Accepted Manuscript Title: The combined and second exposure effect of copper (II) and chlortetracycline on fresh water algae, Chlorella pyrenoidosa and Microcystis aeruginosa Author: Lei Lu Yixiao Wu Huijun Ding Weihao Zhang PII: DOI: Reference:
S1382-6689(15)30007-7 http://dx.doi.org/doi:10.1016/j.etap.2015.06.006 ENVTOX 2274
To appear in:
Environmental Toxicology and Pharmacology
Received date: Accepted date:
12-3-2015 3-6-2015
Please cite this article as: Lu, L., Wu, Y., Ding, H., Zhang, W.,The combined and second exposure effect of copper (II) and chlortetracycline on fresh water algae, Chlorella pyrenoidosa and Microcystis aeruginosa, Environmental Toxicology and Pharmacology (2015), http://dx.doi.org/10.1016/j.etap.2015.06.006 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.
*Highlights (for review)
Highlights The combined toxicity of copper and chlortetracycline on two algae was analyzed.
The biochemical features of both algae were different in the second exposure.
The toxicity of the complex on algae was analyzed in initial and second exposure.
There are significant differences of 96h EC50s between initial and second exposure.
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*Manuscript
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Ⅱ) and chlortetracycline on The combined and second exposure effect of copper (Ⅱ fresh water algae, Chlorella pyrenoidosa and Microcystis aeruginosa
College of Resource and Environmental Science, Wuhan University, Wuhan, PR China.
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Lei Lua, Yixiao Wua, Huijun Dinga, Weihao Zhanga
Correspondence to: Weihao Zhang;
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Tel.: +86-27-68777060
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E-mail: [email protected]
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Abstract In the experiment, Chlorella pyrenoidosa and Microcystis aeruginosa were chosen to test the
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individual, combined and second exposure effect of Cu2+ and chlortetracycline (CTC). The 96h EC50s of each test were calculated, with the ranges of 0.972 µmol/L-15.6 µmol/L (Cu2+),
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29.5 µmol/L-102.5 µmol/L (CTC), 14.4 µmol/L-78.9 µmol/L (mixture). The combined toxicities
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were evaluated with toxicity units (TU) method. The toxicity of complex of Cu2+ and chlortetracycline was analyzed using concentration addition (CA) model. In the initial test, the
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combined effect of the two substances was partly additive to C. pyrenoidosa and antagonistic to M. aeruginosa, while in the second exposure test, the combined effect was synergistic to both algae.
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The biochemical indicators measured in the experiment included chlorophyll fluorescence
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(Fv/Fm), MDA content, SOD activity and content of soluble proteins. When under combined
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stress, the biochemical features of both algae were significantly different between the initial test and the second exposure test.
Keywords Chlorella pyrenoidosa; Microcystis aeruginosa; chlortetracycline; copper; second
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exposure
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1 Introduction Tetracycline antibiotics (TCs) are a group of antibiotics which are widely used around the
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world. TCs along with some heavy metals are often used in animal feed as growth promoters and treatment for diseases, while the metabolism of which is incomplete (Kemper N., 2008). Thus, the
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antibiotics and heavy metals excreted by animals can enter the environment directly or indirectly
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through the emission of aquaculture wastewater. In soils collected from certain feedlots, the concentration of tetracyclines were at range of 4.54–24.66 mg/kg, while the typical heavy metal
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Cu was detected at concentration range of 32.3–730.1 mg/kg (Ji X.L. et al., 2012). In certain piggery and diary effluent, the total Cu concentrations were respectively at ranges of 0.1-1.55 mg/l
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and 0.5-10.5 mg/l (Bolan N.S. et al., 2003), while the total concentration of TCs in the land
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fertilized with organic manures was around 22.9 µg/kg (Wu L.H. et al., 2013). Though the
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wastewater treatment can be conducted, the removal rate of Epi-iso-chlorotetracycline was around 18%(Chang X.S. et al., 2010).Via the transportations among different media in the environment, once released, antibiotics and heavy metals are likely to cause water pollution and affect the
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aquatic system. The detections of antibiotics have been conducted in various environmental media (Christian T. et al., 2003, Tong L. et al., 2009) as well as organisms (Chafer-Pericas C. et al., 2011). Meanwhile the heavy metals can be released anthropogenically and naturally to the environment, the relatively high concentrations of which have been proved to be biologically harmful (Balamurugan K. et al., 2006). With multiple electron donor groups, TCs can react with other organic and inorganic ions, thus the heavy metal ions may affect their behavior in environment. The spatial correlation between antibiotic resistance of bacteria and metal concentration was found in contaminated 3
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streams (Tuckfield R.C. et al., 2008). Recently, the increasing attention has been drawn on the interaction between antibiotics and heavy metals. Oxytetracycline and ciprofloxacin can bound
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with three typical heavy metals, copper, zinc and cadmium, via multiple coordination sites, besides which the antibiotics, heavy metals and their complex acted primarily as concentration
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(Zhang Y. et al., 2012). Tetracycline and Cu2+ can form complex on the surface of the bifunctional
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adsorbent (Ma Y. et al., 2014). Though chlorotetracycline and copper are often used together, the study of their interaction is rare. In addition, the continuous emissions of antibiotics and heavy
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metals are likely to cause multiple stresses to the aquatic system, which means that the study of multiple exposures could be needed.
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In order to figure out whether the multiple stresses of typical heavy metals and veterinary antibiotics can cause heritable implications on the biochemical process of algae and whether the
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combined effects can become different in the second exposure, this study chose Cu2+ and chlortetracycline as stressors and investigated their individual and combined effect on two fresh water algae, C. pyrenoidosa and M. aeruginosa. The combined effects were evaluated and
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analyzed with TU method (Altenburger R. et al., 2003) and CA model (Liu S.S. et al., 2013). Based on those, the second exposure tests were also conducted. 2 Materials and methods 2.1 Chemicals
Chlortetracycline (>95%) was purchased from Kayon, Shanghai China; CuSO4·5H2O and other chemicals were purchased from Sinopharm Chemical Reagent Co., Shanghai China. The antibiotic was dissolved in 0.22 µm filtered sterile Milli-Q water, while the metal solutions were used after sterilized. 4
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2.2 Algae cultivation The tested algae Microcystis aeruginosa (FHACB-315) and Chlorella pyrenoidosa
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(FHACB-10) were purchased from Freshwater Algae Culture Collection at the Institute of Hydrobiology (FACHB-collection), Wuhan China. The experiment used BG11 as liquid culture
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medium. The cultivation was conducted in the PGX-illuminating incubator. The conditions were
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temperature as 25±2℃, light intensity as 2000lx and the light : dark circle as 12h : 12h. The biomass of algae was represented with the optical density under 680nm, before which the linear
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relation between the optical density and the cell number per milliliter had been built. The tests were conducted on the algae in logarithmic phase. Each concentration group had three parallel
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samples. During the experiment, all samples were shaken three times a day artificially and
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changed positions randomly.
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2.3 Toxicity tests 2.3.1 Individual toxicity tests
The experiment used algae in the logarithmic phase with the initial OD680nm around 0.200.
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The concentrations of CuSO4·5H2O were set as 0.08, 0.09, 0.18, 0.58, 1.08, 5.08 mg/L to M. aeruginosa and 0.08, 0.18, 0.58, 1.08, 5.08, 10.08 mg/L to C. pyrenoidosa. The concentration of
negative control was not zero because copper is a trace element required during the growth of the algae. The concentrations of chlortetracycline were set as 0.00, 1.50, 6.00, 12.00, 24.00, 48.00 mg/L to M. aeruginosa and 0.00, 1.00, 10.00, 20.00, 40.00, 60.00 mg/L to C. pyrenoidosa. The
stress time was 96 hours and the biomass of algae was tested every 24 hours. The inhibition rates were calculated with the equations as follows (Kong Q.X. et al., 2010): %I ⁄ 100 5
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Ct: the cell density of treated groups; Co: the cell density of the control; %I: the inhibition rates.
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2.3.2 Combined toxicity tests The concentrations of mixtures were set up depending on the 96h EC50s from the individual
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tests. And the concentrations were set as 0, 0.25, 0.50, 1.00, 1.25, 1.50 times of the combined 96h
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EC50s of the two substances to the two algae.
The effects of the combined toxicity were analyzed using TU method (Altenburger R. et al., 2003). The definition of toxicity units is as follows: ,
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Ci: concentration of the component i; EC50, i: the 96h EC50 of component i when it actions
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individually.
The analysis indicators M and Mo were defined as follows:
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max
(Position of Table 1)
The complex was described using expanded Benesi-Hildebrand equation (Wang R. et al.,
2007). Meanwhile the analysis of its toxicity to the two algae was described using CA model (Liu S.S. et al., 2013).
2.3.3 The second exposure tests The tested algae were first cultivated till their logarithmic phase. Then they were transfered to the treated media, whose concentrations were equal to the 96h EC50s. The OD680nm of the treated groups were around 0.200 and the stress time was 96h. After the stress, the algae were extracted 6
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by centrifugalizing. The centrifugal speed was 5000r/min. The supernatants were abandoned, after which the algae were suspended in sterilized water for 5 min before centrifugalized. The processes
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were repeated twice after that. After the last-time centrifugalizing the algae were suspended in the clean media and cultivated to logarithmic phase. During the procedure, all equipment was sterile.
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The experiment conditions of the second exposure tests were the same as the initial stress tests.
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2.3 Biochemical indicators
The biochemical indicators included chlorophyll fluorescence fv/fm, SOD activity, MDA
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content and soluble protein content. The chlorophyll fluorescence was measured 0.5, 24, 48, 72, 96 h after dosing during each test, while the other indicators were measured after 96-hour stress.
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The chlorophyll fluorescence fv/fm was tested using Handy-PEA (Hansatech Inc., British). The
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SOD activity determination used Nitroblue Tetrazolium Photoreduction method (Hong Y. et al.,
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2008). The content of MDA was measured by thiobarbituric acid method (Sabatini S.E. et al., 2009). The content of soluble protein was determined by Coomassie Brilliant blue G-250 method (Bradford M.M., 1976).
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2.4 Data analysis
The 96h EC50s were analyzed with Originlab software v.8.5 (Originlab Inc., USA). The
statistical analysis was conducted using Statixtical Package for the Social Sciences (SPSS) software v.18.0 (SPSS Inc., USA). 3 Results 3.1 Calculation of 96h EC50s
Table 2 presented the results of the calculation of 96h EC50s. According to the data, M. aeruginosa was more sensitive than C. pyrenoidosa under the individual and combined stress of 7
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the two substances. Table 3 presented the results of the TU method. The combined effect turned out to be partly additive to C. pyrenoidosa and antagonistic to M. aeruginosa in the initial tests; in
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the second exposure tests, the combined effect became synergistic to both algae. This suggested the initial stress may have changed the algae’s biochemical features, so that the results could be
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different in the second exposure tests. As calculated with the B-H equation and the CA model
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(though not presented in the table), only the equivalent complex concentration of the group with the highest inhibition rate to C. pyrenoidosa was positive value (4.50 µmol/L)while the equivalent
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complex concentrations of all experimental groups to M. aeruginosa were negative values in the initial tests; in the second exposure tests, the equivalent complex concentrations of all
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experimental groups to C. pyrenoidosa turned out to be negative values, while on the contrast, the equivalent complex concentrations of the experimental groups with mixture concentrations over
(Position of Table 2)
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28.9 µmol/L were at range of 6.75-9.22 µmol/L.
3.2 Chlorophyll fluorescence
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(Position of Figure 1)
Figure 1 and Figure 2 presented the chlorophyll fluorescence results of C. pyrenoidosa and M.
aeruginosa respectively. As shown (Fig.1), C. pyrenoidosa turned out to be sensitive to the
mixture, for the lowest values showed up at 0.5h after dosing in the initial test and at 24h after dosing in the second exposure test. The different experimental groups’ Fv/Fm values in the second exposure test recovered to the same level as the initial test after 96h, which suggested that the mixture didn’t damage the PSII system in the initial test and that C. pyrenoidosa may have partly blocked the harm via some mechanisms at the first 24h of the second exposure test. According to 8
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the result of Cu2+, the values of Fv/Fm at 0.5h in the second exposure test were relatively larger than those in the initial test, which may suggest that the damage caused by Cu2+ during the initial
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test couldn’t be completely repaired after the removal of Cu2+ at high concentration. On the contrary, the decrease of the Fv/Fm values in the second exposure test of chlortetracycline became
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less notable, for the lowest Fv/Fm values of the initial test reached 0.400 rather than 0.65 in the
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second exposure test and the lowest values showed up at 24h in the initial test, instead of 72h in the second exposure test.
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(Position of Figure 2)
As shown (Fig.2), M. aeruginosa was also sensitive to the mixture, for the lowest value of
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Fv/Fm declined to 0.25 in the initial test and 0.15 in the second exposure test. Furthermore, the
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Fv/Fm values of the groups with concentrations over 0.029mmol/l decreased significantly within
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48h in the second exposure test while only the group at 0.043mmol/l decreased in the initial test. Also, the value of the control group in the second exposure test decreased than that in the initial test. These suggested that the exposure to the mixture weakened the adaptability of M. aeruginosa
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and the damage may be unable to be repaired. For Cu2+, the experimental groups of M. aeruginosa
with concentrations of CuSO4·5H2O under the 0.5 mg/l showed no significant decrease on their Fv/Fm values. In the second exposure test, the non-effect concentration of CuSO4·5H2O elevated to 1.0 mg/l, which suggested that the resistance of M. aeruginosa to Cu2+ improved after the initial
stress. The results of chlortetracycline were similar to those in the tests to C. pyrenoidosa. 3.3 MDA content (Position of Table 4) Table 4 presented the significant differences between the initial and second exposure tests 9
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and Figure 3 presented the results of MDA contents. In the initial tests, the MDA contents of both algae increased with the growing concentrations of the mixture, while there weren’t such
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significant changes in the second exposure tests. When the substances actioned individually, the MDA contents of C. pyrenoidosa increased as the growing concentrations of the substances in the
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initial tests. But in the second exposure tests, the experimental groups’ MDA contents showed to
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remain close to the control, even though the whole MDA level of C. pyrenoidosa under Cu2+ increased during the second exposure test. In the initial individual test of Cu2+, the MDA contents
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of M. aeruginosa didn’t change significantly as the concentrations of CuSO4·5H2O were under 0.18 mg/l and the maximum content showed up at 0.58 mg/l, while in the second exposure test,
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the maximum content showed up at 1.08 mg/l.
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3.4 Soluble protein content
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(Position of Figure 3)
Figure 4 presented the contents of soluble protein. As shown (Fig.4d), under the combined stress, the soluble protein contents of M. aeruginosa generally increased with the growing
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concentrations in both tests, while the increase became less notable in the second exposure test. As to C. pyrenoidosa (Fig.4a), the effect of the mixture was inhibitory to the groups with relatively
higher concentrations in the initial test, while in the second exposure test, the contents of the experimental groups increased compared with the control, which suggested that the inhibitory effect weakened. In the tests of Cu2+ on M. aeruginosa (Fig.4e), the soluble protein contents
slightly decreased at lower concentrations before the increase at the 0.58mg/l (171.0% of the control) in the initial test and at 1.08mg/l (151.2% of the control) in the second exposure test. As to C. pyrenoidosa under the stress of Cu2+ (Fig.4b), the soluble protein contents decreased in the 10
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initial test with the lowest value 50.3% of the control, when in the second exposure test, the inhibition seemed more notable. For the tests of chlortetracycline on M. aeruginosa (Fig.4f), the
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decrease of the content appeared at 12 mg/l in both tests with the lowest value 28.6% of the control in the initial test and the lowest value 74.4% of the control in the second exposure test. As
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to C. pyrenoidosa (Fig.4c), the values of soluble protein contents elevated with the growing
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concentrations in the initial test; the highest value appearing at 40 mg/l reached 141.5% of the control. In the second exposure test, the values of experimental groups decreased with the lowest
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value 62.6% of the control. (Position of Figure 4)
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3.5 SOD activities
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Figure 5 presented the results of SOD activity. As shown, under the combined pressure, the
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SOD activity of C. pyrenoidosa (Fig.5a) generally increased with the growing concentrations in the initial test before the highest value at 0.091mmol/l, which was similar to the trend of the relative SOD activity. In the second exposure test, the highest value appeared at 0.073mmol/l. As
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to M. aeruginosa (Fig.5d), the SOD activity increased with the growing concentrations in both
tests, though the SOD activities in the second exposure test were much lower than those in the initial test, while the relative activities were similar in both tests. When under Cu2+ stress individually, in the initial test (Fig.5b), the SOD activity of C. pyrenoidosa started increasing at 0.58 mg/l, while the relative SOD activities showed a sudden increase at 0.18mg/l then decreased. In the second exposure test, the SOD activity increased and reached the highest value at 5.08 mg/l, while the relative activities kept increasing. As to M. aeruginosa (Fig.5e), the SOD activity reached the highest value at 0.58 mg/l in the initial test and at 1.08 mg/l in the second exposure 11
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test, while the relative SOD activity increased continuously in both tests. When under stress of chlortetracycline, the SOD activity of C. pyrenoidosa (Fig.5c) increased with the growing
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concentrations in both tests, while the increase became less notable in the second exposure test. The result of relative SOD activity was nearly the same, though the value of which was much
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larger in the second exposure test. As to M. aeruginosa (Fig.5f), the SOD activities and relative
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activities showed increase with the growing concentrations in both tests though the increase became less notable in the second exposure test.
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(Position of Figure 5) 4 Discussions
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In the experiment, the initial and second exposure tests about individual and combined effect of Cu2+ and chlortetracycline on Chlorella pyrenoidosa and Microcystis aeruginosa were
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conducted. According to the results, the complex of Cu2+ and chlortetracycline did have effects on both algae. The results of individual tests showed that both algae’s resistance to chlortetracycline improved in the second exposure tests; C. pyrenoidosa’s resistance to Cu2+ weakened while that of
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M. aeruginosa improved in the second exposure tests. Taking the recovery period into
consideration, the results might suggest the possibility of the existence of heritable changes on algae after individual and combined stress of Cu2+ and chlortetracycline. The results of CA model suggested the complex was toxic to C. pyrenoidosa and nontoxic to M. aeruginosa initially, while it became nontoxic to C. pyrenoidosa and toxic to M. aeruginosa in the second exposure tests, which was consistent with the results of TU method. Fv/Fm, as a commonly used indicator of algae, represents the maximum quantum efficiency of PSII photochemistry, the value of which would show an obvious change when algae is under 12
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stress (Baker N.R., 2008). The individual and combined effects caused by the initial stress can also affect the biological characteristics of algae in the second exposure tests. The result of chlorophyll
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fluorescence (Fv/Fm) indicated that the resistance of PSII in both algae towards chlortetracycline was enhanced after the initial stress, while the resistance towards Cu2+ of both algae declined.
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When under the combined stress, the resistance of PSII in C. pyrenoidosa improved after the
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initial stress, which was opposite to M. aeruginosa. Thus, the existence of the two substances in the environment may show some sustainable effects, while the existence of the mixture may cause
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stronger inhibitory effect after multiple stresses.
In the experiment, the biochemical indicators of two algae including MDA content, SOD
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activity, relative SOD activity and soluble protein content were measured after 96-hour stress. The
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the detoxification mechanisms.
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significant changes of the results of the indicators between the two tests indicated the changes of
Malondialdehyde is a commonly analyzed compound when describing liquid peroxidation (Qian H.F. et al., 2009). When algae is under stress, the ROS (reactive oxygen species) generated
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directly or indirectly by the stressor can react with algae’s membrane system, which leads to damages to algae cell and its’ metabolic processes (Sabatini S.E. et al., 2009). The result of the mixture suggested that its initial effect may have changed the mechanism of both algae towards lipid peroxidation. According to the result (Tab.4), to C. pyrenoidosa, the MDA contents in the
second exposure tests were significantly different from those in the initial tests; on the contrast, to M. aeruginosa, only under combined stress the MDA content was significantly different in the second exposure test. The increase of MDA content became less notable in the second exposure test of C. pyrenoidosa, which suggested that the resistance to both substances of C. pyrenoidosa 13
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was enhanced after the initial stress. The increase of the concentration at maximum MDA content of M. aeruginosa suggested that the ability of M. aeruginosa to eliminate ROS may be enhanced
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after the initial stress. In the initial and the second exposure tests of chlortetracycline, the MDA contents of the experimental groups showed no significant difference from the control, which
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might suggest that the effect of chlortetracycline may not generated more ROS than the algae can
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eliminate.
Protein is a kind of structural material for organisms as well as an important group of catalyst
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for the biochemical reactions. Soluble protein is also a commonly used indicator of the changes in metabolism of plants. The oxidative stress can stimulate the increase of soluble protein content
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(Osman M.E.H. et al., 2004, Wu Z.B. et al., 2007). Informed from Tab.4, the soluble protein
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contents of both algae under Cu2+ stress were significantly different between the initial and second
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exposure tests. To M. aeruginos, the results of chlortetracycline tests showed no significant differences between the two tests, while the results of mixture were extremely different, which was exactly opposite to C. pyrenoidosa. The oxidative stress can induce an augmentation of
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protein in algae, which is involved in mechanisms of cellular detoxification via the formation of metal complexes (Sabatini S.E. et al., 2009), while the decrease may suggest the inhibition of the synthesis of soluble protein. Thus, the stress of chlortetracycline can obviously inhibit the synthetic system of M. aeruginosa during both tests; and to C. pyrenoidosa, it can affect the synthesis of soluble protein even after a recovery period and show notable inhibition in the second exposure test. On the contrast, the resistance of both algae toward Cu2+ individually improved after the initial stress. When under combined stress, the resistance of C. pyrenoidosa improved after the initial stress, while M. aeruginosa was negatively influenced by the initial stress. Those 14
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results showed that the multiple stresses can cause different effect to the two algae and the resistance of the two algae to the combined stress was different, which means in a way both the
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situations could affect the composition of the aquatic plankton. Superoxide Dismutase is a family of enzymes, which plays a key role in the elimination of
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the ROS and its secondary products. The ROS and its by-products can interfere in the metabolism
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process and do damages to membrane lipids, protein system, photo-synthetical system and other biosystems (Mittler R. et al., 2004). As shown (Tab.4), the SOD activity of C. pyrenoidosa was
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significantly different between the initial and second exposure tests, while that of M. aeruginosa was extremely different between the two tests only under combined stress. And only when C.
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pyrenoidosa was under the stress of chlortetracycline, the result of relatively SOD activity was
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extremely different between the two tests. According to the results, the sensitivity of M.
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aeruginosa toward ROS and its by-products declined after the initial stresses of different substances, which suggested that though the stressors affected its antioxidant system, M. aeruginosa could recover after the removal of the substances. As to C. pyrenoidosa, the effect
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caused by chlortetracycline enhanced its tolerance towards ROS and its by-products, while the other stressors weakened its resistance, the possible reason of which may be that the damages caused during the initial tests cannot be repaired by C. pyrenoidosa. This may indicate that C.
pyrenoidosa could be more sensitive than M. aeruginosa after multiple stresses. In other work, when under stress of copper, the activities of the antioxidant enzymes tested were not all stimulated despite the increase in lipid peroxidation (Monnet F. et al., 2006), which was consistent with the present result. Besides, the decrease of the MDA content was also consistent with the decrease of SOD activity. 15
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5 Conclusions Altogether, the results showed that under different stress, the recovery abilities of the two
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algae were different, thus, the effect of the combine and multiple stress in the environment may be
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related to the composition changes of the aquatic plankton.
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References Altenburger, R., Nendza, M., Schuurmann, G., 2003. Mixture toxicity and its modeling by
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quantitative structure-activity relationships. Environ Toxicol Chem. 22, 1900-1915. Baker, N.R., 2008. Chlorophyll fluorescence: A probe of photosynthesis in vivo, Annual Review
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of Plant Biology, pp. 89-113.
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Balamurugan, K., Schaffner, W., 2006. Copper homeostasis in eukaryotes: Teetering on a tightrope. BBA Molecular cell research. 1763, 737-746.
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Bolan, N.S., Khan, M.A., Donaldson, J., Adriano, D.C., Matthew, C., 2003. Distribution and bioavailability of copper in farm effluent. Sci Total Environ. 309, 225-236.
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Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities
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Chafer-Pericas, C., Maquieira, A., Puchades, R., Miralles, J., Moreno, A., 2011. Multiresidue determination of antibiotics in feed and fish samples for food safety evaluation. Comparison of immunoassay vs LC-MS-MS. Food Control. 22, 993-999.
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Chang, X.S., Meyer, M.T., Liu, X.Y., Zhao, Q., Chen, H., Chen, J.A., Qiu, Z.Q., Yang, L., Cao, J., Shu, W.Q., 2010. Determination of antibiotics in sewage from hospitals, nursery and slaughter house, wastewater treatment plant and source water in Chongqing region of Three Gorge Reservoir in China. Environ Pollut. 158, 1444-1450. Christian, T., Schneider, R.J., Farber, H.A., Skutlarek, D., Meyer, M.T., Goldbach, H.E., 2003. Determination of antibiotic residues in manure, soil, and surface waters. Acta Hydroch Hydrob. 31, 36-44. Hong, Y., Hu, H.Y., Xie, X., Li, F.M., 2008. Responses of enzymatic antioxidants and 17
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non-enzymatic antioxidants in the cyanobacterium Microcystis aeruginosa to the allelochemical ethyl 2-methyl acetoacetate (EMA) isolated from reed (Phragmites communis). J. Plant Phys. 165,
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1264-1273. Ji, X.L., Shen, Q.H., Liu, F., Ma, J., Xu, G., Wang, Y.L., Wu, M.H., 2012. Antibiotic resistance
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gene abundances associated with antibiotics and heavy metals in animal manures and agricultural
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soils adjacent to feedlots in Shanghai; China. J. Hazard Mater. 235, 178-185.
Kemper, N., 2008. Veterinary antibiotics in the aquatic and terrestrial environment. Ecol Indic. 8,
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Kong, Q.X., Zhu, L.Z., Shen, X.Y., 2010. The toxicity of naphthalene to marine Chlorella vulgaris
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under different nutrient conditions. J. Hazard Mater. 178, 282-286.
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Liu, S.S., Liu, L., Chen, F., 2013. Application of the Concentration Addition Model in the
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Assessment of Chemical Mixture Toxicity. Acta Chim Sinica. 71, 1335-1340. Ma, Y., Zhou, Q., Zhou, S.C., Wang, W., Jin, J., Xie, J.W., Li, A.M., Shuang, C.D., 2014. A bifunctional adsorbent with high surface area and cation exchange property for synergistic
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removal of tetracycline and Cu2+. Chen Eng J. 258, 26-33. Mittler, R., Vanderauwera, S., Gollery, M., Van Breusegem F., 2004. Reactive oxygen gene network of plants. Trends Plant Sci. 9, 490-498. Monnet, F., Bordas, F., Deluchat, V., Baudu, M., 2006. Toxicity of copper excess on the lichen Dermatocarpon luridum: Antioxidant enzyme activities. Chemosphere. 65, 1806-1813. Osman, M.E.H., El-Naggar, A.H., El-Sheekh, M.M., El-Mazally, E.E., 2004. Differential effects of Co2+ and Ni2+ on protein metabolism in Scenedesmus obliquus and Nitzschia perminuta. Environ Toxicol Phar. 16, 169-178. 18
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Qian, H.F., Xu, X.Y., Chen, W., Jiang, H., Jin, Y.X., Liu, W.P., Fu, Z.W., 2009. Allelochemical stress causes oxidative damage and inhibition of photosynthesis in Chlorella vulgaris.
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Chemosphere. 75, 368-375. Sabatini, S.E., Juarez, A.B., Eppis, M.R., Bianchi, L., Luquet, C.M., de Molina M.D.R., 2009.
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Oxidative stress and antioxidant defenses in two green microalgae exposed to copper. Ecotox
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Environ Safe. 72, 1200-1206.
Tong, L., Li, P., Wang, Y.X., Zhu, K.Z., 2009. Analysis of veterinary antibiotic residues in swine
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wastewater and environmental water samples using optimized SPE-LC/MS/MS. Chemosphere. 74, 1090-1097.
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Tuckfield, R.C., McArthur, J.V., 2008. Spatial analysis of antibiotic resistance along metal
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contaminated streams. Microb Ecol. 55, 595-607.
Sin. 23, 1353-1359.
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Wang, R., Yu, Z.W., 2007. Validity and reliability of Benesi-Hildebrand method. Acta Phys-Chim
Wu, L.H., Pan, X., Chen, L.K., Huang, Y.J., Teng, Y., Luo, Y.M., Christie, P., 2013. Occurrence
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and distribution of heavy metals and tetracyclines in agricultural soils after typical land use change in east China. Environ Sci Pollut R. 20, 8342-8354. Wu, Z.B., Deng, P., Wu, X.H., Luo, S., Gao, Y.N., 2007. Allelopathic effects of the submerged macrophyte Potamogeton malaianus on Scenedesmus obliquus. Hydrobiologia. 592, 465-474. Zhang, Y., Cai, X.Y., Lang, X.M., Qiao, X.L., Li, X.H., Chen, J.W., 2012. Insights into aquatic toxicities of the antibiotics oxytetracycline and ciprofloxacin in the presence of metal: Complexation versus mixture. Environ Pollut. 166, 48-56.
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Figure captions
Figure 1 Chlorophyll fluorescence results of C. pyrenoidosa 1) (a), (c), (e) present the results of the initial stress tests; (b), (d), (f) present the results of the second exposure tests. 2) (a), (b) present the results of the mixture; (c), (d) present the results of
Figure 2 Chlorophyll fluorescence results of M. aeruginosa
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Cu2+; (e), (f) present the results of chlortetracycline.
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1) (a), (c), (e) present the results of the initial stress tests; (b), (d), (f) present the results of the
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second exposure tests. 2) (a), (b) present the results of the mixture; (c), (d) present the results of
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ed
Cu2+; (e), (f) present the results of chlortetracycline.
Figure 3 Results of MDA contents
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1) a, b, c present the results of C. pyrenoidosa; d, e, f present the results of M. aeruginosa. 2) MDA1 presents the results of the initial stress test; MDA2 presents the results of the second exposure test. 3) a, d present the results of the mixture; b, e present the results of the stress under Cu2+; c, f present the results of the stress under chlortetracycline.
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Figure 4 Results of soluble protein contents
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1) a, b, c present the results of C. pyrenoidosa; d, e, f present the results of M. aeruginosa. 2) P1 presents the results of the initial stress test; P2 presents the results of the second exposure test. 3) a,
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d present the results of the mixture; b, e present the results of the stress under Cu2+; c, f present the
M
an
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results of the stress under chlortetracycline.
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Figure 5 Results of SOD activities 1) (a), (b), (c) present the results of C. pyrenoidosa; (d), (e), (f) present the results of M.
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aeruginosa. 2) SOD1 presents the SOD activity results of the initial stress test; SOD2 presents the SOD activity results of the second exposure test; SOD/PR1 presents the SOD relative activity results of the initial stress test; SOD/PR2 presents the SOD relative activity results of the second
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exposure test. 3) (a), (d) present the results of the mixture; (b), (e) present the results of the stress under Cu2+; (c), (f) present the results of the stress under chlortetracycline.
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d
M
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Figure1
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d
M
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Figure2
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d
M
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Figure3
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d
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Figure4
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M
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Table
Table 1 Analysis standard of TU method Analysis standard
Simple additive Antagonistic Synergistic Independent Partly additive
M=1 M>Mo MM>1
Ac
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M
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Effect type
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Table 2 The result of 96h EC50
Microcystis aeruginosa
CuSO4•5H2O CTC mixturea CuSO4•5H2O CTC mixturea
R2
EC50
R2
EC50
0.999 0.978 0.998 0.893 0.982 0.961
15.6 mol/l 73.4mol/l 78.9mol/l 0.97mol/l 29.5mol/l 22.3mol/l
0.998 0.995 0.827 0.983 0.941 0.994
11.6mol/l 102.5mol/l 41.9mol/l 2.58mol/l 61.7mol/l 14.4mol/l
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Chlorella pyrenoidosa
Substances
Second exposure testc
Initial stress testb
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Algae species
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a) The mixture represents the experiment of the combined effect; b) all the results of the initial stress tests were fitted using DoseResp model; c) the result of the mixture to C. pyrenoidosa was
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ed
M
result was over the range of the non-linear curve fit.
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fitted using Slogistic1 model, while the result on M. aeruginosa was linear fitted because the
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Table 3 The result of combined effect analysis Initial stress test
Second exposure test
Chlorella pyrenoidosa Microcystis aeruginosa
+a
++c
-b
++
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Algae Species
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a) “+” represents the partly additive effect; b) “-” represents the antagonistic effect; c) “++”
Ac
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ed
M
an
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represents the synergistic effect.
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Table 4 The result of significant differences analysis Sig. a SOD activity
Relative SOD activity
Soluble protein
CuSO4•5H2O CTC mixture CuSO4•5H2O CTC mixture
0.030 0.021 0.047 0.081 0.054 0.001
0.035 0.023 0.027 0.841 0.074 0.001
0.473 0.002 0.444 0.147 0.247 0.857
0.020 0.001 0.536 0.039 0.810 0.001
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Microcystis aeruginosa
MDA content
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Chlorella pyrenoidosa
Substances
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Algae species
a) The analysis was conducted to figure out whether there were significant differences between
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the results of initial test and the second exposure test.
b) The significant differences were calculated with paired-samples t-test; 0.010 < Sig. < 0.050
Ac
ce pt
ed
M
means significantly different; Sig. < 0.01 means extremely different.
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S1382-6689(15)30007-7 http://dx.doi.org/doi:10.1016/j.etap.2015.06.006 ENVTOX 2274
To appear in:
Environmental Toxicology and Pharmacology
Received date: Accepted date:
12-3-2015 3-6-2015
Please cite this article as: Lu, L., Wu, Y., Ding, H., Zhang, W.,The combined and second exposure effect of copper (II) and chlortetracycline on fresh water algae, Chlorella pyrenoidosa and Microcystis aeruginosa, Environmental Toxicology and Pharmacology (2015), http://dx.doi.org/10.1016/j.etap.2015.06.006 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.
*Highlights (for review)
Highlights The combined toxicity of copper and chlortetracycline on two algae was analyzed.
The biochemical features of both algae were different in the second exposure.
The toxicity of the complex on algae was analyzed in initial and second exposure.
There are significant differences of 96h EC50s between initial and second exposure.
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*Manuscript
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Ⅱ) and chlortetracycline on The combined and second exposure effect of copper (Ⅱ fresh water algae, Chlorella pyrenoidosa and Microcystis aeruginosa
College of Resource and Environmental Science, Wuhan University, Wuhan, PR China.
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Lei Lua, Yixiao Wua, Huijun Dinga, Weihao Zhanga
Correspondence to: Weihao Zhang;
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Tel.: +86-27-68777060
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E-mail: [email protected]
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1
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Abstract In the experiment, Chlorella pyrenoidosa and Microcystis aeruginosa were chosen to test the
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individual, combined and second exposure effect of Cu2+ and chlortetracycline (CTC). The 96h EC50s of each test were calculated, with the ranges of 0.972 µmol/L-15.6 µmol/L (Cu2+),
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29.5 µmol/L-102.5 µmol/L (CTC), 14.4 µmol/L-78.9 µmol/L (mixture). The combined toxicities
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were evaluated with toxicity units (TU) method. The toxicity of complex of Cu2+ and chlortetracycline was analyzed using concentration addition (CA) model. In the initial test, the
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combined effect of the two substances was partly additive to C. pyrenoidosa and antagonistic to M. aeruginosa, while in the second exposure test, the combined effect was synergistic to both algae.
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The biochemical indicators measured in the experiment included chlorophyll fluorescence
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(Fv/Fm), MDA content, SOD activity and content of soluble proteins. When under combined
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stress, the biochemical features of both algae were significantly different between the initial test and the second exposure test.
Keywords Chlorella pyrenoidosa; Microcystis aeruginosa; chlortetracycline; copper; second
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exposure
2
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1 Introduction Tetracycline antibiotics (TCs) are a group of antibiotics which are widely used around the
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world. TCs along with some heavy metals are often used in animal feed as growth promoters and treatment for diseases, while the metabolism of which is incomplete (Kemper N., 2008). Thus, the
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antibiotics and heavy metals excreted by animals can enter the environment directly or indirectly
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through the emission of aquaculture wastewater. In soils collected from certain feedlots, the concentration of tetracyclines were at range of 4.54–24.66 mg/kg, while the typical heavy metal
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Cu was detected at concentration range of 32.3–730.1 mg/kg (Ji X.L. et al., 2012). In certain piggery and diary effluent, the total Cu concentrations were respectively at ranges of 0.1-1.55 mg/l
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and 0.5-10.5 mg/l (Bolan N.S. et al., 2003), while the total concentration of TCs in the land
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fertilized with organic manures was around 22.9 µg/kg (Wu L.H. et al., 2013). Though the
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wastewater treatment can be conducted, the removal rate of Epi-iso-chlorotetracycline was around 18%(Chang X.S. et al., 2010).Via the transportations among different media in the environment, once released, antibiotics and heavy metals are likely to cause water pollution and affect the
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aquatic system. The detections of antibiotics have been conducted in various environmental media (Christian T. et al., 2003, Tong L. et al., 2009) as well as organisms (Chafer-Pericas C. et al., 2011). Meanwhile the heavy metals can be released anthropogenically and naturally to the environment, the relatively high concentrations of which have been proved to be biologically harmful (Balamurugan K. et al., 2006). With multiple electron donor groups, TCs can react with other organic and inorganic ions, thus the heavy metal ions may affect their behavior in environment. The spatial correlation between antibiotic resistance of bacteria and metal concentration was found in contaminated 3
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streams (Tuckfield R.C. et al., 2008). Recently, the increasing attention has been drawn on the interaction between antibiotics and heavy metals. Oxytetracycline and ciprofloxacin can bound
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with three typical heavy metals, copper, zinc and cadmium, via multiple coordination sites, besides which the antibiotics, heavy metals and their complex acted primarily as concentration
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(Zhang Y. et al., 2012). Tetracycline and Cu2+ can form complex on the surface of the bifunctional
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adsorbent (Ma Y. et al., 2014). Though chlorotetracycline and copper are often used together, the study of their interaction is rare. In addition, the continuous emissions of antibiotics and heavy
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metals are likely to cause multiple stresses to the aquatic system, which means that the study of multiple exposures could be needed.
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In order to figure out whether the multiple stresses of typical heavy metals and veterinary antibiotics can cause heritable implications on the biochemical process of algae and whether the
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combined effects can become different in the second exposure, this study chose Cu2+ and chlortetracycline as stressors and investigated their individual and combined effect on two fresh water algae, C. pyrenoidosa and M. aeruginosa. The combined effects were evaluated and
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analyzed with TU method (Altenburger R. et al., 2003) and CA model (Liu S.S. et al., 2013). Based on those, the second exposure tests were also conducted. 2 Materials and methods 2.1 Chemicals
Chlortetracycline (>95%) was purchased from Kayon, Shanghai China; CuSO4·5H2O and other chemicals were purchased from Sinopharm Chemical Reagent Co., Shanghai China. The antibiotic was dissolved in 0.22 µm filtered sterile Milli-Q water, while the metal solutions were used after sterilized. 4
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2.2 Algae cultivation The tested algae Microcystis aeruginosa (FHACB-315) and Chlorella pyrenoidosa
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(FHACB-10) were purchased from Freshwater Algae Culture Collection at the Institute of Hydrobiology (FACHB-collection), Wuhan China. The experiment used BG11 as liquid culture
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medium. The cultivation was conducted in the PGX-illuminating incubator. The conditions were
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temperature as 25±2℃, light intensity as 2000lx and the light : dark circle as 12h : 12h. The biomass of algae was represented with the optical density under 680nm, before which the linear
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relation between the optical density and the cell number per milliliter had been built. The tests were conducted on the algae in logarithmic phase. Each concentration group had three parallel
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samples. During the experiment, all samples were shaken three times a day artificially and
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changed positions randomly.
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2.3 Toxicity tests 2.3.1 Individual toxicity tests
The experiment used algae in the logarithmic phase with the initial OD680nm around 0.200.
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The concentrations of CuSO4·5H2O were set as 0.08, 0.09, 0.18, 0.58, 1.08, 5.08 mg/L to M. aeruginosa and 0.08, 0.18, 0.58, 1.08, 5.08, 10.08 mg/L to C. pyrenoidosa. The concentration of
negative control was not zero because copper is a trace element required during the growth of the algae. The concentrations of chlortetracycline were set as 0.00, 1.50, 6.00, 12.00, 24.00, 48.00 mg/L to M. aeruginosa and 0.00, 1.00, 10.00, 20.00, 40.00, 60.00 mg/L to C. pyrenoidosa. The
stress time was 96 hours and the biomass of algae was tested every 24 hours. The inhibition rates were calculated with the equations as follows (Kong Q.X. et al., 2010): %I ⁄ 100 5
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Ct: the cell density of treated groups; Co: the cell density of the control; %I: the inhibition rates.
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2.3.2 Combined toxicity tests The concentrations of mixtures were set up depending on the 96h EC50s from the individual
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tests. And the concentrations were set as 0, 0.25, 0.50, 1.00, 1.25, 1.50 times of the combined 96h
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EC50s of the two substances to the two algae.
The effects of the combined toxicity were analyzed using TU method (Altenburger R. et al., 2003). The definition of toxicity units is as follows: ,
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Ci: concentration of the component i; EC50, i: the 96h EC50 of component i when it actions
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individually.
The analysis indicators M and Mo were defined as follows:
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max
(Position of Table 1)
The complex was described using expanded Benesi-Hildebrand equation (Wang R. et al.,
2007). Meanwhile the analysis of its toxicity to the two algae was described using CA model (Liu S.S. et al., 2013).
2.3.3 The second exposure tests The tested algae were first cultivated till their logarithmic phase. Then they were transfered to the treated media, whose concentrations were equal to the 96h EC50s. The OD680nm of the treated groups were around 0.200 and the stress time was 96h. After the stress, the algae were extracted 6
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by centrifugalizing. The centrifugal speed was 5000r/min. The supernatants were abandoned, after which the algae were suspended in sterilized water for 5 min before centrifugalized. The processes
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were repeated twice after that. After the last-time centrifugalizing the algae were suspended in the clean media and cultivated to logarithmic phase. During the procedure, all equipment was sterile.
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The experiment conditions of the second exposure tests were the same as the initial stress tests.
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2.3 Biochemical indicators
The biochemical indicators included chlorophyll fluorescence fv/fm, SOD activity, MDA
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content and soluble protein content. The chlorophyll fluorescence was measured 0.5, 24, 48, 72, 96 h after dosing during each test, while the other indicators were measured after 96-hour stress.
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The chlorophyll fluorescence fv/fm was tested using Handy-PEA (Hansatech Inc., British). The
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SOD activity determination used Nitroblue Tetrazolium Photoreduction method (Hong Y. et al.,
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2008). The content of MDA was measured by thiobarbituric acid method (Sabatini S.E. et al., 2009). The content of soluble protein was determined by Coomassie Brilliant blue G-250 method (Bradford M.M., 1976).
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2.4 Data analysis
The 96h EC50s were analyzed with Originlab software v.8.5 (Originlab Inc., USA). The
statistical analysis was conducted using Statixtical Package for the Social Sciences (SPSS) software v.18.0 (SPSS Inc., USA). 3 Results 3.1 Calculation of 96h EC50s
Table 2 presented the results of the calculation of 96h EC50s. According to the data, M. aeruginosa was more sensitive than C. pyrenoidosa under the individual and combined stress of 7
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the two substances. Table 3 presented the results of the TU method. The combined effect turned out to be partly additive to C. pyrenoidosa and antagonistic to M. aeruginosa in the initial tests; in
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the second exposure tests, the combined effect became synergistic to both algae. This suggested the initial stress may have changed the algae’s biochemical features, so that the results could be
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different in the second exposure tests. As calculated with the B-H equation and the CA model
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(though not presented in the table), only the equivalent complex concentration of the group with the highest inhibition rate to C. pyrenoidosa was positive value (4.50 µmol/L)while the equivalent
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complex concentrations of all experimental groups to M. aeruginosa were negative values in the initial tests; in the second exposure tests, the equivalent complex concentrations of all
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experimental groups to C. pyrenoidosa turned out to be negative values, while on the contrast, the equivalent complex concentrations of the experimental groups with mixture concentrations over
(Position of Table 2)
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28.9 µmol/L were at range of 6.75-9.22 µmol/L.
3.2 Chlorophyll fluorescence
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(Position of Figure 1)
Figure 1 and Figure 2 presented the chlorophyll fluorescence results of C. pyrenoidosa and M.
aeruginosa respectively. As shown (Fig.1), C. pyrenoidosa turned out to be sensitive to the
mixture, for the lowest values showed up at 0.5h after dosing in the initial test and at 24h after dosing in the second exposure test. The different experimental groups’ Fv/Fm values in the second exposure test recovered to the same level as the initial test after 96h, which suggested that the mixture didn’t damage the PSII system in the initial test and that C. pyrenoidosa may have partly blocked the harm via some mechanisms at the first 24h of the second exposure test. According to 8
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the result of Cu2+, the values of Fv/Fm at 0.5h in the second exposure test were relatively larger than those in the initial test, which may suggest that the damage caused by Cu2+ during the initial
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test couldn’t be completely repaired after the removal of Cu2+ at high concentration. On the contrary, the decrease of the Fv/Fm values in the second exposure test of chlortetracycline became
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less notable, for the lowest Fv/Fm values of the initial test reached 0.400 rather than 0.65 in the
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second exposure test and the lowest values showed up at 24h in the initial test, instead of 72h in the second exposure test.
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(Position of Figure 2)
As shown (Fig.2), M. aeruginosa was also sensitive to the mixture, for the lowest value of
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Fv/Fm declined to 0.25 in the initial test and 0.15 in the second exposure test. Furthermore, the
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Fv/Fm values of the groups with concentrations over 0.029mmol/l decreased significantly within
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48h in the second exposure test while only the group at 0.043mmol/l decreased in the initial test. Also, the value of the control group in the second exposure test decreased than that in the initial test. These suggested that the exposure to the mixture weakened the adaptability of M. aeruginosa
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and the damage may be unable to be repaired. For Cu2+, the experimental groups of M. aeruginosa
with concentrations of CuSO4·5H2O under the 0.5 mg/l showed no significant decrease on their Fv/Fm values. In the second exposure test, the non-effect concentration of CuSO4·5H2O elevated to 1.0 mg/l, which suggested that the resistance of M. aeruginosa to Cu2+ improved after the initial
stress. The results of chlortetracycline were similar to those in the tests to C. pyrenoidosa. 3.3 MDA content (Position of Table 4) Table 4 presented the significant differences between the initial and second exposure tests 9
Page 10 of 31
and Figure 3 presented the results of MDA contents. In the initial tests, the MDA contents of both algae increased with the growing concentrations of the mixture, while there weren’t such
ip t
significant changes in the second exposure tests. When the substances actioned individually, the MDA contents of C. pyrenoidosa increased as the growing concentrations of the substances in the
cr
initial tests. But in the second exposure tests, the experimental groups’ MDA contents showed to
us
remain close to the control, even though the whole MDA level of C. pyrenoidosa under Cu2+ increased during the second exposure test. In the initial individual test of Cu2+, the MDA contents
an
of M. aeruginosa didn’t change significantly as the concentrations of CuSO4·5H2O were under 0.18 mg/l and the maximum content showed up at 0.58 mg/l, while in the second exposure test,
M
the maximum content showed up at 1.08 mg/l.
te
3.4 Soluble protein content
d
(Position of Figure 3)
Figure 4 presented the contents of soluble protein. As shown (Fig.4d), under the combined stress, the soluble protein contents of M. aeruginosa generally increased with the growing
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concentrations in both tests, while the increase became less notable in the second exposure test. As to C. pyrenoidosa (Fig.4a), the effect of the mixture was inhibitory to the groups with relatively
higher concentrations in the initial test, while in the second exposure test, the contents of the experimental groups increased compared with the control, which suggested that the inhibitory effect weakened. In the tests of Cu2+ on M. aeruginosa (Fig.4e), the soluble protein contents
slightly decreased at lower concentrations before the increase at the 0.58mg/l (171.0% of the control) in the initial test and at 1.08mg/l (151.2% of the control) in the second exposure test. As to C. pyrenoidosa under the stress of Cu2+ (Fig.4b), the soluble protein contents decreased in the 10
Page 11 of 31
initial test with the lowest value 50.3% of the control, when in the second exposure test, the inhibition seemed more notable. For the tests of chlortetracycline on M. aeruginosa (Fig.4f), the
ip t
decrease of the content appeared at 12 mg/l in both tests with the lowest value 28.6% of the control in the initial test and the lowest value 74.4% of the control in the second exposure test. As
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to C. pyrenoidosa (Fig.4c), the values of soluble protein contents elevated with the growing
us
concentrations in the initial test; the highest value appearing at 40 mg/l reached 141.5% of the control. In the second exposure test, the values of experimental groups decreased with the lowest
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value 62.6% of the control. (Position of Figure 4)
M
3.5 SOD activities
d
Figure 5 presented the results of SOD activity. As shown, under the combined pressure, the
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SOD activity of C. pyrenoidosa (Fig.5a) generally increased with the growing concentrations in the initial test before the highest value at 0.091mmol/l, which was similar to the trend of the relative SOD activity. In the second exposure test, the highest value appeared at 0.073mmol/l. As
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to M. aeruginosa (Fig.5d), the SOD activity increased with the growing concentrations in both
tests, though the SOD activities in the second exposure test were much lower than those in the initial test, while the relative activities were similar in both tests. When under Cu2+ stress individually, in the initial test (Fig.5b), the SOD activity of C. pyrenoidosa started increasing at 0.58 mg/l, while the relative SOD activities showed a sudden increase at 0.18mg/l then decreased. In the second exposure test, the SOD activity increased and reached the highest value at 5.08 mg/l, while the relative activities kept increasing. As to M. aeruginosa (Fig.5e), the SOD activity reached the highest value at 0.58 mg/l in the initial test and at 1.08 mg/l in the second exposure 11
Page 12 of 31
test, while the relative SOD activity increased continuously in both tests. When under stress of chlortetracycline, the SOD activity of C. pyrenoidosa (Fig.5c) increased with the growing
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concentrations in both tests, while the increase became less notable in the second exposure test. The result of relative SOD activity was nearly the same, though the value of which was much
cr
larger in the second exposure test. As to M. aeruginosa (Fig.5f), the SOD activities and relative
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activities showed increase with the growing concentrations in both tests though the increase became less notable in the second exposure test.
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(Position of Figure 5) 4 Discussions
M
In the experiment, the initial and second exposure tests about individual and combined effect of Cu2+ and chlortetracycline on Chlorella pyrenoidosa and Microcystis aeruginosa were
te
d
conducted. According to the results, the complex of Cu2+ and chlortetracycline did have effects on both algae. The results of individual tests showed that both algae’s resistance to chlortetracycline improved in the second exposure tests; C. pyrenoidosa’s resistance to Cu2+ weakened while that of
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M. aeruginosa improved in the second exposure tests. Taking the recovery period into
consideration, the results might suggest the possibility of the existence of heritable changes on algae after individual and combined stress of Cu2+ and chlortetracycline. The results of CA model suggested the complex was toxic to C. pyrenoidosa and nontoxic to M. aeruginosa initially, while it became nontoxic to C. pyrenoidosa and toxic to M. aeruginosa in the second exposure tests, which was consistent with the results of TU method. Fv/Fm, as a commonly used indicator of algae, represents the maximum quantum efficiency of PSII photochemistry, the value of which would show an obvious change when algae is under 12
Page 13 of 31
stress (Baker N.R., 2008). The individual and combined effects caused by the initial stress can also affect the biological characteristics of algae in the second exposure tests. The result of chlorophyll
ip t
fluorescence (Fv/Fm) indicated that the resistance of PSII in both algae towards chlortetracycline was enhanced after the initial stress, while the resistance towards Cu2+ of both algae declined.
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When under the combined stress, the resistance of PSII in C. pyrenoidosa improved after the
us
initial stress, which was opposite to M. aeruginosa. Thus, the existence of the two substances in the environment may show some sustainable effects, while the existence of the mixture may cause
an
stronger inhibitory effect after multiple stresses.
In the experiment, the biochemical indicators of two algae including MDA content, SOD
M
activity, relative SOD activity and soluble protein content were measured after 96-hour stress. The
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the detoxification mechanisms.
d
significant changes of the results of the indicators between the two tests indicated the changes of
Malondialdehyde is a commonly analyzed compound when describing liquid peroxidation (Qian H.F. et al., 2009). When algae is under stress, the ROS (reactive oxygen species) generated
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directly or indirectly by the stressor can react with algae’s membrane system, which leads to damages to algae cell and its’ metabolic processes (Sabatini S.E. et al., 2009). The result of the mixture suggested that its initial effect may have changed the mechanism of both algae towards lipid peroxidation. According to the result (Tab.4), to C. pyrenoidosa, the MDA contents in the
second exposure tests were significantly different from those in the initial tests; on the contrast, to M. aeruginosa, only under combined stress the MDA content was significantly different in the second exposure test. The increase of MDA content became less notable in the second exposure test of C. pyrenoidosa, which suggested that the resistance to both substances of C. pyrenoidosa 13
Page 14 of 31
was enhanced after the initial stress. The increase of the concentration at maximum MDA content of M. aeruginosa suggested that the ability of M. aeruginosa to eliminate ROS may be enhanced
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after the initial stress. In the initial and the second exposure tests of chlortetracycline, the MDA contents of the experimental groups showed no significant difference from the control, which
cr
might suggest that the effect of chlortetracycline may not generated more ROS than the algae can
us
eliminate.
Protein is a kind of structural material for organisms as well as an important group of catalyst
an
for the biochemical reactions. Soluble protein is also a commonly used indicator of the changes in metabolism of plants. The oxidative stress can stimulate the increase of soluble protein content
M
(Osman M.E.H. et al., 2004, Wu Z.B. et al., 2007). Informed from Tab.4, the soluble protein
d
contents of both algae under Cu2+ stress were significantly different between the initial and second
te
exposure tests. To M. aeruginos, the results of chlortetracycline tests showed no significant differences between the two tests, while the results of mixture were extremely different, which was exactly opposite to C. pyrenoidosa. The oxidative stress can induce an augmentation of
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protein in algae, which is involved in mechanisms of cellular detoxification via the formation of metal complexes (Sabatini S.E. et al., 2009), while the decrease may suggest the inhibition of the synthesis of soluble protein. Thus, the stress of chlortetracycline can obviously inhibit the synthetic system of M. aeruginosa during both tests; and to C. pyrenoidosa, it can affect the synthesis of soluble protein even after a recovery period and show notable inhibition in the second exposure test. On the contrast, the resistance of both algae toward Cu2+ individually improved after the initial stress. When under combined stress, the resistance of C. pyrenoidosa improved after the initial stress, while M. aeruginosa was negatively influenced by the initial stress. Those 14
Page 15 of 31
results showed that the multiple stresses can cause different effect to the two algae and the resistance of the two algae to the combined stress was different, which means in a way both the
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situations could affect the composition of the aquatic plankton. Superoxide Dismutase is a family of enzymes, which plays a key role in the elimination of
cr
the ROS and its secondary products. The ROS and its by-products can interfere in the metabolism
us
process and do damages to membrane lipids, protein system, photo-synthetical system and other biosystems (Mittler R. et al., 2004). As shown (Tab.4), the SOD activity of C. pyrenoidosa was
an
significantly different between the initial and second exposure tests, while that of M. aeruginosa was extremely different between the two tests only under combined stress. And only when C.
M
pyrenoidosa was under the stress of chlortetracycline, the result of relatively SOD activity was
d
extremely different between the two tests. According to the results, the sensitivity of M.
te
aeruginosa toward ROS and its by-products declined after the initial stresses of different substances, which suggested that though the stressors affected its antioxidant system, M. aeruginosa could recover after the removal of the substances. As to C. pyrenoidosa, the effect
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caused by chlortetracycline enhanced its tolerance towards ROS and its by-products, while the other stressors weakened its resistance, the possible reason of which may be that the damages caused during the initial tests cannot be repaired by C. pyrenoidosa. This may indicate that C.
pyrenoidosa could be more sensitive than M. aeruginosa after multiple stresses. In other work, when under stress of copper, the activities of the antioxidant enzymes tested were not all stimulated despite the increase in lipid peroxidation (Monnet F. et al., 2006), which was consistent with the present result. Besides, the decrease of the MDA content was also consistent with the decrease of SOD activity. 15
Page 16 of 31
5 Conclusions Altogether, the results showed that under different stress, the recovery abilities of the two
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algae were different, thus, the effect of the combine and multiple stress in the environment may be
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d
M
an
us
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related to the composition changes of the aquatic plankton.
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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
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quantitative structure-activity relationships. Environ Toxicol Chem. 22, 1900-1915. Baker, N.R., 2008. Chlorophyll fluorescence: A probe of photosynthesis in vivo, Annual Review
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of Plant Biology, pp. 89-113.
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Balamurugan, K., Schaffner, W., 2006. Copper homeostasis in eukaryotes: Teetering on a tightrope. BBA Molecular cell research. 1763, 737-746.
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Bolan, N.S., Khan, M.A., Donaldson, J., Adriano, D.C., Matthew, C., 2003. Distribution and bioavailability of copper in farm effluent. Sci Total Environ. 309, 225-236.
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Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities
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of protein utilizing the principle of protein-dye binding. Anal Biochem. 72, 248-254.
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Chafer-Pericas, C., Maquieira, A., Puchades, R., Miralles, J., Moreno, A., 2011. Multiresidue determination of antibiotics in feed and fish samples for food safety evaluation. Comparison of immunoassay vs LC-MS-MS. Food Control. 22, 993-999.
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Chang, X.S., Meyer, M.T., Liu, X.Y., Zhao, Q., Chen, H., Chen, J.A., Qiu, Z.Q., Yang, L., Cao, J., Shu, W.Q., 2010. Determination of antibiotics in sewage from hospitals, nursery and slaughter house, wastewater treatment plant and source water in Chongqing region of Three Gorge Reservoir in China. Environ Pollut. 158, 1444-1450. Christian, T., Schneider, R.J., Farber, H.A., Skutlarek, D., Meyer, M.T., Goldbach, H.E., 2003. Determination of antibiotic residues in manure, soil, and surface waters. Acta Hydroch Hydrob. 31, 36-44. Hong, Y., Hu, H.Y., Xie, X., Li, F.M., 2008. Responses of enzymatic antioxidants and 17
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non-enzymatic antioxidants in the cyanobacterium Microcystis aeruginosa to the allelochemical ethyl 2-methyl acetoacetate (EMA) isolated from reed (Phragmites communis). J. Plant Phys. 165,
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1264-1273. Ji, X.L., Shen, Q.H., Liu, F., Ma, J., Xu, G., Wang, Y.L., Wu, M.H., 2012. Antibiotic resistance
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gene abundances associated with antibiotics and heavy metals in animal manures and agricultural
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soils adjacent to feedlots in Shanghai; China. J. Hazard Mater. 235, 178-185.
Kemper, N., 2008. Veterinary antibiotics in the aquatic and terrestrial environment. Ecol Indic. 8,
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Kong, Q.X., Zhu, L.Z., Shen, X.Y., 2010. The toxicity of naphthalene to marine Chlorella vulgaris
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under different nutrient conditions. J. Hazard Mater. 178, 282-286.
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Liu, S.S., Liu, L., Chen, F., 2013. Application of the Concentration Addition Model in the
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Assessment of Chemical Mixture Toxicity. Acta Chim Sinica. 71, 1335-1340. Ma, Y., Zhou, Q., Zhou, S.C., Wang, W., Jin, J., Xie, J.W., Li, A.M., Shuang, C.D., 2014. A bifunctional adsorbent with high surface area and cation exchange property for synergistic
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removal of tetracycline and Cu2+. Chen Eng J. 258, 26-33. Mittler, R., Vanderauwera, S., Gollery, M., Van Breusegem F., 2004. Reactive oxygen gene network of plants. Trends Plant Sci. 9, 490-498. Monnet, F., Bordas, F., Deluchat, V., Baudu, M., 2006. Toxicity of copper excess on the lichen Dermatocarpon luridum: Antioxidant enzyme activities. Chemosphere. 65, 1806-1813. Osman, M.E.H., El-Naggar, A.H., El-Sheekh, M.M., El-Mazally, E.E., 2004. Differential effects of Co2+ and Ni2+ on protein metabolism in Scenedesmus obliquus and Nitzschia perminuta. Environ Toxicol Phar. 16, 169-178. 18
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Qian, H.F., Xu, X.Y., Chen, W., Jiang, H., Jin, Y.X., Liu, W.P., Fu, Z.W., 2009. Allelochemical stress causes oxidative damage and inhibition of photosynthesis in Chlorella vulgaris.
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Chemosphere. 75, 368-375. Sabatini, S.E., Juarez, A.B., Eppis, M.R., Bianchi, L., Luquet, C.M., de Molina M.D.R., 2009.
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Oxidative stress and antioxidant defenses in two green microalgae exposed to copper. Ecotox
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Tong, L., Li, P., Wang, Y.X., Zhu, K.Z., 2009. Analysis of veterinary antibiotic residues in swine
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wastewater and environmental water samples using optimized SPE-LC/MS/MS. Chemosphere. 74, 1090-1097.
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Tuckfield, R.C., McArthur, J.V., 2008. Spatial analysis of antibiotic resistance along metal
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Wang, R., Yu, Z.W., 2007. Validity and reliability of Benesi-Hildebrand method. Acta Phys-Chim
Wu, L.H., Pan, X., Chen, L.K., Huang, Y.J., Teng, Y., Luo, Y.M., Christie, P., 2013. Occurrence
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and distribution of heavy metals and tetracyclines in agricultural soils after typical land use change in east China. Environ Sci Pollut R. 20, 8342-8354. Wu, Z.B., Deng, P., Wu, X.H., Luo, S., Gao, Y.N., 2007. Allelopathic effects of the submerged macrophyte Potamogeton malaianus on Scenedesmus obliquus. Hydrobiologia. 592, 465-474. Zhang, Y., Cai, X.Y., Lang, X.M., Qiao, X.L., Li, X.H., Chen, J.W., 2012. Insights into aquatic toxicities of the antibiotics oxytetracycline and ciprofloxacin in the presence of metal: Complexation versus mixture. Environ Pollut. 166, 48-56.
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Figure captions
Figure 1 Chlorophyll fluorescence results of C. pyrenoidosa 1) (a), (c), (e) present the results of the initial stress tests; (b), (d), (f) present the results of the second exposure tests. 2) (a), (b) present the results of the mixture; (c), (d) present the results of
Figure 2 Chlorophyll fluorescence results of M. aeruginosa
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Cu2+; (e), (f) present the results of chlortetracycline.
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1) (a), (c), (e) present the results of the initial stress tests; (b), (d), (f) present the results of the
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second exposure tests. 2) (a), (b) present the results of the mixture; (c), (d) present the results of
ce pt
ed
Cu2+; (e), (f) present the results of chlortetracycline.
Figure 3 Results of MDA contents
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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
1) a, b, c present the results of C. pyrenoidosa; d, e, f present the results of M. aeruginosa. 2) MDA1 presents the results of the initial stress test; MDA2 presents the results of the second exposure test. 3) a, d present the results of the mixture; b, e present the results of the stress under Cu2+; c, f present the results of the stress under chlortetracycline.
Page 21 of 31
Figure 4 Results of soluble protein contents
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1) a, b, c present the results of C. pyrenoidosa; d, e, f present the results of M. aeruginosa. 2) P1 presents the results of the initial stress test; P2 presents the results of the second exposure test. 3) a,
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d present the results of the mixture; b, e present the results of the stress under Cu2+; c, f present the
M
an
us
results of the stress under chlortetracycline.
ed
Figure 5 Results of SOD activities 1) (a), (b), (c) present the results of C. pyrenoidosa; (d), (e), (f) present the results of M.
ce pt
aeruginosa. 2) SOD1 presents the SOD activity results of the initial stress test; SOD2 presents the SOD activity results of the second exposure test; SOD/PR1 presents the SOD relative activity results of the initial stress test; SOD/PR2 presents the SOD relative activity results of the second
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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
exposure test. 3) (a), (d) present the results of the mixture; (b), (e) present the results of the stress under Cu2+; (c), (f) present the results of the stress under chlortetracycline.
Page 22 of 31
Ac ce p
te
d
M
an
us
cr
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Figure1
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Ac ce p
te
d
M
an
us
cr
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Figure2
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Ac ce p
te
d
M
an
us
cr
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Figure3
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Ac ce p
te
d
M
an
us
cr
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Figure4
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Ac
ce
pt
ed
M
an
us
cr
i
Figure5
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Table
Table 1 Analysis standard of TU method Analysis standard
Simple additive Antagonistic Synergistic Independent Partly additive
M=1 M>Mo MM>1
Ac
ce pt
ed
M
an
us
cr
ip t
Effect type
Page 28 of 31
Table 2 The result of 96h EC50
Microcystis aeruginosa
CuSO4•5H2O CTC mixturea CuSO4•5H2O CTC mixturea
R2
EC50
R2
EC50
0.999 0.978 0.998 0.893 0.982 0.961
15.6 mol/l 73.4mol/l 78.9mol/l 0.97mol/l 29.5mol/l 22.3mol/l
0.998 0.995 0.827 0.983 0.941 0.994
11.6mol/l 102.5mol/l 41.9mol/l 2.58mol/l 61.7mol/l 14.4mol/l
ip t
Chlorella pyrenoidosa
Substances
Second exposure testc
Initial stress testb
cr
Algae species
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a) The mixture represents the experiment of the combined effect; b) all the results of the initial stress tests were fitted using DoseResp model; c) the result of the mixture to C. pyrenoidosa was
Ac
ce pt
ed
M
result was over the range of the non-linear curve fit.
an
fitted using Slogistic1 model, while the result on M. aeruginosa was linear fitted because the
Page 29 of 31
Table 3 The result of combined effect analysis Initial stress test
Second exposure test
Chlorella pyrenoidosa Microcystis aeruginosa
+a
++c
-b
++
ip t
Algae Species
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a) “+” represents the partly additive effect; b) “-” represents the antagonistic effect; c) “++”
Ac
ce pt
ed
M
an
us
represents the synergistic effect.
Page 30 of 31
Table 4 The result of significant differences analysis Sig. a SOD activity
Relative SOD activity
Soluble protein
CuSO4•5H2O CTC mixture CuSO4•5H2O CTC mixture
0.030 0.021 0.047 0.081 0.054 0.001
0.035 0.023 0.027 0.841 0.074 0.001
0.473 0.002 0.444 0.147 0.247 0.857
0.020 0.001 0.536 0.039 0.810 0.001
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Microcystis aeruginosa
MDA content
cr
Chlorella pyrenoidosa
Substances
us
Algae species
a) The analysis was conducted to figure out whether there were significant differences between
an
the results of initial test and the second exposure test.
b) The significant differences were calculated with paired-samples t-test; 0.010 < Sig. < 0.050
Ac
ce pt
ed
M
means significantly different; Sig. < 0.01 means extremely different.
Page 31 of 31
