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Title: Assess the combined effects from two kinds of cephalosporins on green
alga (chlorella pyrenoidosa) based on response surface methodology
PII: S0278-6915(15)00046-0
DOI: http://dx.doi.org/doi:10.1016/j.fct.2015.02.007
Reference: FCT 8217
Please cite this article as: Ruixin Guo, Weishu Xie, Jianqiu Chen, Assess the combined effects
from two kinds of cephalosporins on green alga (chlorella pyrenoidosa) based on response
surface methodology, Food and Chemical Toxicology (2015),
http://dx.doi.org/doi:10.1016/j.fct.2015.02.007.
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Assess the combined effects from two kinds of cephalosporins on
methodology
Nanjing, China
*Corresponding author:
Highlights:
RSM has been applied for combined pollution assessment first time.
Abstract: The present work evaluated the combined effects of cefradine and ceftazidime on
the green
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alga Chlorella pyrenoidosa using response surface methodologies (RSM). After a 48 h-
exposure, the
population growth rate (PGR), the chlorophyll-a content and the SOD content of the alga
increased with
increased concentrations of two antibiotics. Though the three responses did not continue to
demonstrate significant increases once antibiotic concentrations exceed a moderate level.
Three twoorder
polynomial regression equations were obtained to describe well the relationship between
the
responses of the alga and the two antibiotics' concentration (R2 = 0.9997, 0.9292 and
0.9039,
respectively). Three 3 D-surface graphs and their contour plots showed directly the
changing trends of
the alga under the combined effects of two antibiotics. This study for the first time employed
the RSM
in ecotoxicology, which indicated that the RSM should be placed under a feasible and a
potential
application prospect in toxicity assessment.
1. Introduction
Green alga, like Chlorella, as the primary producer in freshwater and marine
ecosystem, play a key role in the substance and energy transportation in the food webs
(Jonsson and Aoyama, 2007). Chlorella pyrenoidosa has been named a green, healthy
food by the Food and Agriculture Organization (FAO) and widely applied in the food
industry (Robledo and Freile Pelegrn, 1997). Beside the nutritional value, the green
algae can inhibit the relevant enzymes and therefore exerted a beneficial anti-diabetic
effect as a new generation of drugs (Sun and Chen, 2012). Additionally, it also has
high capacity for photosynthesis and synthesizes neutral lipids for biofuel (Wen et al.,
2014). Due to the value of the green algae in industry, agriculture and pharmacy, the
environmental safety of the aquatic organism should also be attracted more attention.
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Usually, aquatic ecosystems are influenced by many countless stresses, which are
therefore caused widespread attention in recent years for their probable threats to the
aquatic environment and human health finally. China faces serious environmental
chemicals, especially to the antibiotic abuse (Liu and Wong, 2013). On average, the
use of antibiotics in China has been 10 times more than the usage in American.
runoff (Sarmah et al., 2006). Contamination of antibiotics in natural water bodies has
been reported in China (Xu et al., 2007). Disseminated antibiotics usually cause the
ecological problems because of their possible impact on aquatic species as the non-
contaminants due to their continued release into the environment and the permanent
presence (Hernando et al., 2006). Thus, it is necessary to evaluate and assess the
(Chen and Jiang, 2011). The effects of the common antibiotics individually on the
non-target organism are quite well documented recently (Boxall et al., 2003; Halling-
Sorensen, 2000; Lutzhoft et al., 1999; Pan et al., 2008; Robinson et al., 2005).
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different test concentrations of a given substance. In fact, individual chemicals, which
single toxicity testing, the mixture toxicity assessment may better predict the actual
researches focused on the toxicity of the combined antibiotics on diatoms, green algae
The mixture effects were computed by traditional methods such as the combination
index (CI) method, concentration addition (CA) or independent action (IA) method
(Altenburger et al., 2004; Chou, 2006). Based on the calculated toxicity data (e.g.
EC50, EC10), the mixture effect was denoted as antagonistic, additive or synergistic.
These methods, however, less characterize the actual changes of the toxic responses
Response surface methodologies (RSM) were developed since the 50s last
century with the initial aim to optimize the chemical reactions in industry by
sequential involving factors. Thus, the information about the contribution of the
testing factors and their interactions was acquired together (De Schamphelaere et al.,
2003). In addition, the same methodology also could be applied to model any
observed responses that are influenced significantly by the levels of the given factors
(Giloni-Lima et al., 2010). The three-dimensional response and its contour plot could
represent graphically the function of the two factors in ranges (see in Fig. 1). Thus, we
could suggest that the methodology could also be employed to describe the toxic
response that is influenced by the relevant variables. Our previous study employed the
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methodology to indicate the relationship between the behavioral response of rotifers
et al., 2012). Another recent work focused on the neurotoxicity and genotoxicity of
et al., 2013). The recovery parameters such as temperature, time etc. and their
chemical, food and pharmaceutical industry, the employment of RSM in other fields,
especially in toxicity evaluation is limited. Thus, the object of the present work is to
effects of two common antibiotics ceftazidime and cefradine to the freshwater alga C.
pyrenoidosa have been evaluated by RSM. To the best of our knowledge, this is the
first time that of the methodology in the joint toxicity assessment of algae. Our
previous published study indicated that the alga could tolerate cephalosporins at a
given concentration and performed a well removal capacity. The result of the present
study could help us to apply the alga to treat the mixed wastewater of the antibiotics.
The freshwater green alga Chlorella pyrenoidosa were purchased from FACHB-
1C on the photoperiod 12:12 (L:D) with the light at 2000 lx. BG-11 media were
invoked as the culture media, which were adjusted the pH value to 8.0. The cells were
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noted microscopically. Chlorophyll-a and Superoxide dismutase (SOD) were
analyzed using a standard method (Zhang and Huang, 1991). The population growth
rate (PGR, r) of the alga was calculated as the formula (Levasseur et al.,1993):
where N0 and Nt is the algal density at day 0 and day t respectively; t is time in days
when the alga density is maximized. Alga culture without antibiotic was used as a
control.
RSM is a collection from the experimental design and the data analyses (Hanrahan
and Lu, 2006). Thus, the application of the methodology processes in several key
steps as follows:
(1) According to the aim of the given study, the target factors which might
produce major effects should be screened first. In the present study, the possible
cefradine and ceftazidime was selected as two independent factors respectively. The
concentration of the individual antibiotic using in the test was determined by our
composite design (CCD) and Box-Behnken design (BBD) are two common design
models in RSM. CCD often produces the amount of information on the direct effects
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of the tested independent factors and their possible combined effects (Heijerick et al.,
and 0, among them, -values depend on the number of factors. Because of two
antibiotics in the present study, is 1.41 (Bezerra et al., 2008). The factors were
coded as follows:
Xi X0
xi , i = 1, 2, 3, , k
Xi
where xi is the coded value and X0 and Xi is the actual value of the center point and the
ith factor, respectively. Xi indicated change value in every step (Bayraktar, 2001).
Thus, the range and level of the factors in the coded value and actual value are given
in Table 1.
(3) The data obtained were processed statistically to acquire a best fit polynomial
model. The models fitness, the coefficient of determination estimate (R2) should be
design, the model evaluation and the signicant parameters determination using
Analysis of variance (ANOVA) (Faller et al., 2003). Each treatment had three
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The two test factors evaluated in this study were the concentrations of the two
antibiotics. The change in the population growth rate, the chlorophyll-a content and
evaluate the influence of two antibiotics on the population growth rate, the
chlorophyll-a content and the SOD content, the regression equations were obtained by
the RSM software. Before we accepted the regression equations, the significance of
the model and the coefficient of determination estimate (R2) had been evaluated.
Thus, three second-order polynomial models, which obtained the highly significant
(p<0.05, respectively) and the highest values of R2, were fitted to describe the effect of
the two antibiotics and their interaction. ANOVA was implemented to estimate the
significance of the model (see in Table 2). The model for the three responses of the
16.48B2 (R2=0.9997)
(R2=0.9292)
where A and B are the model terms representing the cefradine and ceftazidime
model fits the obtained data. The larger the value of R2, the more relevant the
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independent factors in the model have the contribution to the variation (Fu et al.,
2007). The values of the determination coefficient (R2) of the present three models are
0.9997, 0.9292 and 0.9039, respectively. It revealed that the relationship between the
responses of the alga and two antibiotics concentrations could be described well by
analysis in Table 2 also showed that the three responses of the alga were highly
significantly influenced by two antibiotics (A, B or the second order effect A2, B2, p <
0.01, F-test). However, only the population growth rate was significantly influenced
by the combined effects (AB, p < 0.01, F-test). Additionally, if the model was
accepted, the probability of the lack of fit test has to be larger than 0.05. Test for lack
of fit for the three models showed that a non-significant lack of fit was obtained (p >
0.05, F-test).
The 3D-response surface and the contour plots of three responses of the alga as a
function of two antibiotic concentrations are shown in Fig. 2,3,4. In general, the
population growth rate increased with the concentrations of two antibiotics increased
and then declined. The highest value was obtained when the concentration of
cefradine and ceftazidime at 29.93 and 29.69 mg.L-1, respectively (see in Fig.2). The
population growth was positive in most given antibiotic concentration which implied
that two antibiotic had stimulation impact on algal growth while only a slight
inhibition (change rate was -0.43%) was observed when two antibiotics
content also increased with the concentrations of two antibiotics increased and then
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declined even if at higher concentrations, the significant inhibition was obtained when
the antibiotic concentration was 10.00 mg. L-1 of cefradine and 50.00 mg. L-1 of
when two antibiotics concentrations were at about 30 mg. L-1. The change of the
SOD content was not clear. 27.38 mg.L-1 of cefradine and 26.54 mg.L-1 of ceftazidime
caused the highest increased rate of SOD content (8.13%, see in Fig. 4). However, the
SOD of the alga declined when exposed to about 10 or 50 mg.L-1 of cefradine with 50
mg.L-1 of ceftazidime.
The previous studies about the toxic effect of antibiotics usually focus on the
activity, and the MDA content (Kviderova and Henley, 2005; Pan et al., 2008; Yang
et al., 2013). The antioxidant responses of algae was triggered by antibiotics, which
processes (Liu, et al., 2012). Most of the antibiotics have a negative effect on algae,
in different species also have varied sensitivity to antibiotics (Boxall et al., 2003).
corresponding EC50 values of most antibiotics for M. aeruginosa were less than 0.1
aeruginosa lost its growth capability while the green alga S. obliquus performed a
better adaption under the impact of cefradine (Chen and Guo, 2012). However, the
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adverse effect on S. obliquus was stronger than that on M. aeruginosa with increasing
concentrations of chlortetracycline (Guo and Chen, 2012). Thus, considering the good
tolerance of C. pyrenoidosa under the impact of cephalosporins, the green alga has
well applied to treat the wastewater of four kinds of cephalosporins (Guo and Chen,
2015). The toxic effect of antibiotics was also different between the freshwater
species and marine one. The marine species Tetraselmis chui was more sensitive to
Additionally, antibiotics influenced the organisms not only individually, but also
together as a combined agent (Yang et al., 2008). Thus, the toxicity of antibiotics and
their mixtures have been tested. Because that the compounds in a category usually
have the same mode of action, two or more antibiotics in the identical class are
the present research, the algal population growth, the chlorophyll-a accumulation and
the SOD activity increase occurred when the concentrations of two kinds of
cephalosporins were increasing. The interesting results were that three responses,
however, have not been stimulated continuously when the concentrations of two
antibiotics were higher than the relatively moderate level in the range. A possible
explanation of this phenomenon might be due to competition for uptake or the similar
binding sites, which caused the antagonism on algal population growth. On the other
hand, statistical analysis showed that the chlorophyll-a content and the SOD activity
declined were not due to the interactive effects of the two antibiotics (AB, p > 0.01,
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see in Table 2). The condition of the two antibiotics should be also taken into account.
In the exposure process, the tested antibiotics degraded to new products which
produced the different influence (Yang et al., 2008). During the exposure, cefradine
and ceftazidime might be degraded by the photo or alga to new products with varying
stability and toxicity (Arslan-Alaton and Caglayan, 2006; Cokgor et al., 2006;
Iskender et al., 2007; Paul et al., 2007). Thus, degradation levels of the two antibiotics
are different for each compound, which might cause the chlorophyll-a accumulation
cephalosporins on green alga C. pyrenoidosa. To the best of our knowledge, this study
for the first time employed the response surface methodologies (RSM) in
ecotoxicology. The reference surface plots and contour plots indicated directly the
change trend of the alga when the concentrations of two antibiotics were increasing.
Statistical analysis also could evaluate the reliability of the model and the contribution
of two antibiotics and their interactive effect. Our result indicated that the RSM has a
feasible and potential application in toxicity testing. Further studies are needed to
exclude more species and compounds, which could help the method had a widely
application prospect.
Acknowledgement
This work was supported by The natural science foundation of Jiangsu Province
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Engineering Open Foundation (KF2012008), Doctor Scientific Research Foundation
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Figure 1 Reference surface represent graphically the function of the two factors in
ranges.
Figure 2 Reference surface plots and contour plots as the change rate (%) in the
concentrations. (A) 3D plots for the population change rate, (B) contour plots for the
Figure 3 Reference surface plots and contour plots as the change rate (%) in
concentrations. (A) 3D plots for the change rate of the chlorophyll-a content, (B)
Figure 4 Reference surface plots and contour plots as the change rate (%) in
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antibiotics concentrations. (A) 3D plots for the change rate of the SOD content, (B)
Table 1. Process variables used in the CCD showing the treatment combinations
between the concentrations of two antibiotics. The alga lacking toxic compound was
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Table 2. Summary of p-value for the concentrations of two antibiotics effect on three
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