Article

Embryotoxicity of nitrophenols to the early life stages of zebrafish (Danio rerio)

Toxicology and Industrial Health 1–10 © The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0748233714562444 tih.sagepub.com

Zeynep Ceylan1, Turgay S¸ is¸ man2, Zehra Yazıcı2 ¨ zen Altıkat1 and Aysun O Abstract The nitrophenols (NPs) are water-soluble compounds. These compounds pose a significant health threat since they are priority environmental pollutants. In this study, 2-Nitrophenol (2NP) and 2,4-dinitrophenol (DNP) were examined for embryo and early life stage toxicity in zebrafish (Danio rerio). Acute toxicity and teratogenicity of 2NP and DNP were tested for 4 days using zebrafish embryos. The typical lesions observed were no somite formation, incomplete eye and head development, tail curvature, weak pigmentation (48 hours postfertilization (hpf)), kyphosis, scoliosis, yolk sac deformity, and nonpigmentation (72 hpf). Also, embryo and larval mortality increased and hatching success decreased. The severity of abnormalities and mortalities were concentration- and compound-dependent. Of the compounds tested, 2,4-DNP was found to be highly toxic to the fish embryos following exposure. The median lethal concentrations and median effective concentrations for 2NP are 18.7 mg/L and 7.9 mg/L, respectively; the corresponding values for DNP are 9.65 mg/L and 3.05 mg/L for 48 h. The chorda deformity was the most sensitive endpoint measured. It is suggested that the embryotoxicity may be mediated by an oxidative phosphorylation uncoupling mechanism. This article is the first to describe the teratogenicity and embryotoxicity of two NPs to the early life stages of zebrafish. Keywords Zebrafish, embryotoxicity, nitrophenol, teratogenicity, LC50, EC50

Introduction Nitrophenols (NPs) are water-soluble compounds that are moderately acidic in water as a result of dissociation. NPs are frequently used as raw materials or intermediates in the manufacture of explosives, pharmaceuticals, pesticides, pigments, dyes, wood preservatives, and rubber chemicals. The annual production of 4NP alone is 20,000 million kilogram (Donlon et al., 1996). They are released into the environment from diffuse sources, since these chemicals are frequently used for industrial, agricultural, and defense purposes. NPs pose significant health risks as they are physiologically active and potentially carcinogenic. The good stability and low vapor pressure of NPs together with their polarity are risk factors for their worldwide dispersion through atmospheric precipitations. In largely populated agricultural and industrial countries, NPs were found in rain and snow samples, river water, and sediment (Pocurull et al., 1996). The US Environmental Protection Agency (USEPA) has listed 2NP, 4NP, and

2,4-dinitrophenol (DNP) as ‘‘priority pollutants’’ (USEPA, 1977; Haghighi-Poden et al., 1995) and recommend restricting NP concentrations in fresh waters to below 230 mg/L and salt waters to below 4850 mg/L (USEPA, 1980). Also, 4NP and DNP were listed in the Organisation for Economic Co-operation and Development (OECD) High Production Volume Chemical Table in 1999, meaning that they are produced at levels greater than one thousand tons per year in at least one OECD member country (Koizumi et al., 2001). 4NP has found use in the production of dyes, pigments, 1

Department of Environmental Engineering, Engineering Faculty, Atatu¨rk University, Erzurum, Turkey 2 Department of Biology, Science Faculty, Atatu¨rk University, Erzurum, Turkey Corresponding author: Turgay S¸is¸ man, Department of Biology, Science Faculty, Atatu¨rk University, 25240 Erzurum, Turkey. Email: [email protected]; [email protected]

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medicines, photographic chemicals, and pesticides (ATSDR, 1991). DNP has been used in the production of black sulfur dye, herbicides, pesticides, and wood preservatives (ATSDR, 1995). DNP enters the air, water, and soil primarily from manufacturing releases, spill sites, and ground water seepage from landfills (Harris and Corcoran, 1995). DNP toxicity is consistent in most animals (Harris and Corcoran, 1995). Regarding toxicity, various effects in animals and humans likely due to oxidative phosphorylation uncoupling effects, such as death, hyperthermia, and body weight loss have been reported (ATSDR, 1995). In mammals, sublethal mitochondrial uncoupling by DNP increases -oxidation of fatty acids and leads to loss in adipose tissue (Blaikie et al., 2006; Harper et al., 2001). The zebrafish (Danio rerio) is a suitable model organism in toxicological research due to short spawning intervals (Nagel, 2002) and transparent embryos. The zebrafish in vivo model system has many advantages such as they are easy to maintain and breed, inexpensive, and their rapid assay to screen for teratogenicity (Westerfield, 2000). Zebrafish embryos and larvae have often been used in toxicity tests of environmentally relevant substances (Hollert et al, 2003; Kammann et al., 2006; S¸is¸ man, 2011; S¸is¸ man et al., 2008). Furthermore, the development of zebrafish is similar to that of mammals (Zon and Peterson, 2005). Also, it has been shown that the fish possess orthologs to the majority (86%) of human drug targets (Gunnarsson et al., 2008). Here, the first objective of our study was to determine the median lethal concentrations (LC50) and median effective concentrations (EC50) of 2NP and DNP for 48 hours postfertilization (hpf) embryos of zebrafish. The second objective was to study the teratogenic effects of 2NP and DNP on the development of embryos and larvae. The third objective was to compare the toxicity of the selected NP derivatives.

Materials and methods Chemicals The test chemicals used in this study, 2NP and DNP, were obtained from Sigma (St. Louis, Missouri, USA). Stock solutions were prepared by dissolution in low-conductivity water prepared from a MiliQ water treatment system and were stored in the dark at 5oC before starting the test.

Fish culture and egg production Adult zebrafish were purchased from Research Center of Aquarium Fish, Fisheries Faculty, Atatu¨rk University,

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Turkey, and acclimated for 2 weeks prior to experiments. Fish were held at a density of 20 fish per tank and kept on a 14-h light/10-h dark cycle. Dechlorinated municipal water was maintained at 26+1 C. The tank water was partially changed every week and regularly tested for its quality. Fish were fed twice daily with tetramin flakes. Dry flake food was fed twice daily and live food (Daphnia pulex) was fed once every 2 days. Embryos were obtained from healthy adult fish with a preferable ratio of 1:2 female to male. Four breeding groups were placed separately in a specific spawning aquarium, equipped with a mesh bottom to prevent the eggs from being cannibalized. Spawning was induced on the morning when the light was turned on. Half an hour later, eggs free of macroscopically discernable symptoms of infection and disease were collected, rinsed with an embryo medium and incubated in petri dishes at 26+1 C until a chemical treatment was made.

Embryotoxicity assay In order to determine the LC50 and EC50 zebrafish embryos were exposed to a concentration series of NPs. The experiment doses of 2NP (5, 10, 15, 20, and 25 mg/L) were selected according to the previous work of Lang et al. (1996) and DNP doses (3, 6, 9, 12, and 15 mg/L) were determined according to Marit and Weber (2011). Tris buffer was used as the control. After 2–4 hpf period, blastula stage embryos were collected and selected under a stereomicroscope into a glass petri dish containing Tris buffer. At latest 2.5 hpf, the incubation was started by the addition of fertilized eggs to the test solutions. A total of 20 embryos were placed per group and each experiment was repeated three times. The embryos were exposed in a glass petri dish (10-cm diameter) containing 50-ml test solution at 26+1 C with a 14-h light/10h dark cycle in a precision incubator that works under 96 hpf. Test solutions were renewed every 24 h. The embryos were evaluated and scored for lethal or teratogenic effects using an inverted dissecting microscope (Nikon SMZ1000 (Japan)) equipped with a digital camera device (Nikon DSLR D70s (Japan)) every 12 h. Lethal (coagulation, missing heartbeat, missing somites, missing tail detachment, and missing spontaneous movement) malformations were determined and dead embryos or larvae were removed immediately after each observation. Nonlethal malformations (yolk sac edema, spine malformation, no pigmentation, and incomplete eye development) were

Ceylan et al.

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Table 1. Lethal and teratogenic effects observed in zebrafish embryos depending on the time (Lammer et al., 2009). Exposure time

Lethal end points Coagulation Tail not detachment No somite formation No heartbeat Delayed hatching Sublethal end points Gastrulation Somite formation Eye development Spontaneous movement Blood circulation Heartbeat frequency Pigmentation Edema Teratogenicity end points Head malformation Tail defect Scoliosis Lordosis Modified chorda structure Rachitis Yolk deformation Growth retardation

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96 108/120 h

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also reported. The EC50 were calculated from concentration–effect curves for all endpoints separately as well as for the sum of teratogenic or nonlethal affected embryos. All embryos were staged as described by Kimmel at al. (1995) and lethal and teratogenic effects were recorded according to Lammer et al. (2009) (Table 1). From 48 hpf, embryos started to come out of the chorion asynchronously, and the number of hatched embryos was recorded. At 48 and 96 hpf, the number of larvae displaying morphological abnormalities were recorded. Results were plotted as graphs, and LC50 and EC50 were determined.

Statistical analysis Statistical analysis was performed using Statistical Package for the Social Sciences (SPSS) software (version 15.0). All data were expressed as mean + standard deviation. For the developmental toxicity test, after 48 h of exposure, the LC50, EC50, and the respective 95% confidence intervals (CIs) were determined by probit analysis. After testing for analysis of

variance (ANOVA) assumptions (homogeneity of variances and normality of data), statistical differences in embryo mortality rate and abnormal eggs/ embryos among treatments and stages were evaluated by one-way ANOVA. ANOVA was used for multiple comparisons, followed by Dunnett’s test. Statistics were made on the basis of affected embryos (embryos with lethal and/or teratogenic effects). The level accepted for statistical significance in all cases was p < 0.05.

Results LC50 and EC50 values Calculated LC50 values by the probit analysis program with 95% CI for 2NP and DNP in zebrafish embryos after 48 hpf of exposure were 18.177 mg/L and 9.65 mg/L, respectively. The calculated EC50 values for 48 h of 2NP and DNP were 7.634 mg/L and 3.142 mg/L, respectively. When the values were compared, it was determined that DNP was more toxic than 2NP. For the first time, LC50 and EC50 values of the NPs in zebrafish embryos were determined by the findings of this study (Figures 1 and 2).

Developmental findings Compared to the control, the rate of development in NPs was found to be slow. The completion of gastrulation, completion of somites, formation of optic cups, spontaneous contraction and tail detachment, heartbeat and circulation, retinal and body pigmentation, and hatching occurred at the slowest rate in surviving embryos exposed at the sublethal concentration of 2NP and DNP (Table 2). In terms of hatching success, two NPs were found to significantly reduce the number of hatched embryos with respect to concentration and compound structure (Table 2). In high concentrations, the larvae were found trapped within the chorion and never hatched. Zebrafish embryos kept at high concentrations of the NPs showed a mortality rate more than 50% for 48 h. Also, the mortalities observed at low concentrations of congeners were not found to be significantly different from each other. The frequency of death among the embryos in these groups was increased through the 48-h exposure period. Lethal and teratogenic effects recorded at 96 hpf are listed in Tables 3 and 4. The affected embryo number in the control did not exceed 10% of all embryos. Some of malformations in Table 1 were also observed in embryos exposed to 2NP and DNP. The percentage of affected and dead embryos

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Figure 1. Concentration–response curves used for the calculations of the LC and EC values by the probit analysis: (a) LC curve for 2NP, 95% confidence limits (lower bound 16.169 and upper bound 20.071 mg/L); (b) EC curve for 2NP, 95% confidence limits (lower bound 2.419 and upper bound 10.493 mg/L). LC: lethal concentration; EC: effective concentration; 2NP: 2-nitrophenol.

Figure 2. Concentration–response curves used for the calculations of the LC and EC values by the probit analysis: (a) LC curve for DNP, 95% confidence limits (lower bound 7.832 and upper bound 10.354 mg/L); (b) EC curve for DNP, 95% confidence limits (lower bound 0 and upper bound 4.915 mg/L). LC: lethal concentration; EC: effective concentration.

increased with high 2NP concentrations (Table 3). At the two highest concentrations (20 and 25 mg/L), many early malformations were permanent and resulted in embryo lethality within the next 2 days, reaching 85% lethality at 25 mg/L (Table 3). All 2NP-exposed embryos, which were coagulated at 3 days postfertilization (dpf), showed teratogenic effects at 1 and 2 dpf. A clear concentration–response relationship was detectable for the percentage of embryos with lethal

and teratogenic effects after exposure to DNP (Table 4). At the 3, 6, and 9 mg/L concentrations, teratogenic effects were usually detectable at 3 dpf, such as higher concentrations (12 and 15 mg/L) were seen from 2 dpf. During the earliest stages of development (