Comparison of organics and heavy metals acute toxicities to Vibrio fischeri J. Serb. Chem. Soc. 81 (6) 697–705 (2016) UDC 504.054–039.7+546.47’48’56’76: JSCS–4878 615.9:58.035:639.3 Original scientific paper 697 Comparison of organics and heavy metals acute toxicities to Vibrio fischeri XUEPENG YANG1, JI YAN1, FANGFANG WANG1, JIA XU1, XIANGZHEN LIU2, KE MA1, XIANGMEI HU1 and JIANBIN YE1* 1School of Food and Biological Engineering, Henan Provincial Collaborative Innovation Center for Food Production and Safety, Zhengzhou University of Light Industry, Dongfeng Road, #5, Zhengzhou, 450002, Henan Province, China and 2Technology Center, China Tobacco Henan Industrial Co., Ltd., Zhengzhou, 450000, China (Received 24 November 2015, revised and accepted 15 January 2016) Abstract: Vibrio fischeri bioluminescence inhibition has been widely used to test acute toxicities of metals and organics contaminants. However, the differ- ences of metals and organics acute toxicities to V. fischeri have not been com- pared. Here, four heavy metals (Zn2+, Cu2+, Cd2+ and Cr6+) and five organics (phenol, benzoic acid, p-hydroxybenzoic acid, nitro-benzene and benzene) acute toxicities to V. fischeri were investigated. Heavy metals toxicities to V. fischeri were increased along with the reaction time, while the organics toxi- cities kept the same level in different reaction times. In order to explain the dif- ference, the relative cell death rate of V. fischeri was detected. In metals toxi- cities tests, the bioluminescence inhibition rate of V. fischeri was found to be significantly higher than the relative cell death rate (P < 0.05), while for the organics toxicities tests, the cell death rate was similar to the bioluminescence inhibition rate. These results indicated that organics acute toxicities to V. fisch- eri could reflect the death of cell, but metals acute toxicities to V. fischeri may not lead to the death of cell, just represent the bioluminescence inhibition. Keywords: reaction time; luminescent bacteria; cell death; bioluminescence inhibition; cell death rate; Microtox® test. INTRODUCTION The luminescent bacterium, Vibrio fischeri has been widely applied for the toxicity detection of chemicals, including organics and metals.1–3 Due to its adv- antages of ease use, low cost and high reproducibility, this biotest has been accepted as a quick method of chemicals toxicities assessment in the environ- ment (named as Microtox® test), including wastewater effluent,4 sediment ext- racts5 and contaminated groundwater.6 * Corresponding author. E-mail: happye1986@163.com doi: 10.2298/JSC151124011Y _________________________________________________________________________________________________________________________ (CC) 2016 SCS. All rights reserved. Available on line at www.shd.org.rs/JSCS/ 698 YANG et al. The Microtox® test is based on the luminescence response of the bacterium V. fischeri. The light emission of V. fischeri is an enzyme catalysis reaction, and the process could be concluded as Eq. (1) and (2).7 Given the principle of this bioprocess, toxics that do harmful to any of the factors (O2, aldehyde, flavin reductase, luciferase and NAD(P)H) attending in this process could induce lumi- nescence inhibition, and the toxicities to V. fischeri could be assessed by the luminescence inhibition rate. In the other hand, cells death caused by toxics will also cause the luminescence inhibition. Thus, sporadically studies have issued that chemicals acute toxicities detected by the Microtox® test could either reflect disturbances of biosynthetic pathways8 or death of V. fischeri.9 Flavin reductase 2NAD(P)H H FMN NAD(P) FMNH+ + ⎯⎯⎯⎯⎯⎯⎯→ + (1) Luciferase 2 2 2 FMNH O R CHO FMN H O R COOH hv(490nm) + + − ⎯⎯⎯⎯⎯→ → + + − + (2) Nevertheless, there have been relatively few researches comparing the dif- ference of heavy metals and organics acute toxicities to V. fischeri. When assess- ing the toxicities of metals and organics by Microtox® test, different reaction times were used by experienced-based,10 but the reasons remain unclear. Based on the Microtox® test, a small quantity of studies had demonstrated that heavy metals toxicities to V. fischeri were fickle at different reaction times,11,12 while the organics toxicities kept relatively stable.13 These results in some extent exp- lain the reasonable discrepancy reaction time for detecting heavy metals and organics acute toxicities to V. fischeri. However, systematic comparison of heavy metals and organics acute toxicities to V. fischeri has not been carried out, and the reasons of using different reaction times still need to be further elucidated. In this study, heavy metals and organics acute toxicities to V. fischeri at var- ious reaction times were determined by the Microtox® test. The relative cell death rate was detected following the Microtox® test to explain the possible reasons of the difference between metals and organics. MATERIALS AND METHODS Toxicity assay The bacterium, V. fischeri, used in this study was purchased from the Institute of Soil Science, Chinese Academy of Sciences, Nanjing, China. The toxicity assay was performed according to national standard method of China (Water quality – Determination of the acute toxicity – Luminescent bacteria test. GB/T 15441-1995). Briefly, 5 mg freeze-dry powder of V. fischeri was revived in 1 ml chilled 3 % NaCl, followed by adding 4 ml 3 % NaCl. This solution was served as the working fluid for subsequent test. The toxicity assay was carried out by mixing 1.9 ml testing/control samples (solution only containing 3 % NaCl) and 0.1 ml working fluid of V. fischeri solution, and the light was recorded by the model DXY-2 lumi- nometer after different incubation times. _________________________________________________________________________________________________________________________ (CC) 2016 SCS. All rights reserved. Available on line at www.shd.org.rs/JSCS/ DIFFERENT ACUTE TOXICITIES TO V. fischeri 699 According to the Microtox® test, four kinds of chemicals (K2Cr2O7, ZnSO4⋅7H2O, CuSO4 and CdCl2) were selected as represent metals ions, Cr 6+, Zn2+, Cu2+ and Cd2+, respectively. Five organics were also tested: phenol, benzoic acid, p-hydroxy benzoic acid, nitro-benzene and benzene. These chemicals were purchased from Aladdin (http://www.aladdin-e.com/). These chemicals were selected as common contaminants in the water environment. The EC50 (chemicals concentrations that cause 50 % luminescence inhibition rate) was calculated according to previous research.14 Dimethylsulfoxide (DMSO) was used as the co-solvent for organics according to the method reported in the reference15. Three different reaction times for metals (15, 30 and 45 min) and organics (5, 10 and 15 min) were tested in this study according to the preliminary experiments. Relative cell death rate detection In order to explain the different acute toxicities of metals and organics to V. fischeri, relative cell death rate detection was conducted after the toxicity assay. Following toxicity assay, the bacteria were spread on 12 cm plates containing LB medium with the modification of containing 3 % NaCl and cultivated at 20±1 °C for 48 h. The colony-forming unit (CFU) was used to enumerate the viable cell number. The cell death rate (DR) was calculated with Eq. (3): S C 100(1 )= − C DR C (3) CS is the CFU of testing sample, and CC is the CFU of the control sample without contacting with the toxics. All testing and control samples were carried out in quintuplicate. Statistical analysis All toxicity assays were performed in quintuplicate. The statistical significant of values were calculated by Student’s t-test (mean analysis) at 5 % level of probability using the SPSS 19.0 package software (SPSS International, Chicago, IL, USA). Statistical significant differ- ence was reported when the probability of the result assuming the null hypothesis (P) is less than 0.05. RESULTS AND DISCUSSION Heavy metals toxicities The toxicities of the four kinds of metals (Zn2+, Cu2+, Cd2+ and Cr6+) were detected, as they are common contamination in the environment.16,17 The toxi- cities of four kinds of metals were showed in Fig. 1. Not surprising, the lumen- escence inhibition rate was increased along with the concentration of each metal. Worth to note that, under a certain concentration of metals, luminescence inhi- bition rate was also increased along with the reaction times. EC50 was calculated by the linearity relationship of luminescence inhibition rate and the logarithm of concentration.18 The EC50 at different reaction times were summarized in Table I. From 15 to 45 min, the EC50 of each metal was decreased significantly (P < 0.05). As showed in Fig. 1, the luminescence inhibition rate increased gradually along with the increasing of reaction times. Metals toxicities to V. fischeri were attributed to the metals ions,19 and earlier study demonstrated that the metals toxicities were mostly contributed by the ions that affected the luciferase in the _________________________________________________________________________________________________________________________ (CC) 2016 SCS. All rights reserved. Available on line at www.shd.org.rs/JSCS/ 700 YANG et al. luminescence bioprocess.20 Results in the present study further supported this idea. The toxicities of these heavy metals were closely in agreement with the previous reports.21,22 It was reported that Cd2+ could be adsorbed and trapped to the exo-polysaccharides on the outer layer of the luminescent bacteria.23 This may prevent the interaction between Cd2+ and key enzymes, which resulted in low toxicity of Cd2+ detected by only evaluating luminescence decrease in the present study. Meanwhile, the lower toxicity of Cr6+ in this study could result from Cr6+ being reduced to Cr3+, which has less toxic to the bioenzymes.24 Thus, different luminescence inhibition rate at different exposure times with these metals could partly demonstrates that the decrease of light emission in V. fischeri was due to the enzyme disturbance by heavy metals, and the toxicities were related with how they affect the enzymes rather than cause the cell death. Fig. 1. The luminescence inhibition rate by four different kinds of metals under various concentrations: a), ZnSO4, b) CuSO4, c) CdCl2 and d) K2Cr2O4. _________________________________________________________________________________________________________________________ (CC) 2016 SCS. All rights reserved. Available on line at www.shd.org.rs/JSCS/ DIFFERENT ACUTE TOXICITIES TO V. fischeri 701 TABLE I. Toxicities of heavy metals at different contact times Chemical Contact time, min 15 30 45 EC50 / mg L -1 EC50 / mg L -1 Pa EC50 / mg L -1 Pa (Zn) ZnSO4 0.5±0.02 0.4±0.01 8.5×10 -6 0.3±0.01 4.1×10-8 (Cu) CuSO4 10.8±0.5 8.0±0.4 1.0×10 -5 6.2±0.3 1.1×10-7 (Cd) CdCl2 31.9±1.6 23.6±1.2 1.5×10 -5 18.4±0.9 1.9×10-7 (Cr) K2Cr2O4 160.3±8.0 138.3±6.9 0.002 121.3±6.1 2.4×10 -5 aP value was compared to that of 15 min, represent that whether the toxicities of chemicals were significant dif- ferent between different contact times. The results represent the mean ± SD Organics toxicity Five organics toxicities to V. fischeri were showed in Fig. 2. Similarly, the luminescence inhibition rate increased along with the concentration increasing of organics. The EC50 of all organics at different reaction times were summarized in Table II. Data analysis showed that the EC50 of organics were close at different reaction times (P > 0.153). Unlike heavy metals, reaction time was not a factor for organics acute toxi- cities to V. fischeri. Organics toxicities to V. fischeri could due to different ioniz- ation constant,25 distinctive chemical–physical potential, chemical group, ioniz- ation, logarithm of the 1-octanol/water partition coefficient.26 Previous researches had showed that the different atom and structure can result in much difference in the toxicities of the organics.26–28 But how they affect the V. fischeri cells has been rarely interpreted. Sporadically studies issued that organics involved in the interaction with cell surface receptors, disruption of cell membrane function, or reaction with cellular components could be the reason for the luminescence inhi- bition of V. fischeri.29,30 The harmful that organics do to V. fischeri seem to be irreversible, and could cause the cell dead in short time. Combined with results in this study, the reason of luminescence inhibition by organics could attribute to the cells death. The relative cell death detection In order to identify whether the luminescence inhibition was induced by the cells death, the relative cell death detection was carried out after Microtox® test. Table III showed the relatively death rate of V. fischeri at different luminescence inhibition rate (20, 50 and 90 %). At luminescence inhibition of 20 and 50 %, the cell death rate was close to the luminescence inhibition rate after exposure to organics. The results suggested that the organics acute toxicities to V. fischeri could reflect the death of cells. However, to heavy metals, the relatively death rate of V. fischeri was significantly lower than the luminescence inhibition rate, indicated that heavy metals acute toxicities to V. fischeri may not reflect the cell death, and only caused partly cell death after contacting with the bacteria. _________________________________________________________________________________________________________________________ (CC) 2016 SCS. All rights reserved. Available on line at www.shd.org.rs/JSCS/ 702 YANG et al. Fig. 2. The luminescence inhibition rate by five different kinds of organics under various concentrations: a), phenol, b) benzene, c) benzoic acid, d) nitrobenzene and e) p-hydroxy benzoic acid. The different reasons of luminescence inhibition between heavy metals and organics could partly explain these results. The heavy metals may combine with some important enzymes involved in the luminescence bioprocess without caus- ing the cells death. Organics may cause damage to the cells membrane and _________________________________________________________________________________________________________________________ (CC) 2016 SCS. All rights reserved. Available on line at www.shd.org.rs/JSCS/ DIFFERENT ACUTE TOXICITIES TO V. fischeri 703 induce cells death after contacting for a certain time. Of course, after contacting with higher concentrations of organics and heavy metals, such as at the con- centrations of chemicals that caused 90 % luminescence inhibition, some irrever- sible effect on bacteria growth happened and the cell death rate was almost 90 % for both organics and heavy metals (Table III). TABLE II. Toxicities of aromatics at different contact times Chemical Contact time, min 15 30 45 EC50 / mg L -1 EC50 / mg L -1 Pa EC50 / mg L -1 Pa Phenol 111.5±5.8 110.9±5.5 0.871 107.9±5.0 0.324 Benzene 26.5±1.3 26.1±1.3 0.640 25.8±1.2 0.402 Benzoic acid 10.9±0.5 10.7±0.5 0.545 10.5± 0.5 0.241 Nitro-benzene 61.5±3.1 59.7±2.9 0.371 57.8±5.7 0.238 p-Hydroxy benzoic acid 6.3±0.3 6.1±0.5 0.931 6.0±0.3 0.153 aP value was compared to that of 5 min, represent that whether the toxicities of chemicals were significant dif- ferent between different contact times. The results represent the mean ±SD TABLE III. The relative cell death rate (DR / %) of V. fischeri at different luminescence inhibition rate; The results represent the mean ±SD Chemical Luminescence inhibition rate, % 20 50 90 Zn (ZnSO4) 8.9±0.2 33.8±0.3 91.3±0.6 Cu (CuSO4) 7.6±0.1 30.1±1.2 90.7±1.1 Cd (CdCl2) 10.5±0.4 32.2±0.7 91.5±1.2 Cr (K2Cr2O4) 7.9±0.1 29.8±0.6 92.6±2.2 Phenol 17.5±1.5 45.6±1.2 89.9±1.4 Benzoic acid 18.6±1.1 47.6±2.3 93.5±3.2 Nitrobenzene 19.7±3.2 46.5±0.8 92.6±1.3 p-Hydroxybezoic acid 17.5±1.4 48.1±1.4 91.5±0.9 Benzene 16.8±0.9 50.2±1.1 91.4±1.2 CONCLUSION In summary, systematic comparison of heavy metals and organics acute toxicities to V. fischeri is reported in this study. Metals toxicities detected by luminescence inhibition rate was increased along with the reaction time and was significant higher than the cell death rate, while organics toxicities kept similar at different reaction times and were consistent with the cell death rate. The discrep- ancy results indicated that organics acute toxicities to V. fischeri could represent the cell death, but luminescence inhibition by metals could due to the disturbing of luminescence bioprocess and may not necessary cause the cell death. Acknowledgments. The project was supported in part by Doctor Support Grants of Zhengzhou University of Light Industry, China. _________________________________________________________________________________________________________________________ (CC) 2016 SCS. All rights reserved. Available on line at www.shd.org.rs/JSCS/ 704 YANG et al. И З В О Д ПОРЕЂЕЊЕ ТОКСИЧНОСТИ ОРГАНСКИХ ЈЕДИЊЕЊА И МЕТАЛА ПРЕМА Vibrio fischeri XUEPENG YANG1, JI YAN1, FANGFANG WANG1, JIA XU1, XIANGZHEN LIU2, KE MA1, XIANGMEI HU1 и JIANBIN YE1 1 School of Food and Biological Engineering, Henan Provincial Collaborative Innovation Center for Food Production and Safety, Zhengzhou University of Light Industry, Dongfeng Road, #5, Zhengzhou, 450002, Henan Province, China and 2 Technology Center, China Tobacco Henan Industrial Co., Ltd., Zhengzhou, 450000, China Инхибиција биолуминисценције Vibrio fischeri широко се примењује за испитивање акутних токсичности метала и органских загађујућих материја. Међутим, до сада нису поређене разлике у токсичности ових материја према V. fischeri. У овом раду су испи- тане акутне токсичности четири тешка метала (Zn2+, Cu2+, Cd2+ и Cr6+) и пет органских једињења (фенол, бензоева киселина, p-хидроксибензоева киселина, нитробензен и бен- зен) према V. fischeri. Токсичности тешких метала су се повећавале са реакционим вре- меном, док је токсичност органских једињења остајала на истом нивоу при различитим реакционим временима. Како би се објаснила ова разлика, мерена је релативна брзина умирања ћелија V. fischeri. У тестовима токсичности метала, брзина инхибиције биолу- минисценције код V. fischeri била је значајно већа него релативна смртност ћелија (p < 0,05), док је у тестовима токсичности органских једињења, смртност ћелија била слична брзини инхибиције биолуминисценције. Ови резултати дају индикацију да акут- не токсичности органских једињења према V. fischeri одражавају смрт ћелије, али акутне токсичности метала не морају водити до смрти ћелије, већ само представљају инхиби- цију биолуминисценције. (Примљено 24. новембра 2015, ревидирано и прихваћено 15. јануара 2016) REFERENCE 1. X. Yu, J. Zuo, X. Tang, R. Li, Z. Li, and F. Zhang, J. Hazard. Mater. 266 (2014) 68 2. S.Villa, M.Vighi, and A. Finizio, Chemosphere 108 (2014) 239 3. G. 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