Ecotoxicology and Environmental Safety 111 (2015) 138–145
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Cellular responses and biodegradation of amoxicillin in Microcystis aeruginosa at different nitrogen levels Ying Liu a,n, Feng Wang a, Xiao Chen b, Jian Zhang a, Baoyu Gao a a Shandong Provincial Key Laboratory of Water Pollution Control and Resource Reuse, School of Environmental Science and Engineering, Shandong University, Jinan 250100, PR China b Shandong Urban and Rural Planning Design Institute, Jinan 250013, PR China
ar t ic l e i nf o
a b s t r a c t
Article history: Received 20 August 2014 Received in revised form 7 October 2014 Accepted 8 October 2014
The influence of nitrogen on the interactions between amoxicillin and Microcystis aeruginosa was investigated using a 7-day exposure test. Growth of M. aeruginosa was not significantly (p4 0.05) affected by amoxicillin at the lowest nitrogen level of 0.05 mg L 1, stimulated by 500 ng L 1 of amoxicillin at a moderate nitrogen level of 0.5 mg L 1 and enhanced by 200–500 ng L 1 of amoxicillin at the highest nitrogen level of 5 mg L 1. The generation of reactive oxygen species (ROS) and the synthesis of glutathione S-transferases (GST) and glutathione (GSH) were more sensitive to amoxicillin and were stimulated at all nitrogen levels. At the lowest nitrogen level of 0.05 mg L 1, superoxide dismutase and peroxidase were not effective at eliminating amoxicillin-induced ROS, resulting in the highest malondialdehyde content in M. aeruginosa. The biodegradation of 18.5–30.5% of amoxicillin by M. aeruginosa was coupled to increasing GST activity and GSH content. Elevated nitrogen concentrations significantly enhanced (po0.05) the stimulation effect of amoxicillin on the growth of M. aeruginosa, the antioxidant responses to amoxicillin and the biodegradation of amoxicillin in M. aeruginosa. The nitrogen-dependent hormesis effect of the coexisting amoxicillin contaminant on the M. aeruginosa bloom should be fully considered during the control of M. aeruginosa bloom. & Elsevier Inc. All rights reserved.
Keywords: Combined pollution Hormesis Reactive oxygen species Glutathione conjugation Three-way ANOVA
1. Introduction Cyanobacterial blooms are frequently observed in freshwater environments (Paerl and Huisman, 2009). Excessive growth of cyanobacteria significantly reduces water quality and aquatic ecosystem function, and cyanotoxins pose serious threats to animals and humans (Veldhuis and Wassmann, 2005). Therefore, cyanobacterial blooms have become a worldwide public health and ecological concern. Most studies on cyanobacterial blooms use Microcystis aeruginosa as a model species. M. aeruginosa is one of the most widely distributed cyanobacterial species and produces microcystins (MCs), which is a major group of cyanotoxins (Babica et al., 2006). M. aeruginosa was normally considered to be regulated by conventional factors, including nitrogen (N), phosphorous (P), N:P ratio, light intensity, trace metals, temperature, and pH (Davis et al., 2009; Jiang et al., 2008). In the last two decades, numerous domestic and industrial contaminants were released into aquatic environments from human activities. Some of these contaminants were found to interact with M. aeruginosa, such as n
Corresponding author. Fax: þ86 531 88364513. E-mail address: [email protected] (Y. Liu).
http://dx.doi.org/10.1016/j.ecoenv.2014.10.011 0147-6513/& Elsevier Inc. All rights reserved.
heavy metals, pesticides (Qian et al., 2012), benzene compounds (Perrona and Juneau, 2011) and pharmaceuticals (Hu et al., 2014). Reported interactions were mainly growth inhibition effects of these contaminants at exposure concentrations much higher than the actual contamination levels. Very recently, several studies observed the stimulation effects of anthropogenic contaminants on M. aeruginosa blooms at environmentally relevant concentrations. For instance, an herbicide pentachlorophenol (de Morais et al., 2014) was reported to promote the growth of M. aeruginosa at a concentration of 1 μg L 1. A study performed by our group found that the antibiotic amoxicillin enhanced the growth of M. aeruginosa and the production and release of MCs at its currently detected concentrations in aquatic environments (Liu et al., 2012a). The above studies suggested that the regulation of cyanobacterial blooms by these contaminants became a reality in aquatic environments and should be fully considered during the control of cyanobacterial blooms. However, interaction mechanisms between anthropogenic contaminants and M. aeruginosa remained unclear. Exposure to exogenous chemicals was found to generate reactive oxygen species (ROS), cause oxidative stress and induce antioxidant responses in microalgae (Wang et al., 2011). Antioxidant responses have proven to be correlated with
Y. Liu et al. / Ecotoxicology and Environmental Safety 111 (2015) 138–145
morphological, physiological and biochemical changes in microalgae (Mallick and Mohn, 2000). Wang and Xie (2007) observed that increased antioxidant activities could protect M. aeruginosa from oxidative damage caused by low concentrations of nonylphenols (NPs) and consequently led to stimulated growth as a hormesis response. Additionally, antioxidants, including glutathione S-transferases (GST) and glutathione (GSH), not only participate in the elimination of ROS but also play an important role in the metabolism of exogenous chemicals (Belchik and Xun, 2011). Elevated activities of GST and increased content of GSH, coupled with the degradation of amoxicillin, was found to alleviate the toxicity of amoxicillin in M. aeruginosa (Liu et al., 2012b). The above studies suggested that antioxidant responses may be a promising route for investigating the interaction mechanisms between anthropogenic contaminants and M. aeruginosa. M. aeruginosa in aquatic environments was simultaneously exposed to anthropogenic contaminants and various environmental factors, and the environmental factors have the potential to affect the interactions between M. aeruginosa and anthropogenic contaminants. For instance, low temperature was demonstrated to enhance the inhibition effect of an herbicide, atrazine, on the growth and photosynthesis of M. aeruginosa (Chalifour and Juneau, 2011). Studies on the combined effects of other anthropogenic contaminants and other environmental factors on cyanobacteria were still limited. Nitrogen is usually considered to be the dominant environmental factor regulating cyanobacteria as a major nutrient for growth, and it is also an indispensable element in the molecules of various antioxidants (Downing et al., 2005). Therefore, altered nitrogen concentrations may affect the antioxidant responses in M. aeruginosa under exposure to anthropogenic contaminants. In the present study, amoxicillin was selected as the target chemical, which is a widely used antibiotic with high biological activity, and has been verified to interact with M. aeruginosa at environmentally relevant concentrations (Liu et al., 2012a). The variation of the growth rate, the generation of ROS and the responses of various antioxidants in M. aeruginosa following exposure to amoxicillin, as well as the biodegradation of amoxicillin by M. aeruginosa, were investigated at different nitrogen levels. Combined effects of nitrogen and antibiotic contaminants on M. aeruginosa have not been reported by previous studies. The observed results would contribute to a better understanding of the formation of cyanobacterial bloom with the coexistence of antibiotic contaminants.
2. Materials and methods 2.1. Cellular response test An axenic culture of the cyanobacterium M. aeruginosa PCC7806 was supplied by the Pasteur Culture Collection of Cyanobacteria (Paris, France). Amoxicillin was purchased from SigmaAldrich, Inc. (Shanghai, China), and the stock solution was prepared in methanol and stored at 20 °C prior to use. M. aeruginosa was pre-cultivated under aseptic conditions for two weeks in sterile BG11 medium at 25 71 °C under a 16:8 light:dark cycle provided by cool white fluorescent lights at an intensity of 40 μ mol photons m 2 s 1. The BG11 medium contained nitrate as the source of nitrogen, with a nitrogen concentration of 247 mg L 1. After pre-cultivation, the M. aeruginosa cells reaching the exponential growth phase were collected by centrifugation (4000g, 4 °C, 5 min) and used as the inoculums for the cellular response test and the biodegradation test. Modified BG11 media were used in the cellular response test and the biodegradation test, which were divided into three test groups according to the nitrogen
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concentration in the modified BG11 medium, which were 0.05, 0.5 and 5 mg L 1, respectively. In each test group, 250-mL Erlenmeyer flasks, each containing 150 mL of the modified BG11 medium, were spiked with different concentrations of amoxicillin, each in triplicate. The test concentrations of amoxicillin in each test group were 200 ng L 1 and 500 ng L 1. Amoxicillin was replenished regularly to maintain a stable exposure dose, and the determined test concentrations deviated within an acceptable range (from 9% to þ 12%) compared with their nominal values. The final concentrations of methanol in the test media were below 0.01% (v/v). At each nitrogen level, three flasks containing modified BG11 media with the same nitrogen concentration and 0.01% (v/v) methanol, but without amoxicillin, were prepared as the non-antibiotic-treated control. The initial cell density of M. aeruginosa in each flask was 4 105 cells mL 1. After inoculation, the M. aeruginosa cells were cultured under the same aseptic condition as the pre-cultivation for seven days. The entire experimental apparatus used for the culture of M. aeruginosa and addition of amoxicillin were sterilized by autoclaving at 121 °C for 20 min prior to use. The flasks were shaken well before each sampling. Sampling was conducted in a laminar flow cabinet using sterile apparatus. One milliliter of algal culture was aseptically sampled from each flask every day and used for cell density counting. The specific growth rate was calculated according to Eq. (1):
μ (/day) = (LnX1 − LnX 0 )/(t1 − t0 )
(1)
where X0 and X1 were the cell density at the beginning (t0) and the end (t1) of the selected time interval during the exponential phase of growth, respectively. 2.2. Analysis of ROS and antioxidant responses In the cellular response test, the culture medium was aseptically sampled from each flask after 4 and 7 days of exposure. For the analysis of ROS, the M. aeruginosa cells were collected by centrifugation at 2000g at 4 °C for 10 min, loaded with 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) probes and then measured with a fluorescence plate reader (Bio-TEK, USA), according to the method of Knauert and Knauer (2008), with an excitation filter of 485 nm and an emission filter of 530 nm. DCFHDA is a cell-permeable indicator, which was hydrolyzed by the cellular esterase to form the non-fluorescent DCFH after penetrating into the cells. DCFH is immediately transformed to highly fluorescent 2′,7′-dichlorofluorescein (DCF) in the presence of ROS, and the DCF fluorescence indicates the ROS level. For the analysis of the antioxidants, the M. aeruginosa cells harvested by centrifugation were re-suspended in 1.5 mL of phosphate buffer (100 mM, pH ¼7.4) containing 1% (w/v) of polyvinylpyrrolidone and homogenized on ice by an ultrasonic cell pulverizer at 200 W with total time of 5 min (ultrasonic time: 2 s; rest time: 4 s). The homogenate was then centrifuged at 12,000g at 4 °C for 10 min. The supernatant was used for the determination of antioxidants. The activities of superoxide dismutase (SOD), peroxidase (POD) and GST and the contents of malondialdehyde (MDA) and GSH were determined according to previous studies (Gao and Tam, 2011; Wang et al., 2013a). One unit of SOD activity was defined as the amount of enzyme that caused a 50% decrease in the SODinhibited nitro-blue tetrazolium reduction. One unit of POD activity was defined as the amount of enzyme required for the formation of purpurogallin from pyrogallol in 20 s at pH 6.0. One unit of GST activity was defined as the amount of enzyme that catalyzed the formation of 1 μmol of S-(2.4-dinitrophenyl)-glutathione per minute at 37 °C. The activities of the antioxidant enzymes and the contents of non-enzymatic antioxidants in M. aeruginosa were
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μg per 106 cells,
3.1. Combined effects of amoxicillin and nitrogen on the growth of M. aeruginosa
2.3. Biodegradation test The experimental set-up for the biodegradation test was similar to that for the cellular response test, except for the following: (i) three 1000-mL Erlenmeyer flasks, each containing 400 mL of modified sterile BG11 medium inoculated with M. aeruginosa and spiked with amoxicillin, were prepared for each amoxicillin concentration at each nitrogen level, (ii) amoxicillin was only spiked at the beginning of the test with no replenishment during the entire exposure period, (iii) both an abiotic control and a nonantibiotic-treated control were prepared, and (iv) three flasks containing modified BG11 media spiked with amoxicillin, but without the M. aeruginosa cells, were prepared as the abiotic control for each amoxicillin concentration at each nitrogen level to measure the abiotic loss of amoxicillin from the culture medium. In the biodegradation test, 100 mL of culture medium was sampled after 4 and 7 days of incubation. Sampled medium was separated from the M. aeruginosa cells by filtering through a Whatman GF/B filtration membrane (1 μm). For the determination of the antibiotic concentration in the culture medium, the filtrate was mixed with 0.2 mL of 5% (w/v) ethylene diamine tetraacetic acid (EDTA), and then extracted with an Oasis HLB glass cartridge at a flow rate of 2 mL min 1. Oasis HLB glass cartridges (200 mg, 5 mL) were obtained from Waters Corporation (Massachusetts, USA). The cells harvested by the filtration membrane were freezedried, extracted with 10 mL of acetonitrile under sonication and centrifuged at 4000g at 4 °C for 20 min to remove the cell debris. The extract was also mixed with 0.2 ml of 5% (w/v) EDTA and Milli-Q water to a final volume of 100 mL and then extracted with an Oasis HLB glass cartridge to measure the adsorption of amoxicillin in the M. aeruginosa cells. After extraction, the Agilent 1200 series liquid chromatograph coupled with an Agilent series 6410B triple quadruple mass spectrometer (Agilent, USA) was used for the analysis of amoxicillin. The extraction, cleanup, quantification method and analytical condition of the liquid chromatography– tandem mass spectrometry (LC–MS/MS) system were described in detail by a previous study by our group (Liu et al., 2012b). The biodegradation percentage (BP) of amoxicillin in M. aeruginosa was calculated according to Eq. (2), where Cn was the nominal test concentration of amoxicillin in the culture medium, and Ca, Ct and Cm were the measured concentrations of amoxicillin in the abiotic control, the culture medium and the M. aeruginosa cells, respectively.
BP (%) = (Ca − Ct − Cm )/Cn × 100%
3. Results
(2)
2.4. Statistical analysis Statistical analysis was conducted using the SPSS software (Version 13.0). A parametric two-way analysis of variance (ANOVA) was used to determine the interactions between nitrogen and amoxicillin on the growth rate of M. aeruginosa. A parametric three-way ANOVA was used to determine the influences of the culture time, nitrogen and amoxicillin on the levels of ROS, antioxidants and BP values. A parametric one-way ANOVA coupled with a post-hoc comparison was used to determine the differences in the growth rate, cell density, and the levels of ROS and antioxidants among different amoxicillin concentrations at each nitrogen level.
During the culture of M. aeruginosa, the exponential growth in all of the culture media started from one day after inoculation and lasted for three days. Afterwards, the culture gradually entered the stationary phase. As shown in Fig. 1, the growth rates of the exponential phase increased with increasing nitrogen concentration. At the lowest nitrogen level of 0.05 mg L 1, the growth of M. aeruginosa was not significantly (p 40.05) influenced by amoxicillin. At the moderate nitrogen level of 0.5 mg L 1, the growth was only stimulated by 500 ng L 1 of amoxicillin, with an increase of 20.7% compared to the non-antibiotic-treated control. At the highest nitrogen level of 5 mg L 1, the growth was stimulated by both 200 ng L 1 and 500 ng L 1 of amoxicillin, with respective increases of 18.2% and 25.3% compared to the non-antibiotictreated control. These results indicated that increased nitrogen concentration enhanced the stimulation effect of amoxicillin on the growth of M. aeruginosa. Similar to the growth rate, the cell density of M. aeruginosa was also positively correlated with nitrogen concentration (Table 1). Significant stimulation effect of amoxicillin on cell density (p o0.05) was also observed at test concentration of 500 ng L 1 in culture media with 0.5 mg L 1 of nitrogen and at test concentrations of 200 ng L 1 and 500 ng L 1 in culture media with 5 mg L 1 of nitrogen. Two-way ANOVA analysis (Table 2) presented significant interaction (p o0.05) between nitrogen and amoxicillin on the growth of M. aeruginosa. 3.2. Antioxidant responses to amoxicillin at different nitrogen levels The intensity of DCF fluorescence in Fig. 2a indicates that the ROS level in M. aeruginosa was significantly influenced (p o0.05) by the culture time. The ROS level in the stationary phase (day 7) was higher than that in the exponential phase (day 4) at each nitrogen level and at each amoxicillin concentration. Nitrogen showed no significant effect (p 40.05) on the ROS level. Exposure to amoxicillin caused a significant increase (p o0.05) in ROS levels in the M. aeruginosa cells at each nitrogen level and at each sampling time. The activities of SOD and POD in the non-antibiotic-treated control increased significantly (p o0.05) with 0.5
Non-antibiotic-treated -1 500ng L of AM-treated
-1
200 ng L of AM-treated
b
b
0.4
a -1
expressed in units (U) per 106 cells and respectively.
Growth rate (d )
140
0.3
0.2
b a
a
0.1
0.0 0.05
0.5
5 -1
Test concentration of nitrogen (mg L ) Fig. 1. Effect of amoxicillin (AM) on growth rates of M. aeruginosa at different nitrogen levels. Mean and standard deviation of three replicates are shown. Alphabet letters a and b indicate significant difference in means among test concentrations of AM at each nitrogen level at p o 0.05 according to one-way ANOVA test.
Amoxicillin was not detected in the cells of M. aeruginosa (Cm ¼0), suggesting that the removal of amoxicillin from the
o 0.01 o 0.01 0.491 0.208 0.805 0.661 0.847 48.827 73.319 0.488 1.677 0.062 0.421 0.167 o 0.01 o 0.01 o 0.01 o 0.01 0.04 o 0.01 0.162 107.185 437.897 131.894 23.043 6.602 13.612 1.742 o 0.01 o 0.01 o 0.01 0.01 0.036 0.042 0.207 NA: not available.
p F value p F value p F value
36.239 157.135 88.863 8.551 3.654 2.775 1.557 0.083 0.087 o 0.01 0.316 0.458 0.379 0.738 2.568 2.047 13.221 1.190 0.799 1.084 0.497 o0.01 o0.01 o0.01 0.110 0.534 o0.01 0.873 23.214 101.184 91.191 2.346 0.639 7.142 0.304 o 0.01 o 0.01 o 0.01 0.098 0.064 0.035 0.138 33.758 105.441 56.320 2.481 2.969 2.911 1.863 0.017 0.079 o 0.01 0.603 0.494 o 0.01 0.989 6.236 2.730 84.061 0.513 0.720 6.707 0.075 NA o 0.01 0.03 NA NA 0.025 NA a
3.3. Biodegradation of amoxicillin
NA 872.905 7.883 NA NA 3.622 NA
increasing nitrogen concentrations in the culture media and declined significantly (p o0.05) with the culture time (Fig. 2b and c). The SOD activities showed significant increases (p o0.05) in response to amoxicillin and a positive correlation with the test concentrations of amoxicillin during the entire exposure period at nitrogen levels of 0.5–5 mg L 1. In contrast, no significant alteration in the SOD activity in response to amoxicillin was recorded at the lowest nitrogen level of 0.05 mg L 1 (p 40.05). The POD activity in M. aeruginosa under exposure to amoxicillin varied in a manner similar to that of SOD at each nitrogen level and at each sampling time. Similar to SOD and POD, the GST activity and GSH content in the non-antibiotic-treated control were also positively correlated with nitrogen concentration and negatively correlated with the culture time (Fig. 3a and b). Amoxicillin significantly stimulated (p o0.05) GST activity and GSH content at each nitrogen level during the entire exposure period. The highest GST activity and GSH content were observed in 500 ng L 1 of amoxicillin-treated medium at the highest nitrogen level of 5 mg L 1, with respective increases of 84.6% and 73.8% relative to the non-antibiotic-treated control. The culture time, nitrogen and amoxicillin showed no significant interaction on ROS and antioxidant levels, according to the three-way ANOVA analysis (p 40.05). Instead, significant twoway interactions (p o0.05) between nitrogen and amoxicillin were observed on the ROS level, activities of SOD, POD and GST, as well as content of GSH (Table 2). The MDA content in the non-antibiotic-treated control was not significantly affected (p 40.05) by nitrogen and the culture time (Fig. 3c). No significant alteration of the MDA content was observed in M. aeruginosa under exposure to amoxicillin at the fourth day of exposure (p 40.05). The MDA content was significantly stimulated (po 0.05) by amoxicillin at all of the nitrogen levels at the seventh day of exposure and showed a positive correlation with test concentrations of amoxicillin. The most significant increase of MDA content was observed in 500 ng L 1 of the amoxicillin-treated group at the lowest nitrogen level, which was 1.54fold higher than the non-antibiotic-treated control.
Time Nitrogen Amoxicillin Time Nitrogen Time Amoxicillin Nitrogen Amoxicillin Time Nitrogen Amoxicillin
AM: amoxicillin.
p
17.5 7 0.41
F value
14.7 7 0.47
p
13.6 70.42 16.9 70.31
F value
11.2 7 0.53 13.5 7 0.52
p
Non-antibiotic-treated 200 ng L 1 of AMtreated 500 ng L 1 of AMtreated
F value
8.45 7 0.27
p
7.21 70.37
F value
7.45 7 0.45 7.58 7 0.25
p
6.337 0.39 6.417 0.32
141
a
Non-antibiotic-treated 200 ng L 1 of AMtreated 500 ng L 1 of AMtreated
F value
5.727 0.49
BP value
5.34 7 0.28
GSH content
5.42 7 0.26 5.51 70.35
GST activity
5.187 0.35 5.247 0.42
MDA content
Non-antibiotic-treated 200 ng L 1 of AMtreateda 500 ng L 1 of AMtreated
POD activity
a
7 Days
SOD activity
5
4 Days
ROS level
0.5
Cell density (105 cells mL 1)
Algal growth
0.05
Culture media
Dependent variables
Nitrogen levels (mg L 1)
Factors
Table 1 Cell density of M. aeruginosa after 4 and 7 days of culture in different media at different nitrogen levels.
Table 2 Summary of two-way ANOVA analysis between nitrogen and amoxicillin on algal growth rate and three-way ANOVA analysis among culture time, nitrogen and amoxicillin on ROS and antioxidant levels as well as BP values.
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Y. Liu et al. / Ecotoxicology and Environmental Safety 111 (2015) 138–145
500 ng L of AM-treated
a,b a
c c
a
25
b
a,b
b
b
b
a
a
a
a
20 15 10
c
500 ng L of AM-treated
25
b 20 15
b
c
b
b
a
a,b a
a
b 10
b
c
b a,b
a
a
a 5
5
0
0 0.05
0.5
5
0.05
Day 4
0.5
0.05
5
0.5
0.05
200 ng L of AM-treated
cell)
35
6
b
b
6
b b b
c
500 ng L of AM-treated
-1
25
200 ng L of AM-treated
-1
500 ng L of AM-treated
30
-1
Non-antibiotic-treated
-1
Non-antibiotic-treated
c
b a
b
b
a
b
c c
b
a a a,b a
15
b
a
10
20
5
-1
20
45
0.5
Day 7
Test concentration of nitrogen (mg L )
-1
40
5
Day 4
Day 7
Test concentration of nitrogen (mg L )
SOD activity (U per 10 cell)
200 ng L of AM-treated
-1
6
c
b
b
-1
Non-antibiotic-treated
c
-1
35 30
200 ng L of AM-treated
GST activity (U per 10 cell)
40
30
-1
Non-antibiotic-treated
4
DCF fluorescence intensity per 10 cell
45
b
a
a
a
b
a,b
b
a
10 5 0
0 0.05
0.5
5
0.05
Day 4
0.5
0.05
5
0.5
5
0.05
Day 4
Day 7
0.5
5
Day 7 -1
Test concentration of nitrogen (mg L )
-1
Test concentration of nitrogen (mg L )
10 40
Non-antibiotic-treated
-1
Non-antibiotic-treated
200 ng L of AM-treated
500 ng L of AM-treated
cell)
-1
b b
6
500 ng L of AM-treated
30
c
6
POD activity (U per 10 cell)
-1
200 ng L of AM-treated
-1
b
b
b
b
c
b
20
b
a,b
c
a
5
a,b a
a
b
a
a
a
a
10
0 0.05
0 0.05
0.5
Day 4
5
0.05
0.5
5
Day 7 -1
0.5
Day 4
5
0.05
0.5
5
Day 7 -1
Test concentration of nitrogen (mg L )
Test concentration of nitrogen (mg L ) Fig. 2. Effect of amoxicillin (AM) on (a) ROS level, (b) SOD activity and (c) POD activity of M. aeruginosa at different nitrogen levels after 4 and 7 days of exposure. Mean and standard deviation of three replicates are shown. Alphabet letters a, b and c indicate significant difference in means among test concentrations of AM at each nitrogen level and each exposure time at po 0.05 according to one-way ANOVA test.
culture media was mainly due to biodegradation, rather than accumulation. According to Fig. 4, the biodegradation of amoxicillin in M. aeruginosa was positively correlated with both the nitrogen concentration and the culture time, although it was not significantly affected by the increasing concentration of amoxicillin (p 40.05). At the end of exposure, the respective biodegradation
Fig. 3. Effect of amoxicillin (AM) on (a) GST activity, (b) GSH content and (c) MDA content of M. aeruginosa at different nitrogen levels after 4 and 7 days of exposure. Mean and standard deviation of three replicates are shown. Alphabet letters a, b and c indicate significant difference in means among test concentrations of AM at each nitrogen level and each exposure time at p o 0.05 according to one-way ANOVA test.
percentages (BP) of amoxicillin were 18.5–19.6%, 25.2–26.6% and 29.6–30.5% at nitrogen levels of 0.05, 0.5 and 5 mg L 1. The threeway ANOVA analysis (Table 2) on BP values presented neither significant three-way interaction among the culture time, nitrogen and amoxicillin (p 40.05) nor significant two-way interaction between any two of the three factors (p 40.05).
Y. Liu et al. / Ecotoxicology and Environmental Safety 111 (2015) 138–145
40
-1
200 ng L of AM
-1
500 ng L of AM
BP (%)
30
20
10
0 0.05
0.5
Day 4
5
0.05
0.5
5
Day 7 -1
Test concentration of nitrogen (mg L ) Fig. 4. The biodegradation percentages (BP) of amoxicillin (AM) in M. aeruginosa at different nitrogen levels after 4 and 7 days of exposure. Mean 7standard deviation values of three replicates are shown.
4. Discussion Various researches have demonstrated the ability of the nitrogen supply to positively regulate the growth of M. aeruginosa (Davis et al., 2009). The present study further verified the positive correlation between the nitrogen concentration and the growth rate. Increased nitrogen concentration also contributed to the stimulation effect of amoxicillin on the growth of M. aeruginosa. The action of amoxicillin in prokaryotes depends on the recognition of amoxicillin by intracellular penicillin-binding proteins (PBPs), and synthesis of PBPs is most active during the cell division process (Stevens et al., 1993). Therefore, amoxicillin is mainly effective against cells in the growth phase (Brisson-Noël et al., 1988). With a typical prokaryotic cell structure, cyanobacteria have also been proven to produce PBPs (Marbouty et al., 2009). Therefore, nitrogen may also regulate the responses of M. aeruginosa to amoxicillin via growth rate and synthesis of PBPs. The simulated growth of M. aeruginosa could be regarded as a hormesis effect of amoxicillin at low concentrations. Zhu et al. (2012) also observed similar results, showing that exposure of M. aeruginosa to a mixture of low concentrations of polycyclic aromatic hydrocarbons (PAHs) led to increased growth of M. aeruginosa. The ROS level in M. aeruginosa remained stable at different nitrogen levels. Liu et al. (2007) investigated the effects of nutrients on a microalgae Chattonella marina and similarly observed that the nitrogen supply regulated algal growth but did not affect ROS production. Increased production of ROS was commonly observed in M. aeruginosa under exposure to various environmental stresses, including heavy metals, allelochemicals (Qian et al., 2010), ultraviolet radiation (Jiang and Qiu, 2011) and herbicides (Jin et al., 2012). In this study, exposure to amoxicillin also stimulated ROS production, indicating that amoxicillin led to oxidative stress in M. aeruginosa. Increased ROS levels in response to amoxicillin were observed, even at the lowest nitrogen level. This result indicated that generation of ROS was more sensitive to amoxicillin exposure, compared with the growth of M. aeruginosa. The higher ROS level at a longer exposure time (day 7) indicated that the production of ROS under amoxicillin exposure was time dependent. This result was in accordance with a previous study in which ROS production in M. aeruginosa exposed to catechin and pyrogallic acid also increased with increasing exposure time (Wang et al., 2011). The activities of SOD, POD and GST and the content of GSH on the fourth day of the culture were significantly higher (p o0.05) than that on the seventh day, indicating that synthesis of
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antioxidants was related to the growth phase of M. aeruginosa. Synthesis of antioxidants was also related to nitrogen. The activities of SOD, POD and GST and the content of GSH in M. aeruginosa were positively correlated with nitrogen concentration at each sampling time. Nitrogen is an indispensable element in the molecular structure of protein, and has been proven to positively regulate the synthesis of enzymes and peptides in M. aeruginosa (Downing et al., 2005). Therefore, the nitrogen-dependent synthesis of these antioxidants could be explained by their molecular structures. The antioxidants SOD and POD played an important role in eliminating ROS and protecting the microalgae from oxidative damage (Blokhina et al., 2003). SOD converts superoxide radicals into hydrogen peroxide, and POD converts hydrogen peroxide into water and oxygen (Cho and Seo, 2005). Increased activities of SOD and POD were found to protect M. aeruginosa from low doses of gamma irradiation (Zheng et al., 2012). The stimulation of SOD activity was reported to be responsible for the tolerance of a diatom Phaeodactylum tricornutum to antibiotic exposure (Yang et al., 2013). Shen et al. (2014) observed that SOD and POD could eliminate pesticide-induced ROS from a green alga Chlorella vulgaris. In the present study, responses of SOD and POD to amoxicillin indicated that these two enzymes did not participate in eliminating ROS from M. aeruginosa at the lowest nitrogen level. Increased activities of SOD and POD under exposure to amoxicillin were observed at higher nitrogen levels. These results indicated that the antioxidant responses to amoxicillin were enhanced by increasing nitrogen concentration. Due to the stimulation of SOD and POD, M. aeruginosa was more adaptive to amoxicillin at higher nitrogen levels, as indicated by the increased growth rate. GST and GSH could also scavenge ROS from the microalgae (Belchik and Xun, 2011). Wang et al. (2013a) found that GSH participated in eliminating ROS from the green alga Selenastrum capricornutum and was stimulated under exposure to heavy metals and PAHs. Mofeed and Mosleh (2013) observed increased GST activity and GSH content in the green alga Scenedesmus obliquus under oxidative stresses caused by two pesticides, fenhexamid and atrazine. Following exposure to amoxicillin, increased activities of GST and elevated synthesis of GSH were observed at all of the nitrogen levels. This result suggested that GSH and GST were more sensitive than SOD and POD in responding to oxidative stress caused by amoxicillin. Wang and Xie (2007) examined the effects of NPs on the antioxidant system of M. aeruginosa and similarly observed that GSH and GST contributed more to eliminating ROS than SOD. Wang et al. (2013b) also found that GSH was more sensitive to oxidative stresses in Chlorella sp. exposed to estradiol and ethinylestradiol, compared with SOD and POD. Along with the elimination of ROS, GST also played an important role in the metabolism of exogenous chemicals by catalyzing the conjugation of exogenous chemicals with GSH for the purpose of detoxification (Belchik and Xun, 2011). The biodegradation of amoxicillin coupled with stimulated GST activity and GSH content further verified the contribution of GST and GSH to the detoxification process of M. aeruginosa. The variation of BP values indicated that the biodegradation of amoxicillin by M. aeruginosa was enhanced by nitrogen. The positive correlation between nitrogen and the synthesis of GST and GSH may explain the elevated BP values with increasing nitrogen concentrations. Stimulated growth and increased cell density of M. aeruginosa at higher nitrogen levels could also lead to increased biodegradation because more algal cells participate in the biodegradation process. Stoichev et al. (2011) reported a biodegradation ability of less than 12% in M. aeruginosa exposed to an antibiotic minocycline at an initial concentration of 2 μM. A previous study also observed 7-day BP values of 12.5%–32.9% for spiramycin in M. aeruginosa (Liu et al., 2012b). The above comparison indicated that amoxicillin
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was more degradable than minocycline and comparable to spiramycin for M. aeruginosa. Studies on the biodegradation of other antibiotics in M. aeruginosa were still limited. Bioaccumulation of amoxicillin in M. aeruginosa was not found in this study, possibly due to the hydrophilicity of this antibiotic. As a product of lipid peroxidation, increased MDA content is a commonly used indicator of oxidative damage (Wang et al., 2013b). Increased MDA content at the seventh day of exposure indicated that amoxicillin caused lipid peroxidation in M. aeruginosa at all of the nitrogen levels. The amoxicillin-induced MDA content increased with decreasing nitrogen concentrations. This result was in accordance with the antioxidant responses. More significant antioxidant responses were observed at higher nitrogen levels, such that the amoxicillin-induced oxidative damage was alleviated. Similar to ROS, the formation of MDA was also time dependent. Kong et al. (2013) also similarly reported that the increase of MDA content in actinomycetes-treated M. aeruginosa gradually increased with time. The lower antioxidant levels in the stationary phase compared with the exponential phase was another possible reason for the increased formation of MDA in the stationary phase. Reported nitrogen concentrations were 0.03–0.6 mg L 1 in moderately polluted waters and up to 100 mg L 1 in heavily polluted waters (Polyak et al., 2013). Currently detected concentrations of amoxicillin in aquatic environments were below 622 ng L 1 (Zuccato et al., 2010). The test concentrations of nitrogen and amoxicillin in this study covered their actual contamination levels. Observed results indicated coexisting amoxicillin contaminant would aggravate the pollution of M. aeruginosa via the growth stimulation effect, when the contamination concentration of nitrogen was Z0.5 mg L 1. In this case, amoxicillin should also be removed from aquatic environments during the control of M. aeruginosa bloom. At the lowest nitrogen level of 0.05 mg L 1, coexisting amoxicillin had no significant effect on the pollution of M. aeruginosa, while M. aeruginosa would alleviate the pollution of amoxicillin via biodegradation. Significant interactions between nitrogen and amoxicillin on the antioxidant system of M. aeruginosa, as indicated by the three-way ANOVA analysis, still deserve further investigation, in order to fully interpret the mechanisms of combined action of nitrogen and amoxicillin.
5. Conclusions At current contamination levels of 0.05–5 mg L 1 for nitrogen and 200–500 ng L 1 for amoxicillin, the two factors significantly interact with each other (po 0.05) on the growth of M. aeruginosa, the generation of ROS and the biodegradation of amoxicillin. Elevated nitrogen concentrations significantly enhanced (p o0.05) the detoxification of amoxicillin via antioxidants, including SOD, POD, GST and GSH, and consequently led to stimulated growth of M. aeruginosa as an adaptive response to amoxicillin stress at higher nitrogen levels of 0.5–5 mg L 1. At the lowest nitrogen level of 0.05 mg L 1, non-participation of SOD and POD in eliminating ROS may explain the high lipid peroxidation level in M. aeruginosa exposed to amoxicillin. The biodegradation of amoxicillin was also positively correlated with nitrogen concentration, possibly via the regulation of GSH conjugation. The coexistence of nitrogen and amoxicillin contaminants with M. aeruginosa would pose an increased threat to aquatic environments and should be considered during the control of cyanobacterial bloom.
Acknowledgments This work was supported by National Natural Science Foundation of China (51209125) and partly by Research Foundation for the Doctoral Program of Higher Education of China (20110131120014) and Promotive Research Foundation for Young and Middle-aged Scientists of Shandong Province of China (2013BSE27073).
References Babica, P., Bláha, L., Maršálek, B., 2006. Exploring the natural role of microcystins – a review of effects on photoautotrophic organisms. J. Phycol. 42, 9–20. Belchik, S.M., Xun, L., 2011. S-glutathionyl-(chloro) hydroquinone reductases: a new class of glutathione transferases functioning as oxidoreductases. Drug Metab. Rev. 43, 307–316. Blokhina, O., Virolainen, E., Fagerstedt, K.V., 2003. Antioxidants, oxidative damage, and oxygen deprivation stress: a review. Ann. Bot. Lond. 91, 179–194. Brisson-Noël, A., Trieu-Cuot, P., Courvalin, P.J., 1988. Mechanism of action of amoxicillin and other macrolidesJ. Antimicrob. Chemother. 22 (Suppl. B), S13–S23. Chalifour, A., Juneau, P., 2011. Temperature-dependent sensitivity of growth and photosynthesis of Scenedesmus obliquus, Navicula pelliculosa and two strains of Microcystis aeruginosa to the herbicide atrazine. Aquat. Toxicol. 103, 9–17. Cho, U.H., Seo, N.H., 2005. Oxidative stress in Arabidopsis thaliana exposed to cadmium is due to hydrogenperoxide accumulation. Plant Sci. 168, 113–120. Davis, T.W., Berry, D.L., Boyer, G.L., Gobler, C.J., 2009. The effects of temperature and nutrients on the growth and dynamics of toxic and non-toxic strains of Microcystis during cyanobacteria blooms. Harmful Algae 8, 715–725. de Morais, P., Stoichev, T., Basto, M.C.P., Ramos, V., Vasconcelos, V.M., Vasconcelos, M.T.S.D., 2014. Cyanobacterium Microcystis aeruginosa response to pentachlorophenol and comparison with that of the microalga Chlorella vulgaris. Water Res 52, 63–72. Downing., T.G., Sember, C.S., Gehringer, M.M., Leukes, W., 2005. Medium N:P ratios and specific growth rate comodulate microcystin and protein content in Microcystis aeruginosa PCC7806 and M. aeruginosa UV027. Microb. Ecol. 49, 468–473. Gao, Q.T., Tam, N.F.Y., 2011. Growth, photosynthesis and antioxidant responses of two microalgal species, Chlorella vulgaris and Selenastrum capricornutum, to nonylphenol stress. Chemosphere 82, 346–354. Hu, X., Liu, Y.G., Zeng, G.M., Hu, X.J., Wang, Y.Q., Zeng, X.X., 2014. Effects of limonene stress on the growth of and microcystin release by the freshwater cyanobacterium Microcystis aeruginosa FACHB-905. Ecotoxicol. Environ. Saf. 105, 121–127. Jiang, Y., Ji, B., Wong, R.N.S., Wong, M.H., 2008. Statistical study on the effects of environmental factors on the growth and microcystins production of bloomforming cyanobacterium – Microcystis aeruginosa. Harmful Algae 7, 127–136. Jiang, H.B., Qiu, B.S., 2011. Inhibition of photosynthesis by UV-B exposure and its repair in the bloom-forming cyanobacterium Microcystis aeruginosa. J. Appl. Phycol. 23, 691–696. Jin, Z.P., Luo, K., Zhang, S., Zheng, Q., Yang, H., 2012. Bioaccumulation and catabolism of prometryne in green algae. Chemosphere 87, 278–284. Knauert, S., Knauer, K., 2008. The role of reactive oxygen species in copper toxicity to two freshwater green algae. J. Phycol. 44, 311–319. Kong, Y., Zou, P., Yang, Q., Xu, X.Y., Miao, L.H., Zhu, L., 2013. Physiological responses of Microcystis aeruginosa under the stress of antialgal actinomycetes. J. Hazard. Mater. 262, 274–280. Liu, W.H., Au, D.W.T., Anderson, D.M., Lam, P.K.S., Wu, R.S.S., 2007. Effects of nutrients, salinity, pH and light:dark cycle on the production of reactive oxygen species in the alga Chattonella marina. J. Exp. Mar. Biol. Ecol. 346, 76–86. Liu, Y., Gao, B.Y., Yue, Q.Y., Guan, Y.T., Wang, Y., Huang, L.H., 2012a. Influences of two antibiotic contaminants on the production, release and toxicity of microcystins. Ecotoxicol. Environ. Saf. 77, 79–87. Liu, Y., Guan, Y.T., Gao, B.Y., Yue, Q.Y., 2012b. Antioxidant responses and degradation of two antibiotic contaminants in Microcystis aeruginosa. Ecotoxicol. Environ. Saf. 86, 23–30. Mallick, N., Mohn, F.H., 2000. Reactive oxygen species: response of algal cells. J. Plant Physiol. 157, 183–193. Marbouty, M., Mazouni, K., Saguez, C., Cassier-Chauvat, C., Chauvat, F., 2009. Characterization of the Synechocystis strain PCC 6803 penicillin-binding proteins and cytokinetic proteins FtsQ and FtsW and their network of interactions with ZipN. J. Bacteriol. 191, 5123–5133. Mofeed, J., Mosleh, Y.Y., 2013. Toxic responses and antioxidative enzymes activity of Scenedesmus obliquus exposed to fenhexamid and atrazine, alone and inmixture. Ecotoxicol. Environ. Safe 95, 234–240. Paerl, H.W., Huisman, J., 2009. Climate change: a catalyst for global expansion of harmful cyanobacterial blooms. Environ. Microbiol. Rep 1, 27–37. Perrona, M.C., Juneau, P., 2011. Effect of endocrine disrupters on photosystem II energy fluxes of green algae and cyanobacteria. Environ. Res. 11, 520–529.
Y. Liu et al. / Ecotoxicology and Environmental Safety 111 (2015) 138–145
Polyak, Y., Zaytseva, T., Medvedeva, N., 2013. Response of toxic cyanobacterium Microcystis aeruginosa to environmental pollution. Water Air Soil Pollut. 224, 1494–1508. Qian, H.F., Pan, X.J., Chen, J., Zhou, D.M., Chen, Z.G., Zhang, L., Fu, Z.W., 2012. Analyses of gene expression and physiological changes in Microcystis aeruginosa reveal the phytotoxicities of three environmental pollutants. Ecotoxicology 21, 847–859. Qian, H.F., Yu, S.Q., Sun, Z.Q., Xie, X.C., Liu, W.P., Fu, Z.W., 2010. Effects of copper sulfate, hydrogen peroxide and N-phenyl-2-naphthylamine on oxidative stress and the expression of genes involved photosynthesis and microcystin disposition in Microcystis aeruginosa. Aquat. Toxicol. 99, 405–412. Shen, Y.F., Liu, L., Gong, Y.X., Zhu, B., Liu, G.L., Wang, G.X., 2014. Potential toxic effect of trifloxystrobin on cellular microstructure, mRNA expression and antioxidant enzymes in Chlorella vulgaris. Environ. Toxicol. Pharmacol. 37, 1040–1047. Stevens, D.L., Yan, S., Bryant, A.E., 1993. Penicillin-binding protein expression at different growth stages determines penicillin efficacy in vitro and in vivo: an explanation for the inoculum effect. J. Infect. Dis. 167, 1401–1405. Stoichev, T., Baptista, M.S., Basto, M.C.P., Vasconcelos, V.M., Vasconcelos, M.T.S.D., 2011. Effects of minocycline and its degradation products on the growth of Microcystis aeruginosa,. Ecotoxicol. Environ. Saf. 74, 219–224. Veldhuis, M.J.W., Wassmann, P., 2005. Bloom dynamics and biologicalcontrol of a high biomass HAB species in European coastal waters: a Phaeocystis case study. Harmful Algae 4, 805–809. Wang, P., Luo, L.J., Ke, L., Luan, T.G., Tam, N.F.Y., 2013a. Combined toxicity of polycyclic aromatic hydrocarbons and heavy metals to biochemical and antioxidant
145
responses of free and immobilized Selenastrum capricornutum. Environ. Toxicol. Chem. 32, 673–683. Wang, P., Wong, M.H., Tam, N.F.Y., 2013b. Antioxidant responses of two microalgae, Selenastrum capricornutum and Chlorella sp., to estradiol and ethinylestradiol. J. Appl. Phycol. 25, 891–903. Wang, J., Xie, P., 2007. Antioxidant enzyme activities of Microcystis aeruginosa in response to nonylphenols and degradation of nonylphenols by M. aeruginosa. Environ. Geochem. Health 29, 375–383. Wang, J., Zhu, J.Y., Liu, S.P., Liu, B.Y., Gao, Y.N., Wu., Z.B., 2011. Generation of reactive oxygen species in cyanobacteria and green algae induced by allelochemicals of submerged macrophytes. Chemosphere 85, 977–982. Yang, C.Y., Zhou, J., Liu, S.J., Fan, P., Wang, W.H., Xia, C.H., 2013. Allelochemical induces growth and photosynthesis inhibition, oxidative damage in marine diatom Phaeodactylum tricornutum. J. Exp. Mar. Biol. Ecol. 444, 16–23. Zheng, B.G., Zheng, Z., Zhang, J.B., Luo, X.Z., Liu, Q., Wang, J.Q., Zhao, Y.F., 2012. The removal of Microcystis aeruginosa in water by gamma-ray irradiation. Sep. Purif. Technol. 85, 165–170. Zhu, X.Z., Kong, H.L., Gao, Y.Z., Wu, M.F., Kong, F.X., 2012. Low concentrations of polycyclic aromatic hydrocarbons promote the growth of Microcystis aeruginosa. J. Hazard. Mater. 237, 371–375. Zuccato, E., Castiglioni, S., Bagnati, R., Melis, M., Fanelli, R., 2010. Source, occurrence and fate of antibiotics in the Italian aquatic environment. J. Hazard. Mater. 179, 1042–1048.
Contents lists available at ScienceDirect
Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Cellular responses and biodegradation of amoxicillin in Microcystis aeruginosa at different nitrogen levels Ying Liu a,n, Feng Wang a, Xiao Chen b, Jian Zhang a, Baoyu Gao a a Shandong Provincial Key Laboratory of Water Pollution Control and Resource Reuse, School of Environmental Science and Engineering, Shandong University, Jinan 250100, PR China b Shandong Urban and Rural Planning Design Institute, Jinan 250013, PR China
ar t ic l e i nf o
a b s t r a c t
Article history: Received 20 August 2014 Received in revised form 7 October 2014 Accepted 8 October 2014
The influence of nitrogen on the interactions between amoxicillin and Microcystis aeruginosa was investigated using a 7-day exposure test. Growth of M. aeruginosa was not significantly (p4 0.05) affected by amoxicillin at the lowest nitrogen level of 0.05 mg L 1, stimulated by 500 ng L 1 of amoxicillin at a moderate nitrogen level of 0.5 mg L 1 and enhanced by 200–500 ng L 1 of amoxicillin at the highest nitrogen level of 5 mg L 1. The generation of reactive oxygen species (ROS) and the synthesis of glutathione S-transferases (GST) and glutathione (GSH) were more sensitive to amoxicillin and were stimulated at all nitrogen levels. At the lowest nitrogen level of 0.05 mg L 1, superoxide dismutase and peroxidase were not effective at eliminating amoxicillin-induced ROS, resulting in the highest malondialdehyde content in M. aeruginosa. The biodegradation of 18.5–30.5% of amoxicillin by M. aeruginosa was coupled to increasing GST activity and GSH content. Elevated nitrogen concentrations significantly enhanced (po0.05) the stimulation effect of amoxicillin on the growth of M. aeruginosa, the antioxidant responses to amoxicillin and the biodegradation of amoxicillin in M. aeruginosa. The nitrogen-dependent hormesis effect of the coexisting amoxicillin contaminant on the M. aeruginosa bloom should be fully considered during the control of M. aeruginosa bloom. & Elsevier Inc. All rights reserved.
Keywords: Combined pollution Hormesis Reactive oxygen species Glutathione conjugation Three-way ANOVA
1. Introduction Cyanobacterial blooms are frequently observed in freshwater environments (Paerl and Huisman, 2009). Excessive growth of cyanobacteria significantly reduces water quality and aquatic ecosystem function, and cyanotoxins pose serious threats to animals and humans (Veldhuis and Wassmann, 2005). Therefore, cyanobacterial blooms have become a worldwide public health and ecological concern. Most studies on cyanobacterial blooms use Microcystis aeruginosa as a model species. M. aeruginosa is one of the most widely distributed cyanobacterial species and produces microcystins (MCs), which is a major group of cyanotoxins (Babica et al., 2006). M. aeruginosa was normally considered to be regulated by conventional factors, including nitrogen (N), phosphorous (P), N:P ratio, light intensity, trace metals, temperature, and pH (Davis et al., 2009; Jiang et al., 2008). In the last two decades, numerous domestic and industrial contaminants were released into aquatic environments from human activities. Some of these contaminants were found to interact with M. aeruginosa, such as n
Corresponding author. Fax: þ86 531 88364513. E-mail address: [email protected] (Y. Liu).
http://dx.doi.org/10.1016/j.ecoenv.2014.10.011 0147-6513/& Elsevier Inc. All rights reserved.
heavy metals, pesticides (Qian et al., 2012), benzene compounds (Perrona and Juneau, 2011) and pharmaceuticals (Hu et al., 2014). Reported interactions were mainly growth inhibition effects of these contaminants at exposure concentrations much higher than the actual contamination levels. Very recently, several studies observed the stimulation effects of anthropogenic contaminants on M. aeruginosa blooms at environmentally relevant concentrations. For instance, an herbicide pentachlorophenol (de Morais et al., 2014) was reported to promote the growth of M. aeruginosa at a concentration of 1 μg L 1. A study performed by our group found that the antibiotic amoxicillin enhanced the growth of M. aeruginosa and the production and release of MCs at its currently detected concentrations in aquatic environments (Liu et al., 2012a). The above studies suggested that the regulation of cyanobacterial blooms by these contaminants became a reality in aquatic environments and should be fully considered during the control of cyanobacterial blooms. However, interaction mechanisms between anthropogenic contaminants and M. aeruginosa remained unclear. Exposure to exogenous chemicals was found to generate reactive oxygen species (ROS), cause oxidative stress and induce antioxidant responses in microalgae (Wang et al., 2011). Antioxidant responses have proven to be correlated with
Y. Liu et al. / Ecotoxicology and Environmental Safety 111 (2015) 138–145
morphological, physiological and biochemical changes in microalgae (Mallick and Mohn, 2000). Wang and Xie (2007) observed that increased antioxidant activities could protect M. aeruginosa from oxidative damage caused by low concentrations of nonylphenols (NPs) and consequently led to stimulated growth as a hormesis response. Additionally, antioxidants, including glutathione S-transferases (GST) and glutathione (GSH), not only participate in the elimination of ROS but also play an important role in the metabolism of exogenous chemicals (Belchik and Xun, 2011). Elevated activities of GST and increased content of GSH, coupled with the degradation of amoxicillin, was found to alleviate the toxicity of amoxicillin in M. aeruginosa (Liu et al., 2012b). The above studies suggested that antioxidant responses may be a promising route for investigating the interaction mechanisms between anthropogenic contaminants and M. aeruginosa. M. aeruginosa in aquatic environments was simultaneously exposed to anthropogenic contaminants and various environmental factors, and the environmental factors have the potential to affect the interactions between M. aeruginosa and anthropogenic contaminants. For instance, low temperature was demonstrated to enhance the inhibition effect of an herbicide, atrazine, on the growth and photosynthesis of M. aeruginosa (Chalifour and Juneau, 2011). Studies on the combined effects of other anthropogenic contaminants and other environmental factors on cyanobacteria were still limited. Nitrogen is usually considered to be the dominant environmental factor regulating cyanobacteria as a major nutrient for growth, and it is also an indispensable element in the molecules of various antioxidants (Downing et al., 2005). Therefore, altered nitrogen concentrations may affect the antioxidant responses in M. aeruginosa under exposure to anthropogenic contaminants. In the present study, amoxicillin was selected as the target chemical, which is a widely used antibiotic with high biological activity, and has been verified to interact with M. aeruginosa at environmentally relevant concentrations (Liu et al., 2012a). The variation of the growth rate, the generation of ROS and the responses of various antioxidants in M. aeruginosa following exposure to amoxicillin, as well as the biodegradation of amoxicillin by M. aeruginosa, were investigated at different nitrogen levels. Combined effects of nitrogen and antibiotic contaminants on M. aeruginosa have not been reported by previous studies. The observed results would contribute to a better understanding of the formation of cyanobacterial bloom with the coexistence of antibiotic contaminants.
2. Materials and methods 2.1. Cellular response test An axenic culture of the cyanobacterium M. aeruginosa PCC7806 was supplied by the Pasteur Culture Collection of Cyanobacteria (Paris, France). Amoxicillin was purchased from SigmaAldrich, Inc. (Shanghai, China), and the stock solution was prepared in methanol and stored at 20 °C prior to use. M. aeruginosa was pre-cultivated under aseptic conditions for two weeks in sterile BG11 medium at 25 71 °C under a 16:8 light:dark cycle provided by cool white fluorescent lights at an intensity of 40 μ mol photons m 2 s 1. The BG11 medium contained nitrate as the source of nitrogen, with a nitrogen concentration of 247 mg L 1. After pre-cultivation, the M. aeruginosa cells reaching the exponential growth phase were collected by centrifugation (4000g, 4 °C, 5 min) and used as the inoculums for the cellular response test and the biodegradation test. Modified BG11 media were used in the cellular response test and the biodegradation test, which were divided into three test groups according to the nitrogen
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concentration in the modified BG11 medium, which were 0.05, 0.5 and 5 mg L 1, respectively. In each test group, 250-mL Erlenmeyer flasks, each containing 150 mL of the modified BG11 medium, were spiked with different concentrations of amoxicillin, each in triplicate. The test concentrations of amoxicillin in each test group were 200 ng L 1 and 500 ng L 1. Amoxicillin was replenished regularly to maintain a stable exposure dose, and the determined test concentrations deviated within an acceptable range (from 9% to þ 12%) compared with their nominal values. The final concentrations of methanol in the test media were below 0.01% (v/v). At each nitrogen level, three flasks containing modified BG11 media with the same nitrogen concentration and 0.01% (v/v) methanol, but without amoxicillin, were prepared as the non-antibiotic-treated control. The initial cell density of M. aeruginosa in each flask was 4 105 cells mL 1. After inoculation, the M. aeruginosa cells were cultured under the same aseptic condition as the pre-cultivation for seven days. The entire experimental apparatus used for the culture of M. aeruginosa and addition of amoxicillin were sterilized by autoclaving at 121 °C for 20 min prior to use. The flasks were shaken well before each sampling. Sampling was conducted in a laminar flow cabinet using sterile apparatus. One milliliter of algal culture was aseptically sampled from each flask every day and used for cell density counting. The specific growth rate was calculated according to Eq. (1):
μ (/day) = (LnX1 − LnX 0 )/(t1 − t0 )
(1)
where X0 and X1 were the cell density at the beginning (t0) and the end (t1) of the selected time interval during the exponential phase of growth, respectively. 2.2. Analysis of ROS and antioxidant responses In the cellular response test, the culture medium was aseptically sampled from each flask after 4 and 7 days of exposure. For the analysis of ROS, the M. aeruginosa cells were collected by centrifugation at 2000g at 4 °C for 10 min, loaded with 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) probes and then measured with a fluorescence plate reader (Bio-TEK, USA), according to the method of Knauert and Knauer (2008), with an excitation filter of 485 nm and an emission filter of 530 nm. DCFHDA is a cell-permeable indicator, which was hydrolyzed by the cellular esterase to form the non-fluorescent DCFH after penetrating into the cells. DCFH is immediately transformed to highly fluorescent 2′,7′-dichlorofluorescein (DCF) in the presence of ROS, and the DCF fluorescence indicates the ROS level. For the analysis of the antioxidants, the M. aeruginosa cells harvested by centrifugation were re-suspended in 1.5 mL of phosphate buffer (100 mM, pH ¼7.4) containing 1% (w/v) of polyvinylpyrrolidone and homogenized on ice by an ultrasonic cell pulverizer at 200 W with total time of 5 min (ultrasonic time: 2 s; rest time: 4 s). The homogenate was then centrifuged at 12,000g at 4 °C for 10 min. The supernatant was used for the determination of antioxidants. The activities of superoxide dismutase (SOD), peroxidase (POD) and GST and the contents of malondialdehyde (MDA) and GSH were determined according to previous studies (Gao and Tam, 2011; Wang et al., 2013a). One unit of SOD activity was defined as the amount of enzyme that caused a 50% decrease in the SODinhibited nitro-blue tetrazolium reduction. One unit of POD activity was defined as the amount of enzyme required for the formation of purpurogallin from pyrogallol in 20 s at pH 6.0. One unit of GST activity was defined as the amount of enzyme that catalyzed the formation of 1 μmol of S-(2.4-dinitrophenyl)-glutathione per minute at 37 °C. The activities of the antioxidant enzymes and the contents of non-enzymatic antioxidants in M. aeruginosa were
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μg per 106 cells,
3.1. Combined effects of amoxicillin and nitrogen on the growth of M. aeruginosa
2.3. Biodegradation test The experimental set-up for the biodegradation test was similar to that for the cellular response test, except for the following: (i) three 1000-mL Erlenmeyer flasks, each containing 400 mL of modified sterile BG11 medium inoculated with M. aeruginosa and spiked with amoxicillin, were prepared for each amoxicillin concentration at each nitrogen level, (ii) amoxicillin was only spiked at the beginning of the test with no replenishment during the entire exposure period, (iii) both an abiotic control and a nonantibiotic-treated control were prepared, and (iv) three flasks containing modified BG11 media spiked with amoxicillin, but without the M. aeruginosa cells, were prepared as the abiotic control for each amoxicillin concentration at each nitrogen level to measure the abiotic loss of amoxicillin from the culture medium. In the biodegradation test, 100 mL of culture medium was sampled after 4 and 7 days of incubation. Sampled medium was separated from the M. aeruginosa cells by filtering through a Whatman GF/B filtration membrane (1 μm). For the determination of the antibiotic concentration in the culture medium, the filtrate was mixed with 0.2 mL of 5% (w/v) ethylene diamine tetraacetic acid (EDTA), and then extracted with an Oasis HLB glass cartridge at a flow rate of 2 mL min 1. Oasis HLB glass cartridges (200 mg, 5 mL) were obtained from Waters Corporation (Massachusetts, USA). The cells harvested by the filtration membrane were freezedried, extracted with 10 mL of acetonitrile under sonication and centrifuged at 4000g at 4 °C for 20 min to remove the cell debris. The extract was also mixed with 0.2 ml of 5% (w/v) EDTA and Milli-Q water to a final volume of 100 mL and then extracted with an Oasis HLB glass cartridge to measure the adsorption of amoxicillin in the M. aeruginosa cells. After extraction, the Agilent 1200 series liquid chromatograph coupled with an Agilent series 6410B triple quadruple mass spectrometer (Agilent, USA) was used for the analysis of amoxicillin. The extraction, cleanup, quantification method and analytical condition of the liquid chromatography– tandem mass spectrometry (LC–MS/MS) system were described in detail by a previous study by our group (Liu et al., 2012b). The biodegradation percentage (BP) of amoxicillin in M. aeruginosa was calculated according to Eq. (2), where Cn was the nominal test concentration of amoxicillin in the culture medium, and Ca, Ct and Cm were the measured concentrations of amoxicillin in the abiotic control, the culture medium and the M. aeruginosa cells, respectively.
BP (%) = (Ca − Ct − Cm )/Cn × 100%
3. Results
(2)
2.4. Statistical analysis Statistical analysis was conducted using the SPSS software (Version 13.0). A parametric two-way analysis of variance (ANOVA) was used to determine the interactions between nitrogen and amoxicillin on the growth rate of M. aeruginosa. A parametric three-way ANOVA was used to determine the influences of the culture time, nitrogen and amoxicillin on the levels of ROS, antioxidants and BP values. A parametric one-way ANOVA coupled with a post-hoc comparison was used to determine the differences in the growth rate, cell density, and the levels of ROS and antioxidants among different amoxicillin concentrations at each nitrogen level.
During the culture of M. aeruginosa, the exponential growth in all of the culture media started from one day after inoculation and lasted for three days. Afterwards, the culture gradually entered the stationary phase. As shown in Fig. 1, the growth rates of the exponential phase increased with increasing nitrogen concentration. At the lowest nitrogen level of 0.05 mg L 1, the growth of M. aeruginosa was not significantly (p 40.05) influenced by amoxicillin. At the moderate nitrogen level of 0.5 mg L 1, the growth was only stimulated by 500 ng L 1 of amoxicillin, with an increase of 20.7% compared to the non-antibiotic-treated control. At the highest nitrogen level of 5 mg L 1, the growth was stimulated by both 200 ng L 1 and 500 ng L 1 of amoxicillin, with respective increases of 18.2% and 25.3% compared to the non-antibiotictreated control. These results indicated that increased nitrogen concentration enhanced the stimulation effect of amoxicillin on the growth of M. aeruginosa. Similar to the growth rate, the cell density of M. aeruginosa was also positively correlated with nitrogen concentration (Table 1). Significant stimulation effect of amoxicillin on cell density (p o0.05) was also observed at test concentration of 500 ng L 1 in culture media with 0.5 mg L 1 of nitrogen and at test concentrations of 200 ng L 1 and 500 ng L 1 in culture media with 5 mg L 1 of nitrogen. Two-way ANOVA analysis (Table 2) presented significant interaction (p o0.05) between nitrogen and amoxicillin on the growth of M. aeruginosa. 3.2. Antioxidant responses to amoxicillin at different nitrogen levels The intensity of DCF fluorescence in Fig. 2a indicates that the ROS level in M. aeruginosa was significantly influenced (p o0.05) by the culture time. The ROS level in the stationary phase (day 7) was higher than that in the exponential phase (day 4) at each nitrogen level and at each amoxicillin concentration. Nitrogen showed no significant effect (p 40.05) on the ROS level. Exposure to amoxicillin caused a significant increase (p o0.05) in ROS levels in the M. aeruginosa cells at each nitrogen level and at each sampling time. The activities of SOD and POD in the non-antibiotic-treated control increased significantly (p o0.05) with 0.5
Non-antibiotic-treated -1 500ng L of AM-treated
-1
200 ng L of AM-treated
b
b
0.4
a -1
expressed in units (U) per 106 cells and respectively.
Growth rate (d )
140
0.3
0.2
b a
a
0.1
0.0 0.05
0.5
5 -1
Test concentration of nitrogen (mg L ) Fig. 1. Effect of amoxicillin (AM) on growth rates of M. aeruginosa at different nitrogen levels. Mean and standard deviation of three replicates are shown. Alphabet letters a and b indicate significant difference in means among test concentrations of AM at each nitrogen level at p o 0.05 according to one-way ANOVA test.
Amoxicillin was not detected in the cells of M. aeruginosa (Cm ¼0), suggesting that the removal of amoxicillin from the
o 0.01 o 0.01 0.491 0.208 0.805 0.661 0.847 48.827 73.319 0.488 1.677 0.062 0.421 0.167 o 0.01 o 0.01 o 0.01 o 0.01 0.04 o 0.01 0.162 107.185 437.897 131.894 23.043 6.602 13.612 1.742 o 0.01 o 0.01 o 0.01 0.01 0.036 0.042 0.207 NA: not available.
p F value p F value p F value
36.239 157.135 88.863 8.551 3.654 2.775 1.557 0.083 0.087 o 0.01 0.316 0.458 0.379 0.738 2.568 2.047 13.221 1.190 0.799 1.084 0.497 o0.01 o0.01 o0.01 0.110 0.534 o0.01 0.873 23.214 101.184 91.191 2.346 0.639 7.142 0.304 o 0.01 o 0.01 o 0.01 0.098 0.064 0.035 0.138 33.758 105.441 56.320 2.481 2.969 2.911 1.863 0.017 0.079 o 0.01 0.603 0.494 o 0.01 0.989 6.236 2.730 84.061 0.513 0.720 6.707 0.075 NA o 0.01 0.03 NA NA 0.025 NA a
3.3. Biodegradation of amoxicillin
NA 872.905 7.883 NA NA 3.622 NA
increasing nitrogen concentrations in the culture media and declined significantly (p o0.05) with the culture time (Fig. 2b and c). The SOD activities showed significant increases (p o0.05) in response to amoxicillin and a positive correlation with the test concentrations of amoxicillin during the entire exposure period at nitrogen levels of 0.5–5 mg L 1. In contrast, no significant alteration in the SOD activity in response to amoxicillin was recorded at the lowest nitrogen level of 0.05 mg L 1 (p 40.05). The POD activity in M. aeruginosa under exposure to amoxicillin varied in a manner similar to that of SOD at each nitrogen level and at each sampling time. Similar to SOD and POD, the GST activity and GSH content in the non-antibiotic-treated control were also positively correlated with nitrogen concentration and negatively correlated with the culture time (Fig. 3a and b). Amoxicillin significantly stimulated (p o0.05) GST activity and GSH content at each nitrogen level during the entire exposure period. The highest GST activity and GSH content were observed in 500 ng L 1 of amoxicillin-treated medium at the highest nitrogen level of 5 mg L 1, with respective increases of 84.6% and 73.8% relative to the non-antibiotic-treated control. The culture time, nitrogen and amoxicillin showed no significant interaction on ROS and antioxidant levels, according to the three-way ANOVA analysis (p 40.05). Instead, significant twoway interactions (p o0.05) between nitrogen and amoxicillin were observed on the ROS level, activities of SOD, POD and GST, as well as content of GSH (Table 2). The MDA content in the non-antibiotic-treated control was not significantly affected (p 40.05) by nitrogen and the culture time (Fig. 3c). No significant alteration of the MDA content was observed in M. aeruginosa under exposure to amoxicillin at the fourth day of exposure (p 40.05). The MDA content was significantly stimulated (po 0.05) by amoxicillin at all of the nitrogen levels at the seventh day of exposure and showed a positive correlation with test concentrations of amoxicillin. The most significant increase of MDA content was observed in 500 ng L 1 of the amoxicillin-treated group at the lowest nitrogen level, which was 1.54fold higher than the non-antibiotic-treated control.
Time Nitrogen Amoxicillin Time Nitrogen Time Amoxicillin Nitrogen Amoxicillin Time Nitrogen Amoxicillin
AM: amoxicillin.
p
17.5 7 0.41
F value
14.7 7 0.47
p
13.6 70.42 16.9 70.31
F value
11.2 7 0.53 13.5 7 0.52
p
Non-antibiotic-treated 200 ng L 1 of AMtreated 500 ng L 1 of AMtreated
F value
8.45 7 0.27
p
7.21 70.37
F value
7.45 7 0.45 7.58 7 0.25
p
6.337 0.39 6.417 0.32
141
a
Non-antibiotic-treated 200 ng L 1 of AMtreated 500 ng L 1 of AMtreated
F value
5.727 0.49
BP value
5.34 7 0.28
GSH content
5.42 7 0.26 5.51 70.35
GST activity
5.187 0.35 5.247 0.42
MDA content
Non-antibiotic-treated 200 ng L 1 of AMtreateda 500 ng L 1 of AMtreated
POD activity
a
7 Days
SOD activity
5
4 Days
ROS level
0.5
Cell density (105 cells mL 1)
Algal growth
0.05
Culture media
Dependent variables
Nitrogen levels (mg L 1)
Factors
Table 1 Cell density of M. aeruginosa after 4 and 7 days of culture in different media at different nitrogen levels.
Table 2 Summary of two-way ANOVA analysis between nitrogen and amoxicillin on algal growth rate and three-way ANOVA analysis among culture time, nitrogen and amoxicillin on ROS and antioxidant levels as well as BP values.
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500 ng L of AM-treated
a,b a
c c
a
25
b
a,b
b
b
b
a
a
a
a
20 15 10
c
500 ng L of AM-treated
25
b 20 15
b
c
b
b
a
a,b a
a
b 10
b
c
b a,b
a
a
a 5
5
0
0 0.05
0.5
5
0.05
Day 4
0.5
0.05
5
0.5
0.05
200 ng L of AM-treated
cell)
35
6
b
b
6
b b b
c
500 ng L of AM-treated
-1
25
200 ng L of AM-treated
-1
500 ng L of AM-treated
30
-1
Non-antibiotic-treated
-1
Non-antibiotic-treated
c
b a
b
b
a
b
c c
b
a a a,b a
15
b
a
10
20
5
-1
20
45
0.5
Day 7
Test concentration of nitrogen (mg L )
-1
40
5
Day 4
Day 7
Test concentration of nitrogen (mg L )
SOD activity (U per 10 cell)
200 ng L of AM-treated
-1
6
c
b
b
-1
Non-antibiotic-treated
c
-1
35 30
200 ng L of AM-treated
GST activity (U per 10 cell)
40
30
-1
Non-antibiotic-treated
4
DCF fluorescence intensity per 10 cell
45
b
a
a
a
b
a,b
b
a
10 5 0
0 0.05
0.5
5
0.05
Day 4
0.5
0.05
5
0.5
5
0.05
Day 4
Day 7
0.5
5
Day 7 -1
Test concentration of nitrogen (mg L )
-1
Test concentration of nitrogen (mg L )
10 40
Non-antibiotic-treated
-1
Non-antibiotic-treated
200 ng L of AM-treated
500 ng L of AM-treated
cell)
-1
b b
6
500 ng L of AM-treated
30
c
6
POD activity (U per 10 cell)
-1
200 ng L of AM-treated
-1
b
b
b
b
c
b
20
b
a,b
c
a
5
a,b a
a
b
a
a
a
a
10
0 0.05
0 0.05
0.5
Day 4
5
0.05
0.5
5
Day 7 -1
0.5
Day 4
5
0.05
0.5
5
Day 7 -1
Test concentration of nitrogen (mg L )
Test concentration of nitrogen (mg L ) Fig. 2. Effect of amoxicillin (AM) on (a) ROS level, (b) SOD activity and (c) POD activity of M. aeruginosa at different nitrogen levels after 4 and 7 days of exposure. Mean and standard deviation of three replicates are shown. Alphabet letters a, b and c indicate significant difference in means among test concentrations of AM at each nitrogen level and each exposure time at po 0.05 according to one-way ANOVA test.
culture media was mainly due to biodegradation, rather than accumulation. According to Fig. 4, the biodegradation of amoxicillin in M. aeruginosa was positively correlated with both the nitrogen concentration and the culture time, although it was not significantly affected by the increasing concentration of amoxicillin (p 40.05). At the end of exposure, the respective biodegradation
Fig. 3. Effect of amoxicillin (AM) on (a) GST activity, (b) GSH content and (c) MDA content of M. aeruginosa at different nitrogen levels after 4 and 7 days of exposure. Mean and standard deviation of three replicates are shown. Alphabet letters a, b and c indicate significant difference in means among test concentrations of AM at each nitrogen level and each exposure time at p o 0.05 according to one-way ANOVA test.
percentages (BP) of amoxicillin were 18.5–19.6%, 25.2–26.6% and 29.6–30.5% at nitrogen levels of 0.05, 0.5 and 5 mg L 1. The threeway ANOVA analysis (Table 2) on BP values presented neither significant three-way interaction among the culture time, nitrogen and amoxicillin (p 40.05) nor significant two-way interaction between any two of the three factors (p 40.05).
Y. Liu et al. / Ecotoxicology and Environmental Safety 111 (2015) 138–145
40
-1
200 ng L of AM
-1
500 ng L of AM
BP (%)
30
20
10
0 0.05
0.5
Day 4
5
0.05
0.5
5
Day 7 -1
Test concentration of nitrogen (mg L ) Fig. 4. The biodegradation percentages (BP) of amoxicillin (AM) in M. aeruginosa at different nitrogen levels after 4 and 7 days of exposure. Mean 7standard deviation values of three replicates are shown.
4. Discussion Various researches have demonstrated the ability of the nitrogen supply to positively regulate the growth of M. aeruginosa (Davis et al., 2009). The present study further verified the positive correlation between the nitrogen concentration and the growth rate. Increased nitrogen concentration also contributed to the stimulation effect of amoxicillin on the growth of M. aeruginosa. The action of amoxicillin in prokaryotes depends on the recognition of amoxicillin by intracellular penicillin-binding proteins (PBPs), and synthesis of PBPs is most active during the cell division process (Stevens et al., 1993). Therefore, amoxicillin is mainly effective against cells in the growth phase (Brisson-Noël et al., 1988). With a typical prokaryotic cell structure, cyanobacteria have also been proven to produce PBPs (Marbouty et al., 2009). Therefore, nitrogen may also regulate the responses of M. aeruginosa to amoxicillin via growth rate and synthesis of PBPs. The simulated growth of M. aeruginosa could be regarded as a hormesis effect of amoxicillin at low concentrations. Zhu et al. (2012) also observed similar results, showing that exposure of M. aeruginosa to a mixture of low concentrations of polycyclic aromatic hydrocarbons (PAHs) led to increased growth of M. aeruginosa. The ROS level in M. aeruginosa remained stable at different nitrogen levels. Liu et al. (2007) investigated the effects of nutrients on a microalgae Chattonella marina and similarly observed that the nitrogen supply regulated algal growth but did not affect ROS production. Increased production of ROS was commonly observed in M. aeruginosa under exposure to various environmental stresses, including heavy metals, allelochemicals (Qian et al., 2010), ultraviolet radiation (Jiang and Qiu, 2011) and herbicides (Jin et al., 2012). In this study, exposure to amoxicillin also stimulated ROS production, indicating that amoxicillin led to oxidative stress in M. aeruginosa. Increased ROS levels in response to amoxicillin were observed, even at the lowest nitrogen level. This result indicated that generation of ROS was more sensitive to amoxicillin exposure, compared with the growth of M. aeruginosa. The higher ROS level at a longer exposure time (day 7) indicated that the production of ROS under amoxicillin exposure was time dependent. This result was in accordance with a previous study in which ROS production in M. aeruginosa exposed to catechin and pyrogallic acid also increased with increasing exposure time (Wang et al., 2011). The activities of SOD, POD and GST and the content of GSH on the fourth day of the culture were significantly higher (p o0.05) than that on the seventh day, indicating that synthesis of
143
antioxidants was related to the growth phase of M. aeruginosa. Synthesis of antioxidants was also related to nitrogen. The activities of SOD, POD and GST and the content of GSH in M. aeruginosa were positively correlated with nitrogen concentration at each sampling time. Nitrogen is an indispensable element in the molecular structure of protein, and has been proven to positively regulate the synthesis of enzymes and peptides in M. aeruginosa (Downing et al., 2005). Therefore, the nitrogen-dependent synthesis of these antioxidants could be explained by their molecular structures. The antioxidants SOD and POD played an important role in eliminating ROS and protecting the microalgae from oxidative damage (Blokhina et al., 2003). SOD converts superoxide radicals into hydrogen peroxide, and POD converts hydrogen peroxide into water and oxygen (Cho and Seo, 2005). Increased activities of SOD and POD were found to protect M. aeruginosa from low doses of gamma irradiation (Zheng et al., 2012). The stimulation of SOD activity was reported to be responsible for the tolerance of a diatom Phaeodactylum tricornutum to antibiotic exposure (Yang et al., 2013). Shen et al. (2014) observed that SOD and POD could eliminate pesticide-induced ROS from a green alga Chlorella vulgaris. In the present study, responses of SOD and POD to amoxicillin indicated that these two enzymes did not participate in eliminating ROS from M. aeruginosa at the lowest nitrogen level. Increased activities of SOD and POD under exposure to amoxicillin were observed at higher nitrogen levels. These results indicated that the antioxidant responses to amoxicillin were enhanced by increasing nitrogen concentration. Due to the stimulation of SOD and POD, M. aeruginosa was more adaptive to amoxicillin at higher nitrogen levels, as indicated by the increased growth rate. GST and GSH could also scavenge ROS from the microalgae (Belchik and Xun, 2011). Wang et al. (2013a) found that GSH participated in eliminating ROS from the green alga Selenastrum capricornutum and was stimulated under exposure to heavy metals and PAHs. Mofeed and Mosleh (2013) observed increased GST activity and GSH content in the green alga Scenedesmus obliquus under oxidative stresses caused by two pesticides, fenhexamid and atrazine. Following exposure to amoxicillin, increased activities of GST and elevated synthesis of GSH were observed at all of the nitrogen levels. This result suggested that GSH and GST were more sensitive than SOD and POD in responding to oxidative stress caused by amoxicillin. Wang and Xie (2007) examined the effects of NPs on the antioxidant system of M. aeruginosa and similarly observed that GSH and GST contributed more to eliminating ROS than SOD. Wang et al. (2013b) also found that GSH was more sensitive to oxidative stresses in Chlorella sp. exposed to estradiol and ethinylestradiol, compared with SOD and POD. Along with the elimination of ROS, GST also played an important role in the metabolism of exogenous chemicals by catalyzing the conjugation of exogenous chemicals with GSH for the purpose of detoxification (Belchik and Xun, 2011). The biodegradation of amoxicillin coupled with stimulated GST activity and GSH content further verified the contribution of GST and GSH to the detoxification process of M. aeruginosa. The variation of BP values indicated that the biodegradation of amoxicillin by M. aeruginosa was enhanced by nitrogen. The positive correlation between nitrogen and the synthesis of GST and GSH may explain the elevated BP values with increasing nitrogen concentrations. Stimulated growth and increased cell density of M. aeruginosa at higher nitrogen levels could also lead to increased biodegradation because more algal cells participate in the biodegradation process. Stoichev et al. (2011) reported a biodegradation ability of less than 12% in M. aeruginosa exposed to an antibiotic minocycline at an initial concentration of 2 μM. A previous study also observed 7-day BP values of 12.5%–32.9% for spiramycin in M. aeruginosa (Liu et al., 2012b). The above comparison indicated that amoxicillin
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was more degradable than minocycline and comparable to spiramycin for M. aeruginosa. Studies on the biodegradation of other antibiotics in M. aeruginosa were still limited. Bioaccumulation of amoxicillin in M. aeruginosa was not found in this study, possibly due to the hydrophilicity of this antibiotic. As a product of lipid peroxidation, increased MDA content is a commonly used indicator of oxidative damage (Wang et al., 2013b). Increased MDA content at the seventh day of exposure indicated that amoxicillin caused lipid peroxidation in M. aeruginosa at all of the nitrogen levels. The amoxicillin-induced MDA content increased with decreasing nitrogen concentrations. This result was in accordance with the antioxidant responses. More significant antioxidant responses were observed at higher nitrogen levels, such that the amoxicillin-induced oxidative damage was alleviated. Similar to ROS, the formation of MDA was also time dependent. Kong et al. (2013) also similarly reported that the increase of MDA content in actinomycetes-treated M. aeruginosa gradually increased with time. The lower antioxidant levels in the stationary phase compared with the exponential phase was another possible reason for the increased formation of MDA in the stationary phase. Reported nitrogen concentrations were 0.03–0.6 mg L 1 in moderately polluted waters and up to 100 mg L 1 in heavily polluted waters (Polyak et al., 2013). Currently detected concentrations of amoxicillin in aquatic environments were below 622 ng L 1 (Zuccato et al., 2010). The test concentrations of nitrogen and amoxicillin in this study covered their actual contamination levels. Observed results indicated coexisting amoxicillin contaminant would aggravate the pollution of M. aeruginosa via the growth stimulation effect, when the contamination concentration of nitrogen was Z0.5 mg L 1. In this case, amoxicillin should also be removed from aquatic environments during the control of M. aeruginosa bloom. At the lowest nitrogen level of 0.05 mg L 1, coexisting amoxicillin had no significant effect on the pollution of M. aeruginosa, while M. aeruginosa would alleviate the pollution of amoxicillin via biodegradation. Significant interactions between nitrogen and amoxicillin on the antioxidant system of M. aeruginosa, as indicated by the three-way ANOVA analysis, still deserve further investigation, in order to fully interpret the mechanisms of combined action of nitrogen and amoxicillin.
5. Conclusions At current contamination levels of 0.05–5 mg L 1 for nitrogen and 200–500 ng L 1 for amoxicillin, the two factors significantly interact with each other (po 0.05) on the growth of M. aeruginosa, the generation of ROS and the biodegradation of amoxicillin. Elevated nitrogen concentrations significantly enhanced (p o0.05) the detoxification of amoxicillin via antioxidants, including SOD, POD, GST and GSH, and consequently led to stimulated growth of M. aeruginosa as an adaptive response to amoxicillin stress at higher nitrogen levels of 0.5–5 mg L 1. At the lowest nitrogen level of 0.05 mg L 1, non-participation of SOD and POD in eliminating ROS may explain the high lipid peroxidation level in M. aeruginosa exposed to amoxicillin. The biodegradation of amoxicillin was also positively correlated with nitrogen concentration, possibly via the regulation of GSH conjugation. The coexistence of nitrogen and amoxicillin contaminants with M. aeruginosa would pose an increased threat to aquatic environments and should be considered during the control of cyanobacterial bloom.
Acknowledgments This work was supported by National Natural Science Foundation of China (51209125) and partly by Research Foundation for the Doctoral Program of Higher Education of China (20110131120014) and Promotive Research Foundation for Young and Middle-aged Scientists of Shandong Province of China (2013BSE27073).
References Babica, P., Bláha, L., Maršálek, B., 2006. Exploring the natural role of microcystins – a review of effects on photoautotrophic organisms. J. Phycol. 42, 9–20. Belchik, S.M., Xun, L., 2011. S-glutathionyl-(chloro) hydroquinone reductases: a new class of glutathione transferases functioning as oxidoreductases. Drug Metab. Rev. 43, 307–316. Blokhina, O., Virolainen, E., Fagerstedt, K.V., 2003. Antioxidants, oxidative damage, and oxygen deprivation stress: a review. Ann. Bot. Lond. 91, 179–194. Brisson-Noël, A., Trieu-Cuot, P., Courvalin, P.J., 1988. Mechanism of action of amoxicillin and other macrolidesJ. Antimicrob. Chemother. 22 (Suppl. B), S13–S23. Chalifour, A., Juneau, P., 2011. Temperature-dependent sensitivity of growth and photosynthesis of Scenedesmus obliquus, Navicula pelliculosa and two strains of Microcystis aeruginosa to the herbicide atrazine. Aquat. Toxicol. 103, 9–17. Cho, U.H., Seo, N.H., 2005. Oxidative stress in Arabidopsis thaliana exposed to cadmium is due to hydrogenperoxide accumulation. Plant Sci. 168, 113–120. Davis, T.W., Berry, D.L., Boyer, G.L., Gobler, C.J., 2009. The effects of temperature and nutrients on the growth and dynamics of toxic and non-toxic strains of Microcystis during cyanobacteria blooms. Harmful Algae 8, 715–725. de Morais, P., Stoichev, T., Basto, M.C.P., Ramos, V., Vasconcelos, V.M., Vasconcelos, M.T.S.D., 2014. Cyanobacterium Microcystis aeruginosa response to pentachlorophenol and comparison with that of the microalga Chlorella vulgaris. Water Res 52, 63–72. Downing., T.G., Sember, C.S., Gehringer, M.M., Leukes, W., 2005. Medium N:P ratios and specific growth rate comodulate microcystin and protein content in Microcystis aeruginosa PCC7806 and M. aeruginosa UV027. Microb. Ecol. 49, 468–473. Gao, Q.T., Tam, N.F.Y., 2011. Growth, photosynthesis and antioxidant responses of two microalgal species, Chlorella vulgaris and Selenastrum capricornutum, to nonylphenol stress. Chemosphere 82, 346–354. Hu, X., Liu, Y.G., Zeng, G.M., Hu, X.J., Wang, Y.Q., Zeng, X.X., 2014. Effects of limonene stress on the growth of and microcystin release by the freshwater cyanobacterium Microcystis aeruginosa FACHB-905. Ecotoxicol. Environ. Saf. 105, 121–127. Jiang, Y., Ji, B., Wong, R.N.S., Wong, M.H., 2008. Statistical study on the effects of environmental factors on the growth and microcystins production of bloomforming cyanobacterium – Microcystis aeruginosa. Harmful Algae 7, 127–136. Jiang, H.B., Qiu, B.S., 2011. Inhibition of photosynthesis by UV-B exposure and its repair in the bloom-forming cyanobacterium Microcystis aeruginosa. J. Appl. Phycol. 23, 691–696. Jin, Z.P., Luo, K., Zhang, S., Zheng, Q., Yang, H., 2012. Bioaccumulation and catabolism of prometryne in green algae. Chemosphere 87, 278–284. Knauert, S., Knauer, K., 2008. The role of reactive oxygen species in copper toxicity to two freshwater green algae. J. Phycol. 44, 311–319. Kong, Y., Zou, P., Yang, Q., Xu, X.Y., Miao, L.H., Zhu, L., 2013. Physiological responses of Microcystis aeruginosa under the stress of antialgal actinomycetes. J. Hazard. Mater. 262, 274–280. Liu, W.H., Au, D.W.T., Anderson, D.M., Lam, P.K.S., Wu, R.S.S., 2007. Effects of nutrients, salinity, pH and light:dark cycle on the production of reactive oxygen species in the alga Chattonella marina. J. Exp. Mar. Biol. Ecol. 346, 76–86. Liu, Y., Gao, B.Y., Yue, Q.Y., Guan, Y.T., Wang, Y., Huang, L.H., 2012a. Influences of two antibiotic contaminants on the production, release and toxicity of microcystins. Ecotoxicol. Environ. Saf. 77, 79–87. Liu, Y., Guan, Y.T., Gao, B.Y., Yue, Q.Y., 2012b. Antioxidant responses and degradation of two antibiotic contaminants in Microcystis aeruginosa. Ecotoxicol. Environ. Saf. 86, 23–30. Mallick, N., Mohn, F.H., 2000. Reactive oxygen species: response of algal cells. J. Plant Physiol. 157, 183–193. Marbouty, M., Mazouni, K., Saguez, C., Cassier-Chauvat, C., Chauvat, F., 2009. Characterization of the Synechocystis strain PCC 6803 penicillin-binding proteins and cytokinetic proteins FtsQ and FtsW and their network of interactions with ZipN. J. Bacteriol. 191, 5123–5133. Mofeed, J., Mosleh, Y.Y., 2013. Toxic responses and antioxidative enzymes activity of Scenedesmus obliquus exposed to fenhexamid and atrazine, alone and inmixture. Ecotoxicol. Environ. Safe 95, 234–240. Paerl, H.W., Huisman, J., 2009. Climate change: a catalyst for global expansion of harmful cyanobacterial blooms. Environ. Microbiol. Rep 1, 27–37. Perrona, M.C., Juneau, P., 2011. Effect of endocrine disrupters on photosystem II energy fluxes of green algae and cyanobacteria. Environ. Res. 11, 520–529.
Y. Liu et al. / Ecotoxicology and Environmental Safety 111 (2015) 138–145
Polyak, Y., Zaytseva, T., Medvedeva, N., 2013. Response of toxic cyanobacterium Microcystis aeruginosa to environmental pollution. Water Air Soil Pollut. 224, 1494–1508. Qian, H.F., Pan, X.J., Chen, J., Zhou, D.M., Chen, Z.G., Zhang, L., Fu, Z.W., 2012. Analyses of gene expression and physiological changes in Microcystis aeruginosa reveal the phytotoxicities of three environmental pollutants. Ecotoxicology 21, 847–859. Qian, H.F., Yu, S.Q., Sun, Z.Q., Xie, X.C., Liu, W.P., Fu, Z.W., 2010. Effects of copper sulfate, hydrogen peroxide and N-phenyl-2-naphthylamine on oxidative stress and the expression of genes involved photosynthesis and microcystin disposition in Microcystis aeruginosa. Aquat. Toxicol. 99, 405–412. Shen, Y.F., Liu, L., Gong, Y.X., Zhu, B., Liu, G.L., Wang, G.X., 2014. Potential toxic effect of trifloxystrobin on cellular microstructure, mRNA expression and antioxidant enzymes in Chlorella vulgaris. Environ. Toxicol. Pharmacol. 37, 1040–1047. Stevens, D.L., Yan, S., Bryant, A.E., 1993. Penicillin-binding protein expression at different growth stages determines penicillin efficacy in vitro and in vivo: an explanation for the inoculum effect. J. Infect. Dis. 167, 1401–1405. Stoichev, T., Baptista, M.S., Basto, M.C.P., Vasconcelos, V.M., Vasconcelos, M.T.S.D., 2011. Effects of minocycline and its degradation products on the growth of Microcystis aeruginosa,. Ecotoxicol. Environ. Saf. 74, 219–224. Veldhuis, M.J.W., Wassmann, P., 2005. Bloom dynamics and biologicalcontrol of a high biomass HAB species in European coastal waters: a Phaeocystis case study. Harmful Algae 4, 805–809. Wang, P., Luo, L.J., Ke, L., Luan, T.G., Tam, N.F.Y., 2013a. Combined toxicity of polycyclic aromatic hydrocarbons and heavy metals to biochemical and antioxidant
145
responses of free and immobilized Selenastrum capricornutum. Environ. Toxicol. Chem. 32, 673–683. Wang, P., Wong, M.H., Tam, N.F.Y., 2013b. Antioxidant responses of two microalgae, Selenastrum capricornutum and Chlorella sp., to estradiol and ethinylestradiol. J. Appl. Phycol. 25, 891–903. Wang, J., Xie, P., 2007. Antioxidant enzyme activities of Microcystis aeruginosa in response to nonylphenols and degradation of nonylphenols by M. aeruginosa. Environ. Geochem. Health 29, 375–383. Wang, J., Zhu, J.Y., Liu, S.P., Liu, B.Y., Gao, Y.N., Wu., Z.B., 2011. Generation of reactive oxygen species in cyanobacteria and green algae induced by allelochemicals of submerged macrophytes. Chemosphere 85, 977–982. Yang, C.Y., Zhou, J., Liu, S.J., Fan, P., Wang, W.H., Xia, C.H., 2013. Allelochemical induces growth and photosynthesis inhibition, oxidative damage in marine diatom Phaeodactylum tricornutum. J. Exp. Mar. Biol. Ecol. 444, 16–23. Zheng, B.G., Zheng, Z., Zhang, J.B., Luo, X.Z., Liu, Q., Wang, J.Q., Zhao, Y.F., 2012. The removal of Microcystis aeruginosa in water by gamma-ray irradiation. Sep. Purif. Technol. 85, 165–170. Zhu, X.Z., Kong, H.L., Gao, Y.Z., Wu, M.F., Kong, F.X., 2012. Low concentrations of polycyclic aromatic hydrocarbons promote the growth of Microcystis aeruginosa. J. Hazard. Mater. 237, 371–375. Zuccato, E., Castiglioni, S., Bagnati, R., Melis, M., Fanelli, R., 2010. Source, occurrence and fate of antibiotics in the Italian aquatic environment. J. Hazard. Mater. 179, 1042–1048.
