Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

Maternal-embryonic metabolic and antioxidant response of Chapalichthys pardalis (Teleostei: Goodeidae) induced by exposure to 3,4-dichloroaniline


Chapalichthys pardalis is a viviparous fish, microendemic to the Tocumbo Region in the state of Michoacán, Mexico. Despite the peculiar type of reproduction of goodeid fish and their mother-embryo interaction, the effects on embryos induced by maternal exposure to aquatic xenobiotics are still unknown. The objective of the present work was to determine the maternal-embryonic metabolic and antioxidant response of C. pardalis exposed to 3,4-dichloroaniline (3,4-DCA), a compound considered highly noxious to the environment because of its high toxicity and persistence, which has been used as reference toxicant in toxicological bioassays. We determined the median lethal concentration (LC50, 96 h) and then exposed pregnant females to 3.3, 2.5, and 0.5 mg L−1 of 3,4-DCA (equivalent to LC1, LC0.01, and LC50/10, respectively) during 21 days. We assessed the activity of antioxidant enzymes like superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), macromolecules content (proteins, lipids, carbohydrates), glucose, and lactate concentration, as well as the oxidative damage, by measuring thiobarbituric acid reactive substances, and protein oxidation. To interpret results, we used the integrated biomarker response (IBRv2). The average LC50 was of 5.18 mg L−1 (4.8–5.5 mg L−1; p = 0.05). All females exposed to concentrations of 3.3 and 2.5 mg L−1 lost 100% of the embryos during the bioassay, whereas those exposed to 0.5 mg L−1 showed alterations in the antioxidant activity and oxidative damage, being the embryos and the maternal liver the most affected, with IBRv2 values of 10.09 and 9.21, respectively. Damage to macromolecules was greater in embryos and the maternal liver, with IBRv2 of 16.14 and 8.40, respectively. We conclude that exposure to xenobiotics, like 3,4-DCA, in species with a marked maternal-embryonic interaction represents a potential risk for the development and survival of the descendants, thereby, potentially affecting the future of the population.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3


  1. Abramov JP, Wells PG (2011) Embryonic catalase protects against endogenous and phenytoin enhanced DNA oxidation and embryopathies in acatalasemic and human catalase expressing mice. FASEB J 25(7):2188–2200. doi:10.1096/fj.11-182444fj.11-182444

  2. Aparicio-Simón B, Piñón M, Racotta R, Racotta IS (2010) Neuroendocrine and metabolic responses of Pacific whiteleg shrimp Litopenaeus vannamei exposed to acute handling stress. Aquaculture 298:308–314. doi:10.1016/j.aquaculture.2009.10.016

  3. Arellano-Aguilar O, Macías-Garcia C (2009) Effects of methyl parathion exposure on development and reproduction in the viviparous fish Girardinichthys multiradiatus. Environ Toxicol 24(2):178–186. doi:10.1002/tox.20414

  4. Arzate-Cárdenas MA, Martínez-Jerónimo F (2012) Energy resource reallocation in Daphnia schoedleri (Anomopoda: Daphniidae) reproduction induced by exposure to hexavalent chromium. Chemosphere 87:326–332. doi:10.1016/j.chemosphere.2011.12.014

  5. Bagnyukova TV, Chahrak OI, Lushchak VI (2006) Coordinated response of goldfish antioxidant defenses to environmental stress. Aquat Toxicol 78(4):325–331. doi:10.1016/j.aquatox.2006.04.005

  6. Barton BA (2002) Stress in fishes: a diversity of responses with particular reference to changes in circulating corticosteroids. Integr Comp Biol 42:517–525. doi:10.1093/icb/42.3.517

  7. Begum G, Vijayaraghavan S (1999) Effect of acute exposure of the organophosphate insecticide Rogor on some biochemical aspect of Clarias batrachus (Linn). Environ Res 80:80–83. doi:10.1006/enrs.1998.3871

  8. Beliaeff B, Burgeot T (2002) Integrated biomarker response: a useful tool forecological risk assessment. Environ Toxicol Chem 21(6):1316–1322. doi:10.1002/etc.5620210629

  9. Bhavan PS, Geraldine P (1997) Alterations in concentrations of protein, carbohydrate, glycogen, free sugar and lipid in the prawn Macrobrachium malcolmsonii on exposure to sub lethal concentrations of endosulfan. Pestic Biochem Physiol 58:89–101. doi:10.1006/pest.1997.2287

  10. Buege JA, Aust SD (1978) Microsomal lipid peroxidation. Methods Enzymol 52:302–310. doi:10.1016/S0076-6879(78)52032-6

  11. Call DJ, Poirier SH, Knuth ML, Harting SL, Lindberg CA (1987) Toxicity of 3,4-dichloroaniline to fathead minnows, Pimephales promelas, in acute and early life-stage exposure. Bull Environ Contam Toxicol 38(2):352–358. doi:10.1007/BF01606686

  12. Cano-Europa E, López-Galindo G, Hernández-García A, Blas-Valdivia V, Gallardo-Casas C, Vargas-Lascari M, Buitrón-Ortíz R (2008) Lidocaine affect the redox environment and the antioxidant enzymatic system causing oxidative stress in the hippocampus and amygdala of adult of rats. Life Sci 83:681–685. doi:10.1016/j.lfs.2008.09.005

  13. Cattaneo R, Moraes BS, Loro VL, Menezes CC, Sartori GMS, Clasen B, de Avila LA, Marchesan E, Zanella R (2012) Tissue biochemical alterations of Cyprinus carpio exposed to commercial herbicide containing clomazone under rice-fieldconditions. Arch Environ Contam Toxicol 62:97–106. doi:10.1007/s00244-011-9669-8

  14. Chance B, Sies H, Boveris A (1979) Hydroperoxide metabolism in mammalian organs. Physiol Rev 59(3):527–605

  15. Commission of the European Communities (1994) Commission Regulation (EC) No. 1179/94 of 25 May 1994 concerning the first list of priority substances as foreseen under council regulation (EEC) No. 793/93, Official J Eur Commun, Brussels, 0003–0004

  16. Crapo JD, McCord JM, Fridovich I (1978) Preparation and assay of superoxide dismutases. Methods Enzymol 53:382–393. doi:10.1016/S0076-6879(78)53044-9

  17. Crossland NO (1990) A review of the fate and toxicity of 3,4-dichloroaniline in aquatic environments. Chemosphere 21(12):1489–1497. doi:10.1016/0045-6535(90)90054-W

  18. Da Fonseca MB, Glusczak L, Moraes BS, Menezes CC, Pretto A, Tierno MA, Loro VL (2008) The 2,4-D herbicide effects on acetylcholinesterase activity and metabolic parameters of piava freshwater fish (Leporinus obtusidens). Ecotoxicol Environ Saf 69:416–420. doi:10.1016/j.ecoenv.2007.08.006

  19. De Coen WM, Janssen CR (1997) The use of biomarkers in Daphnia magna toxicity testing: IV. Cellular energy allocation: a new methodology to assess the energy budget of toxicant-stressed Daphnia populations. J Aquat Ecosyst Stress Recover 6:43–55. doi:10.1023/A:1008228517955

  20. De La Vega-Salazar MY, Martínez-Tabche L, Macías-García C (1997) Bioaccumulation of methyl parathion and its toxicology in several species of the freshwater community in Ignacio Ramirez dam in Mexico. Ecotoxicol Environ Saf 38(1):53–62. doi:10.1006/eesa.1997.1551

  21. De Zwart LL, Meerman JHN, Commandeur JNM, Vermeulen NPE (1999) Biomarkers of free radical damage: applications in experimental animals and in humans. Free Radic Biol Med 26:1–2. doi:10.1016/S0891-5849(98)00196-8

  22. Doadrio I, Domínguez O (2004) Phylogenetic relationships within the fish family Goodeidae based on cytochrome b sequence data. Mol Phylogenet Evol 31:416–430. doi:10.1016/j.ympev.2003.08.022

  23. Domínguez-Domínguez O, Pérez-Ponce de León G (2007) Los goodeidos peces endémicos del centro de México. CONABIO Biodiversitas 75:12–15

  24. Domínguez-Domínguez O, Zambrano L, Escalera-Vázquez LH, Pérez-Rodríguez R, Pérez-Ponce de León G (2008) Cambio en la distribución de goodeidos (Osteichthyes: Cyprinodontiformes: Goodeidae) en cuencas hidrológicas del centro de México. Rev Mex Biodiv 79(2):501–512

  25. Dubois M, Gilles K, Hamilton JK, Rebers PA, Smith F (1956) Colorimetric method for the determination of sugars and related substances. Nature 4265:167–168. doi:10.1021/ac60111a017

  26. Dzul-Caamal R, Olivares-Rubio HF, Medina-Segura CG, Vega-López A (2012) Endangered mexican fish special protection: diagnosis of habitat fragmentation, protection, and future—a review. In book: endangered species: habitat, protection and ecological significance. Nova Sci Publ 1(6), pp 109–130

  27. Dzul-Caamal R, Olivares-Rubio HF, López-Tapia P, Vega-López A (2013) Pro-oxidant and antioxidant response elicited by CH2Cl2, CHCl3 and BrCHCl2 in Goodea gracilis using non-invasive methods. Comp Biochem Physiol A Mol Integr Physiol 165:515–527. doi:10.1016/j.cbpa.2013.03.005

  28. Feng M, He Q, Meng L, Zhang X, Sun P, Wang Z (2015) Evaluation of single and joint toxicity of perfluorooctane sulfonate, perfluorooctanoic acid, and copper to Carassius auratus using oxidative stress biomarkers. Aquat Toxicol 161:108–116. doi:10.1016/j.aquatox.2015.01.025

  29. Guilhermino L, Soares AMVM, Carvalho AP, Lopes MC (1998) Acute effects of 3,4-dichloroaniline on blood of male Wistar rats. Chemosphere 37(4):619–632. doi:10.1016/S0045-6535(98)00087-3

  30. Gupta SK, Shriwastav A, Kumari S, Ansari FA, Malik A, Bux F, Bhaskar S, Kuldeep B, Faizal B (2015) Phycoremediation of emerging contaminants. In: algae and environmental sustainability. 7, pp 129–146. doi:10.1007/978-81-322-2641-3_11. Springer, New Delhi, India

  31. Hernández-López J (2001) Diseño de técnicas para la cuantificación de moléculas plasmáticas de camarón. Tesis Doctoral en Ciencias. Centro de Investigación en Alimentación y Desarrollo, A.C. Hermosillo, Sonora, pp 104

  32. Javed M, Usmani N (2015) Stress response of biomolecules (carbohydrate, protein and lipid profiles) in fish Channa punctatus inhabiting river polluted by thermal power plant effluent. Saudi J Biol Sci 22(2):237–242. doi:10.1016/j.sjbs.2014.09.021

  33. Jentoft S, Aastveit AH, Torjesen PA, Andersen Ø (2005) Effects of stress on growth, cortisol and glucose levels in non domesticated Eurasian perch (Perca fluviatilis) and domesticated rainbow trout (Oncorhynchus mykiss). Comp Biochem Physiol A Mol Intregr Physiol 141:353–358. doi:10.1016/j.cbpb.2005.06.006

  34. Lardinois OM, Mestdagh MM, Rouxhet PG (1996) Reversible inhibition and irreversible inactivation of catalase in presence of hydrogen peroxide. Biochim Biophys Acta 2:222–238. doi:10.1016/0167-4838(96)00043-X

  35. Lei XG, Evenson JK, Thompson KM, Sunde RA (1995) Glutathione peroxidase and phospholipid hydroperoxide glutathione peroxidase are differentially regulated in rats by dietary selenium. J Nutr 125:1438–1446

  36. Lerapetritou MG, Georgopoulos PG, Roth CM, Androulakis LP (2009) Tissue-level modeling of xenobiotic metabolism in liver: an emerging tool for enabling clinical translational research. Clin Transl Sci 2(3):228–237. doi:10.1111/j.1752-8062.2009.00092.x

  37. Levine RL, Williams JA, Stadtman EP, Shacter E (1994) Carbonyl assays for determination of oxidatively modified proteins. Methods Enzymol 233:346–357. doi:10.1016/S0076-6879(94)33040-9

  38. Livingstone DR (2001) Contaminated-stimulated reactive oxygen species production and oxidative damage in aquatic organisms. Mar Pollut Bull 42:656–666. doi:10.1016/S0025-326X(01)00060-1

  39. López-López E, Favari LE, Martínez-Tabche L, Madrigal M, Soto C (2003) Hazard assessment of a mixture of pollutants from a sugar industry to three fish species of western Mexico by the responses of enzymes and lipid peroxidation. Bull Environ Contam Toxicol 70:739–745. doi:10.1007/s00128-003-0045-x

  40. López-López E, Sedeño-Díaz JE, Soto C, Favari L (2011) Responses of antioxidant enzymes, lipid peroxidation, and Na+/K+-ATPase in liver of the fish Goodea atripinnis exposed to Lake Yuriria water. Fish Physiol Biochem 37(3):511–522. doi:10.1007/s10695-010-9453-0

  41. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275

  42. Lu GH, Qi PD, Chen W (2013) Integrated biomarker responses of Carassius auratus exposed to BDE-47, BDE-99 and their mixtures. Int J Environ Res 7:807–816

  43. Martinez-Porchas M, Martinez-Cordova LR, Ramos-Enriquez R (2009) Cortisol and glucose: reliable stress indicators of fish stress? Pan-Am J Aquat Sci 4:158–178

  44. Monteiro M, Quintaneiro C, Pastorinho M, Pereira ML, Morgado F, Guilhermino L, Soares AM (2006) Acute effects of 3,4-dichloroaniline on biomarkers and spleen histology of the common goby Pomatoschistus microps. Chemosphere 62:1333–1339. doi:10.1016/j.chemosphere.2005.07.038

  45. NOM-062-ZOO (1999). Norma Oficial Mexicana. Especificaciones técnicas para la producción, cuidado y uso de los animales de laboratorio, Secretaría de Agricultura, Ganadería, Desarrollo Rural, Pesca y Alimentación (SAGARPA), México. pp 58

  46. Olivares-Rubio HF, Martínez-Torres ML, Domínguez-López ML, García-Latorre EA, Vega-López A (2013) Pro-oxidant and antioxidant responses in the liver and kidney of wild Goodea gracilis and their relation with halomethanes bioactivation. J Fish Physiol Biochem 39:1603–1617. doi:10.1007/s10695-013-9812-8

  47. Organisation for Economic Cooperation and Development (1992) Fish acute toxicity test. OECD Guideline for Testing of Chemicals No. 203, Paris, France, pp 9. doi: 10.1787/20745761

  48. Organisation for Economic Cooperation and Development (2012) Validation report (Phase 2) for zebrafish embryo toxicity test. Series on testing and assessment No. 179, Paris, France. pp 57. ENV/JM/MONO (2012)25

  49. Parvez S, Raisuddin S (2005) Protein carbonyls: novel biomarkers of exposure to oxidative stress-inducing pesticides in freshwater fish Channa punctata (Bloch). Environ Toxicol Pharmacol 20(1):112–117. doi:10.1016/j.etap.2004.11.002

  50. Radi R, Turrens JF, Chang LY, Bush KM, Crapo JD, Freeman BA (1991) Detection of catalase in rat heart mitochondria. J Biol Chem 266:22028–22034

  51. Repetto M, Boveris A, Semprine J (2012) Lipid peroxidation: chemical mechanism, biological implications and analytical determination. INTECH Open Access Publisher Book, Croatia, pp 1–30. doi:10.5772/4594e3

  52. Roex EWM, Keijzers R, Van Gestel CAM (2003) Acetylcholinesterase and increased food consumption rate in the zebrafish, Danio rerio, after chronic exposure to parathion. Aquat Toxicol 64:451–460. doi:10.1016/S0166-445X(03)00100-0

  53. Saborido-Rey F (2008) Ecología de la reproducción y potencial reproductivo en las poblaciones de peces marinos. Curso doutoramenton do bieno. Universidad de Vigo, Pontevedra, Spain, pp 71

  54. Sanchez W, Burgeot T, Porcher JM (2013) A novel “integrated biomarker response” calculation based on reference deviation concept. Environ Sci Pollut Res Int 20(5):2721–2725. doi:10.1007/s11356-012-1359-1

  55. Sancho E, Ferrando MD, Andreu E (1996) Physiological stress responses of Anguilla anguilla to fenitrothion. J Environ Sci Health B 31(1):87–98. doi:10.1080/03601239609372976

  56. Sancho E, Villarroel MJ, Andreu E, Ferrando MD (2009) Disturbances in energy metabolism of Daphnia magna after exposure to tebuconazole. Chemosphere 74(9):1171–1178. doi:10.1016/j.chemosphere.2008.11.076

  57. Schindler JF (2003) Scavenger receptors facilitate protein transport in the trophotaenial placenta of the goodeid fish, Ameca splendens (Teleostei: Atheriniformes). J Exp Zool A Comp Exp Biol 299A(2):197–212. doi:10.1002/jez.a.10281

  58. Schindler JF, de Vries U (1987) Maternal-embryonic relationships in the goodeid teleost, Xenoophorus captivus. Cell Tissue Res 247(2):325–338. doi:10.1007/BF00218314

  59. Smolders R, Bervoets L, De Coen W, Blust R (2004) Cellular energy allocation in zebra mussels exposed along a pollution gradient: linking cellular effects to higher levels of biological organization. Environ Pollut 129:99–112. doi:10.1016/j.envpol.2003.09.027

  60. Sreenivasa J, Renuka M, Suneetha Y, Srinivasulu M (2016) Evaluation of antioxidant defence system during xenobiotic induced oxidative stress in freshwater fish Oreochromis mossambicus. Int J Fish Aquat Sci 4:379–385

  61. Srinivas AU, Vutukuru S, Suma C, Madhavi KR, Venkateswara RJ, Anjaneyulu Y (2006) Acute effects of copper on superoxide dismutase, catalase and lipid peroxidation in the freshwater teleost fish, Esomus danricus. Fish Physiol Biochem 32:221–229. doi:10.1007/s10695-006-9004-x

  62. Stephan CE (1977) Methods for calculating an LC50. In: Mayer FI, Hamelink JL (eds) Aquatic toxicology and hazard evaluation. ASTM STP 634. American Society for Testing and Materials, Philadelphia, pp 65–84

  63. Tejeda-Vera R, López-López E, Sedeño-Díaz JE (2007) Biomarkers and bioindicators of the health condition of Ameca splendens and Goodea atripinnis (Pisces: Goodeaidae) in the Ameca River, Mexico. Environ Int 33(4):521–531. doi:10.1016/j.envint.2006.11.018

  64. Tessier AJ, Henry LL, Clyde CE (1983) Starvation in Daphnia: energy reserves and reproductive allocation. Limnol Oceanogr 28:667–676. doi:10.4319/lo.1983.28.4.0667

  65. Turner CL (1947) Viviparity in teleost fishes. Sci Mon 65:508–518

  66. United States Environmental Protection Agency (2002) Methods for measuring the acute toxicity of effluents and receiving waters to freshwater and marine organisms. Quinta edition. U.S. EPA Office of Water, Washington, p 266 EPA-821-R-02-012

  67. Uribe MC, De la Rosa-Cruz G, García-Alarcón A (2014) Branchial placenta in the viviparous teleost Ilyodon whitei (Goodeidae). J Morphol 275(12):1406–1417. doi:10.1002/jmor.20315

  68. Vega-López A, Martínez-Tabche L, Galar-Martínez M (2007) Toxic effects of waterborne polychlorinated biphenyls and sex differences in an endangered goodeid fish (Girardinichthys viviparus). Environ Int 33(4):540–545. doi:10.1016/j.envint.2006.09.002

  69. Vega-López A, Ayala-López G, Posadas-Espadas BP, Olivares-Rubio HF, Dzul-Caamal R (2013) Relations of oxidative stress in freshwater phytoplankton with heavy metals and polycyclic aromatic hydrocarbons. Comp Biochem Physiol A Mol Integr Physiol 165(4):498–507. doi:10.1016/j.cbpa.2013.01.026

  70. Webb SA, Graves JA, Macias-Garcia C, Magurran AE, Foighil ÓD, Ritchie MG (2004) Molecular phylogeny of the livebearing Goodeidae (Cyprinodontiformes). Mol Phylogene Evo 30(3):527–544. doi:10.1016/S1055-7903(03)00257-4

  71. West TG, Arthur PG, Suarez RK, Doll CJ, Hochachka PW (1993) In vivo utilization of glucose by heart and locomotory muscles of exercising rainbow trout (Oncorhynchus mykiss). J Exp Biol 177:63–79

  72. Wintrobe MM (1981) Clinical hematology, 8th edn. Lea and Febiger, Philadelphia

  73. Wootton RJ (1990) Ecology of teleost fishes. Chapman & Hall, London, p 404

  74. Wourms JP (1981) Viviparity: the maternal-fetal relationship in fishes. Am Zool 21(2):473–515. doi:10.1093/icb/21.2.473 473-515

  75. Wourms JP, Lombardi J (1992) Reflections on the evolution of piscine viviparity. Am Zool 32(2):276–293. doi:10.1093/icb/32.2.276 276-293

  76. Wu Y, Zhou Q (2013) Silver nanoparticles cause oxidative damage and histological changes in medaka (Oryzias latipes) after 14 days of exposure. Environ Toxicol Chem 32(1):165–173. doi:10.1002/etc.2038

  77. Xia J, Zhao HZ, Lu GH (2013) Effects of selected metal oxide nanoparticles on multiple biomarkers in Carassius auratus. Biomed Environ Sci 26(9):742–749. doi:10.3967/0895-3988.2013.09.005

  78. Xu P, Zeng G, Huang D, Liu L, Zhao M, Lai C, Li N, Wei Z, Huang C, Zhang C (2016) Metal bioaccumulation, oxidative stress and antioxidant defenses in Phanerochaete chrysosporium response to Cd exposure. Ecol Eng 87:150–156. doi:10.1016/j.ecoleng.2015.11.029

  79. Zhang B, Lin S (2009) Effects of 3,4-dichloroaniline on testicle enzymes as biological markers in rats. Biomed Environ Sci 22(1):40–43. doi:10.1016/S0895-3988(09)60020-9

  80. Zhu B, Liu T, Hu X, Wang G (2013) Developmental toxicity of 3,4-dichloroaniline on rare minnow (Gobiocypris rarus) embryos and larvae. Chemosphere 90(3):1132–1139. doi:10.1016/j.chemosphere.2012.09.021

  81. Zöllner N, Kirsch K (1962) Microdetermination of lipids by the sulphophosphovanillin reaction. Zeitschrift fur die gesamte experimentelle medizine in schliesslich experimenteller chirurgie 135:545–561

Download references


A. L. Carbajal-Hernández had a scholarship from Consejo Nacional de Ciencia y Tecnología (CONACYT) and Beca de Estímulo Institucional de Formación de Investigadores of the Instituto Politécnico Nacional (BEIFI-IPN). Fernando Martínez-Jerónimo is a fellow of the Sistema de Estímulo al Desempeño de los Investigadores (EDI) and the Comisión de Operación y Fomento de Actividades Académicas (COFAA) of the Instituto Politécnico Nacional. The authors are thankful to Dr. Omar Domínguez-Domínguez, Laboratorio de Biología Acuática of the Universidad Michoacana de San Nicolás de Hidalgo, for the kind donation of C. pardalis.

Author information

Correspondence to Fernando Martínez-Jerónimo.

Ethics declarations

All the procedures and the management and of test organisms were in accordance to that approved by the Institutional Ethics Committee.

Conflict of interest

The authors declare that they have no conflict of interest.

Additional information

Responsible editor: Philippe Garrigues

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Carbajal-Hernández, A.L., Valerio-García, R.C., Martínez-Ruíz, E.B. et al. Maternal-embryonic metabolic and antioxidant response of Chapalichthys pardalis (Teleostei: Goodeidae) induced by exposure to 3,4-dichloroaniline. Environ Sci Pollut Res 24, 17534–17546 (2017).

Download citation


  • Transgenerational effects
  • Goodeidae
  • Endemic fish
  • Herbicides
  • Aquatic ecotoxicology