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Individual and joint toxic effects of pentachlorophenol and bisphenol A on the development of zebrafish (Danio rerio) embryo
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Ecotoxicology and Environmental Safety 71 (2008) 774–780 www.elsevier.com/locate/ecoenv
Individual and joint toxic effects of pentachlorophenol and bisphenol A on the development of zebrafish (Danio rerio) embryo Zhenghua Duana, Lin Zhua,b,, Lingyan Zhua,b, Kun Yaoa, Xiaoshan Zhua a
College of Environmental Science and Engineering, Nankai University, Tianjin 300071, PR China Tianjin Key Laboratory of Environmental Remediation and Pollution Control, TEDA 300253, PR China
b
Received 20 June 2007; received in revised form 21 January 2008; accepted 27 January 2008 Available online 21 March 2008
Abstract Investigation of the toxicological effects of pentachlorophenol (PCP) and bisphenol A (BPA) alone and in combination was carried out following the method of the early life stage (ELS) test on zebrafish embryos. Both chemicals revealed lethal and sub-lethal effects, such as no blood flow, cardiac edema, delayed hatching, and tail malformations. According to their median effective concentrations (EC50 values) in the single exposure, the toxic level of PCP was about two orders of magnitude higher than that of BPA. Result of the joint action modes varied depending on different endpoints. Synergistic action was observed based on the endpoint of 24 h mortality and antagonistic effect displayed based on the endpoint of 72 h cardiac edema. It was also found that the toxicity of PCP would be enhanced with the addition of BPA even below its no observed effect concentration (NOEC) level at the endpoint of 32 h with no blood flow, and the level of the increase was influenced by the toxic unit (TU) ratio. r 2007 Elsevier Inc. All rights reserved. Keywords: Joint toxicity; Pentachlorophenol; Bisphenol A; Zebrafish embryo
1. Introduction The individual toxicity of most chemicals is widely studied and well understood. However, chemicals usually exist in the environment as mixtures and information regarding the joint effects of multi-pollutants as well as their toxic mechanisms remain insufficient. Pentachlorophenol (PCP) has been used as an industrial antiseptic and biocide for many decades since the 1960s. Due to its proven carcinogenicity and toxicity, as well as the existence of a large number of known PCPcontaminated sites, PCP has been designated as a ‘‘priority toxic pollutant’’ by the United States Environmental Protection Agency (EPA) under the guidance of the Clean Water Act in 1972. Bisphenol A (BPA) is another common chemical that has been widely used in the chemical industry in the manufacturing of epoxy- and polyester-styrene resins. Studies have testified that BPA was potentially toxic to Corresponding author at: College of Environmental Science and Engineering, Nankai University, Tianjin 300071, PR China. Fax: +86 22 23508807. E-mail address: [email protected] (L. Zhu).
0147-6513/$ - see front matter r 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.ecoenv.2008.01.021
embryos and has been found to cause genetic defects (Bindhumol et al., 2003). BPA can also be converted to DNA binding metabolites in vitro (Atkinson and Roy, 1995). Since these two chemicals are extensively used in daily life and industrial applications, their potential toxicities have recently sparked attention. These chemicals usually exist in the environment as mixtures. Huang et al. (2001) have shown that the concentrations of PCP and BPA were 3.61–2010 and 5.57–29.80 ng L1, respectively, in river water and seawater in China. Previous studies have mostly focused on the individual toxicities of these chemicals and there is no report so far about their joint toxicity effects. Zebrafish (Danio rerio) has gained merits as a model species over the past few years because of its short growth period, high fecundity, and transparent eggs (Hallare et al., 2004; Reimers et al., 2004). The early life stage (ELS) test using zebrafish embryos is widely used in investigating the toxicity and teratogenicity of chemicals known to cause significant impact on environmental and human health (Van Leeuwen et al., 1990; Nagel, 2002). Furthermore, as the zebrafish holds many similar cellular and physiological characteristics with higher vertebrates,
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the toxicological results of zebrafish embryonic development can be easily compared with those of mammalian development (Marguerie et al., 2006; Reimers et al., 2004). Therefore, the zebrafish embryo is a perfect model to study the developmental toxicity of vertebrates and the ELS test has become an effective alternative method of adult fish testing. In the present study, PCP and BPA were chosen as examples to examine their individual and joint effects on the development of zebrafish embryo. The difference between their individual toxicities was investigated by their quantum chemical descriptors. The mechanisms of joint effects and their influence factors were also studied according to biochemical reactions and membrane transport processes. 2. Materials and methods 2.1. Chemicals PCP (C6Cl5OH, purity499.50%) and BPA (C15H16O2, purity499.00%) were purchased from ALDRICH company (Unite States). Their structural formulas are as shown: CH3
OH Cl
Cl
Cl
Cl
HO
C
775
treatment solution (PCP, BPA, or PCP/BPA solutions). The remaining four wells were filled with 2 mL reconstituted water instead of treatment solution to act as controls. In addition, 1% ethanol (1 mL diluted in 1 L reconstituted water) was used as a solvent control in another multi-well plate. All of the wells were covered with transparent plastic film and placed in an incubator at 2671 1C with a 10/14-h light/dark cycle. Based on preliminary experiments, it was found that if the embryos were exposed after gastrula (8 h post-fertilization) stage, the lethal toxicity of chemicals to the embryos decreased greatly and more severe sub-lethal toxic effects were observed. Therefore, two additional exposures were designed in the following experiments: (1) early cleavage exposure (exposure immediately after fertilization, 0 hpf exposure) to study the endpoint of mortality, and (2) gastrula exposure (exposure after 8 h postfertilization, 8 hpf exposure) to study the endpoints of sub-lethal effects. The developmental status of zebrafish embryos were observed with an inverted microscope (8–50 ) (IMT 2, Olympus Corporation, Tokyo, Japan) and documented photographically at specific times after fertilization (time points ¼ 0, 8, 24, 32, and 72 h). Endpoints used for assessing developmental toxicity were recorded and described for embryos for both the control and treated groups. The endpoints included 24 h mortality (the embryo had no heart beat; 0 hpf exposure), 32 h no with blood flow (the embryo had heart beat, but the blood in the vessels and stopped flowing; 8 hpf exposure), 72 h cardiac edema (8 hpf exposure, Figs. 1C and D), 72 h delayed hatching (8 hpf exposure), and 72 h tail deformations (tail damaged or broken; 8 hpf exposure, Figs. 1E and F).
2.4. Equitoxicity test OH
CH3
Bisphenol A
Cl
Pentachlorophenol All tests were performed with reconstituted purified water in accordance with ISO (International Standard Organization) standard of 7346-3:1996. The pH value of the water was kept at 8.0 and the hardness was equivalent to about 150 mg L1 of calcium carbonate (CaCO3). The exposure concentrations in the studies ranged between 0.01–1.00 mg L1 for PCP and 2.00–25.00 mg L1 for BPA. Therefore, 1 L PCP stock solution at the concentration of 1.00 mg L1 was prepared in reconstituted purified water together with 1 mL ethanol as co-solvent. BPA stock solution at the concentration of 25.00 mg L1 was prepared in a similar way. The stock solutions were then diluted to the specific test concentrations with reconstituted water.
For a single chemical, the concentration at its EC50 is defined as 1 toxic unit (TU, dimensionless). The toxicity of the chemical at other concentrations is expressed in TU by dividing the concentrations by its EC50 value. A mixture is equitoxic if each component is present at the same fraction of its own EC50 (or LC50) value, i.e. the ratios of the concentrations of the chemicals to their EC50 (or LC50) values are equal. The equitoxicity test in this study consisted of three simultaneous treatment series: (A) PCP alone, (B) BPA alone, and (C) equitoxic mixtures of PCP and BPA. The endpoints of 24 h mortality and 72 h delayed hatching were selected to study the joint effects. Parallel experiments of three replicates each were conducted for each treatment level. Basically, when two chemicals were applied simultaneously to a living system or unit, three types of joint effects was observed. They are additive toxicity effect (usually upon dose addition), synergism (greater than additive toxicity), and antagonism (less than additive toxicity) (Hertzberg and MacDonell, 2002). The type of joint action for a specific binary mixture of toxicants is determined by the additive index (M) or the sum of TUs, which is calculated using the following equation: M¼
2.2. Zebrafish Adult zebrafish was second generation in our laboratory, and the parents were obtained from an aquarium in Tianjin, China. The maintenance of brood fish strictly followed the OECD (Organization for Economic Cooperation and Development) guideline for fish embryo toxicity test (OECD, 1998). Fish eggs were collected and rinsed three times in reconstituted water to remove any residue on the egg’s surface. Fertilized eggs were selected for exposure experiments after visual inspection with a stereomicroscope (8–50 ). Studies involving zebrafish embryo were conducted in accordance with national and institutional guidelines for the protection of human subjects and animal welfare.
2.3. Toxicity test The embryonic toxicity test was developed by Nagel (2002). Twentyfour eggs were transferred into a 24-well multi-well plate such that each well contained one embryo. Twenty wells were filled with 2 mL of
Zð1Þ Zð2Þ þ , EC50 ð1Þ EC50 ð2Þ
where Z(1) and Z(2) denote the individual concentrations of the mixture in which the combined concentrations resulted in a 50% response. When M ¼ 1, the joint effect would be a simple addition, when M41, it would be antagonism and Mo1, it is synergism (Hsieh et al., 2006). A mixture of the two toxicants with different sums of TUs was tested. The total TUs varied in the range of 0.25–2.60. According to the number of embryos which displayed toxicity effects, M and its 95% confidence interval (CI) at 50% response were calculated.
2.5. Unequitoxicty test A pre-screening experiment for single exposure was conducted to determine the range of exposure concentrations for PCP and BPA. Based on the results of single exposure, it was found that PCP was more toxic than BPA (it will be discussed later). To evaluate the combined toxicity of PCP with the presence of BPA, two toxicity experiments were performed for comparison based on the endpoint of 32 h no blood flow. One was
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Fig. 1. Observed normal and abnormal zebrafish embryo and larva fish. (A) Normal embryo, 48 hpf; (B) normal larva fish after hatching, 72 hpf; (C) cardiac edema in the embryo, 48 hpf, 15.00 mg L1 BPA; (D) cardiac edema after hatching, 72 hpf, 15.00 mg L1 BPA; (E) tail deformations in the embryo, 48 hpf, 20.00 mg L1 BPA; and (F) tail deformations after hatching, 72 hpf, 20.00 mg L1 BPA. tested with PCP alone and the other was a combination of PCP and BPA. For all combined groups, the total toxic levels were kept at a constant TU of 0.26, while the concentration ratios of PCP and BPA differed; the concentrations of PCP varied around its EC50 value, whereas those of BPA remained at a level below its no observed effect concentrations (NOEC). The TU ratios (TU1:TU2 ¼ PCP:BPA) were selected to be 1/5, 1/3, 1, 3, and 5. For individual exposure of PCP, the concentrations of PCP were the same as those in the combined mixture with zero concentrations of BPA.
2.7. Statistical analysis Results from microscopic examinations of the embryos were categorized according to the types and severity of the toxic endpoints. The 50% effect concentrations for mortality (LC50) and sub-lethality (EC50) were calculated using Sigmoid Fit by ORIGIN 7.0 (OriginLab, US). Comparison tests between groups were carried out by one-way ANOVA. A p-value of o0.05 was considered as a statistically significant.
3. Results 2.6. Chemical analysis
3.1. Individual toxicities of PCP and BPA Due to the adsorption of organic chemicals on the inner wall of plastic containers, the actual chemical concentration determined in the solution may be lower than the nominal value. After 72 h single exposure of PCP or BPA (0 hpf exposure), 2 mL water was removed from testing chambers and analyzed by high pressure liquid chromatography (HPLC, Waters 1525). It was found that actual concentrations of both PCP and BPA were less than their nominal concentrations as expected. The linear regression between the actual (Y) and nominal (X) concentrations was calculated for PCP and BPA following the equations Y ¼ 0.85X+0.03 (R2 ¼ 0.9956) and Y ¼ 0.71X+1.78 (R2 ¼ 0.9549), respectively. Based on the above equations, the actual concentrations of PCP and BPA in the study were determined and reported.
No effect was observed in the zebrafish embryos when 1% ethanol was used as a solvent control (data not shown). In the individual exposure of zebrafish embryos to PCP, dose–response curves were obtained as shown in Fig. 2A. The median lethal concentration (LC50) after 24 h was 0.76 mg L1. The median effect concentrations (EC50) were 0.19 mg L1 for no blood flow at 32 hpf, 0.08 mg L1 for cardiac edema at 72 hpf, and 0.09 mg L1 for delayed hatching at 72 hpf. According to the EC50 values of every
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sub-lethal endpoint, the most sensitive toxicological endpoint induced by PCP was cardiac edema at 72 hpf. In the individual exposure of zebrafish embryos to BPA, the median lethal concentration (LC50) after 24 h was 16.75 mg L1 (Fig. 2B). All embryos were dead when exposed to 25.00 mg L1. The no effect concentration (NOEC) for all sub-lethal endpoints was 2.00 mg L1. The median effect concentrations (EC50) were 23.05 mg L1 for no blood flow at 32 hpf, 20.87 mg L1 for cardiac edema at 72 hpf, and 13.81 mg L1 for delayed hatching at 72 hpf
777
(Fig. 2B). Embryos exposed to high concentrations of BPA (415.00 mg L1) showed tail deformations (Figs. 1E and F). Therefore, the sensitive endpoint induced by BPA was delayed hatching at 72 hpf. Based on the results of single exposure, it was found PCP was much more toxic than BPA on the development of zebrafish embryos. For instance, based on the endpoint of 32 h with no blood flow, the EC50 value of BPA (23.05 mg L1) was about 121 times as high as that of PCP (0.19 mg L1). Based on the endpoint of 72 h cardiac
Fig. 2. Dose–response relations for PCP (A) and BPA (B). Death—24 h death; blood—32 h with no blood flow; edema—72 h cardiac edema; hatch—72 h delayed hatching.
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Z. Duan et al. / Ecotoxicology and Environmental Safety 71 (2008) 774–780 Table 1 Joint effects of PCP and BPA to zebrafish embryos at equitoxicity level Endpoints
Aa
Bb
PCP BPA
PCP BPA
24 h mortality 0.76 72 h cardiac 0.08 edema a
16.75 0.22 20.87 0.07
M
4.75 18.9
95%CI
Joint effect
0.57 0.48–0.66 Synergism 1.78 1.56–2.10 Antagonism
EC50 of individual exposure (mg L1). Combined concentrations resulted in a 50% response (mg L1).
b
Fig. 3. Effects of PCP and BPA with equitoxicity. Death—24 h death; edema—72 h cardiac edema.
edema, the EC50 value of BPA (20.87 mg L1) was about 260 times that of PCP (0.08 mg L1). Hence, the toxicity of PCP to zebrafish embryos is about two orders of magnitude higher than that of BPA. 3.2. Joint action with equitoxicity Two representative endpoints, 24 h mortality and 72 h cardiac edema in the embryonic development of zebrafish, were selected for the combined equitoxicity study. For the endpoint of 24 h mortality, mortality of the embryos showed significant dose–response relationship with TU (range from 0.25 to 1.00), and all embryos were dead when TU was higher than 1.00 (Fig. 3). For the endpoint of 72 h cardiac edema, the percentage of cardiac edema also displayed significant sigmoid relationship with TU (range from 1.00 to 2.50). No cardiac edema effect detected in all embryos when TUp1.00 (Fig. 3). As shown in Table 1, 0.57 TU induced a 50% toxic response for 24 h mortality. However, TU that induced 50% toxic response for 72 h cardiac edema was 1.78. Judging from the 95% CI for M, the mixtures displayed synergistic joint action based on the endpoint of 24 h mortality, whereas antagonistic joint action was observed based on the endpoint of 72 h cardiac edema. 3.3. Joint action with unequitoxicity As shown in Fig. 2, according to the endpoint of 32 h with no blood flow, the dose–response relationship was significant at an individual exposure of PCP. When BPA was added, even at a level below its NOEC, the observed toxicity effects increased significantly (Fig. 4). All p-values between the toxicities of each chemical alone and their combined exposures were statistically significant at less than 0.05. The combined effect differed significantly even when the concentrations of PCP and BPA remained at
Fig. 4. Effects of PCP alone and in combination with BPA based on the endpoint of 32 h with no blood flow. Corresponding with the concentrations of PCP and BPA at the forward direction of X-axis, TU ratios (TU1/ TU2 ¼ PCP: BPA) were 1/5, 1/3, 1, 3, and 5. In single exposures, the concentrations of PCP were the same as those in combined exposures, whereas all the concentrations of BPA were zero.
constant total TU value. The combined toxicity increased with the concentration of PCP, attained the highest percentage of toxicity when PCP was 0.10 mg L1 and BPA was 2.16 mg L1 (TU1/TU2 ¼ 1:1), and then decreased with the increase of PCP concentration (Fig. 4). 4. Discussion 4.1. Action mechanisms of individual toxicity In this study, it was found that both PCP and BPA, either alone or in a mixture, had profound effects on the developmental stage of zebrafish embryos. Besides death, there were several other sub-lethal effects, such as no blood flow, cardiac edema, delayed hatching, and tail deformations. Our data showed that the toxicity of PCP was much higher than that of BPA. These results were in good agreement with those reported in previous studies. As compared with BPA, PCP displayed higher toxicity in all organisms shown in Table 2 (Repetto et al., 2001; Johnson and Finley, 1998; Staples et al., 1998). These could be due
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to their different quantum chemical descriptors. A chemical exerts its toxic effects to organisms in two major steps: (1) transport-associated process which is mainly controlled by the parameter of the octanol–water partition coefficient (log Kow value of the compound); (2) biochemical reactionassociated process which is described by total energy (TE) of the molecule. It was found that the effective concentrations or even the lethal concentrations (in mol L1), such as the EC50 or LC50 values, are related to the log Kow values of each compound (Schwarzenbach et al., 2003). Zhang et al. (2000) have also concluded that TE is the key parameter in determining the toxicity of the group of chlorinated aromatic compounds to Selenastrum capricornutum. The quantum chemical descriptors (log Kow and TE values) of PCP and BPA were calculated using the Chemoffice 6.0 software with the algorithm of MOPAC-AM1. Log Kow values were about 5.74 for PCP and 3.40 for BPA, indicating that at the same concentrations, it was much easier for PCP to permeate through the biological membrane and combine with target organs. In addition, the TE values were about 2970.5 eV for PCP and 2774.91 eV for BPA, suggesting that the molecular of PCP was more active than that of BPA. It has been reported that PCP has enhanced toxicity than that of other phenols due to its specific effects, for example, interference with the energy transduction of cells by interfering with the electrochemical proton gradient (Escher et al., 2001). Therefore, PCP exhibited a higher toxicity than BPA in the organisms discussed above. 4.2. Action mechanisms of joint exposure Joint action modes were different depending on the toxicity endpoints. In the study of equitoxicity, synergistic joint action mode was observed based on 24 h mortality when the exposure concentrations were 0.10–1.00 mg L1 for PCP and 2.16–21.60 mg L1 for BPA, and antagonistic joint action mode was observed based on 72 h cardiac edema when the exposure concentrations were 0.01–0.10 mg L1 for PCP and 2.62–26.20 mg L1 for BPA. Death might result to the combined effects of the chemicals. Any changes in the membrane that induce the abnormal embryonic development would finally bring the embryo to death. PCP and BPA induce cellular death via Table 2 Toxicity data for PCP and BPA to several organisms Results (mg L1) Organism
Daphnia magna Pimephales promelas Oncorhynchus mykiss a
Test type
48 h EC50 96 h LC50 96 h LC50
Endpoint
Immobilization Mortality Mortality
Referred by Repetto et al., 2001. Referred by Staples et al., 1998. c Referred by Johnson and Finley, 1998. b
PCP
BPA
0.40a 0.21c 0.05c
3.90b 4.70b 4.00b
779
different mechanisms. PCP induces the embryonic death mainly through its binding to mitochondrial proteins and inhibiting the mitochondrial ATPase activity. As a result, both the formations of ATP and ADP are prevented (Pande et al., 1999). However, when the embryo was exposed to BPA, lipid peroxidation was induced by free radical generation via metabolic redox cycling between the quinone and hydroquinone forms of BPA (Chitra et al., 2003). When the embryos were exposed to a mixture of PCP and BPA mixture, both damage mechanisms were activated, and the embryonic membrane would be break down more easily. A synergism effect might then be demonstrated. The antagonism observed at the endpoint of 72 h cardiac edema could be explained with a competitive site theory (Vranken et al., 1988). Some active sites might exist which could induce the effect of cardiac edema. When PCP and BPA existed as a mixture and the concentrations of BPA in the combined solutions were about 20 times that of PCP, there were more chances for BPA ions to occupy the limited active sites, thereby suppressing the binding of PCP to the active sites. However, the toxicity of BPA was lower than that of PCP at the same exposure concentration, which would finally lead to the decrease of total toxicity. Therefore, suggesting that the joint toxicity effect would be antagonism. 4.3. Influences on the joint effect In this study, it was found that the toxicity of PCP at the endpoint of 32 h with no blood flow was enhanced with the addition of BPA to even below its NOEC level, indicating that BPA has a toxicity effect when treated in mixtures even though in concentrations below the NOEC of single dose BPA. Therefore, synergistic effects were exhibited at this endpoint. When embryos were exposed in a mixture of PCP and BPA at the same total toxicity level (0.26 TU), it was presumed that the joint toxicity would be constant. However, results in this study indicated that the joint toxicity is influenced by the TU ratio and can be changed accordingly. The toxicity of PCP in a single exposure increased with its concentration at the endpoint of 32 h with no blood flow. When BPA was added to the PCP solution while the total toxicity level remained constant, the exhibited toxicity was enhanced in all of the test concentration range. As discussed previously, PCP and BPA had different toxicity mechanisms. When PCP and BPA existed as a mixture, both mechanisms were activated in embryos and enhanced toxicity thus resulted. Addition of BPA into PCP solution resulted in an increase of the toxicity, which was affected by both of the PCP and BPA concentrations. The relative increase of toxicity was defined as the ratio of [combined toxicity single toxicity of PCP]/[single toxicity of PCP]. Date showed that while the TU ratio increased and the concentration of BPA in the mixture decreased, the relative degree of toxicity decreased as well (Table 3). On the other
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Table 3 The relation between the concentrations of BPA and the relative increase of toxicity TU ratio Concentrations of BPA in the mixture (mg L1) Combined toxicitya Single toxicity of PCPa Relative increase of toxicityb
1/5
1/3
1
3
5
3.6
3.25
2.16
1.08
0.72
35 0
50 5
70 15
55 20
40 35
–
9
3.67
1.75
0.14
a
The toxicity denoted the effective percent (%) of 32 h with no blood flow, as derived from Fig. 4. b The relative increase of toxicity was defined as the ratio of [combined toxicitysingle toxicity of PCP]/[single toxicity of PCP].
hand, the single toxicity of PCP increased consistently with the increase of PCP concentration. Thus, there would be a specific TU ratio at which the mixture displayed the highest toxicity. In this present study, it was found that the combined toxicity peaked at TU1/TU2 ¼ 1:1 (PCP was 0.10 mg L1 and BPA was 2.16 mg L1). 5. Conclusions PCP and BPA were both toxic to the development of zebrafish embryos, and the toxicity level of PCP was about two orders of magnitude higher than that of BPA. Joint action modes varied depending on different endpoints. Synergistic action was displayed based on the endpoint of 24 h mortality, but antagonistic effect was exhibited based on the endpoint of 72 h cardiac edema. Combined effects would be influenced by the TU ratios. Acknowledgments This work was funded by Natural Science foundation of China (approval no.30340091). We would like to thank everyone who contributes to this study, especially Liuming Pan and Si Gao for their editorial assistance. This study involving zebrafish embryos was conducted in accordance with national and institutional guidelines for the protection of human subjects and animal welfare. References Atkinson, A., Roy, D., 1995. In vitro conversion of environmental estrogenic chemical bisphenol A to DNA binding metabolite(s). Biochem. Biophys. Res. Commun. 210 (2), 424–433.
Bindhumol, V., Chitra, K.C., Mathur, P.P., 2003. Bisphenol A induces reactive oxygen species generation in the liver of male rats. Toxicology 188 (2–3), 117–124. Chitra, K.C., Latchoumycandane, C., Mathur, P.P., 2003. Induction of oxidative stress by bisphenol A in the epididymal sperm of rats. Toxicology 185 (1–2), 119–127. Escher, B.I., Hunziker, R.W., Schwarzenbach, R.P., 2001. Interaction of phenolic uncouplers in binary mixture: concentration-additive and synergistic effects. Environ. Sci. Technol. 35 (19), 3905–3941. Hallare, A.V., Ko¨hler, H.R., Triebskorn, R., 2004. Developmental toxicity and stress protein responses in zebrafish embryos after exposure to diclofenac and its solvent, DMSO. Chemosphere 56 (7), 659–666. Hertzberg, R.C., MacDonell, M.M., 2002. Synergy and other ineffective mixture risk definitions. Sci. Total Environ. 288 (1–2), 31–42. Hsieh, S.H., Tsai, K.P., Chen, C.Y., 2006. The combined toxic effects of nonpolar narcotic chemicals to Pseudokirchneriella subcapitata. Water Res. 40 (10), 1957–1964. Huang, Y.R., Shi, J.H., Zhou, L., Di, Y., Quan, H., 2001. Environmental monitoring and governance in the East Asian coastal hydrosphere. Endocrine Disrupter Compounds (EDCs) in Water 2001 Report, China. Johnson, W.W., Finley, M.T., 1998. Handbook of Acute Toxicity of Chemicals to Fish and Aquatic Invertebrates. US Department of Interior, Fish and Wildlife Service, Washington DC, p. 65. Marguerie, A., Buckley, C., Fleming, A., 2006. Validation of an embryotoxicology screen using zebrafish. Toxicol. Lett. 164 (Suppl. 1), 220. Nagel, R., 2002. DarT: the embryo test with the zebrafish Danio rerio–a general model in ecotoxicology and toxicology. ALTEX 19 (Suppl. 1), 38–48. OECD, 1998. Fish, short-term toxicity test on embryo and sax-fry stages. OECD Guideline for Testing of Chemicals, OECD TG212, Paris. Pande, M., Cameron, J.A., Vig, P.J.S., Ali, S.F., Desaiah, D., 1999. Inhibition of calcium ATPase by phencyclidine in rat brain. Mol. Cell. Biochem. 194 (1–2), 173–177. Reimers, M.J., Flockton, A.R., Tanguay, R.L., 2004. Ethanol- and acetaldehyde-mediated developmental toxicity in zebrafish. Neurotoxicol. Teratol. 26 (6), 769–781. Repetto, G., Jos, A., Hazen, M.J., Molero, M.L., del Peso, A., Salguero, M., Castillo, P.D., Rodriguez-Vicente, M.C., Repetto, M., 2001. A test battery for the ecotoxicological evaluation of pentachlorophenol. Toxicol. In Vitro 15 (4–5), 503–509. Schwarzenbach, R.P., Gschwend, P.M., Imboden, D.M., 2003. Environmental Organic Chemistry. Wiley, New Jersey, USA, pp. 375–380. Staples, C.A., Dorn, P.B., Klecka, G.M., O’Block, S.T., Harris, L.R., 1998. A review of the environmental fate, effects, and exposures of bisphenol A. Chemosphere 36 (10), 2149–2173. Van Leeuwen, C.J., Grooteiaar, E.M., Niebeek, G., 1990. Fish embryos as teratogenicity screens: a comparison of embryotoxicity between fish and birds. Ecotoxicol. Environ. Saf. 20 (1), 42–52. Vranken, G., Tire, C., Heip, C., 1988. The toxicity of paired metal mixtures to the nematode monhystera disjuncta. Mar. Environ. Res. 26 (3), 161–179. Zhang, A.Q., Chen, R.Q., Wei, D.B., Wang, L.S., 2000. QSAR research of chlorinated aromatic compound toxicity of Selenastrum capricornutum. China Environ. Sci. 20 (2), 102–105.
Ecotoxicology and Environmental Safety 71 (2008) 774–780 www.elsevier.com/locate/ecoenv
Individual and joint toxic effects of pentachlorophenol and bisphenol A on the development of zebrafish (Danio rerio) embryo Zhenghua Duana, Lin Zhua,b,, Lingyan Zhua,b, Kun Yaoa, Xiaoshan Zhua a
College of Environmental Science and Engineering, Nankai University, Tianjin 300071, PR China Tianjin Key Laboratory of Environmental Remediation and Pollution Control, TEDA 300253, PR China
b
Received 20 June 2007; received in revised form 21 January 2008; accepted 27 January 2008 Available online 21 March 2008
Abstract Investigation of the toxicological effects of pentachlorophenol (PCP) and bisphenol A (BPA) alone and in combination was carried out following the method of the early life stage (ELS) test on zebrafish embryos. Both chemicals revealed lethal and sub-lethal effects, such as no blood flow, cardiac edema, delayed hatching, and tail malformations. According to their median effective concentrations (EC50 values) in the single exposure, the toxic level of PCP was about two orders of magnitude higher than that of BPA. Result of the joint action modes varied depending on different endpoints. Synergistic action was observed based on the endpoint of 24 h mortality and antagonistic effect displayed based on the endpoint of 72 h cardiac edema. It was also found that the toxicity of PCP would be enhanced with the addition of BPA even below its no observed effect concentration (NOEC) level at the endpoint of 32 h with no blood flow, and the level of the increase was influenced by the toxic unit (TU) ratio. r 2007 Elsevier Inc. All rights reserved. Keywords: Joint toxicity; Pentachlorophenol; Bisphenol A; Zebrafish embryo
1. Introduction The individual toxicity of most chemicals is widely studied and well understood. However, chemicals usually exist in the environment as mixtures and information regarding the joint effects of multi-pollutants as well as their toxic mechanisms remain insufficient. Pentachlorophenol (PCP) has been used as an industrial antiseptic and biocide for many decades since the 1960s. Due to its proven carcinogenicity and toxicity, as well as the existence of a large number of known PCPcontaminated sites, PCP has been designated as a ‘‘priority toxic pollutant’’ by the United States Environmental Protection Agency (EPA) under the guidance of the Clean Water Act in 1972. Bisphenol A (BPA) is another common chemical that has been widely used in the chemical industry in the manufacturing of epoxy- and polyester-styrene resins. Studies have testified that BPA was potentially toxic to Corresponding author at: College of Environmental Science and Engineering, Nankai University, Tianjin 300071, PR China. Fax: +86 22 23508807. E-mail address: [email protected] (L. Zhu).
0147-6513/$ - see front matter r 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.ecoenv.2008.01.021
embryos and has been found to cause genetic defects (Bindhumol et al., 2003). BPA can also be converted to DNA binding metabolites in vitro (Atkinson and Roy, 1995). Since these two chemicals are extensively used in daily life and industrial applications, their potential toxicities have recently sparked attention. These chemicals usually exist in the environment as mixtures. Huang et al. (2001) have shown that the concentrations of PCP and BPA were 3.61–2010 and 5.57–29.80 ng L1, respectively, in river water and seawater in China. Previous studies have mostly focused on the individual toxicities of these chemicals and there is no report so far about their joint toxicity effects. Zebrafish (Danio rerio) has gained merits as a model species over the past few years because of its short growth period, high fecundity, and transparent eggs (Hallare et al., 2004; Reimers et al., 2004). The early life stage (ELS) test using zebrafish embryos is widely used in investigating the toxicity and teratogenicity of chemicals known to cause significant impact on environmental and human health (Van Leeuwen et al., 1990; Nagel, 2002). Furthermore, as the zebrafish holds many similar cellular and physiological characteristics with higher vertebrates,
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the toxicological results of zebrafish embryonic development can be easily compared with those of mammalian development (Marguerie et al., 2006; Reimers et al., 2004). Therefore, the zebrafish embryo is a perfect model to study the developmental toxicity of vertebrates and the ELS test has become an effective alternative method of adult fish testing. In the present study, PCP and BPA were chosen as examples to examine their individual and joint effects on the development of zebrafish embryo. The difference between their individual toxicities was investigated by their quantum chemical descriptors. The mechanisms of joint effects and their influence factors were also studied according to biochemical reactions and membrane transport processes. 2. Materials and methods 2.1. Chemicals PCP (C6Cl5OH, purity499.50%) and BPA (C15H16O2, purity499.00%) were purchased from ALDRICH company (Unite States). Their structural formulas are as shown: CH3
OH Cl
Cl
Cl
Cl
HO
C
775
treatment solution (PCP, BPA, or PCP/BPA solutions). The remaining four wells were filled with 2 mL reconstituted water instead of treatment solution to act as controls. In addition, 1% ethanol (1 mL diluted in 1 L reconstituted water) was used as a solvent control in another multi-well plate. All of the wells were covered with transparent plastic film and placed in an incubator at 2671 1C with a 10/14-h light/dark cycle. Based on preliminary experiments, it was found that if the embryos were exposed after gastrula (8 h post-fertilization) stage, the lethal toxicity of chemicals to the embryos decreased greatly and more severe sub-lethal toxic effects were observed. Therefore, two additional exposures were designed in the following experiments: (1) early cleavage exposure (exposure immediately after fertilization, 0 hpf exposure) to study the endpoint of mortality, and (2) gastrula exposure (exposure after 8 h postfertilization, 8 hpf exposure) to study the endpoints of sub-lethal effects. The developmental status of zebrafish embryos were observed with an inverted microscope (8–50 ) (IMT 2, Olympus Corporation, Tokyo, Japan) and documented photographically at specific times after fertilization (time points ¼ 0, 8, 24, 32, and 72 h). Endpoints used for assessing developmental toxicity were recorded and described for embryos for both the control and treated groups. The endpoints included 24 h mortality (the embryo had no heart beat; 0 hpf exposure), 32 h no with blood flow (the embryo had heart beat, but the blood in the vessels and stopped flowing; 8 hpf exposure), 72 h cardiac edema (8 hpf exposure, Figs. 1C and D), 72 h delayed hatching (8 hpf exposure), and 72 h tail deformations (tail damaged or broken; 8 hpf exposure, Figs. 1E and F).
2.4. Equitoxicity test OH
CH3
Bisphenol A
Cl
Pentachlorophenol All tests were performed with reconstituted purified water in accordance with ISO (International Standard Organization) standard of 7346-3:1996. The pH value of the water was kept at 8.0 and the hardness was equivalent to about 150 mg L1 of calcium carbonate (CaCO3). The exposure concentrations in the studies ranged between 0.01–1.00 mg L1 for PCP and 2.00–25.00 mg L1 for BPA. Therefore, 1 L PCP stock solution at the concentration of 1.00 mg L1 was prepared in reconstituted purified water together with 1 mL ethanol as co-solvent. BPA stock solution at the concentration of 25.00 mg L1 was prepared in a similar way. The stock solutions were then diluted to the specific test concentrations with reconstituted water.
For a single chemical, the concentration at its EC50 is defined as 1 toxic unit (TU, dimensionless). The toxicity of the chemical at other concentrations is expressed in TU by dividing the concentrations by its EC50 value. A mixture is equitoxic if each component is present at the same fraction of its own EC50 (or LC50) value, i.e. the ratios of the concentrations of the chemicals to their EC50 (or LC50) values are equal. The equitoxicity test in this study consisted of three simultaneous treatment series: (A) PCP alone, (B) BPA alone, and (C) equitoxic mixtures of PCP and BPA. The endpoints of 24 h mortality and 72 h delayed hatching were selected to study the joint effects. Parallel experiments of three replicates each were conducted for each treatment level. Basically, when two chemicals were applied simultaneously to a living system or unit, three types of joint effects was observed. They are additive toxicity effect (usually upon dose addition), synergism (greater than additive toxicity), and antagonism (less than additive toxicity) (Hertzberg and MacDonell, 2002). The type of joint action for a specific binary mixture of toxicants is determined by the additive index (M) or the sum of TUs, which is calculated using the following equation: M¼
2.2. Zebrafish Adult zebrafish was second generation in our laboratory, and the parents were obtained from an aquarium in Tianjin, China. The maintenance of brood fish strictly followed the OECD (Organization for Economic Cooperation and Development) guideline for fish embryo toxicity test (OECD, 1998). Fish eggs were collected and rinsed three times in reconstituted water to remove any residue on the egg’s surface. Fertilized eggs were selected for exposure experiments after visual inspection with a stereomicroscope (8–50 ). Studies involving zebrafish embryo were conducted in accordance with national and institutional guidelines for the protection of human subjects and animal welfare.
2.3. Toxicity test The embryonic toxicity test was developed by Nagel (2002). Twentyfour eggs were transferred into a 24-well multi-well plate such that each well contained one embryo. Twenty wells were filled with 2 mL of
Zð1Þ Zð2Þ þ , EC50 ð1Þ EC50 ð2Þ
where Z(1) and Z(2) denote the individual concentrations of the mixture in which the combined concentrations resulted in a 50% response. When M ¼ 1, the joint effect would be a simple addition, when M41, it would be antagonism and Mo1, it is synergism (Hsieh et al., 2006). A mixture of the two toxicants with different sums of TUs was tested. The total TUs varied in the range of 0.25–2.60. According to the number of embryos which displayed toxicity effects, M and its 95% confidence interval (CI) at 50% response were calculated.
2.5. Unequitoxicty test A pre-screening experiment for single exposure was conducted to determine the range of exposure concentrations for PCP and BPA. Based on the results of single exposure, it was found that PCP was more toxic than BPA (it will be discussed later). To evaluate the combined toxicity of PCP with the presence of BPA, two toxicity experiments were performed for comparison based on the endpoint of 32 h no blood flow. One was
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Fig. 1. Observed normal and abnormal zebrafish embryo and larva fish. (A) Normal embryo, 48 hpf; (B) normal larva fish after hatching, 72 hpf; (C) cardiac edema in the embryo, 48 hpf, 15.00 mg L1 BPA; (D) cardiac edema after hatching, 72 hpf, 15.00 mg L1 BPA; (E) tail deformations in the embryo, 48 hpf, 20.00 mg L1 BPA; and (F) tail deformations after hatching, 72 hpf, 20.00 mg L1 BPA. tested with PCP alone and the other was a combination of PCP and BPA. For all combined groups, the total toxic levels were kept at a constant TU of 0.26, while the concentration ratios of PCP and BPA differed; the concentrations of PCP varied around its EC50 value, whereas those of BPA remained at a level below its no observed effect concentrations (NOEC). The TU ratios (TU1:TU2 ¼ PCP:BPA) were selected to be 1/5, 1/3, 1, 3, and 5. For individual exposure of PCP, the concentrations of PCP were the same as those in the combined mixture with zero concentrations of BPA.
2.7. Statistical analysis Results from microscopic examinations of the embryos were categorized according to the types and severity of the toxic endpoints. The 50% effect concentrations for mortality (LC50) and sub-lethality (EC50) were calculated using Sigmoid Fit by ORIGIN 7.0 (OriginLab, US). Comparison tests between groups were carried out by one-way ANOVA. A p-value of o0.05 was considered as a statistically significant.
3. Results 2.6. Chemical analysis
3.1. Individual toxicities of PCP and BPA Due to the adsorption of organic chemicals on the inner wall of plastic containers, the actual chemical concentration determined in the solution may be lower than the nominal value. After 72 h single exposure of PCP or BPA (0 hpf exposure), 2 mL water was removed from testing chambers and analyzed by high pressure liquid chromatography (HPLC, Waters 1525). It was found that actual concentrations of both PCP and BPA were less than their nominal concentrations as expected. The linear regression between the actual (Y) and nominal (X) concentrations was calculated for PCP and BPA following the equations Y ¼ 0.85X+0.03 (R2 ¼ 0.9956) and Y ¼ 0.71X+1.78 (R2 ¼ 0.9549), respectively. Based on the above equations, the actual concentrations of PCP and BPA in the study were determined and reported.
No effect was observed in the zebrafish embryos when 1% ethanol was used as a solvent control (data not shown). In the individual exposure of zebrafish embryos to PCP, dose–response curves were obtained as shown in Fig. 2A. The median lethal concentration (LC50) after 24 h was 0.76 mg L1. The median effect concentrations (EC50) were 0.19 mg L1 for no blood flow at 32 hpf, 0.08 mg L1 for cardiac edema at 72 hpf, and 0.09 mg L1 for delayed hatching at 72 hpf. According to the EC50 values of every
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sub-lethal endpoint, the most sensitive toxicological endpoint induced by PCP was cardiac edema at 72 hpf. In the individual exposure of zebrafish embryos to BPA, the median lethal concentration (LC50) after 24 h was 16.75 mg L1 (Fig. 2B). All embryos were dead when exposed to 25.00 mg L1. The no effect concentration (NOEC) for all sub-lethal endpoints was 2.00 mg L1. The median effect concentrations (EC50) were 23.05 mg L1 for no blood flow at 32 hpf, 20.87 mg L1 for cardiac edema at 72 hpf, and 13.81 mg L1 for delayed hatching at 72 hpf
777
(Fig. 2B). Embryos exposed to high concentrations of BPA (415.00 mg L1) showed tail deformations (Figs. 1E and F). Therefore, the sensitive endpoint induced by BPA was delayed hatching at 72 hpf. Based on the results of single exposure, it was found PCP was much more toxic than BPA on the development of zebrafish embryos. For instance, based on the endpoint of 32 h with no blood flow, the EC50 value of BPA (23.05 mg L1) was about 121 times as high as that of PCP (0.19 mg L1). Based on the endpoint of 72 h cardiac
Fig. 2. Dose–response relations for PCP (A) and BPA (B). Death—24 h death; blood—32 h with no blood flow; edema—72 h cardiac edema; hatch—72 h delayed hatching.
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Z. Duan et al. / Ecotoxicology and Environmental Safety 71 (2008) 774–780 Table 1 Joint effects of PCP and BPA to zebrafish embryos at equitoxicity level Endpoints
Aa
Bb
PCP BPA
PCP BPA
24 h mortality 0.76 72 h cardiac 0.08 edema a
16.75 0.22 20.87 0.07
M
4.75 18.9
95%CI
Joint effect
0.57 0.48–0.66 Synergism 1.78 1.56–2.10 Antagonism
EC50 of individual exposure (mg L1). Combined concentrations resulted in a 50% response (mg L1).
b
Fig. 3. Effects of PCP and BPA with equitoxicity. Death—24 h death; edema—72 h cardiac edema.
edema, the EC50 value of BPA (20.87 mg L1) was about 260 times that of PCP (0.08 mg L1). Hence, the toxicity of PCP to zebrafish embryos is about two orders of magnitude higher than that of BPA. 3.2. Joint action with equitoxicity Two representative endpoints, 24 h mortality and 72 h cardiac edema in the embryonic development of zebrafish, were selected for the combined equitoxicity study. For the endpoint of 24 h mortality, mortality of the embryos showed significant dose–response relationship with TU (range from 0.25 to 1.00), and all embryos were dead when TU was higher than 1.00 (Fig. 3). For the endpoint of 72 h cardiac edema, the percentage of cardiac edema also displayed significant sigmoid relationship with TU (range from 1.00 to 2.50). No cardiac edema effect detected in all embryos when TUp1.00 (Fig. 3). As shown in Table 1, 0.57 TU induced a 50% toxic response for 24 h mortality. However, TU that induced 50% toxic response for 72 h cardiac edema was 1.78. Judging from the 95% CI for M, the mixtures displayed synergistic joint action based on the endpoint of 24 h mortality, whereas antagonistic joint action was observed based on the endpoint of 72 h cardiac edema. 3.3. Joint action with unequitoxicity As shown in Fig. 2, according to the endpoint of 32 h with no blood flow, the dose–response relationship was significant at an individual exposure of PCP. When BPA was added, even at a level below its NOEC, the observed toxicity effects increased significantly (Fig. 4). All p-values between the toxicities of each chemical alone and their combined exposures were statistically significant at less than 0.05. The combined effect differed significantly even when the concentrations of PCP and BPA remained at
Fig. 4. Effects of PCP alone and in combination with BPA based on the endpoint of 32 h with no blood flow. Corresponding with the concentrations of PCP and BPA at the forward direction of X-axis, TU ratios (TU1/ TU2 ¼ PCP: BPA) were 1/5, 1/3, 1, 3, and 5. In single exposures, the concentrations of PCP were the same as those in combined exposures, whereas all the concentrations of BPA were zero.
constant total TU value. The combined toxicity increased with the concentration of PCP, attained the highest percentage of toxicity when PCP was 0.10 mg L1 and BPA was 2.16 mg L1 (TU1/TU2 ¼ 1:1), and then decreased with the increase of PCP concentration (Fig. 4). 4. Discussion 4.1. Action mechanisms of individual toxicity In this study, it was found that both PCP and BPA, either alone or in a mixture, had profound effects on the developmental stage of zebrafish embryos. Besides death, there were several other sub-lethal effects, such as no blood flow, cardiac edema, delayed hatching, and tail deformations. Our data showed that the toxicity of PCP was much higher than that of BPA. These results were in good agreement with those reported in previous studies. As compared with BPA, PCP displayed higher toxicity in all organisms shown in Table 2 (Repetto et al., 2001; Johnson and Finley, 1998; Staples et al., 1998). These could be due
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to their different quantum chemical descriptors. A chemical exerts its toxic effects to organisms in two major steps: (1) transport-associated process which is mainly controlled by the parameter of the octanol–water partition coefficient (log Kow value of the compound); (2) biochemical reactionassociated process which is described by total energy (TE) of the molecule. It was found that the effective concentrations or even the lethal concentrations (in mol L1), such as the EC50 or LC50 values, are related to the log Kow values of each compound (Schwarzenbach et al., 2003). Zhang et al. (2000) have also concluded that TE is the key parameter in determining the toxicity of the group of chlorinated aromatic compounds to Selenastrum capricornutum. The quantum chemical descriptors (log Kow and TE values) of PCP and BPA were calculated using the Chemoffice 6.0 software with the algorithm of MOPAC-AM1. Log Kow values were about 5.74 for PCP and 3.40 for BPA, indicating that at the same concentrations, it was much easier for PCP to permeate through the biological membrane and combine with target organs. In addition, the TE values were about 2970.5 eV for PCP and 2774.91 eV for BPA, suggesting that the molecular of PCP was more active than that of BPA. It has been reported that PCP has enhanced toxicity than that of other phenols due to its specific effects, for example, interference with the energy transduction of cells by interfering with the electrochemical proton gradient (Escher et al., 2001). Therefore, PCP exhibited a higher toxicity than BPA in the organisms discussed above. 4.2. Action mechanisms of joint exposure Joint action modes were different depending on the toxicity endpoints. In the study of equitoxicity, synergistic joint action mode was observed based on 24 h mortality when the exposure concentrations were 0.10–1.00 mg L1 for PCP and 2.16–21.60 mg L1 for BPA, and antagonistic joint action mode was observed based on 72 h cardiac edema when the exposure concentrations were 0.01–0.10 mg L1 for PCP and 2.62–26.20 mg L1 for BPA. Death might result to the combined effects of the chemicals. Any changes in the membrane that induce the abnormal embryonic development would finally bring the embryo to death. PCP and BPA induce cellular death via Table 2 Toxicity data for PCP and BPA to several organisms Results (mg L1) Organism
Daphnia magna Pimephales promelas Oncorhynchus mykiss a
Test type
48 h EC50 96 h LC50 96 h LC50
Endpoint
Immobilization Mortality Mortality
Referred by Repetto et al., 2001. Referred by Staples et al., 1998. c Referred by Johnson and Finley, 1998. b
PCP
BPA
0.40a 0.21c 0.05c
3.90b 4.70b 4.00b
779
different mechanisms. PCP induces the embryonic death mainly through its binding to mitochondrial proteins and inhibiting the mitochondrial ATPase activity. As a result, both the formations of ATP and ADP are prevented (Pande et al., 1999). However, when the embryo was exposed to BPA, lipid peroxidation was induced by free radical generation via metabolic redox cycling between the quinone and hydroquinone forms of BPA (Chitra et al., 2003). When the embryos were exposed to a mixture of PCP and BPA mixture, both damage mechanisms were activated, and the embryonic membrane would be break down more easily. A synergism effect might then be demonstrated. The antagonism observed at the endpoint of 72 h cardiac edema could be explained with a competitive site theory (Vranken et al., 1988). Some active sites might exist which could induce the effect of cardiac edema. When PCP and BPA existed as a mixture and the concentrations of BPA in the combined solutions were about 20 times that of PCP, there were more chances for BPA ions to occupy the limited active sites, thereby suppressing the binding of PCP to the active sites. However, the toxicity of BPA was lower than that of PCP at the same exposure concentration, which would finally lead to the decrease of total toxicity. Therefore, suggesting that the joint toxicity effect would be antagonism. 4.3. Influences on the joint effect In this study, it was found that the toxicity of PCP at the endpoint of 32 h with no blood flow was enhanced with the addition of BPA to even below its NOEC level, indicating that BPA has a toxicity effect when treated in mixtures even though in concentrations below the NOEC of single dose BPA. Therefore, synergistic effects were exhibited at this endpoint. When embryos were exposed in a mixture of PCP and BPA at the same total toxicity level (0.26 TU), it was presumed that the joint toxicity would be constant. However, results in this study indicated that the joint toxicity is influenced by the TU ratio and can be changed accordingly. The toxicity of PCP in a single exposure increased with its concentration at the endpoint of 32 h with no blood flow. When BPA was added to the PCP solution while the total toxicity level remained constant, the exhibited toxicity was enhanced in all of the test concentration range. As discussed previously, PCP and BPA had different toxicity mechanisms. When PCP and BPA existed as a mixture, both mechanisms were activated in embryos and enhanced toxicity thus resulted. Addition of BPA into PCP solution resulted in an increase of the toxicity, which was affected by both of the PCP and BPA concentrations. The relative increase of toxicity was defined as the ratio of [combined toxicity single toxicity of PCP]/[single toxicity of PCP]. Date showed that while the TU ratio increased and the concentration of BPA in the mixture decreased, the relative degree of toxicity decreased as well (Table 3). On the other
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Table 3 The relation between the concentrations of BPA and the relative increase of toxicity TU ratio Concentrations of BPA in the mixture (mg L1) Combined toxicitya Single toxicity of PCPa Relative increase of toxicityb
1/5
1/3
1
3
5
3.6
3.25
2.16
1.08
0.72
35 0
50 5
70 15
55 20
40 35
–
9
3.67
1.75
0.14
a
The toxicity denoted the effective percent (%) of 32 h with no blood flow, as derived from Fig. 4. b The relative increase of toxicity was defined as the ratio of [combined toxicitysingle toxicity of PCP]/[single toxicity of PCP].
hand, the single toxicity of PCP increased consistently with the increase of PCP concentration. Thus, there would be a specific TU ratio at which the mixture displayed the highest toxicity. In this present study, it was found that the combined toxicity peaked at TU1/TU2 ¼ 1:1 (PCP was 0.10 mg L1 and BPA was 2.16 mg L1). 5. Conclusions PCP and BPA were both toxic to the development of zebrafish embryos, and the toxicity level of PCP was about two orders of magnitude higher than that of BPA. Joint action modes varied depending on different endpoints. Synergistic action was displayed based on the endpoint of 24 h mortality, but antagonistic effect was exhibited based on the endpoint of 72 h cardiac edema. Combined effects would be influenced by the TU ratios. Acknowledgments This work was funded by Natural Science foundation of China (approval no.30340091). We would like to thank everyone who contributes to this study, especially Liuming Pan and Si Gao for their editorial assistance. This study involving zebrafish embryos was conducted in accordance with national and institutional guidelines for the protection of human subjects and animal welfare. References Atkinson, A., Roy, D., 1995. In vitro conversion of environmental estrogenic chemical bisphenol A to DNA binding metabolite(s). Biochem. Biophys. Res. Commun. 210 (2), 424–433.
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