakai et al., 2000; Gao et al., 2011). It was reported for the first time that alle- lochemical at such a low concentration added repeatedly could Fig. 3. Effects of N-phenyl-1-naphthylamine (NPN) on the cellular membrane integrity exert so potent inhibitory effects. It indicated that a continual of M. aeruginosa in different exposure patterns. Significant differences between groups release of low-dosage allelochemicals by aquatic plants could cause on the same day are indicated with different letters (a, b) (p < 0.01). 736 Y.N. Gao et al. / Chemosphere 174 (2017) 732e738 reduction after 10 days. This is consistent with the trend of the HSE indicated that the PSII maximum photochemistry efficiency in group in the present study, indicated a transitory damage to cya- M. aeruginosa was totally inhibited by lowedosage repeated nobacterial cell membrane under initial single exposure pattern. exposure of NPN by the end of experiment, whereas a transitory However, cell membrane damage by NPN was more serious in the inhibition followed by a gradual recovery was observed in the HSE LRE group, which proved that repeated exposure of low-dosage group. NPN exerted destructive effects on cyanobacterial cell membranes FPSII denotes operational photochemical efficiency of open PSII during a longer period compared to single exposure. The big dif- centers under a given light acclimation status (Campbell et al., ferences between two exposure groups further indicated that a 1998). Reduction in FPSII means a decline of light-harvesting ca- continual and cumulative effect from low-dosage allelochemicals pacity and a block of the electron transfer process (Zhou et al., to cyanobacteria was more fatal than one-off attack even at much 2013). As shown in Fig. 4B, FPSII in M. aeruginosa cells was among higher concentration. 0.33e0.45 for the control, and its slight decrease on day 9 was in accordance with the growth curve changes in Fig. 2. Its time- 3.3. Photosynthesis inhibition dependent variation under two exposure patterns was signifi- cantly different. The percentage of FPSII in the HSE group decreased It has been demonstrated that PSII in cyanobacteria was a target sharply to 78.59% on day 2, with a continuous declination till day 4 site of most allelochemicals and environmental stress (Leu et al., when the percentage fell to 60.71%, but followed by an obvious rise F 2002; Zhu et al., 2010). Fv/Fm, FPSII, NPQ and ETRmax are four to 88.65% on day 9. PSII in the LRE group was 94.23% on day 2, fell key parameters to characterize the PSII status. In the present study, rapidly to 60.87% on day 3, 22.53% on day 4, and lower than 10% on the four chlorophyll fluorescence parameters in M. aeruginosa were day 9. measured. Fig. 4 shows that these four parameters were signifi- NPQ is a process to dissipate excess excitation energy in the cantly inhibited by allelochemical NPN, the extent of which was chlorophyll pigment bed of PSII by a non-radiative pathway, which dependent on exposure patterns. Fv/Fm is representing maximal serves to protect the photosynthetic apparatus from photo-damage efficiency of PSII photochemistry in cyanobacterial cells, i.e. the (Joshua et al., 2005; Bailey and Grossman, 2008). A significant quantum efficiency if all PSII centers were open (Campbell et al., elevation of NPQ under stress indicated that excessive photons 1998; Maxwell and Johnson, 2000). A marked decrease of Fv/Fm were partly consumed in non-photochemical process, e.g. heat was easily recorded after cyanobacterial cells were exposed to bi- dissipation, which was a part of the protection and recovery stra- otic and abiotic stress e.g. UV-C, algicides, or insufficient inorganic tegies (Tao et al., 2013). As shown in Fig. 4C, NPQ value in the phosphorus (Tao et al., 2013; Wang et al., 2010, 2016). Fig. 4A shows control was among 0.12 to 0.19 with a fluctuation during the that Fv/Fm of M. aeruginosa cells in the control ranged from 0.49 to experiment, which was partly because NPQ was more sensitive 0.61 during the experiment, which was in accordance with the than other chlorophyll fluorescence parameters (Campbell et al., previous research (Yang et al., 2013). Fv/Fm value in the HSE group 1998; Zhu et al., 2010). However, there were much bigger differ- was 85.14% of that in the control on day 2, and the biggest differ- ences between the control and the treatments. NPQ value in the ence from the control appeared on day 3 with a percentage of HSE group was 97.56% and 96.15% of the control on day 2 and day 3, 77.98%. It indicated significant inhibition on Fv/Fm by NPN single dropped dramatically to 78.26% and 64.70% on day 4 and day 5, exposure. However, a slight recovery was observed for this group respectively. A slight elevation of NPQ value was observed on day 9 with the percentage increasing to 89.31% on day 9. For LRE group, a with 103.14% of the control. Whereas there was an obvious increase time variation trend was obviously different. Although there were of NPQ value on day 2 and day 3 in the LRE group, but a dramatical slightly positive effects of NPN on Fv/Fm in M. aeruginosa on day 2, drop to below zero from day 4 to day 9, which indicated that PSII the Fv/Fm value decreased significantly to 73.28% of the control on photo-protective system of M. aeruginosa was affected severely by day 3, fell rapidly to 20.1% on day 4, and then remained around zero low-dosage NPN repeated exposure. later. The notable differences between two exposure patterns The maximum relative electron transport rate of PSII (ETRmax),

Fig. 4. Effects of N-phenyl-1-naphthylamine (NPN) on the chlorophyll fluorescence parameters (Fv/Fm, FPSII, NPQ and ETRmax) of M. aeruginosa under different exposure patterns. Significant differences between groups are indicated with * or ** on the same day (p < 0.05 or p < 0.01). Y.N. Gao et al. / Chemosphere 174 (2017) 732e738 737

NPN contents in the LRE group increased from 353.71 mgL 1 on day 1e1043.95 mgL 1 on day 5 during dosing period. Then, it declined to 692.72 mgL 1 and 534.72 mgL 1 on day 7 and day 9, respectively. In the HSE group, high NPN contents at initial stage caused an obvious damage to cyanobacterial cells, which was gradually recovered along with the decrease of NPN contents in culture so- lutions, and it indicated a positive correlation between NPN con- tents and inhibitory ratio on the growth and physiology of M. aeruginosa. However, time variation of NPN contents in the LRE group was not totally in accordance with that of inhibition effects. Specifically, NPN contents increased during the initial five days and exceeded those in the HSE group after day 4, followed by a slight decrease. Whereas its inhibition ratios on cyanobacterial growth and photosynthesis activity were the same as those in the HSE group on day 3, and then increased rapidly to 77.75% for growth, 79.9.% for Fv/Fm, 77.47% for FPSII, more than 100% for NPQ and 89.07% on day 4, which were remained and even enhanced during the later five days. On the other hand, the highest concentration in the LRE group was detected as 1043.95 mgL 1 on day 5, which was 1 Fig. 5. Fitted residual dynamic of NPN in culture solutions under different exposure significantly lower than that in the HSE group, 2036.08 mgL on patterns. day 1. However, the highest inhibition ratio on growth and physi- ological endpoints for the HSE group was much lower than that for the LRE group during the experiment. In other words, NPN contents which is also closely associated with carbon assimilation activity in in the LRE group maintained a relatively lower but more stable level photosynthesis process (Wang et al., 2010). Pronounced differences during the experiment, which caused strong inhibition on the of time-dependent variation patterns for ETRmax occurred be- M. aeruginosa cells. It is indicated that continual exposure must be a tween the two treatments (Fig. 4D). A successive growth from key factor for stronger inhibition in the LRE group during a longer m 2 1 e m 2 1 65.35 mol m s on day 1 143.30 mol m s on day 9, indi- period, and sustained stress on test organisms by low-dosage cated a gradually increasing photosynthesis capacity for allelochemicals could be a potent mode for aquatic plants to sup- M. aeruginosa in the control. In contrast, ETRmax value maintained press cyanobacterial development through allelopathy under nat- and the percentage compared to the control decreased from 71.08% ural conditions. As for allelopathy research methodology, more e fi on day 2 48.18% on day 4, exhibited a signi cant inhibition initially attention should be paid to the investigation into the effects of by NPN single exposure. A slight recovery in the end was recorded allelochemicals on target photosynthetic plankton under continual with the percentage of 67.57% on day 9, suggesting a reversible exposure at low-dosage levels during a long term to reveal mech- photosynthesis inhibition under NPN single exposure even at a anisms of allelopathy in aquatic ecosystems. high dosage. For LRE group, ETRmax on day 2 was 86.12% of the control, but followed by a sharply reduction from 50.39% on day 3e10.93% on day 4, a notable drop to 3.10% and 2.47% of the control 4. Conclusions on day 5 and day 9, respectively. The variation trend of ETRmax for LRE group provided further evidence that PSII electron transport of The effects of NPN on M. aeruginosa under two exposure pat- M. aeruginosa cells was thoroughly inhibited under repeated terns, i.e. HSE and LRE, were compared in the present study. It was exposure of NPN. shown that a transitory toxicity on M. aeruginosa at initial stage was Changes of the four parameters were in consistent with each followed by a gradual recovery in the HSE pattern as per routine other, revealing that PSII photosynthesis activity of M. aeruginosa toxicity assay. However, NPN toxicity in terms of the detrimental was severely inhibited by low-dosage NPN repeated exposure, but effects on the cyanobacteria growth, cellular membrane integrity inhibited transitorily by high-dosage NPN single exposure during a and PSII photochemical activity was more severe and remained for 9-day period. From day 3, Fv/Fm, FPSII and ETRmax of M. aeruginosa a longer period in the LRE pattern simulating continual release of in the LRE group were inhibited significantly and inhibition ratios plant allelochemicals. were higher than those in the HSE group. Residual dynamics of NPN during the experiment indicated a rapid decline of NPN content over time in the HSE group, which was 3.4. Residual dynamic of NPN in culture solutions accompanied by descending inhibition activity on the M. aeruginosa growth and gradual recovery of its physiological NPN concentrations in culture solutions were detected every functions. NPN contents in the LRE group remained at a relatively other day during the experiment for both exposure patterns, and lower but more stable level to exert sustained stress and cause their changes over time were fitted. The fitting curves and equa- serious damage to M. aeruginosa cells during the experiment. tions are shown in Fig. 5 and Table 1, respectively. Mean value of The present study provided evidence that continual release of detected NPN concentrations in the HSE group decreased gradually low-dosage allelochemicals to exert sustained stress was an effec- from 2036.08 mgL 1 on day 1e88.32 mgL 1 on day 9. While median tive mode for aquatic plants to inhibit cyanobacteria through

Table 1 Fitting equations of NPN content changes (y) over time (x) in two treatments.

Treatments Equation R2

High-dosage single exposure y ¼ 3480.96 exp(x/8.04)1083.18 0.9822 Low-dosage repeated exposure Y ¼ 444.21 þ (2187.63/(2.94 sqrt(p/2))) exp(2 (x5.06)/2.94)2) 0.9134 738 Y.N. Gao et al. / Chemosphere 174 (2017) 732e738 allelopathy. Low-dosage continual exposure pattern of alle- Mulderij, G., Mau, B., de Senerpont Domis, L.N., Smolders, A.J.P., Van Donk, E., 2009. fi lochemicals should also be used as the methodology in the future Interaction between the macrophyte Stratiotes aloides and lamentous algae: does it indicate allelopathy? Aquat. Ecol. 43, 305e312. research of allelopathy. Nakai, S., Inoue, Y., Hosomi, M., 2000. Myriophyllum spicatum-released allelopathic polyphenols inhibiting growth of blue-green algae Microcystis aeruginosa. e Acknowledgements Water Res. 34, 3026 3032. Nakai, S., Inoue, Y., Hosomi, M., Murakami, A., 1999. Growth inhibition of blue-green algae by allelopathic effects of macrophytes. Water Sci. Technol. 39, 47e53. This work was supported by National Natural Science Founda- Nakai, S., Yamada, S., Hosomi, M., 2005. Anti-cyanobacterial fatty acids released tion of China (31500380), Major Science and Technology Program from Myriophyllum spicatum. Hydrobiologia 543, 71e78. fi Nakai, S., Zou, G., Okuda, T., Nishijima, W., Hosomi, M., Okada, M., 2012. Polyphenols in Henan Province (152102210289), Key Scienti c Research Project and fatty acids responsible for anti-cyanobacterial allelopathic effects of sub- of Colleges and Universities in Henan Province (15A240001), the merged macrophyte Myriophyllum spicatum. Water Sci. Technol. 66, 993e999. Youth Science Fund of Henan Normal University (2014QK25), and Narwal, S.S., 2006. Allelopathy in ecological sustainable agriculture. In: fi Reigosa, M.J., Pedrol, N., Gonzalez, L. (Eds.), Allelopathy: a Physiological Process Doctoral Scienti c Research Start-up Foundation of Henan Normal with Ecological Implications. Springer, The Netherlands, p. 548. 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