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Comparative biotoxicity of N-Phenyl-1-naphthylamine and N-Phenyl-2-naphthylamine on cyanobacteria Microcystis aeruginosa
Accepted Manuscript Comparative biotoxicity of N-Phenyl-1-naphthylamine and N-Phenyl-2-naphthylamine on cyanobacteria Microcystis aeruginosa Long Cheng, Yan He, Yun Tian, Biyun Liu, Yongyuan Zhang, Qiaohong Zhou, Zhenbin Wu PII:
S0045-6535(17)30298-9
DOI:
10.1016/j.chemosphere.2017.02.110
Reference:
CHEM 18873
To appear in:
ECSN
Received Date: 10 October 2016 Revised Date:
4 February 2017
Accepted Date: 21 February 2017
Please cite this article as: Cheng, L., He, Y., Tian, Y., Liu, B., Zhang, Y., Zhou, Q., Wu, Z., Comparative biotoxicity of N-Phenyl-1-naphthylamine and N-Phenyl-2-naphthylamine on cyanobacteria Microcystis aeruginosa, Chemosphere (2017), doi: 10.1016/j.chemosphere.2017.02.110. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Comparative biotoxicity of N-Phenyl-1-naphthylamine and
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N-Phenyl-2-naphthylamine on cyanobacteria Microcystis aeruginosa
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Long Cheng a,b, Yan He a,b, Yun Tian a,b, Biyun Liu a,*, Yongyuan Zhang a, Qiaohong
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Zhou a,*, Zhenbin Wua
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a
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Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China;
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b
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*Correspondence: [email protected] (B. Liu)
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State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of
University of Chinese Academy of Sciences, Beijing 100049, China
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[email protected] (Q. Zhou)
Abstract: N-Phenyl-1-naphthylamine (P1NA) and N-Phenyl-2-naphthylamine (P2NA)
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are both widely used as antioxidant and plant secondary metabolites. In this study, growth,
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esterase, photosynthetic activity and cell membrane integrity were used as biomarkers to
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compare biotoxicity of P1NA and P2NA on Microcystis aeruginosa. According to the
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results, a dose-response relationship was observed only between P1NA concentrations and
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growth inhibition. The EC50 (48 h) of P1NA calculated from growth inhibition was 16.62
1）
µM, while that of P2NA was not detected. When the esterase and photosynthetic activity
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were applied to evaluate the biotoxicity, it was found that a concentration of 20 µM P1NA,
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P2NA caused reduction of esterase activity and Fv/Fm of M. aeruginosa to 22.2 and 3.3%,
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97.5 and 92.1%, respectively, after 48 h exposure. The percentage of membrane-damaged
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cells was increased as P1NA exposure concentration increased, but that was not detected
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when exposure to P2NA. The difference substituted position in the molecular structure of
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P1NA and P2NA leads to different toxicological properties and only P1NA was found
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highly toxic to M. aeruginosa. The toxicity is due to that only P1NA can be
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biotransformed to 1,4-naphthoquinone, which could induce overproduction of
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intracellular ROS as well as result in oxidative damage and growth inhibition of test
2）
organism.
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Keywords:
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aeruginosa; Toxic effects; Reactive oxygen species
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1. Introduction
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N-Phenyl-2-naphthylamine;
Microcystis
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N-Phenyl-1-naphthylamine (P1NA) serves as an antioxidant in various lubrication
31
oils and also as a protective agent in rubbers as well as rubber mixtures. (Koennecker et
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al., 1998). It was estimated that the annual production of P1NA was 5800-6300 tons from
33
1986 to 1990 in the world (Koennecker et al., 1998). The distribution of P1NA was
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predicted approximately 29% to water, 34% to sediment, 36% to soil, and less than 1%
35
each to air, suspended sediment, and biota in the environment using a Level II fugacity
3）
model (Mackay and Models, 2001; Koennecker et al., 1998). N-Phenyl-2-naphthylamine
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(P2NA), the isomer of P1NA, also used as an additive jet oils and it is a byproduct in the
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manufacture of P1NA (Winder and Balouet, 2002). It was reported that P1NA was
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detected in river freshwater (2-7 µg L-1) and sediment (1-5 mg kg-1) near a small specialty
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chemicals manufacturing ( Koennecker et al., 1998). And P2NA has been detected in
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sediments and freshwater in the proximity of chemical production sites in mg kg-1 and µg
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L-1 concentrations (Brack et al., 1999; Altenburger et al., 2006). Studies performed according to standard protocols yielded LD50 of P1NA in male
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and female Wistar rats of >5000 mg kg-1 body weight (Koennecker et al., 1998). Acute
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oral toxicity test of P2NA indicated that its LD50 in rats and in mice were 8730 mg kg-1
4）
and 1450 mg kg-1 body weight (NTP, 1988). Due to P1NA and P2NA’s lipophilicity with
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octanol-water partition coefficient, log KOW of 4.2 and 4.38, respectively, they may be
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expected to have a considerable bio-accumulative potential in aquatic environments and
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be toxic to aquatic organisms. The studies from laboratory tests with ciliates, Daphnia,
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and fish indicated that P1NA was highly toxic to aquatic species. EC50 (48 h) for the
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inhibition of cell proliferation of freshwater ciliates (Tetrahymena pyriformis), LC50 (48 h)
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with young Daphnia magna and LC50 (96 h) in a flow-through system for rainbow trout
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(Oncorhynchus mykiss) are, respectively, 2 mg L-1 (Epstein et al., 1967), 0.30 mg L-1, and
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0.44-0.74 mg L-1 (Sikka et al., 1981). Phytoplankton is the fundamental part in the
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aquatic ecosystems. However, there is little research about the toxicity of P1NA on
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phytoplankton.
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Submerged plants can secrete secondary metabolites to inhibit photosynthetic
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plankton in order to compete for light and nutrition dominance in the water environment.
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This phenomenon is called allelopathy and the active substances are known as
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allelochemicals (Rice, 1984). In recent years, significant progress in the allelopathic
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effect of submerged macrophyte on cyanobacteria has been made in both laboratory and
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field study. In respect of the mechanism of allelopathy, it was reported that aquatic plants
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release allelochemicals continually with low concentration, and caused persistent stress
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on target organisms. Under this condition, the intracellular signaling radicals (ROS, NO)
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were induced, as well as, the programmed cell death (PCD) was trigger in M. aeruginosa
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which was confirmed by the hallmarks of PCD, caspase-3-like protease activity, DNA
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fragmentation, and destruction of cell ultrastructure (He et al., 2016). The engineering
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application of allelopathy through reconstructing the aquatic vegetation in the restoration
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of the subtropical eutrophic lake was studied. Ma et al. (2009) reported that
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transformation of water in Yuehu Lake (66 ha), a shallow eutrophic lake in China, from
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turbid algae green to a transparent state, was achieved by restoring the submerged
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macrophyte community while the nitrogen and phosphorus are at the level of
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eutrophication. Thus, it attracted the great interest of researchers to study the mechanism
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of allelopathy. P1NA and P2NA as a kind of allelochemicals have been isolated and
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identified in axenic as well as noaxenic root exudates of Eichhornia crassipes
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(Sultankhodzhaev and Tadzhibaev, 1976; Sun et al., 1993). These two substances have
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also been detected in the culture medium of three submerged macrophytes (Elodea
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nuttallii, Hydrilla verticillata and Vallisneria spiralis) by GC-MS (Gao et al., 2010).
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Qian et al. (2009, 2010) studied the toxicity of allelochemical P2NA to Chlorella vulgaris
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and M. aeruginosa. The only difference in molecular structure between P1NA and P2NA
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is that anilino-N substitutes at 1 and 2 positions of the naphthyl group. No information is
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available about whether the toxicity of P1NA and P2NA on cyanobacteria is different and
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how the toxicity is produced.
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The present study, M. aeruginosa, a dominant species of cyanobacterial blooming,
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was chosen as a target species. The physiological and photosynthetic parameters i.e.
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growth inhibition, esterase activity, cell membrane integrity, and photosynthetic activity
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were used as biomarkers to compare the toxicity between P1NA and P2NA. Moreover,
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reactive oxygen specious (ROS) level was used to evaluate the toxicity mechanism. The
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aim of this study was to (1) provide ecotoxicological information for evaluating the
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toxicity of P1NA and P2NA to the cyanobacteria (M. aeruginosa) in future ecological risk
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assessments and regulation of P1NA and P2NA use and discharges in aquatic ecosystems,
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(2) elucidate toxicity and inhibition mechanism of P1NA and P2NA on cyanobacteria to
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provide the base for further study the mechanism of allelopathic mechanism.
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2. Materials and methods
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2.1 Cultures conditions
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Cyanobacteria, Microcystis aeruginosa FACHB 905 was provided by the Institute
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of Hydrobiology of the Chinese Academy of Sciences. M. aeruginosa was cultured in
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autoclaved BG11 media (Rippka et al., 1979) at 25 ±0.5
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intensity of 18 µmol m-2 s-1 with a 12:12 h light: dark cycle. The cultures were shaken
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three times per day by hands.
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in an incubator under a light
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2.2 Chemicals N-phenyl-1-naphthylamine (98.0%) and N-phenyl-2-naphthylamine (97.0%) were
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commercially
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2´,7´-dichlorodihydrofluorescein diacetate (DCFH-DA), fluorescein diacetate (FDA) and
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dimethyl sulfoxide (DMSO, 99%) were all obtained from Sigma (St. Louis, Missouri,
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USA). Propidium Iodide (PI) was purchased from Sangon Co. Ltd (shanghai, China).
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2.3 Toxicity assays
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2.3.1 Experimental design
from
the
same
company
(TCI,
Tokyo,
Japan).
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Conical flasks (250 mL) and culture medium were prepared and autoclaved. 100 mL
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culture media was added to each bottle. P1NA and P2NA were added with the
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concentration of 0, 5, 10, 15, 20 µM, respectively. Then M. aeruginosa was inoculated
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into each flask with the initial density of 1 × 106 cells mL−1 when it was at the
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exponential growth phase. The samples were taken after 48 and 72 h for the measurement
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of growth and other physiological parameters. The stock solutions of P1NA and P2NA
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were prepared with DMSO which in test solution was less than 0.1% (v/v). 0.1% DMSO
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had no effects on the growth of M. aeruginosa (FACHB-905) (Gao et al., 2011). The
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experimental conditions are the same to the cyanobacteria culture conditions shown
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above. All the experiments were performed in triplicate.
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2.3.2 Measurement of growth inhibition
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The cells density was measured by spectrophotometry at 680 nm. The regression
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equation between cell density (y×103 cells mL-1) and OD680 (x) was calculated as follow:
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y = 19735x – 33 (R2 = 0.9999). The inhibition ratio (IR) of cyanobacterial growth was
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calculated according to the following Eq. (1):
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IR = ( − )⁄ × 100%
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(1)
where N0 and Ns (cells mL-1) represent cell density in the control and treatment groups,
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respectively.
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2.3.3 Measurement of esterase activity
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The fluorescein diacetate (FDA) assay has been used to indicate the metabolic
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activity of cells (Dunker et al., 2013; Xiao et al., 2010, 2011). 5 µL of 1 g L-1 FDA was
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added into 500 µL cells samples in a final concentration of 10 mg L-1 incubated for 15
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min at room temperature in darkness. Then cells were analyzed by flow cytometer FACS
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Verse (BD Biosciences, Franklin Lakes, New Jersey, USA) with excitation and emission
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wavelength of 488 nm and 527/32 nm. As depicted by Hadjoudja et al. (2009), the
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changes of esterase activity exposure to P1NA and P2NA were showed as the decrease in
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the proportion of cells in the S2 regions (normal activity status) compared with control
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cells. Every sample was collected 10000 events.
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2.3.4 Detection of cell membrane integrity
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Cell membrane integrity was assessed by fluorescent dye PI which could interact
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with nucleic acids to produce red fluorescence when the cell membrane was damaged
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(Xiao et al., 2011). For the measurement of M. aeruginosa cell membrane integrity, 5 µL
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of PI working solution was added to 500 µL of the sample with the final concentration of
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15 µM and the sample was stained for 15 min in the dark at room temperature.
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Fluorescence was collected through 586/42 nm with 488 nm laser. Data were expressed
144
as stained cell percentages.
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2.3.5 Detection of intracellular ROS level
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The ROS level in M. aeruginosa was measured using the cell permeable indicator
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2´,7´-dichlorodihydrofluorescein diacetate (DCFH-DA) according to He et al. (2016).
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The samples of cells were added with a final concentration of 10 µM DCFH-DA and
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pre-incubation at 25°C in dark for 1 h. Sample fluorescence was measured using flow
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cytometer with excitation and emission wavelength of 488 nm and 527/32 nm. To prove
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the role of ROS in the P1NA induced growth inhibition, the BG11 culture mediums were
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added with ROS scavenger ascorbic acid (0.5, 1 and 2 mM) and then adjusted to pH 7.1
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with 1 M NaOH. The cells were pre-treated for 1 h and then exposed to 15 µM P1NA for
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24 h. Growth inhibition in test organism was detected.
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2.3.6 Photosynthesis activity analysis
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Maximum quantum yield (Fv/Fm), maximal electron transport rate (ETRmax) and
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light use efficiency ratio (α) were selected to represent the M. aeruginosa photosynthesis
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status. These values were measured using phyto-PAM analyzer (Walz, Effeltrich,
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Germany) (Maxwell and Johnson, 2000).
1）0
2.3.7 Determination of P1NA and P2NA concentrations and metabolites
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For analyzing the concentration of P1NA and P2NA, 2 mL culture samples were
1）2
extracted by equivalent ethyl acetate and then the extracts were dehydrated by anhydrous
1）3
sodium sulfate. The extracts being diluted 20 times were then transferred to auto-sampler
1）4
vials for gas chromatography–mass spectrometry (GC–MS) (Agilent 6890N/5973Inert,
1）5
Santa Clara, California, USA). The GC temperature profile consisted of an initial holding
1））
time of 2 min at 40
1）7
reaching a final temperature of 320
1）8
P2NA were 13.5 min and 13.9 min, respectively. The standard curve was prepared and the
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equation for relation between peak area (y) and concentration (x)(µM) (0-1 µM) was as
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follows: y = 70781x – 2533.9 (R2 = 0.991) and y = 268031x – 5141.2 (R2 = 0.997) for
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P1NA and P2NA, respectively. The metabolites were qualitatively analyzed in M.
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aeruginosa exposed to 20 µM of P1NA, P2NA and control group respectively. The
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metabolites were determined by GC–MS, briefly as follows: 25 mL culture samples
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(exposure for 72 h) were adjusted to pH 2 with 1 M HCl and extracted three times with
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an equivalent volume of ethyl acetate. The following sample handling and determination
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condition were the same to the Lu et al. (2016).
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2.4 Statistical analysis
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followed by a gradual increase in temperature of 20
min−1,
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Flow cytometric analysis data were analyzed using FlowJo software (Tree Star
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Software, San Carlos, California, USA). Data are presented in mean ± SD. One-way
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ANOVA followed by LSD (when homogeneity of variance) or Tamhane's T2 (when
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heterogeneity of variance) post hoc test was used to test for the differences between
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individual means among P1NA or P2NA treatment at different concentrations.
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Independent samples t-tests were used to compare the differences individual means when
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at the same P1NA and P2NA concentration. Differences were considered to be significant
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at p < 0.05. EC50 values were determined with probit regression. All the statistical
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analyses were performed using SPSS 18.0 software (IBM Corporation, Armonk, New
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York, USA).
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3 Results and discussion
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3.1 Real concentrations of P1NA and P2NA concentrations during experiment
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In order to determine the real concentrations and stability of P1NA and P2NA, the
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concentrations in culture medium were quantitatively analyzed at 0, 24, 48 and 72 h. The
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results are shown in Fig 1. For nominal concentrations of 5, 10, 15, 20 µM, the real
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exposure concentrations of P1NA were 4.75±0.018, 11.64±1.86, 14.89±0.27, 20.15±0.86
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µM, respectively. The real exposure concentration of P2NA were 4.17±0.11, 10.62±0.48,
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12.73±1.46, 19.90±0.17 µM, respectively. It can be found that the actually determining
19）
concentrations are close to the nominal concentrations. The average percentage error of
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P1NA and P2NA was 5.7% and 9.6% between measured concentrations and nominal
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values. The concentration of P1NA and P2NA decreased gradually over time in biotic and
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abiotic conditions. And the decomposition rate of P1NA is faster than that of P2NA. For
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instance, 20 µM of P1NA and P2NA in the culture medium were reduced by 59.2% and
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35.8% within 72 h. The half-life of P1NA was 24-50 h, while that of P2NA was 66-120 h
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which was out of our experimental period calculated from the degradation patterns at 5,
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10, 15, and 20 µM.
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We speculated that P1NA and P2NA concentrations decreased possibly due to the
205
reason that these substances can be adsorbed and metabolized by cyanobacteria or
20）
photochemically decomposed in the culture medium. Sikka et al. (1981) demonstrated
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that P1NA was degraded with a half-life ranging from 4 to 11 days in water inoculated
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with domestic sewage and lake water, respectively. A further experiment conducted in
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aqueous solution, indicated that P1NA could be directly photochemically degraded (Sikka
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et al. 1981). Meanwhile, on the basis of in vitro studies, metabolism of P1NA and P2NA
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likely occurs primarily via hydroxylation (Deutsche Forschungsgemeinschaft, 2015;
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Koennecker et al., 1998; Weiss et al., 2007). 5 µM
10
15 µM
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10 µM
P1NA
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20 µM
P2NA 15
10
5
5
0
0
20 µM in abiotic conditions
20
0 1
2
3
0
1
2
3
Time(day)
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Fig 1. Real concentrations of P1NA and P2NA (µM) in the culture medium, during the experimental period.
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Data are presented in mean ± SD.
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3.2 Effects of P1NA and P2NA on M. aeruginosa growth Cell growth inhibition is commonly used as a valid biomarker to evaluate the
218
phytotoxicity on cyanobacteria as it shows. Because it is an integrating parameter
219
showing toxicants inhibitory effects on all cellular metabolism (Dewez et al., 2008). The
220
inhibition effects of P1NA and P2NA on M. aeruginosa growth were studied after 48 and
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72 h (Fig 2). It can be seen that when exposure to P1NA, the growth inhibition of M.
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aeruginosa increased with an increasing concentration of P1NA. After 48 h, the inhibition
223
rates in the group of 5, 10, 15, 20 µM of P1NA were 4.0%, 6.9%, 40.6%, 69.4%,
224
respectively. However, those of P2NA were 0.3%, 3.1%, 0.6%, 0.3%, respectively. The
225
inhibition rates were 5.0%, 14.1%, 46.1%, 74.1%, respectively, exposure by P1NA after
22）
72 h. A significant dose-response and time-dependent relationship were displayed in
227
P1NA group, while these relationships for P2NA were not observed in the experiment.
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Growth inhibition rates of P1NA exposed to 10, 15, 20 µM were higher than that of P2NA
229
at 48h, and the differences were 3.8%, 40.0%, 69.0%, respectively. It showed significant
230
differences (p< 0.05) with the exposure time for 48 h in the group of 10, 15 and 20 µM,
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and with the exposure time for 72 h in the group of 15 and 20 µM, respectively.
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The EC50 (48 h) value (Table S1) of P1NA calculated from growth inhibition was
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16.616 µM (3.64 mg/L), while that of P2NA was not detected. The results showed that
234
only P1NA was highly toxic to M. aeruginosa. Koennecker et al., (1998) reported that
235
P1NA was low toxic to the animal but highly toxic to aquatic organisms. An EC50 (48h) of
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2 mg L-1 of P1NA was measured for the inhibition of cell proliferation of freshwater
237
ciliates Tetrahymena pyriformis (Epstein et al., 1967). LC50 (48h) for young and adult
238
Daphnia magna were in the range of 0.30-0.68 mg L-1 (Koennecker et al., 1998). The
239
toxicity of P1NA to M. aeruginosa was similar to that of protozoa, which was lower than
240
that of cladoceran zooplankton. However, several studies have reported that P2NA is a
241
highly toxic substance to C. vulgaris (Qian et al., 2009), M. aeruginosa (Qian et al., 2010)
242
and Cylindrospermopsis raciborskii (Liu et al., 2015). The toxicity of P2NA may be
243
affected by the strain of the test organism or the culture conditions. The difference needs
244
to be furtherly studied in the future.
48h
***
**
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40
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5
10
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A a
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B a
a
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a
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60
P2NA
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***
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20
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P1NA
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100
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40
20
0
20
B c A a
b
A
0
5
10
c
15
c
20
P1NA/P2NA Concentration(µM)
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Fig 2. Effect of P1NA and P2NA on growth inhibition in M. aeruginosa. * (P<0.05), ** (P<0.01), and ***
247
(P<0.001) indicate significant differences between P1NA and P2NA. Different small letters in each figure in
248
the same type of growth inhibition represent statistically significant differences (p<0.05) compared to the
249
corresponding controls. Data are presented in mean ± SD.
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3.3 Impact on esterase activity
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The esterase activity in M. aeruginosa exposed to P1NA and P2NA for 48 and 72 h
252
were shown in Fig 3. It could be found that the esterase activity significantly decreased in
253
the group of 5 µM and 10 µM exposure to P1NA at 48 h. And then the esterase activity
254
drastically reduced with the increase of P1NA concentration, it declined to 70.2% and
255
22.2% of the proportion of cells in the S2 regions (normal activity status) (Hadjoudja et al.
25）
2009) with 15 µM and 20 µM of concentrations exposure to P1NA. The similar pattern
257
could be found in the group exposure for 72 h, while it did not further lower the esterase
258
activity. As opposed to P1NA, the esterase activity was invariant as P2NA concentrations
259
increased at 48 and 72 h.
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80
80
48 h
60
40
0
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5
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2）0
P2NA
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% S2 FDA Fluorescence
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P1NA
10
72 h
60
40
20
0 15
20
0
5
10
15
20
P1NA/P2NA Concentration (µM)
Fig 3. Effect of P1NA and P2NA on esterase activity in M. aeruginosa. Data are presented in mean ± SD.
Esterase activity was commonly used to evaluate the metabolic status of the cell
2）3
because it was a rapid and sensitive endpoint (Valiente Moro et al., 2012), and usually, a
2）4
decline in enzyme activity indicated the presence of stress (Hadjoudja et al., 2009; Regel
2）5
et al., 2002). Herbicide paraquat and copper could result in reducing esterase activity due
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to oxidative stress and showed a dose-response relationship in acute toxicity test for
2）7
phytoplankton (Hadjoudja et al., 2009; Prado et al., 2012). It indicated that the decline in
2）8
esterase activity exposure to P1NA being similar to paraquat and copper. And it gave an
2）9
information not only to be helpful to promptly determine the physiological state of cells
270
but also to evaluate the toxicity of pollutants. The change of esterase activity along with
271
growth inhibition suggested that the toxicity of P1NA and P2NA to M. aeruginosa had an
272
obvious difference.
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3.4 Inhibition of photosynthetic activity
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In the present study, maximum quantum yield (Fv/Fm), maximal electron transport
275
rate (ETRmax) and light use efficiency ratio (α) were used to estimate the efficiency of
27）
PSII in M. aeruginosa. PSII efficiency in M. aeruginosa exposure to P1NA and P2NA are
277
shown in Fig 4. The results showed that Fv/Fm has significantly decreased in the group
278
of 15 and 20 µM P1NA exposure for 48 and 72 h (p < 0.05) (Fig 3A, 3B). It was different
279
when exposure to P2NA. At 48 h, Fv/Fm was significantly decreased in the group of 10,
280
15 and 20 µM P2NA (p<0.05). But every concentration of the P2NA treatment group did
281
not have any differences at 72 h (p> 0.05) which was consistent with the trend of growth
282
inhibition. There was a significant difference between P1NA and P2NA in the group of 15
283
and 20 µM at 48 and 72 h. The effects on ETRmax and α are shown in Fig 4C, 4D and
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Fig 4E, 4F, respectively. P1NA treatment led to a significant decrease in ETRmax or α
285
values at high concentration, while exposure of P2NA slightly reduced them. It was much
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similar to the change of Fv/Fm values. The P1NA caused a severe toxicity on PSII of the cyanobacteria M. aeruginosa, as
288
introduced above. These results were consistent with the growth and esterase activity of
289
M. aeruginosa. It is known that the value of Fv/Fm characterizes the content of
290
photochemically active centers of PSII and indicates the current balance between
291
processes of light destruction and the reparation of PSII (Brack and Frank, 1998).
292
Similarly, some kinds of exogenous substances such as tetracycline and natural
293
flavonoids were found to inhibit growth and reduce Fv/Fm values in M. aeruginosa and
294
Selenastrum capricornutum (Huang et al., 2015; Yang et al., 2013). Allelochemicals
295
identified from Myriophyllum spicatum, including pyrogallic acid, gallic acid, ellagic acid
29）
and (+)-catechin caused significant reductions of PSII efficiency and whole electron
297
transport chain activities in M. aeruginosa (Zhu et al., 2010). The decrease of the
298
ETRmax suggested that stress resulting from exposure to P1NA and P2NA had inhibitory
299
effects on the donor side of PSII in M. aeruginosa. Blockage of electron transfer chain
300
accompanied by a decrease in Fv/Fm (Han et al., 2008; Liu et al., 2015). In M.
301
aeruginosa, the initial effect concentrations showing a significant difference between
302
P1NA and P2NA for ETRmax (5 µM) was lower than that for Fv/Fm (10 µM) for 48 h.
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The disparity between the responses of Fv/Fm and ETRmax reflects inhibition
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downstream of PS II, suggesting that the down-regulation of PS II electron transport is a
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result of inhibition of Calvin cycle enzymes and Rubisco (Han et al., 2008; Yu et al.,
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2002). But in general, though P2NA can slightly weaken the photosynthesis of M.
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aeruginosa, it is no doubt that the toxicity is different between P1NA and P2NA in M.
308
aeruginosa.
Fv/Fm--maximal quantum yield (A) 48h
∗
0.6 0.5
A a
A a
A
∗∗∗
∗∗
b
0.5
B
0.4
0.2
0.2
0.1 5
10
15
(B)
B a
∗∗∗
∗∗
a
a
C
0.1
C 0
A a
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0.0
AB a
0.4
0.3
P2NA
72h
0.6
bc
c
P1NA
0.7
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0.0
20
0
5
D 10
15
20
2
140 120 100
ETRmax--maximal electron transport ratio(m s) 140
A a
∗∗ 48h
∗ A
A b
b
60 40
B
b
∗∗∗
5
10
∗∗
AB
b
∗
72h
A a
A b
∗
A c
AC C
0.25 0.20
15
b
b
48h (E)
∗∗
∗∗∗ bc
c
20 0
D 0
5
10
309 310
5
15 P1NA
P2NA
72h (F) 0.25
A a
A b
A b
∗
B
0.20
20
∗∗∗ b
b
0.15 0.10
0.05
0
c
C b
40
0.30
0.10
0.00
∗∗∗
60
20
B
0.15
(D)
B
α--light use efficiency ratio 0.30
P2NA
80
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0
A a
100
C
20 0
∗
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80
(C)
P1NA
0.05
C
C 10
15
20
0.00
0
5
10
15
20
P1NA/P2NA Concentration (µM) Fig 4. Effects of P1NA and P2NA on Fv/Fm (A, B), ETRmax (C, D) and α (E, F) in M. aeruginosa. (F). *
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(P<0.05), ** (P<0.01), and *** (P<0.001) indicate significant differences at the same concentration of
312
P1NA and P2NA. Different small letters in each figure in the same type of Fv/Fm, ETRmax or α represent
313
statistically significant differences (p<0.05) compared to the corresponding controls without P1NA or
314
P2NA.
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3.5 Effect on cell membrane integrity
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The percentage of PI-stained cells of M. aeruginosa exposure to P1NA and P2NA
317
are presented in Fig 5. After 48 h, percentage of cells stained by PI exposed to 5, 10, 15,
318
20 µM, of P1NA were 7.0%, 7.6%, 21.3%, 61.4%, respectively. However, that of P2NA
319
were 3.2%, 3.2%, 3.4%, 3.3%, respectively. It showed that as P1NA concentration
320
increased, the PI positive cell percentage was increased in a dose-response relationship
321
and it had a significant difference in every P1NA concentration being tested compared to
322
the control (p < 0.05). However, there was no change in the integrity of the cell
323
membrane in M. aeruginosa when exposure to P2NA (P > 0.05). And it all showed a
324
significant difference in same exposure concentration between P1NA and P2NA (p <
325
0.05).
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Cells stained by PI is a recognized method being used to probe cell membrane
327
integrity for the assessment of the environmental impact of toxic contaminants in aquatic
328
systems. Herbicide paraquat and allelochemical tannic acid have been reported to lead
329
damages to the membrane integrity of phytoplankton cells indicating a reduction in the
330
percentage of viable cells exposed to that agent (Eigemann et al., 2013; Prado et al.,
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2009). Studies reported that aquatic plants such as E. crassipes (Sultankhodzhaev and
332
Tadzhibaev, 1976; Sun et al., 1993), Elodea nuttallii, Hydrilla verticillata and Vallisneria
333
spiralis (Gao et al., 2010) could secrete P1NA and P2NA, which showed high allelopathic
334
activity. Xiao et al. (2010) indicated that assay of cell membrane integrity was a novel
335
and fast method to determine whether the antialgal agents were algicidal (killing algae) or
33）
algistatic (preventing algal growth). This paper suggested that P1NA has the nature of
337
killing cyanobacteria together with growth assays and cell membrane integrity tests. The
338
studies indicated that P1NA is the allelochemical which shows higher inhibitory activities
339
to cyanobacteria than that of P2NA.
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P1NA
80
∗∗∗
48h
D
40
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60
∗∗∗
∗∗∗
20
A a a 0
B a 5
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0
∗∗∗
B
10
a
∗∗∗
40
C
C
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PI Positive (%)
60
∗∗∗
72h
D
340
P2NA
80
a
a
15
20
∗ A a
B a
0
5
∗∗∗ B
a
a
a
0 20
10
15
20
P1NA/P2NA Concentration (µM)
341
Fig 5. Effect of P1NA and P2NA on cell membrane integrity in M. aeruginosa. * (P<0.05), ** (P<0.01), and
342
*** (P<0.001) indicate significant differences between P1NA and P2NA. Different small letters in each
343
figure in the same type of growth inhibition represent statistically significant differences (p<0.05)
344
compared to the corresponding controls. Data are presented in mean ± SD.
345
A comparison of the sensitivity tested by different endpoints including growth
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inhibition, esterase activity, cell membrane integrity and the efficiency of photosynthesis
347
was performed. And the EC50 values of P1NA were given in Table S1. The EC50 values
348
based on growth inhibition, esterase activity, cell membrane integrity and Fv/Fm were
349
16.616, 17.445, 16.099, 19.604 µM, respectively, which were not far from each other.
350
These results suggested that the parameters being tested were effective endpoints in acute
351
toxicity test. However, EC50 values of P2NA was not detected in same conditions in this
352
study. All results shown above indicated that the acute toxicity test of P1NA and P2NA in
353
M. aeruginosa exhibited significant difference.
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3.6 Toxic mechanism of P1NA
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Oxidative damage induced by ROS has been proved to be the reason resulting in
35）
the toxic effect of chemical toxicants such as anthraquinone derivative (Schrader et al.,
357
2005) and pyrogallic acid (Lu et al., 2016) to phytoplankton. To reveal the possible
358
mechanism of the different toxicity between P1NA and P2NA, the intracellular ROS level
359
was measured. From Fig 6, the remarkable increase in intracellular ROS levels with the
3）0
increase of P1NA concentration was observed after 48h exposure (p < 0.05). The ROS
3）1
level in the group of 5, 10, 15, 20 µM P1NA were 1.31, 1.75, 2.46, 2.83 times higher than
3）2
that of control, respectively. However, a significant dose-response relationship was not
3）3
found in the group exposure to P2NA, in which slight lower level than the control was
3）4
observed.
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P1NA
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P2NA
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1.0
0.5
0.0 0
5
10
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ROS relative intisity
2.5
15
20
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3）5 3））
Fig 6. Intracellular ROS level in M. aeruginosa exposure to P1NA and P2NA for 48 h. Relative ROS level
3）7
was reflected by the mean fluorescence intensity of DCF. Data are presented in mean ± SD.
ROS including O2·-, H2O2 and ·OH are produced by the normal metabolic process
3）9
and maintain a dynamic balance with the antioxidant system in organisms. The
370
overproduction of ROS in phytoplankton was in response to toxicants with redox-cycling
371
property and adverse environmental stresses (Wang et al., 2012; Yang et al., 2011). It was
372
reported that the acute increase of ROS induced by gramine could be a potential cause of
373
the growth inhibition of cyanobacteria (Hong et al., 2009). In addition, it has been also
374
demonstrated that the overproduction of ROS affected cell growth or proliferation. For
375
example, Wang et al. (2011) reported that allelochemicals (+)-catechin and pyrogallic
37）
acid can induce the generation of intracellular ROS to inhibit the growth of two
377
freshwater algae.
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As is known that high level of ROS can directly damage proteins, nucleic acids, and
379
cell membrane (Yu et al., 2007), finally leading to growth inhibition and cell death. In
380
order to evaluate the effects of ROS level on the growth inhibition, the decrease of
381
esterase activity, Fv/Fm, and cell membrane integrity, the relevance between ROS and
382
these four parameters was analyzed. The regression curve and correlation coefficients (R2)
383
were given in Table S2. The R2 between ROS and these four parameters mentioned above
384
were 0.892, 0.781, 0.694, 0.694, respectively. It suggested that increased ROS level was
385
the major mechanism contributing to the growth inhibition of M. aeruginosa exposure to
38）
P1NA under the experimental conditions. In addition, the regression analysis was done for
387
the relationships between the growth inhibition effect and ROS, esterase activity, Fv/Fm
388
and cell membrane integrity.
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To furtherly verify the role of ROS in the growth inhibition of M. aeruginosa
390
exposed to P1NA, the protective effect of ascorbic acid, a kind of free radical scavenger,
391
was investigated. As illustrated in Fig S1, growth inhibition induced by 15 µM P1NA in
392
M. aeruginosa was significantly alleviated after the treatments of ascorbic acid. Ascorbic
393
acid was the widely and water-soluble antioxidant for the prevention or minimization of
394
damages caused by ROS in plants (Khan and Ashraf, 2008). Lu et al. (2016)
395
demonstrated that ascorbic acid could decrease the DNA strand breaks of M. aeruginosa
39）
exposed to pyrogallic acid. The result of ROS scavenger experiments demonstrated that
397
the excessive formation of ROS induced by P1NA was the main cause leading to growth
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inhibition in M. aeruginosa. In vitro studies have demonstrated that P1NA was primarily metabolized via
400
hydroxylation (Koennecker et al., 1998) to form Mono- and dihydroxy-metabolites
401
(Sikka et al., 1981; Wolff, 1992). However, only the monohydroxy-metabolites
402
6-hydroxy-N-phenyl-2-naphthylamine and 4′- hydroxy-N-phenyl-2-naphthylamine were
403
detected after exposure with P2NA (Deutsche Forschungsgemeinschaft, 2015; Weiss et al.,
404
2007). Dihydroxy-metabolites can form quinone while monohydroxy-metabolites not. As
405
summarized previously (O’brien, 1991), oxidative stress arises when the quinone is
40）
reduced by reductases to a semiquinone radical which reduces oxygen to superoxide
407
radicals and reforms the quinone. Wang et al. (2011) reported that polyphenolic
408
allelochemicals such as (+)-catechin and pyrogallic acid could elevate cellular ROS level
409
through futile redox cycles, which amplify the generation of O2.- with the expense of
410
NAD(P)H and greatly enhance the toxicity to the target organisms. The results suggested
411
that the generation of ROS was related to the biotransformation metabolites of P1NA.
412
Therefore, the metabolites of P1NA and P2NA in M. aeruginosa were detected by GC/MS.
413
1,4-naphthoquinone was found in M. aeruginosa exposure to P1NA, while it was not
414
detected in P2NA and control groups (Fig S2). We speculated P1NA metabolized to form
415
1,4-dihydroxynaphthalene firstly. It was similar to (+)-catechin and pyrogallic acid which
41）
may cause autoxidation or enzymatic oxidation to easily form semiquinone radical in the
417
target organism. The reactive semiquinone radical is able to react with O2 to produce
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o-quinone form of 1,4-dihydroxynaphthalene accompanying the production of O2.-.
419
Dismutation of O2.- produces H2O2, and then H2O2 can be reduced via the Fenton-type
420
reaction in the presence of transition metals to form the ·OH. Therefore, it is reasonable
421
to expect that the metabolites of P1NA may undergo futile redox cycling in the presence
422
of intracellular reductants such as NAD(P)H (O’brien, 1991) and induce ROS production
423
in M. aeruginosa. A possible pathway of ROS generation in M. aeruginosa induced by
424
P1NA could be proposed as shown in Fig 7. In brief, it was the first time to demonstrate
425
that P1NA exhibited remarkably stronger growth inhibition effects in M. aeruginosa than
42）
its isomer P2NA. The different property in inducing excessive ROS between P1NA and
427
P2NA was the important reason to exhibit different toxicity in M. aeruginosa. The
428
formation of 1,4-naphthoquinone was the key factor to generate excessive ROS.
429
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Fig 7. A possible pathway of ROS production induced by P1NA in M. aeruginosa.
431
4 Conclusion
432
The results of comparing the toxicity of P1NA and P2NA indicated that only P1NA
433
was highly toxic to M. aeruginosa. The 48 h EC50 values based on growth inhibition were
24
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16.616 µM, while that of P2NA was not detected in same conditions. The intracellular
435
ROS levels were significantly increased in M. aeruginosa exposure to P1NA, while it was
43）
not found in the group of P2NA. Attention should be paid to the influence of P1NA on
437
other aquatic organisms and aquatic ecosystem. In the research field of allelopathy, the
438
toxic effect of P1NA on phytoplankton should be investigated as the main object
439
compared with P2NA.
440
Acknowledgements
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The study was financially supported by the National Nature Science Foundation of
442
China (grant number 51178452, 50909091 and 31123001), the Major Science and
443
Technology Program for Water Pollution Control and Treatment of China 12th Five-Year
444
Plan (grant number 2012ZX07101007-005), and the State Key Laboratory of Freshwater
445
Ecology and Biotechnology Program (grant number 2013FB20). The authors thank
44）
Liping Zhang and Yan Wang at Institute of Hydrobiology, Chinese Academy of Sciences
447
for assistance in using GC-MS and flow cytometer.
448
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ACCEPTED MANUSCRIPT Highlights: ● P1NA and P2NA with similar molecular structure, only P1NA was found high toxic to M. aeruginosa.
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● ROS induced by P1NA could the reason of the growth inhibition of M. aeruginosa.
● The formation of 1,4-naphthoquinone was the key factor to generate excessive
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S0045-6535(17)30298-9
DOI:
10.1016/j.chemosphere.2017.02.110
Reference:
CHEM 18873
To appear in:
ECSN
Received Date: 10 October 2016 Revised Date:
4 February 2017
Accepted Date: 21 February 2017
Please cite this article as: Cheng, L., He, Y., Tian, Y., Liu, B., Zhang, Y., Zhou, Q., Wu, Z., Comparative biotoxicity of N-Phenyl-1-naphthylamine and N-Phenyl-2-naphthylamine on cyanobacteria Microcystis aeruginosa, Chemosphere (2017), doi: 10.1016/j.chemosphere.2017.02.110. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Comparative biotoxicity of N-Phenyl-1-naphthylamine and
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N-Phenyl-2-naphthylamine on cyanobacteria Microcystis aeruginosa
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Long Cheng a,b, Yan He a,b, Yun Tian a,b, Biyun Liu a,*, Yongyuan Zhang a, Qiaohong
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Zhou a,*, Zhenbin Wua
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a
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Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China;
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b
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*Correspondence: [email protected] (B. Liu)
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State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of
University of Chinese Academy of Sciences, Beijing 100049, China
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[email protected] (Q. Zhou)
Abstract: N-Phenyl-1-naphthylamine (P1NA) and N-Phenyl-2-naphthylamine (P2NA)
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are both widely used as antioxidant and plant secondary metabolites. In this study, growth,
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esterase, photosynthetic activity and cell membrane integrity were used as biomarkers to
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compare biotoxicity of P1NA and P2NA on Microcystis aeruginosa. According to the
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results, a dose-response relationship was observed only between P1NA concentrations and
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growth inhibition. The EC50 (48 h) of P1NA calculated from growth inhibition was 16.62
1）
µM, while that of P2NA was not detected. When the esterase and photosynthetic activity
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were applied to evaluate the biotoxicity, it was found that a concentration of 20 µM P1NA,
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P2NA caused reduction of esterase activity and Fv/Fm of M. aeruginosa to 22.2 and 3.3%,
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97.5 and 92.1%, respectively, after 48 h exposure. The percentage of membrane-damaged
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cells was increased as P1NA exposure concentration increased, but that was not detected
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when exposure to P2NA. The difference substituted position in the molecular structure of
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P1NA and P2NA leads to different toxicological properties and only P1NA was found
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highly toxic to M. aeruginosa. The toxicity is due to that only P1NA can be
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biotransformed to 1,4-naphthoquinone, which could induce overproduction of
25
intracellular ROS as well as result in oxidative damage and growth inhibition of test
2）
organism.
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Keywords:
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aeruginosa; Toxic effects; Reactive oxygen species
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1. Introduction
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N-Phenyl-2-naphthylamine;
Microcystis
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N-Phenyl-1-naphthylamine (P1NA) serves as an antioxidant in various lubrication
31
oils and also as a protective agent in rubbers as well as rubber mixtures. (Koennecker et
32
al., 1998). It was estimated that the annual production of P1NA was 5800-6300 tons from
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1986 to 1990 in the world (Koennecker et al., 1998). The distribution of P1NA was
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predicted approximately 29% to water, 34% to sediment, 36% to soil, and less than 1%
35
each to air, suspended sediment, and biota in the environment using a Level II fugacity
3）
model (Mackay and Models, 2001; Koennecker et al., 1998). N-Phenyl-2-naphthylamine
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(P2NA), the isomer of P1NA, also used as an additive jet oils and it is a byproduct in the
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manufacture of P1NA (Winder and Balouet, 2002). It was reported that P1NA was
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detected in river freshwater (2-7 µg L-1) and sediment (1-5 mg kg-1) near a small specialty
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chemicals manufacturing ( Koennecker et al., 1998). And P2NA has been detected in
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sediments and freshwater in the proximity of chemical production sites in mg kg-1 and µg
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L-1 concentrations (Brack et al., 1999; Altenburger et al., 2006). Studies performed according to standard protocols yielded LD50 of P1NA in male
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and female Wistar rats of >5000 mg kg-1 body weight (Koennecker et al., 1998). Acute
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oral toxicity test of P2NA indicated that its LD50 in rats and in mice were 8730 mg kg-1
4）
and 1450 mg kg-1 body weight (NTP, 1988). Due to P1NA and P2NA’s lipophilicity with
47
octanol-water partition coefficient, log KOW of 4.2 and 4.38, respectively, they may be
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expected to have a considerable bio-accumulative potential in aquatic environments and
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be toxic to aquatic organisms. The studies from laboratory tests with ciliates, Daphnia,
50
and fish indicated that P1NA was highly toxic to aquatic species. EC50 (48 h) for the
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inhibition of cell proliferation of freshwater ciliates (Tetrahymena pyriformis), LC50 (48 h)
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with young Daphnia magna and LC50 (96 h) in a flow-through system for rainbow trout
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(Oncorhynchus mykiss) are, respectively, 2 mg L-1 (Epstein et al., 1967), 0.30 mg L-1, and
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0.44-0.74 mg L-1 (Sikka et al., 1981). Phytoplankton is the fundamental part in the
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aquatic ecosystems. However, there is little research about the toxicity of P1NA on
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phytoplankton.
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Submerged plants can secrete secondary metabolites to inhibit photosynthetic
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plankton in order to compete for light and nutrition dominance in the water environment.
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This phenomenon is called allelopathy and the active substances are known as
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allelochemicals (Rice, 1984). In recent years, significant progress in the allelopathic
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effect of submerged macrophyte on cyanobacteria has been made in both laboratory and
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field study. In respect of the mechanism of allelopathy, it was reported that aquatic plants
）3
release allelochemicals continually with low concentration, and caused persistent stress
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on target organisms. Under this condition, the intracellular signaling radicals (ROS, NO)
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were induced, as well as, the programmed cell death (PCD) was trigger in M. aeruginosa
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which was confirmed by the hallmarks of PCD, caspase-3-like protease activity, DNA
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fragmentation, and destruction of cell ultrastructure (He et al., 2016). The engineering
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application of allelopathy through reconstructing the aquatic vegetation in the restoration
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of the subtropical eutrophic lake was studied. Ma et al. (2009) reported that
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transformation of water in Yuehu Lake (66 ha), a shallow eutrophic lake in China, from
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turbid algae green to a transparent state, was achieved by restoring the submerged
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macrophyte community while the nitrogen and phosphorus are at the level of
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eutrophication. Thus, it attracted the great interest of researchers to study the mechanism
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of allelopathy. P1NA and P2NA as a kind of allelochemicals have been isolated and
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identified in axenic as well as noaxenic root exudates of Eichhornia crassipes
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(Sultankhodzhaev and Tadzhibaev, 1976; Sun et al., 1993). These two substances have
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also been detected in the culture medium of three submerged macrophytes (Elodea
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nuttallii, Hydrilla verticillata and Vallisneria spiralis) by GC-MS (Gao et al., 2010).
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Qian et al. (2009, 2010) studied the toxicity of allelochemical P2NA to Chlorella vulgaris
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and M. aeruginosa. The only difference in molecular structure between P1NA and P2NA
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is that anilino-N substitutes at 1 and 2 positions of the naphthyl group. No information is
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available about whether the toxicity of P1NA and P2NA on cyanobacteria is different and
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how the toxicity is produced.
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The present study, M. aeruginosa, a dominant species of cyanobacterial blooming,
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was chosen as a target species. The physiological and photosynthetic parameters i.e.
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growth inhibition, esterase activity, cell membrane integrity, and photosynthetic activity
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were used as biomarkers to compare the toxicity between P1NA and P2NA. Moreover,
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reactive oxygen specious (ROS) level was used to evaluate the toxicity mechanism. The
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aim of this study was to (1) provide ecotoxicological information for evaluating the
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toxicity of P1NA and P2NA to the cyanobacteria (M. aeruginosa) in future ecological risk
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assessments and regulation of P1NA and P2NA use and discharges in aquatic ecosystems,
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(2) elucidate toxicity and inhibition mechanism of P1NA and P2NA on cyanobacteria to
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provide the base for further study the mechanism of allelopathic mechanism.
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2. Materials and methods
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2.1 Cultures conditions
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Cyanobacteria, Microcystis aeruginosa FACHB 905 was provided by the Institute
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of Hydrobiology of the Chinese Academy of Sciences. M. aeruginosa was cultured in
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autoclaved BG11 media (Rippka et al., 1979) at 25 ±0.5
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intensity of 18 µmol m-2 s-1 with a 12:12 h light: dark cycle. The cultures were shaken
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three times per day by hands.
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in an incubator under a light
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2.2 Chemicals N-phenyl-1-naphthylamine (98.0%) and N-phenyl-2-naphthylamine (97.0%) were
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commercially
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2´,7´-dichlorodihydrofluorescein diacetate (DCFH-DA), fluorescein diacetate (FDA) and
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dimethyl sulfoxide (DMSO, 99%) were all obtained from Sigma (St. Louis, Missouri,
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USA). Propidium Iodide (PI) was purchased from Sangon Co. Ltd (shanghai, China).
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2.3 Toxicity assays
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2.3.1 Experimental design
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same
company
(TCI,
Tokyo,
Japan).
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Conical flasks (250 mL) and culture medium were prepared and autoclaved. 100 mL
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culture media was added to each bottle. P1NA and P2NA were added with the
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concentration of 0, 5, 10, 15, 20 µM, respectively. Then M. aeruginosa was inoculated
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into each flask with the initial density of 1 × 106 cells mL−1 when it was at the
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exponential growth phase. The samples were taken after 48 and 72 h for the measurement
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of growth and other physiological parameters. The stock solutions of P1NA and P2NA
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were prepared with DMSO which in test solution was less than 0.1% (v/v). 0.1% DMSO
11）
had no effects on the growth of M. aeruginosa (FACHB-905) (Gao et al., 2011). The
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experimental conditions are the same to the cyanobacteria culture conditions shown
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above. All the experiments were performed in triplicate.
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2.3.2 Measurement of growth inhibition
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The cells density was measured by spectrophotometry at 680 nm. The regression
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equation between cell density (y×103 cells mL-1) and OD680 (x) was calculated as follow:
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y = 19735x – 33 (R2 = 0.9999). The inhibition ratio (IR) of cyanobacterial growth was
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calculated according to the following Eq. (1):
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IR = ( − )⁄ × 100%
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(1)
where N0 and Ns (cells mL-1) represent cell density in the control and treatment groups,
12）
respectively.
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2.3.3 Measurement of esterase activity
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The fluorescein diacetate (FDA) assay has been used to indicate the metabolic
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activity of cells (Dunker et al., 2013; Xiao et al., 2010, 2011). 5 µL of 1 g L-1 FDA was
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added into 500 µL cells samples in a final concentration of 10 mg L-1 incubated for 15
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min at room temperature in darkness. Then cells were analyzed by flow cytometer FACS
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Verse (BD Biosciences, Franklin Lakes, New Jersey, USA) with excitation and emission
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wavelength of 488 nm and 527/32 nm. As depicted by Hadjoudja et al. (2009), the
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changes of esterase activity exposure to P1NA and P2NA were showed as the decrease in
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the proportion of cells in the S2 regions (normal activity status) compared with control
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cells. Every sample was collected 10000 events.
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2.3.4 Detection of cell membrane integrity
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Cell membrane integrity was assessed by fluorescent dye PI which could interact
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with nucleic acids to produce red fluorescence when the cell membrane was damaged
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(Xiao et al., 2011). For the measurement of M. aeruginosa cell membrane integrity, 5 µL
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of PI working solution was added to 500 µL of the sample with the final concentration of
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15 µM and the sample was stained for 15 min in the dark at room temperature.
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Fluorescence was collected through 586/42 nm with 488 nm laser. Data were expressed
144
as stained cell percentages.
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2.3.5 Detection of intracellular ROS level
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The ROS level in M. aeruginosa was measured using the cell permeable indicator
147
2´,7´-dichlorodihydrofluorescein diacetate (DCFH-DA) according to He et al. (2016).
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The samples of cells were added with a final concentration of 10 µM DCFH-DA and
149
pre-incubation at 25°C in dark for 1 h. Sample fluorescence was measured using flow
150
cytometer with excitation and emission wavelength of 488 nm and 527/32 nm. To prove
151
the role of ROS in the P1NA induced growth inhibition, the BG11 culture mediums were
152
added with ROS scavenger ascorbic acid (0.5, 1 and 2 mM) and then adjusted to pH 7.1
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with 1 M NaOH. The cells were pre-treated for 1 h and then exposed to 15 µM P1NA for
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24 h. Growth inhibition in test organism was detected.
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2.3.6 Photosynthesis activity analysis
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Maximum quantum yield (Fv/Fm), maximal electron transport rate (ETRmax) and
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light use efficiency ratio (α) were selected to represent the M. aeruginosa photosynthesis
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status. These values were measured using phyto-PAM analyzer (Walz, Effeltrich,
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Germany) (Maxwell and Johnson, 2000).
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2.3.7 Determination of P1NA and P2NA concentrations and metabolites
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For analyzing the concentration of P1NA and P2NA, 2 mL culture samples were
1）2
extracted by equivalent ethyl acetate and then the extracts were dehydrated by anhydrous
1）3
sodium sulfate. The extracts being diluted 20 times were then transferred to auto-sampler
1）4
vials for gas chromatography–mass spectrometry (GC–MS) (Agilent 6890N/5973Inert,
1）5
Santa Clara, California, USA). The GC temperature profile consisted of an initial holding
1））
time of 2 min at 40
1）7
reaching a final temperature of 320
1）8
P2NA were 13.5 min and 13.9 min, respectively. The standard curve was prepared and the
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equation for relation between peak area (y) and concentration (x)(µM) (0-1 µM) was as
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follows: y = 70781x – 2533.9 (R2 = 0.991) and y = 268031x – 5141.2 (R2 = 0.997) for
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P1NA and P2NA, respectively. The metabolites were qualitatively analyzed in M.
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aeruginosa exposed to 20 µM of P1NA, P2NA and control group respectively. The
173
metabolites were determined by GC–MS, briefly as follows: 25 mL culture samples
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(exposure for 72 h) were adjusted to pH 2 with 1 M HCl and extracted three times with
175
an equivalent volume of ethyl acetate. The following sample handling and determination
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condition were the same to the Lu et al. (2016).
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2.4 Statistical analysis
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followed by a gradual increase in temperature of 20
min−1,
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Flow cytometric analysis data were analyzed using FlowJo software (Tree Star
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Software, San Carlos, California, USA). Data are presented in mean ± SD. One-way
180
ANOVA followed by LSD (when homogeneity of variance) or Tamhane's T2 (when
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heterogeneity of variance) post hoc test was used to test for the differences between
182
individual means among P1NA or P2NA treatment at different concentrations.
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Independent samples t-tests were used to compare the differences individual means when
184
at the same P1NA and P2NA concentration. Differences were considered to be significant
185
at p < 0.05. EC50 values were determined with probit regression. All the statistical
18）
analyses were performed using SPSS 18.0 software (IBM Corporation, Armonk, New
187
York, USA).
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3 Results and discussion
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3.1 Real concentrations of P1NA and P2NA concentrations during experiment
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In order to determine the real concentrations and stability of P1NA and P2NA, the
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concentrations in culture medium were quantitatively analyzed at 0, 24, 48 and 72 h. The
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results are shown in Fig 1. For nominal concentrations of 5, 10, 15, 20 µM, the real
193
exposure concentrations of P1NA were 4.75±0.018, 11.64±1.86, 14.89±0.27, 20.15±0.86
194
µM, respectively. The real exposure concentration of P2NA were 4.17±0.11, 10.62±0.48,
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12.73±1.46, 19.90±0.17 µM, respectively. It can be found that the actually determining
19）
concentrations are close to the nominal concentrations. The average percentage error of
197
P1NA and P2NA was 5.7% and 9.6% between measured concentrations and nominal
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values. The concentration of P1NA and P2NA decreased gradually over time in biotic and
199
abiotic conditions. And the decomposition rate of P1NA is faster than that of P2NA. For
200
instance, 20 µM of P1NA and P2NA in the culture medium were reduced by 59.2% and
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35.8% within 72 h. The half-life of P1NA was 24-50 h, while that of P2NA was 66-120 h
202
which was out of our experimental period calculated from the degradation patterns at 5,
203
10, 15, and 20 µM.
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We speculated that P1NA and P2NA concentrations decreased possibly due to the
205
reason that these substances can be adsorbed and metabolized by cyanobacteria or
20）
photochemically decomposed in the culture medium. Sikka et al. (1981) demonstrated
207
that P1NA was degraded with a half-life ranging from 4 to 11 days in water inoculated
208
with domestic sewage and lake water, respectively. A further experiment conducted in
209
aqueous solution, indicated that P1NA could be directly photochemically degraded (Sikka
210
et al. 1981). Meanwhile, on the basis of in vitro studies, metabolism of P1NA and P2NA
211
likely occurs primarily via hydroxylation (Deutsche Forschungsgemeinschaft, 2015;
212
Koennecker et al., 1998; Weiss et al., 2007). 5 µM
10
15 µM
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15
10 µM
P1NA
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20 µM
P2NA 15
10
5
5
0
0
20 µM in abiotic conditions
20
0 1
2
3
0
1
2
3
Time(day)
213 214
Fig 1. Real concentrations of P1NA and P2NA (µM) in the culture medium, during the experimental period.
215
Data are presented in mean ± SD.
11
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21）
3.2 Effects of P1NA and P2NA on M. aeruginosa growth Cell growth inhibition is commonly used as a valid biomarker to evaluate the
218
phytotoxicity on cyanobacteria as it shows. Because it is an integrating parameter
219
showing toxicants inhibitory effects on all cellular metabolism (Dewez et al., 2008). The
220
inhibition effects of P1NA and P2NA on M. aeruginosa growth were studied after 48 and
221
72 h (Fig 2). It can be seen that when exposure to P1NA, the growth inhibition of M.
222
aeruginosa increased with an increasing concentration of P1NA. After 48 h, the inhibition
223
rates in the group of 5, 10, 15, 20 µM of P1NA were 4.0%, 6.9%, 40.6%, 69.4%,
224
respectively. However, those of P2NA were 0.3%, 3.1%, 0.6%, 0.3%, respectively. The
225
inhibition rates were 5.0%, 14.1%, 46.1%, 74.1%, respectively, exposure by P1NA after
22）
72 h. A significant dose-response and time-dependent relationship were displayed in
227
P1NA group, while these relationships for P2NA were not observed in the experiment.
228
Growth inhibition rates of P1NA exposed to 10, 15, 20 µM were higher than that of P2NA
229
at 48h, and the differences were 3.8%, 40.0%, 69.0%, respectively. It showed significant
230
differences (p< 0.05) with the exposure time for 48 h in the group of 10, 15 and 20 µM,
231
and with the exposure time for 72 h in the group of 15 and 20 µM, respectively.
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The EC50 (48 h) value (Table S1) of P1NA calculated from growth inhibition was
233
16.616 µM (3.64 mg/L), while that of P2NA was not detected. The results showed that
234
only P1NA was highly toxic to M. aeruginosa. Koennecker et al., (1998) reported that
235
P1NA was low toxic to the animal but highly toxic to aquatic organisms. An EC50 (48h) of
12
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2 mg L-1 of P1NA was measured for the inhibition of cell proliferation of freshwater
237
ciliates Tetrahymena pyriformis (Epstein et al., 1967). LC50 (48h) for young and adult
238
Daphnia magna were in the range of 0.30-0.68 mg L-1 (Koennecker et al., 1998). The
239
toxicity of P1NA to M. aeruginosa was similar to that of protozoa, which was lower than
240
that of cladoceran zooplankton. However, several studies have reported that P2NA is a
241
highly toxic substance to C. vulgaris (Qian et al., 2009), M. aeruginosa (Qian et al., 2010)
242
and Cylindrospermopsis raciborskii (Liu et al., 2015). The toxicity of P2NA may be
243
affected by the strain of the test organism or the culture conditions. The difference needs
244
to be furtherly studied in the future.
48h
***
**
60
C
C
40
0
5
10
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A a
A a
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0
*
B a
a
15
a
*** D
80
D
60
P2NA
72h
***
80
20
245
P1NA
100
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Growth Inhibition (%)
100
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23）
40
20
0
20
B c A a
b
A
0
5
10
c
15
c
20
P1NA/P2NA Concentration(µM)
24）
Fig 2. Effect of P1NA and P2NA on growth inhibition in M. aeruginosa. * (P<0.05), ** (P<0.01), and ***
247
(P<0.001) indicate significant differences between P1NA and P2NA. Different small letters in each figure in
248
the same type of growth inhibition represent statistically significant differences (p<0.05) compared to the
249
corresponding controls. Data are presented in mean ± SD.
250
3.3 Impact on esterase activity
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The esterase activity in M. aeruginosa exposed to P1NA and P2NA for 48 and 72 h
252
were shown in Fig 3. It could be found that the esterase activity significantly decreased in
253
the group of 5 µM and 10 µM exposure to P1NA at 48 h. And then the esterase activity
254
drastically reduced with the increase of P1NA concentration, it declined to 70.2% and
255
22.2% of the proportion of cells in the S2 regions (normal activity status) (Hadjoudja et al.
25）
2009) with 15 µM and 20 µM of concentrations exposure to P1NA. The similar pattern
257
could be found in the group exposure for 72 h, while it did not further lower the esterase
258
activity. As opposed to P1NA, the esterase activity was invariant as P2NA concentrations
259
increased at 48 and 72 h.
M AN U
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80
80
48 h
60
40
0
2）1 2）2
5
AC C
0
EP
20
2）0
P2NA
100
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% S2 FDA Fluorescence
100
P1NA
10
72 h
60
40
20
0 15
20
0
5
10
15
20
P1NA/P2NA Concentration (µM)
Fig 3. Effect of P1NA and P2NA on esterase activity in M. aeruginosa. Data are presented in mean ± SD.
Esterase activity was commonly used to evaluate the metabolic status of the cell
2）3
because it was a rapid and sensitive endpoint (Valiente Moro et al., 2012), and usually, a
2）4
decline in enzyme activity indicated the presence of stress (Hadjoudja et al., 2009; Regel
2）5
et al., 2002). Herbicide paraquat and copper could result in reducing esterase activity due
14
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to oxidative stress and showed a dose-response relationship in acute toxicity test for
2）7
phytoplankton (Hadjoudja et al., 2009; Prado et al., 2012). It indicated that the decline in
2）8
esterase activity exposure to P1NA being similar to paraquat and copper. And it gave an
2）9
information not only to be helpful to promptly determine the physiological state of cells
270
but also to evaluate the toxicity of pollutants. The change of esterase activity along with
271
growth inhibition suggested that the toxicity of P1NA and P2NA to M. aeruginosa had an
272
obvious difference.
273
3.4 Inhibition of photosynthetic activity
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2））
In the present study, maximum quantum yield (Fv/Fm), maximal electron transport
275
rate (ETRmax) and light use efficiency ratio (α) were used to estimate the efficiency of
27）
PSII in M. aeruginosa. PSII efficiency in M. aeruginosa exposure to P1NA and P2NA are
277
shown in Fig 4. The results showed that Fv/Fm has significantly decreased in the group
278
of 15 and 20 µM P1NA exposure for 48 and 72 h (p < 0.05) (Fig 3A, 3B). It was different
279
when exposure to P2NA. At 48 h, Fv/Fm was significantly decreased in the group of 10,
280
15 and 20 µM P2NA (p<0.05). But every concentration of the P2NA treatment group did
281
not have any differences at 72 h (p> 0.05) which was consistent with the trend of growth
282
inhibition. There was a significant difference between P1NA and P2NA in the group of 15
283
and 20 µM at 48 and 72 h. The effects on ETRmax and α are shown in Fig 4C, 4D and
284
Fig 4E, 4F, respectively. P1NA treatment led to a significant decrease in ETRmax or α
285
values at high concentration, while exposure of P2NA slightly reduced them. It was much
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28）
similar to the change of Fv/Fm values. The P1NA caused a severe toxicity on PSII of the cyanobacteria M. aeruginosa, as
288
introduced above. These results were consistent with the growth and esterase activity of
289
M. aeruginosa. It is known that the value of Fv/Fm characterizes the content of
290
photochemically active centers of PSII and indicates the current balance between
291
processes of light destruction and the reparation of PSII (Brack and Frank, 1998).
292
Similarly, some kinds of exogenous substances such as tetracycline and natural
293
flavonoids were found to inhibit growth and reduce Fv/Fm values in M. aeruginosa and
294
Selenastrum capricornutum (Huang et al., 2015; Yang et al., 2013). Allelochemicals
295
identified from Myriophyllum spicatum, including pyrogallic acid, gallic acid, ellagic acid
29）
and (+)-catechin caused significant reductions of PSII efficiency and whole electron
297
transport chain activities in M. aeruginosa (Zhu et al., 2010). The decrease of the
298
ETRmax suggested that stress resulting from exposure to P1NA and P2NA had inhibitory
299
effects on the donor side of PSII in M. aeruginosa. Blockage of electron transfer chain
300
accompanied by a decrease in Fv/Fm (Han et al., 2008; Liu et al., 2015). In M.
301
aeruginosa, the initial effect concentrations showing a significant difference between
302
P1NA and P2NA for ETRmax (5 µM) was lower than that for Fv/Fm (10 µM) for 48 h.
303
The disparity between the responses of Fv/Fm and ETRmax reflects inhibition
304
downstream of PS II, suggesting that the down-regulation of PS II electron transport is a
305
result of inhibition of Calvin cycle enzymes and Rubisco (Han et al., 2008; Yu et al.,
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2002). But in general, though P2NA can slightly weaken the photosynthesis of M.
307
aeruginosa, it is no doubt that the toxicity is different between P1NA and P2NA in M.
308
aeruginosa.
Fv/Fm--maximal quantum yield (A) 48h
∗
0.6 0.5
A a
A a
A
∗∗∗
∗∗
b
0.5
B
0.4
0.2
0.2
0.1 5
10
15
(B)
B a
∗∗∗
∗∗
a
a
C
0.1
C 0
A a
M AN U
0.3
0.0
AB a
0.4
0.3
P2NA
72h
0.6
bc
c
P1NA
0.7
SC
0.7
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30）
0.0
20
0
5
D 10
15
20
2
140 120 100
ETRmax--maximal electron transport ratio(m s) 140
A a
∗∗ 48h
∗ A
A b
b
60 40
B
b
∗∗∗
5
10
∗∗
AB
b
∗
72h
A a
A b
∗
A c
AC C
0.25 0.20
15
b
b
48h (E)
∗∗
∗∗∗ bc
c
20 0
D 0
5
10
309 310
5
15 P1NA
P2NA
72h (F) 0.25
A a
A b
A b
∗
B
0.20
20
∗∗∗ b
b
0.15 0.10
0.05
0
c
C b
40
0.30
0.10
0.00
∗∗∗
60
20
B
0.15
(D)
B
α--light use efficiency ratio 0.30
P2NA
80
EP
0
A a
100
C
20 0
∗
120
TE D
80
(C)
P1NA
0.05
C
C 10
15
20
0.00
0
5
10
15
20
P1NA/P2NA Concentration (µM) Fig 4. Effects of P1NA and P2NA on Fv/Fm (A, B), ETRmax (C, D) and α (E, F) in M. aeruginosa. (F). *
17
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(P<0.05), ** (P<0.01), and *** (P<0.001) indicate significant differences at the same concentration of
312
P1NA and P2NA. Different small letters in each figure in the same type of Fv/Fm, ETRmax or α represent
313
statistically significant differences (p<0.05) compared to the corresponding controls without P1NA or
314
P2NA.
315
3.5 Effect on cell membrane integrity
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311
The percentage of PI-stained cells of M. aeruginosa exposure to P1NA and P2NA
317
are presented in Fig 5. After 48 h, percentage of cells stained by PI exposed to 5, 10, 15,
318
20 µM, of P1NA were 7.0%, 7.6%, 21.3%, 61.4%, respectively. However, that of P2NA
319
were 3.2%, 3.2%, 3.4%, 3.3%, respectively. It showed that as P1NA concentration
320
increased, the PI positive cell percentage was increased in a dose-response relationship
321
and it had a significant difference in every P1NA concentration being tested compared to
322
the control (p < 0.05). However, there was no change in the integrity of the cell
323
membrane in M. aeruginosa when exposure to P2NA (P > 0.05). And it all showed a
324
significant difference in same exposure concentration between P1NA and P2NA (p <
325
0.05).
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SC
31）
Cells stained by PI is a recognized method being used to probe cell membrane
327
integrity for the assessment of the environmental impact of toxic contaminants in aquatic
328
systems. Herbicide paraquat and allelochemical tannic acid have been reported to lead
329
damages to the membrane integrity of phytoplankton cells indicating a reduction in the
330
percentage of viable cells exposed to that agent (Eigemann et al., 2013; Prado et al.,
18
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2009). Studies reported that aquatic plants such as E. crassipes (Sultankhodzhaev and
332
Tadzhibaev, 1976; Sun et al., 1993), Elodea nuttallii, Hydrilla verticillata and Vallisneria
333
spiralis (Gao et al., 2010) could secrete P1NA and P2NA, which showed high allelopathic
334
activity. Xiao et al. (2010) indicated that assay of cell membrane integrity was a novel
335
and fast method to determine whether the antialgal agents were algicidal (killing algae) or
33）
algistatic (preventing algal growth). This paper suggested that P1NA has the nature of
337
killing cyanobacteria together with growth assays and cell membrane integrity tests. The
338
studies indicated that P1NA is the allelochemical which shows higher inhibitory activities
339
to cyanobacteria than that of P2NA.
M AN U
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P1NA
80
∗∗∗
48h
D
40
TE D
60
∗∗∗
∗∗∗
20
A a a 0
B a 5
AC C
0
∗∗∗
B
10
a
∗∗∗
40
C
C
EP
PI Positive (%)
60
∗∗∗
72h
D
340
P2NA
80
a
a
15
20
∗ A a
B a
0
5
∗∗∗ B
a
a
a
0 20
10
15
20
P1NA/P2NA Concentration (µM)
341
Fig 5. Effect of P1NA and P2NA on cell membrane integrity in M. aeruginosa. * (P<0.05), ** (P<0.01), and
342
*** (P<0.001) indicate significant differences between P1NA and P2NA. Different small letters in each
343
figure in the same type of growth inhibition represent statistically significant differences (p<0.05)
344
compared to the corresponding controls. Data are presented in mean ± SD.
345
A comparison of the sensitivity tested by different endpoints including growth
19
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inhibition, esterase activity, cell membrane integrity and the efficiency of photosynthesis
347
was performed. And the EC50 values of P1NA were given in Table S1. The EC50 values
348
based on growth inhibition, esterase activity, cell membrane integrity and Fv/Fm were
349
16.616, 17.445, 16.099, 19.604 µM, respectively, which were not far from each other.
350
These results suggested that the parameters being tested were effective endpoints in acute
351
toxicity test. However, EC50 values of P2NA was not detected in same conditions in this
352
study. All results shown above indicated that the acute toxicity test of P1NA and P2NA in
353
M. aeruginosa exhibited significant difference.
354
3.6 Toxic mechanism of P1NA
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34）
Oxidative damage induced by ROS has been proved to be the reason resulting in
35）
the toxic effect of chemical toxicants such as anthraquinone derivative (Schrader et al.,
357
2005) and pyrogallic acid (Lu et al., 2016) to phytoplankton. To reveal the possible
358
mechanism of the different toxicity between P1NA and P2NA, the intracellular ROS level
359
was measured. From Fig 6, the remarkable increase in intracellular ROS levels with the
3）0
increase of P1NA concentration was observed after 48h exposure (p < 0.05). The ROS
3）1
level in the group of 5, 10, 15, 20 µM P1NA were 1.31, 1.75, 2.46, 2.83 times higher than
3）2
that of control, respectively. However, a significant dose-response relationship was not
3）3
found in the group exposure to P2NA, in which slight lower level than the control was
3）4
observed.
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P1NA
3.0
P2NA
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2.0
1.5
1.0
0.5
0.0 0
5
10
SC
ROS relative intisity
2.5
15
20
M AN U
P1NA/P2NA Concentration (µM)
3）5 3））
Fig 6. Intracellular ROS level in M. aeruginosa exposure to P1NA and P2NA for 48 h. Relative ROS level
3）7
was reflected by the mean fluorescence intensity of DCF. Data are presented in mean ± SD.
ROS including O2·-, H2O2 and ·OH are produced by the normal metabolic process
3）9
and maintain a dynamic balance with the antioxidant system in organisms. The
370
overproduction of ROS in phytoplankton was in response to toxicants with redox-cycling
371
property and adverse environmental stresses (Wang et al., 2012; Yang et al., 2011). It was
372
reported that the acute increase of ROS induced by gramine could be a potential cause of
373
the growth inhibition of cyanobacteria (Hong et al., 2009). In addition, it has been also
374
demonstrated that the overproduction of ROS affected cell growth or proliferation. For
375
example, Wang et al. (2011) reported that allelochemicals (+)-catechin and pyrogallic
37）
acid can induce the generation of intracellular ROS to inhibit the growth of two
377
freshwater algae.
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As is known that high level of ROS can directly damage proteins, nucleic acids, and
379
cell membrane (Yu et al., 2007), finally leading to growth inhibition and cell death. In
380
order to evaluate the effects of ROS level on the growth inhibition, the decrease of
381
esterase activity, Fv/Fm, and cell membrane integrity, the relevance between ROS and
382
these four parameters was analyzed. The regression curve and correlation coefficients (R2)
383
were given in Table S2. The R2 between ROS and these four parameters mentioned above
384
were 0.892, 0.781, 0.694, 0.694, respectively. It suggested that increased ROS level was
385
the major mechanism contributing to the growth inhibition of M. aeruginosa exposure to
38）
P1NA under the experimental conditions. In addition, the regression analysis was done for
387
the relationships between the growth inhibition effect and ROS, esterase activity, Fv/Fm
388
and cell membrane integrity.
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To furtherly verify the role of ROS in the growth inhibition of M. aeruginosa
390
exposed to P1NA, the protective effect of ascorbic acid, a kind of free radical scavenger,
391
was investigated. As illustrated in Fig S1, growth inhibition induced by 15 µM P1NA in
392
M. aeruginosa was significantly alleviated after the treatments of ascorbic acid. Ascorbic
393
acid was the widely and water-soluble antioxidant for the prevention or minimization of
394
damages caused by ROS in plants (Khan and Ashraf, 2008). Lu et al. (2016)
395
demonstrated that ascorbic acid could decrease the DNA strand breaks of M. aeruginosa
39）
exposed to pyrogallic acid. The result of ROS scavenger experiments demonstrated that
397
the excessive formation of ROS induced by P1NA was the main cause leading to growth
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398
inhibition in M. aeruginosa. In vitro studies have demonstrated that P1NA was primarily metabolized via
400
hydroxylation (Koennecker et al., 1998) to form Mono- and dihydroxy-metabolites
401
(Sikka et al., 1981; Wolff, 1992). However, only the monohydroxy-metabolites
402
6-hydroxy-N-phenyl-2-naphthylamine and 4′- hydroxy-N-phenyl-2-naphthylamine were
403
detected after exposure with P2NA (Deutsche Forschungsgemeinschaft, 2015; Weiss et al.,
404
2007). Dihydroxy-metabolites can form quinone while monohydroxy-metabolites not. As
405
summarized previously (O’brien, 1991), oxidative stress arises when the quinone is
40）
reduced by reductases to a semiquinone radical which reduces oxygen to superoxide
407
radicals and reforms the quinone. Wang et al. (2011) reported that polyphenolic
408
allelochemicals such as (+)-catechin and pyrogallic acid could elevate cellular ROS level
409
through futile redox cycles, which amplify the generation of O2.- with the expense of
410
NAD(P)H and greatly enhance the toxicity to the target organisms. The results suggested
411
that the generation of ROS was related to the biotransformation metabolites of P1NA.
412
Therefore, the metabolites of P1NA and P2NA in M. aeruginosa were detected by GC/MS.
413
1,4-naphthoquinone was found in M. aeruginosa exposure to P1NA, while it was not
414
detected in P2NA and control groups (Fig S2). We speculated P1NA metabolized to form
415
1,4-dihydroxynaphthalene firstly. It was similar to (+)-catechin and pyrogallic acid which
41）
may cause autoxidation or enzymatic oxidation to easily form semiquinone radical in the
417
target organism. The reactive semiquinone radical is able to react with O2 to produce
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o-quinone form of 1,4-dihydroxynaphthalene accompanying the production of O2.-.
419
Dismutation of O2.- produces H2O2, and then H2O2 can be reduced via the Fenton-type
420
reaction in the presence of transition metals to form the ·OH. Therefore, it is reasonable
421
to expect that the metabolites of P1NA may undergo futile redox cycling in the presence
422
of intracellular reductants such as NAD(P)H (O’brien, 1991) and induce ROS production
423
in M. aeruginosa. A possible pathway of ROS generation in M. aeruginosa induced by
424
P1NA could be proposed as shown in Fig 7. In brief, it was the first time to demonstrate
425
that P1NA exhibited remarkably stronger growth inhibition effects in M. aeruginosa than
42）
its isomer P2NA. The different property in inducing excessive ROS between P1NA and
427
P2NA was the important reason to exhibit different toxicity in M. aeruginosa. The
428
formation of 1,4-naphthoquinone was the key factor to generate excessive ROS.
429
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430
Fig 7. A possible pathway of ROS production induced by P1NA in M. aeruginosa.
431
4 Conclusion
432
The results of comparing the toxicity of P1NA and P2NA indicated that only P1NA
433
was highly toxic to M. aeruginosa. The 48 h EC50 values based on growth inhibition were
24
ACCEPTED MANUSCRIPT
16.616 µM, while that of P2NA was not detected in same conditions. The intracellular
435
ROS levels were significantly increased in M. aeruginosa exposure to P1NA, while it was
43）
not found in the group of P2NA. Attention should be paid to the influence of P1NA on
437
other aquatic organisms and aquatic ecosystem. In the research field of allelopathy, the
438
toxic effect of P1NA on phytoplankton should be investigated as the main object
439
compared with P2NA.
440
Acknowledgements
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434
The study was financially supported by the National Nature Science Foundation of
442
China (grant number 51178452, 50909091 and 31123001), the Major Science and
443
Technology Program for Water Pollution Control and Treatment of China 12th Five-Year
444
Plan (grant number 2012ZX07101007-005), and the State Key Laboratory of Freshwater
445
Ecology and Biotechnology Program (grant number 2013FB20). The authors thank
44）
Liping Zhang and Yan Wang at Institute of Hydrobiology, Chinese Academy of Sciences
447
for assistance in using GC-MS and flow cytometer.
448
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ACCEPTED MANUSCRIPT Highlights: ● P1NA and P2NA with similar molecular structure, only P1NA was found high toxic to M. aeruginosa.
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● ROS induced by P1NA could the reason of the growth inhibition of M. aeruginosa.
● The formation of 1,4-naphthoquinone was the key factor to generate excessive
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ROS.