Accepted Manuscript

Title: Assess the combined effects from two kinds of cephalosporins on green
alga (chlorella pyrenoidosa) based on response surface methodology

Author: Ruixin Guo, Weishu Xie, Jianqiu Chen

PII: S0278-6915(15)00046-0
DOI: http://dx.doi.org/doi:10.1016/j.fct.2015.02.007
Reference: FCT 8217

To appear in: Food and Chemical Toxicology

Received date: 29-7-2014

Accepted date: 2-2-2015

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

green alga (Chlorella pyrenoidosa) based on response surface

methodology

Ruixin Guo, Weishu Xie and Jianqiu Chen*

Department of Environmental Science, China Pharmaceutical University, 210009

Nanjing, China

*Corresponding author:

Jianqiu Chen: Department of Environmental Science, China Pharmaceutical

University, 210009, Nanjing, China

Tel.: + 86 25 86185190, Fax: + 86 25 86185190. E-mail address: cjqalga@163.com

Highlights:

RSM has been applied for combined pollution assessment first time.

Combined effects of cephalosporins on alga are reported first time.

Our work provides a new method in environmental combined pollution.

Abstract: The present work evaluated the combined effects of cefradine and ceftazidime on
the green

Page 1 of 21
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.

Keywords: Chlorella pyrenoidosa, toxicity, combined effect, cefradine, ceftazidime, RSM

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.

Page 2 of 21
 Usually, aquatic ecosystems are influenced by many countless stresses, which are

anthropogenic origin. Pharmaceuticals and personal care products (PPCPs) have

therefore caused widespread attention in recent years for their probable threats to the

aquatic environment and human health finally. China faces serious environmental

pollution by PPCPs due to the growing production and consumptions of the

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.

Antibiotics may be distributed in the environment mainly from excretion, medical

waste, wastewater treatment facilities, agricultural antibiotic application and overland

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-

target organisms. Antibiotics are often considered to be pseudopersistant

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

effects of antibiotics on aquatic environment's safety, especially on the algae.

Alga is sensitive to most contaminants, even if at a relatively low concentration

(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).

However, effects of contaminants on aquatic organisms are usually evaluated by

ecotoxicological standard testing, in which the model organisms are exposed to

3

Page 3 of 21
different test concentrations of a given substance. In fact, individual chemicals, which

disseminate in aquatic environments usually occur in mixtures. Thus, opposed to the

single toxicity testing, the mixture toxicity assessment may better predict the actual

exposures of an organism in the environments (Chen et al., 2014). Although recent

researches focused on the toxicity of the combined antibiotics on diatoms, green algae

and cyanobacterium (Gonzlez-Pleiter et al., 2013; Hagenbuch and Pinckney, 2012).

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

of alga under the given compounds in ranges.

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
4

Page 4 of 21
methodology to indicate the relationship between the behavioral response of rotifers

and the exposure concentration and time (concentration-time-response analyses) (Guo

et al., 2012). Another recent work focused on the neurotoxicity and genotoxicity of

the herbicide Roundup on the common carp Cyprinus carpio L (Gholami-Seyedkolaei

et al., 2013). The recovery parameters such as temperature, time etc. and their

optimization were calculated by RSM. However, opposed to the widely application in

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

expand the application range of the methodology in ecotoxicology. The combined

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.

2. Materials and methods

2.1. Test organisms

The freshwater green alga Chlorella pyrenoidosa were purchased from FACHB-

collection, Chinese Academy of Sciences. Cells of the alga were cultivated at 26

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

Page 5 of 21
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.

2.2. Experimental design and data analyses

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

combined effects of two antibiotics should be assessed. Thus, the concentration of

cefradine and ceftazidime was selected as two independent factors respectively. The

concentration of the individual antibiotic using in the test was determined by our

preliminary study (Guo and Chen, 2015).

(2) An appropriate experimental design model should be chosen. Central

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

Page 6 of 21
of the tested independent factors and their possible combined effects (Heijerick et al.,

2003). Thus, the concentrations of two antibiotics are studied at ve levels: , 1

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

evaluated before acceptation.

Design-Expert version 7.1.6 (Stat-Ease, USA) was employed in the experiment

design, the model evaluation and the signicant parameters determination using

Analysis of variance (ANOVA) (Faller et al., 2003). Each treatment had three

replications. The statistical significance was confirmed if p<0.05.

3. Result and discussion

Page 7 of 21
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

superoxide dismutase (SOD) content of the alga at 48 h were considered as the

response parameters under the combined effect of cefradine and ceftazidime. To

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

alga was given as follows:

The population growth rate change = 33.55-0.28A-0.28B-0.27AB-16.48A2 -

16.48B2 (R2=0.9997)

The chlorophyll-a content change = 126.33+9.26A-15.13B -61.12A2 -37.37B2

(R2=0.9292)

The SOD content change = 8.08-0.42A-2.11B-9.66A2-4.92B2 (R2=0.9039)

where A and B are the model terms representing the cefradine and ceftazidime

concentration, respectively. When R2 indicated the matched-degree of the empirical

model fits the obtained data. The larger the value of R2, the more relevant the
8

Page 8 of 21
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

the three suggested polynomial models, respectively. In addition, the statistical

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

concentrations was up to about 50 mg. L-1. Additionally, although the chlorophyll-a

content also increased with the concentrations of two antibiotics increased and then
9

Page 9 of 21
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

ceftazidime. The highest chlorophyll-a content was observed (increased 128.08%)

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

single effect on alga including population growth inhibition, photosynthesis, SOD

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

also participate in the regulation of algal growth and numerous physiological

processes (Liu, et al., 2012). Most of the antibiotics have a negative effect on algae,

which are concentration and category-dependent. In addition, algae or cyanobacteria

in different species also have varied sensitivity to antibiotics (Boxall et al., 2003).

Microcystis aeruginosa was observed to be more sensitive to most antibiotics than

other algal species such as Selenastrum capricornutum. In the testing, the

corresponding EC50 values of most antibiotics for M. aeruginosa were less than 0.1

mg.L-1 (Halling-Sorensen, 2000). Our previous research also indicated that M.

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
10

Page 10 of 21
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

three phenicol antibiotics chloramphenicol, florfenicol and thiamphenicol than the

freshwater alga C. pyrenoidosa (Lai et al., 2009).

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

administered to increase the toxicity as a combined effect (Broderius et al., 2005). In

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,
11

Page 11 of 21
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

and the SOD activity declined by oneself.

In conclusion, the research demonstrates the combined effect of two kinds of

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

Youth Fund (BK20130646, BK20140653), Jiangsu Key Lab of Environmental

12

Page 12 of 21
Engineering Open Foundation (KF2012008), Doctor Scientific Research Foundation

from China Pharmaceutical University (2012ZJ13002), National Undergraduate

Training Programs for Innovation and Entrepreneurship (02640556), the Fundamental

Research Funds for the Central Universities (JKQZ2013009, JKPZ2013017), Qing

Lan project (2014).

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Zhang, Z.S., Huang, X.F., 1991. Method for study on freshwater plankton. Science

Press, Beijing.

Figure captions list

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

population growth rate of Chlorella pyrenoidosa as a function of two antibiotics

concentrations. (A) 3D plots for the population change rate, (B) contour plots for the

population change rate.

Figure 3 Reference surface plots and contour plots as the change rate (%) in

chlorophyll-a content of Chlorella pyrenoidosa as a function of two antibiotics

concentrations. (A) 3D plots for the change rate of the chlorophyll-a content, (B)

contour plots for the change rate of the chlorophyll-a content.

Figure 4 Reference surface plots and contour plots as the change rate (%) in

superoxide dismutase (SOD) content of Chlorella pyrenoidosa as a function of two

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antibiotics concentrations. (A) 3D plots for the change rate of the SOD content, (B)

contour plots for the change rate of the SOD content.

Table 1. Process variables used in the CCD showing the treatment combinations

between the concentrations of two antibiotics. The alga lacking toxic compound was

tested as the control group.

Coded setting levels Actual levels
Treatment
X1a X2 b X1 a X2 b
1 0 0 30 30
2 1 -1 50 10
3 0 0 30 30
4 -1.414 0 1.7 30
5 1.414 0 58.3 30
6 0 -1.414 30 1.7
7 -1 1 10 50
8 0 0 30 30
9 0 1.414 30 58.3
10 -1 -1 10 10

a: cefradine concentration (mg.L-1)

b: ceftazidime concentration (mg.L-1)

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Table 2. Summary of p-value for the concentrations of two antibiotics effect on three

responses of Chlorella pyrenoidosa.

Response
Source The population growth The chlorophyll-a The superoxide
rate content dismutase (SOD)
Model <0.01 <0.01 <0.01
A <0.01 >0.05 >0.05
B <0.01 >0.05 >0.05
A2 <0.01 <0.01 <0.01
2
B <0.01 <0.01 <0.01
AB <0.01 >0.05 >0.05
Lack of fit >0.05 >0.05 >0.05

A: cefradine concentration (mg.L-1)

B: ceftazidime concentration (mg.L-1)

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