The Embryotoxicity of Some Phenol Derivatives on Zebrafish, Danio rerio

Document Type: Research Paper


1 Atatürk University Engineering Faculty, Department of Environment Engineering, Erzurum, Turkey

2 Atatürk University Science Faculty, Department of Biology, Erzurum, Turkey


The existence of toxicants in ecosystems has been increased dramatically in recent years, especially in aquatic environments. Phenols and chlorinated phenol derivatives are toxic industrial compounds. Phenols and derivatives are known to be environmental contaminants. In the present study, 2,4-Dichlorophenol, 2-Chlorophenol and substituted phenol were tested for embryotoxicity and mortality in a four-day period using zebrafish, Danio rerio embryos. Tested phenol derivatives caused teratogenicity and embryo mortality in the embryos. The semi static 48-h LC50 (median lethal concentration) value for Substituted-phenol was 13.850 mg L-1; the corresponding values for 2-Chlorophenol and 2,4-Dichlorophenol were 8.378 mg L-1 and 6.558 mg L-1, respectively. The endpoints are incomplete eyes, head and tail, heart and chorda deformity, yolk sac edema, tail curvature, shrunken eyes, lordosis, delayed hatching, weak pigmentation, heart edema and non-pigmentation after exposure to the compounds. 2,4-Dichlorophenol was found to be more toxic than the others. This paper is the first to describe the relative toxicity of a suite of phenols in the early life stages of zebrafish.


Arana, J, Tello-Rendon, E, Dona-Rodriguez, JM, Valdes do Campo, C, Herrera-Melidan, JA, Gonzalez-Diaz, O & Perez-Pena, J 2001, Highly concentrated phenolic wastewater treatment by heterogeneous and homogeneous photocatalysis: mechanism study by FTIR-ATR. Water Science and Technology, 44: 229–236.

Bernadini, G, Spinelli, O, Presutti, C, Vismara, C, Bolzacchini, E, Orlando, M, Settimi, R 1996, Evaluation of the developmental toxicity of pesticide MCPA and its contaminants phenol and chlorocresol. Environmental Toxicology and Chemistry, 15: 754–760.

Birge, WJ, Black, JA & Kuehne, R 1980, Effects of Organic Compounds on Amphibian Reproduction. 121. Water Resources Institute, University of Kentucky, Lexington, KY, USA.

Blum, DJW & Speece, RE 1991, A database of chemical toxicity to environmental bacteria and its use in interspecies comparisons and correlations. Research Journal of the Water Pollution Control Federation,63: 198-207.

Bolognesi, C, Perrone, E, Roggieri, P, Pampanın, DM, Sciutto, A 2006, Assessment of micronuclei induction in peripheral erythrocytes of fish to xenobiotics under controlled conditions. Aquatic Toxicology, 78: 93-98.

Bucher, F & Hofer, R 1993, Histopathological effects of sublethal exposure to phenol on two variously pre-stressed populations of bullhead (Cottus gobioL.). Environmental Contamination and Toxicology, 51: 309-316.

Colgan, PW, Cross, JA, Johansen, PH 1982, Guppy behavior during exposure to a sublethal concentration of phenol. Bulletin Environmental Contamination and Toxicology, 28: 20–27.

Czaplicka, M 2004, Sources and transformations of chlorophenols in the natural environment. Science of the Total Environment, 332: 21–39.

Dauble, DD, Barraclough, SA, Bean, RM & Fallon, WE 1983, Chronic effect of coal-liquid dispersions on fathead minnows and rainbow trout. Transactions of the American Fisheries Society, 112: 712–719.

Dec, J, Haider, K, Bollag, JM 2003, Release of substituents from phenolic compounds during oxidative coupling reactions. Chemosphere, 52: 549–556.

Dumpert, K 1987, Embryotoxic effects of environmental chemicals: test with the South African Clawed Toad (Xenopus laevis). Ecotoxicology and Environmental Safety, 13: 324–338.

Fogels, A & Sprague, JF1977, Comparative short-term tolerance of zebrafish, flagfish, and rainbow trout to five poisons including potential reference toxicants. Water Research,11: 811–817.

Gami, AA 2014, Phenol and its toxicity. Journal of Environmental Microbiology and Toxicology, 2: 11-24.

Han, SK, Ichikawa, K & Utsumi, H 1998, Quantitative analysis for the enhancement of hydroxyl radical generation by phenols during ozonation of water. Water Research, 32: 3261–3266.

Holcombe, GW, Benoi,t DA, Hammermeister, DE, Leonard, EN & Johnson, RD1995, Acute and long-term effects of nine chemicals on the Japanese medaka (Oryzias latipes). Archives Environmental Contamination and Toxicology, 28: 287–297.

Hori, TSF, Avilez, IM, Inoue, LK & Moraes, G 2006, Metabolical changes induced by chronic phenol exposure in matrinxã Brycon amazonicus (teleostei: characidae) juveniles. Comparative Biochemistry and Physiology C, 143: 67-72.

House, WA, Leach, D, Long, JL, Cranwell, P, Smith, C, Bharwaj, L, Meharg, A, Ryland, G, Orr, DO & Wright, J 1997, Micro-organic compounds in the Humber Rivers. Science of the Total Environment, 194–195: 357–371.

Jin, X, Gao, J, Zha, J, Xu, Y, Wang, Z, Giesy, JP & Richardson, KL 2012, A tiered ecological risk assessment of three chlorophenols in Chinese surface waters. Environmental Science and Pollution Research, 19: 1544–1554.

Kimmel, CB, Ballard, WW, Kimmel, SR, Ullmann, B & Schilling, TF 1995, Stages of embryonic development of the zebrafish. Developmental Dynamics, 203: 253–310.

Kishino, T & Kobayashi, K 1995, Relation between toxicity and accumulation of chlorophenols at various pH, and their absorption mechanism in fish. Water Research, 29: 431-42.

Kobayashi, K, Akitake, H & Manabe, K 1979, Relation between toxicity and accumulation of various chlorophenols in Goldfish. Bulletin of the Japanese Society for the Science of Fish, 45: 172-175.

Kondaiah, K & Murty, AS 1994, Avoidance behaviour test as an alternative to acute toxicity test. Bulletin Environmental Contamination and Toxicology, 53: 836-43.

Krijgsheld, KR & Van der Gen, A 1986, Assessment of the impact of the emission of certain organochlorine compounds on the aquatic environment. Chemosphere, 15: 825-860.

Kumar, V & Mukherjee, D 1988, Phenol and sulfide induced changes in the ovary and liver of sexually maturing common carp, Cyprinus carpio. Aquatic Toxicology, 13: 53-59.

Lammer, E, Carr, GJ, Wendler, K, Rawlings, JM, Belanger, SE & Braunbeck, T 2009, Is the fish embryo toxicity test (FET) with the zebrafish (Danio rerio) a potential alternative for the fish acute toxicity test? Comparative Biochemistry and Physiology-Part C: Toxicology and Pharmacology, 149: 196–209.

Livingstone, DR 1998, The fate of organic xenobiotics in aquatic ecosystems: quantitative and qualitative differences in biotransformation by invertebrates and fish. Comparative Biochemistry and Physiology A, 120: 43–49.

Livingstone, DR 2001, Contaminant-stimulated reactive oxygen species production and oxidative damage in aquatic organisms. Marine Pollution Bulletin, 42: 656–666.

Luo, YSu, Y, Wang, XR & Tian, Y 2006, 2-chlorophenol induced ROS generation in freshwater fish Carassius auratus based on the EPR method. Chemosphere, 65: 1064–1073.

Luo, Y, Su, Y, Wang, XR & Tian, Y 2008, 2-chlorophenol induced hydroxyl radical production in mitochondria in Carassius auratus and oxidative stress – An electron paramagnetic resonance study. Chemosphere, 71: 1260-1268.

Ma, Y, Han, J, Guo, Y, Lam, PKS, Wu, RSS, Giesy, JP, Zhang, X & Zhou, B 2012, Disruption of endocrine function in in vitro H295R cell-based and in in vivo assay in zebrafish by 2,4-dichlorophenol. Aquatic Toxicology, 106-107: 173-181.

McLeay, DJ 1976, A rapid method for measuring the acute toxicity of pulp mill effluents and other toxicants to salmonid fish at ambient room temperature. Journal of the Fisheries Research Board of Canada, 233: 1303-1331.

Moraes, FD, Figueiredo, JSL, Rossi, PA, Venturini, FP & Moraes, G 2015, Acute toxicity and sublethal effects of phenol on hematological parameters of channel catfish Ictalurus punctatus and pacu Piaractus mesopotamicus. Ecotoxicology and Environmental Containation, 10: 31-36.

Murray, AR, Kisin, E, Castranova, V, Kommineni, C, Gunther, MR & Shvedova, AA 2007, Phenol-induced in vivo oxidative stress in skin: Evidence for enhanced free radical generation, thiol oxidation, and antioxidant depletion. Chemical Research in Toxicology, 20: 1769-1777.

Nagel, R 2002, DarT: The embryo test with the zebrafish Danio rerio-a general model in ecotoxicology and toxicology. Altex, 19: 38–48.

Padilla, S, Hunter, DL, Padnos, B, Frady, S & MavPhail, RC 2011, Assessing locomotor activity in larval zebrafish: Influence of extrinsic and intrinsic variables. Neurotoxicology and Teratology, 33: 624–630.

Paisio, CE, Agostini, E, Gonzales, PS & Bertuzzi, ML 2009, Lethal and teratogenic effects of phenol on Bufo arenarum embryos. Journal of Hazardous Materials, 167: 64-68.

Prati, M, Biganzoli, E, Boracchi, M, Tesauro, M, Monetti, C & Bernardini, G 2000, Ecotoxicological soil evaluation by FETAX. Chemosphere, 41: 1621–1628.

Pera-Titus, M, Garcya-Molina, V, Banos, MA, Gimenez, J & Esplugas, S 2004, Degradation of chlorophenols by means of advanced oxidation processes: a general review. Applied Catalysis B: Environmental, 47: 219–256.

Phipps, GL, Holcombe, GW & Fiandt, JT1981, Acute toxicity of phenol and substituted phenols to the fathead minnow. Bulletin Environmental Contamination and Toxicology, 26: 585–593.

Qiao, M, Wang, C, Huang, S, Wang, D & Wang, Z 2006, Composition, sources, and potential toxicological significance of PAHs in the surface sediments of the Meiliang Bay, Taihu Lake, China. Environment International, 32: 28–33.

Ramamoorthy, S & Ramamoorthy, S 1997, Chlorinated organic compounds in the environment: regulatory and monitoring assessment. CRC press.

Rand, GM, Wells, PG & Mccarty, LS 1995, Introduction to aquatic toxicology. In: Rand, G.M. (ed), Fundamentals of aquatic toxicology, 2nd edition. Boca Raton, FL: CRC Press.

Rice, PJ, Drewes, CD, Klunertanz, TM, Bradrury, SP & Coats, JR 1997, Acute toxicity and behavioral effects of Chlorpyrifos, Permethrin, Phenol, Strychnine, and 2,4-Dinitrophenol to 30-day-old Japanese Medaka (Oryzias latipes). Environmental Toxicology and Chemistry, 16: 696–704.

Roche, H & Bogé, G 2000, In vivo effects of phenolic compounds on blood parameters of a marine fish (Dicentrarchuslabrax). Comparative Biochemistry and Physiology-Part C: Toxicology and Pharmacology, 125: 345-353.

Saha, NC, Bhunia, F & Kavıraj, A 1999, Toxicity of phenol to fish and aquatic ecosystems. Bulletin Environmental Contamination and Toxicology, 63: 195-202.

Sawle, AD, Wit, E, Whale, G & Cossins, AR 2010, An information-rich alternative, chemicals testing strategy using a high definition toxicogenomics and zebrafish (Danio rerio) embryos. Toxicological Sciences, 118: 128-139.

Scow, K, Goyer, M & Perwak, J 1982, Exposure and risk assessment for chlorinated phenols (2-chlorophenol, 2, 4-dichlorophenol, 2, 4, 6-trichlorophenol). Cambridge, MA: Arthur D. Little. EPA, 440: 4-85.

Shackelford, WM & Keith, LH, 1976, Frequency of organic compounds identified in water (Vol. 1). Environmental Protection Agency, Office of Research and Development, Environmental Research Laboratory, Analytical Chemistry Branch.

Stringer, R & Johnston, P 2001, Chlorine and the environment: an overview of the chlorine industry. Springer Science & Business Media.

USEPA, 1980a, Ambient Water Quality Criteria for 2-chlorophenol.Report No. EPA No: 440/5-80-034. U.S. Environmental Protection Agency, Office of Water Regulations and Standards, Criteria and Standards Division, Washington, DC.

USEPA, 1980b, Ambient Water Quality Criteria for 2,4-Dichlorophenol. Report No. EPA/ 440/5-80-042. U.S. Environmental Protection Agency, Office of Water Regulations and Standards, Criteria and Standards Division, Washington, DC.

USEPA 1989, Generalized methodology for Conducting Industrial reduction evaluations TREs, EPA 600/2-88/070.

Westerfield, M 2007, The Zebrafish Book. A Guide for the Laboratory Use of Zebrafish (Danio rerio). 5th Edition, University of Oregon Press, Eugene.

Yin, D, Gu, Y, Li, Y, Wang, X & Zhao, Q 2006, Pentachlorophenol treatment in vivo elevates point mutation rate in zebrafish p53 gene. Mutation Research, 609: 92-101.

Zhang, Y, Liu, M, Liu, J, Wang, X, Wang, C, Ai, W, Chen, S & Wang, H 2018, Combined toxicity of triclosan, 2, 4-dichlorophenol and 2, 4, 6-trichlorophenol to zebrafish (Danio rerio). Environmental Toxicology and Pharmacology, 57: 9-18.