Jin Zhang*,
Meng-ting Tao,
Chongchong Song and
Bai Sun
Key Laboratory of Water Pollution Control and Wastewater Resource of Anhui Province, College of Environment and Energy Engineering, Anhui Jianzhu University, Hefei 230601, PR China. E-mail: ginnzy@163.com; Tel: +86-180-1958-0589
First published on 26th March 2020
A large number of antibiotics are entering the aquatic environment accompanying human and animal excreta, which will threaten the survival of aquatic organisms and even human health. It has been found that binary mixtures of aminoglycoside (AG) exhibit additive action and can be evaluated well by a classical model, concentration addition (CA) in our past study. Therefore, to investigate the toxicity interaction within multi-component mixtures of AG antibiotics, five antibiotics, kanamycin sulfate (KAN), neomycin sulfate (NEO), tobramycin (TOB), streptomycin sulfate (STS), and gentamicin sulfate (GEN), were selected to construct five-component mixture systems by a uniform design ray method. The toxic effects (luminescence inhibition) of single antibiotic and five-antibiotic mixture systems towards a photobacterium Vibrio qinghaiensis sp.-Q67 (V. qinghaiensis) in different exposure time (0.25, 2, 4, 8, and 12 h) were determined by the time-dependent microplate toxicity analysis method. The concentration-effect data were fitted by a nonlinear least square method, toxicity interaction within mixture systems was analyzed by a CA model, and the interaction intensity was characterized by deviation from the CA model (dCA). Besides, the toxicity mechanism of five antibiotics and their five-component mixtures to V. qinghaiensis was analyzed by electron microscopy. The results show that toxicity of five antibiotics and their five-component mixture systems to V. qinghaiensis is time-dependent and has strong long-term toxicity. Different from binary AG antibiotic mixture systems, five-antibiotic mixture systems exhibit obviously time-dependent synergism. In addition, toxicity of the five-antibiotic mixtures can be 1.4 times higher than that of the mixtures without synergisms at the same concentration level. According to dCA, synergism intensity (dCA) curves of rays move slowly from the high concentration region to the medium or lower one and the maximum dCA values also increase, decrease, or first increase, then decrease with the lengthening of exposure time. The inhibition activity and synergism intensity of mixture rays have good correlation with the concentration ratios of STS, the key component for synergism. The cell morphology of V. qinghaiensis indicates the strong toxicity of five antibiotics and their mixture rays is not due to the destruction of cell structure, but the inhibition of the light-emitting activity of the photobacterium.
Exposure time and interaction, synergism and antagonism, between mixture components may increase the toxicity of pollutants at a certain concentration.5–10 Zhang et al.11 found that four AG antibiotics and their mixtures,ampumycin sulfate, dihydrostreptomycin, kanamycin sulfate and neomycin salt, have obvious time-dependent toxicity to Vibrio qinghaiensis sp.-Q67 (V. qinghaiensis). Liu et al.12 found that the binary mixture of imidacloprid and pirimicarb showed synergism. Tang et al.13 studied the combined toxicity of ternary mixtures of heavy metals, ionic liquids and pesticides, and found that there was obvious synergism in the ternary mixture system. Therefore, the effects of exposure time and interaction between mixture components should be considered in order to accurately assess the environmental risk of AG antibiotics.
Usually, analysis of toxicity interaction within mixtures is relative to a standard reference model, so it is very important to select a suitable reference model when evaluating the toxicity (CA) is considered to be a relatively conservative model for predicting mixture toxicity14 and widely used by many scholars.15–18 However, CA model cannot quantitatively evaluate the degree of synergism or antagonism. Deviation from CA model (dCA) can visualize the difference between experimental observation and model prediction. The greater the absolute value of dCA is, the greater the degree of interaction between the components of the mixture system.
Some studies found that toxicity and toxicity interaction of complex mixture system may be closely related to a key component.19 Fan et al.20 found that polymyxin B sulfate may be the key component of antagonism between ionic liquids and antibiotic. Zhang et al.21 found that 1-butyl-3-methylimidazolium-octyl sulfate could induce antagonism in quaternary ionic liquid mixtures. So whether there are key components in AG antibiotics mixture system is also the concern of this paper.
As stated above, this study aims to investigate toxicity interaction, synergism or antagonism, within multi-component AG antibiotic mixtures. To do so, five AG antibiotics, kanamycin sulfate (KAN), neomycin sulfate (NEO), tobramycin (TOB), streptomycin sulfate (STS), gentamicin sulfate (GEN), were selected as the research objects, and V. qinghaiensis as the tested organisms to explore the toxicity characteristics of AG antibiotics and their mixtures. The five-component mixture systems of the five antibiotics were constructed by uniform design method.22–24 The inhibition data of single antibiotic and its five-component mixtures to V. qinghaiensis at different exposure time (0.25, 2, 4, 8, and 12 h) were determined by time-dependent microplate toxicity analysis method.25–27 The concentration-effect data were fitted by nonlinear least square method, the toxicity interaction of mixture systems was analysed by concentration addition model (CA) with 95% confidence interval,17–22,28 and the degree of toxicity interaction was characterized by deviation from CA model (dCA). Besides, the luminescence inhibition mechanism of five antibiotics and their five-component mixtures to V. qinghaiensis was preliminarily determined by observing the cell morphology.29,30 The experimental data obtained would provide a reference for the assessment of the ecological risk of the AG antibiotics.
Name | Abbr. | MW | CAS RN | Stock (mol L−1) | f |
---|---|---|---|---|---|
Kanamycin sulfate | KAN | 582.58 | 25389-94-0 | 1.10 × 10−5 | 0.68 |
Neomycin sulfate | NEO | 908.880 | 1405-10-3 | 1.12 × 10−5 | 0.68 |
Tobramycin | TOB | 467.514 | 32986.56-4 | 2.19 × 10−6 | 0.68 |
Streptomycin sulfate | STS | 1457.38 | 3810-47-0 | 6.56 × 10−7 | 0.68 |
Gentamicin sulfate | GEN | 575.67 | 1405-41-0 | 3.44 × 10−5 | 0.68 |
![]() | (1) |
Ray | PKAN | PNEO | PTOB | PSTS | PGEN |
---|---|---|---|---|---|
R1 | 1.70 × 10−2 | 1.21 × 10−1 | 1.60 × 10−2 | 9.00 × 10−3 | 8.37 × 10−1 |
R2 | 3.30 × 10−2 | 3.44 × 10−1 | 4.60 × 10−2 | 1.00 × 10−3 | 5.76 × 10−1 |
R3 | 6.50 × 10−2 | 5.59 × 10−1 | 6.00 × 10−3 | 1.10 × 10−2 | 3.60 × 10−1 |
R4 | 2.53 × 10−1 | 1.16 × 10−1 | 7.00 × 10−2 | 4.00 × 10−3 | 5.57 × 10−1 |
R5 | 3.22 × 10−1 | 4.00 × 10−1 | 6.00 × 10−3 | 3.00 × 10−2 | 2.42 × 10−1 |
R6 | 2.99 × 10−1 | 5.83 × 10−1 | 2.80 × 10−2 | 6.00 × 10−3 | 8.40 × 10−2 |
R7 | 1.49 × 10−1 | 3.72 × 10−1 | 2.70 × 10−2 | 1.00 × 10−2 | 4.41 × 10−1 |
E = 1/(1 + exp(−α − β × log10(c))) | (2) |
E = 1 − exp(−exp(α + β × log10(c))) | (3) |
![]() | (4) |
To further characterize the degree of toxicity interaction, dCA was introduced as the evaluation criteria. Mathematically, dCA can be formulated as:
dCA = |EOBS − EPRD,CA| | (5) |
V. qinghaiensis bacterial suspension during logarithmic growth period was placed in a series of conical flasks. Then, five AG antibiotics and their five-component mixtures were added to the conical flasks with V. qinghaiensis bacterial suspension, respectively. The final liquid volume of conical flask was 40 mL. In each conical, the concentrations of each antibiotics and mixtures in the suspension were equal to the EC50 of each antibiotic and their five-component mixtures in 12 h. At the same time, Milli-Q water was added as blank control. All the conical flasks were cultured in a constant temperature incubator at 22 ± 1 °C for 12 h. After 12 h, the cell morphology of V. qinghaiensis in bacterial suspension of each conical flask was observed by electron microscope. The specific steps for the preparation of electron microscope samples refer to the relevant literatures.29,30
From Fig. 1, five AG antibiotics showed strong time-dependent inhibition activity to V. qinghaiensis, and the time characteristics of the CRCs of five antibiotics were similar. Within 2 h, the luminescence inhibition rates of the five antibiotics were very small, and their t–CRCs were almost straight lines, but the inhibition rates increased greatly with the further extension of the exposure time, and the t–CRCs became the standard S-shaped. The inhibition activity of the five antibiotics was the highest when the exposure time was 12 h, and the fastest increase rate of inhibition activity was within 2–4 h. This indicates that the five AG antibiotics with the level of experimental concentrations have no obviously short-term toxicity but strong long-term toxicity. Therefore, the effects of exposure time cannot be ignored in the environmental risk assessment antibiotics.5,7,8
From Fig. S1,† CRCs of seven rays in the five-component mixture system was also time-dependent to V. qinghaiensis, the inhibition activity increases with the prolongation of the exposure time, and the time characteristics of CRCs of seven mixture rays were similar to those of a single antibiotic.
The toxicity of a mixture may be related to some component's concentration ratios.31,32 In this study, pEC50 (−lgEC50) values of seven mixture rays at 12 h were linearly fitted with the concentration ratios of each component. The results showed that there was a good linear relationship between pEC50 values of seven mixture rays at 12 h and the concentration ratio of STS (Fig. 2), which indicates that the long-term toxicity of each mixture ray depends on the concentration ratios of STS in the mixture system, and the long-term toxicity of the five-component mixture system can be predicted by using this linear function relationship (pEC50 = 22.42PSTS + 6.04, R = 0.9060).
![]() | ||
Fig. 2 The relationship between pEC50 values of seven mixture rays at 12 h and the concentration ratio of STS. |
From Fig. 3, CA prediction curves located below the confidence intervals of experimental observations from 4 to 12 h, and five out of seven rays in five-component mixture systems showed obvious time-dependent synergism. R2 and R4 had no obvious synergism in the whole exposure time and exhibit classical additive action (Fig. S2†). From Fig. 1 and S2,† the five antibiotics and their five-component mixtures have no obvious toxicity within 2 h probably due the experimental concentrations are well below the acute concentration range. In addition, V. qinghaiensis grows very slow in the first stage of exposure time (0.25 h and 2 h). Therefore, the five-component mixtures don't have a synergism at the early stage of exposure (0.25 h and 2 h). It was also noticed that the five rays are time-dependent synergism while R2 and R4 rays with additive action, though the seven rays have the same components. So, the reason mixture rays with the same component exhibit different toxicity interaction are mainly due to effects of the concentration ratios.32,33
It can be seen from Fig. 4 that the synergism intensity of the five-component mixture rays with different concentration ratios varies with lengthening of exposure time. From 4 to 12 h, dCA curves of rays move slowly from high concentration region to the medium or lower one, which is probably due to the enough contact between mixture components and bacteria therein. The maximum dCA values also vary with time. From 4 to 12 h, the maximum dCA value of R1 increases gradually with time lengthening, while that of R6 gradually reduces. As for R3, R5 and R7, the maximum dCA value first decrease, then increase. Among the five rays in Fig. 4, R5 shows the strongest synergism with the maximum dCA value of 40%, which means that the toxicity R5 can be at least 1.4 times stronger than that of rays without synergisms. The maximum dCA values of the rest of rays in Fig. 4 are higher than 30%.
By comparing the concentration ratios of seven mixture rays in Table 2, it is found that the synergism in the mixture system is probably correlated with STS. The higher the concentration ratio of STS in the mixture system is, the more obvious synergism in the system. The concentration ratios of STS in R2 and R4 are the smallest compared with other rays, and there is no obvious synergism. The concentration ratio order of STS in the seven rays is: R5 > R3 > R7 > R1 > R6 > R4 > R2, and the synergism intensity (dCA value) order of seven rays is also: R5 > R3 > R7 > R1 > R6 > R4 (R2). However, no significant correlations are found between the synergism of five-component mixture systems and the concentration ratios of other four antibiotics, which indicates that STS may be the key component in five-component mixture systems of AG antibiotics and the time-dependent synergism of the five-component mixtures is induced by the component STS.19–21
As can be seen from Fig. 5, most of the cell morphology of V. qinghaiensis was maintained in the normal state. Therefore, the toxic effects of the five antibiotics and their five-component mixtures on V. qinghaiensis are not caused by destroying cell morphology, but inhibition of luminescence.
Footnote |
† Electronic supplementary information (ESI) available. See DOI: 10.1039/d0ra00915f |
This journal is © The Royal Society of Chemistry 2020 |