Quantifying Organ-Specific Toxicity of Dietary Heavy Metals in Zebrafish Using a Histopathological Scoring Index | ||
| Journal of Applied Veterinary Sciences | ||
| Articles in Press, Corrected Proof, Available Online from 20 August 2025 PDF (750.17 K) | ||
| Document Type: Original Article | ||
| DOI: 10.21608/javs.2025.394052.1640 | ||
| Authors | ||
| Evangelia Gouva1; Markos N. Kolygas2; Cosmas I. Nathanailides* 1; Ioannis Skoufos1; Athina Tzora1; Ioannis Paschos1; Ioannis S. Pappas2; Fotini Athanassopoulou2 | ||
| 1Faculty of Agriculture, University of Ioannina, Arta, Greece | ||
| 2Faculty of Veterinary Medicine, University of Thessaly, Karditsa, Greece | ||
| Abstract | ||
| This study investigates sublethal effects of eight dietary heavy metals—copper (Cu), zinc (Zn), iron (Fe), cobalt (Co), chromium (Cr), aluminum (Al), manganese (Mn), and molybdenum (Mo)—on juvenile zebrafish (Danio rerio). Over a three-month period, fish were fed diets supplemented with each metal at two concentrations (1 mg/kg and 5 mg/kg), resulting in 16 exposure groups plus a control. Histological sections of gills, intestine, axial muscle, liver, kidney, and brain were evaluated across 49 histopathological parameters. A Metal Impact Index (MI) was used to quantify organ-specific toxicity. Both tested concentrations induced significant alterations in multiple organs, with severity varying by metal and tissue. Gill tissues exhibited the highest MI values, with Cu, Al, and Cr causing epithelial hyperplasia, lamellar degeneration, and pillar cell collapse. Cr exposure led to significant intestinal damage, while Fe caused the most prominent muscle pathology. Liver lesions (vacuolation, necrosis) increased with Cu, Cr, and Fe, and kidney damage (tubular degeneration) was evident in Cr- and Fe-treated groups. Brain tissue showed no significant MI variation. Hierarchical clustering based on cumulative MI scores grouped Cu, Cr, and Fe as high-toxicity metals, particularly in gills, liver, and kidney, while Mn, Zn, Al, and Mo formed lower-toxicity clusters. The MI approach provides a standardized framework for detecting early organ-specific toxicity and may support aquaculture health monitoring, environmental risk assessment, and fish welfare management in metal-contaminated ecosystems. | ||
| Keywords | ||
| Aquaculture; Fish; Gills; Heavy Metals; Toxicity | ||
| References | ||
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AHMAD-BAKER, A. M., MANSOUR, M. A., AL-HARBI, M. S., AL-GHANIM, K. A., AL-MISNED, F., MAHBOOB, S., and AHMAD, Z., 2025. Histopathological Study of The Acute and Chronic Toxic Effects of Dimethyl Mercury on Liver and Kidney of Male Albino Rats. Journal of Applied Veterinary Sciences, 10 (2): 137 – 143. https://javs.journals.ekb.eg/article_419354.html
AHMED, N. M., GHANNAM, H. E., TAYEL, S. I., and MOHAMED, M. Y., 2020. Biochemical and histopathological alterations of Oreochromis niloticus fish related to heavy metals in Lake Nasser, Egypt. Egyptian Journal of Histology, 43(4), 1059–1069. https://dx.doi.org/10.21608/ejh.2020.23337.1246
AHMED, I., ZAKIYA, A., and FAZIO, F., 2022. Effects of aquatic heavy metal intoxication on the level of hematocrit and hemoglobin in fishes: A review. Frontiers in Environmental Science, 10, 919204. https://doi.org/10.3389/fenvs.2022.919204
ALI, H., KHAN, E., and ILAHI, I., 2019. Environmental chemistry and ecotoxicology of hazardous heavy metals: Environmental persistence, toxicity, and bioaccumulation. Journal of Chemistry, 2019(1), Article 6730305. https://doi.org/10.1155/2019/6730305
BAMBINO, K., and CHU, J., 2017. Zebrafish in toxicology and environmental health. Current Topics in Developmental Biology, 124, 331–367. https://doi.org/10.1016/bs.ctdb.2016.10.007
CHEN, X., LIU, S., DING, Q., TEAME, T., YANG, Y., RAN, C., ZHANG, Z., and ZHOU, Z., 2023. Research advances in the structure, function, and regulation of the gill barrier in teleost fish. Water Biology and Security, 2(2), Article 100139. https://doi.org/10.1016/j.watbs.2023.100139
CRAIG, P. M., GALUS, M., WOOD, C. M., and MCCLELLAND, G. B., 2009. Dietary iron alters waterborne copper-induced gene expression in soft water acclimated zebrafish. American Journal of Physiology-Regulatory, Integrative and Comparative Physiology, 296(2), R362–R373. https://doi.org/10.1152/ajpregu.90581.2008
DANE, H., and ŞIŞMAN, T., 2020. A morpho-histopathological study in the digestive tract of three fish species influenced by heavy metal pollution. Chemosphere, 242, Article 125212. https://doi.org/10.1016/j.chemosphere.2019.125212
DANG, F., WANG, W. X., and RAINBOW, P. S., 2012. Unifying prolonged copper exposure, accumulation, and toxicity from food and water in a marine fish. Environmental Science and Technology, 46(6), 3465–3471. https://doi.org/10.1021/es203951z
DIRECTIVE 2002/32/EC OF THE EUROPEAN PARLIAMENT AND OF THE COUNCIL, 2002. On undesirable substances in animal feed. Official Journal of the European Communities, L 140, 10–21.
DURAI, U., ATHISUYAMBULINGAM, M., VISWAMBARAN, G., and BEEVI, F. S. M., 2021. Cytopathological changes of selected tissues in Asian sea bass, Lates calcarifer (Bloch) exposed to mercury. Annals of the Romanian Society for Cell Biology, 25, 1749–1772.
EL-SHERIF, A. A. S., SHABAAN, A. M., ABDO, M. H., ABDEL LATIF, A. K. M., and GOHER, M. E., 2025. Water feature and heavy metal pollution indices in Lake Qarun, Fayoum, Egypt. Egyptian Journal of Aquatic Biology and Fisheries, 29(3), 119–148. https://dx.doi.org/10.21608/ejabf.2025.425777
ENEJI, I. S., SHA’ATO, R., and ANNUNE, P. A., 2011. Bioaccumulation of heavy metals in fish (Tilapia zilli and Clarias gariepinus) organs from River Benue, North-Central Nigeria. Pakistan Journal of Analytical and Environmental Chemistry, 12, 25–31.
FERNANDO, V. K., PERERA, I. C., DANGALLE, C. D., PREMAWANSA, S., and WIJESINGHE, M. R., 2015. Histological alterations in the body wall of the tropical earthworm Eudrilus eugeniae exposed to hexavalent chromium. Bulletin of Environmental Contamination and Toxicology, 94, 744–748. https://doi.org/10.1007/s00128-015-1480-1
FERRARI, S., and CRIBARI-NETO, F., 2004. Beta regression for modelling rates and proportions. Journal of Applied Statistics, 31, 799–815. https://doi.org/10.1080/0266476042000214501
GOUVA, E., NATHANAILIDES, C., PASCHOS, I., ATHANASSOPOULOU, F., and PAPPAS, I. S., 2020a. Comparative study of the effects of heavy metals on embryonic development of zebrafish. Aquaculture Research, 51, 3255–3267. https://doi.org/10.1111/are.14660
GOUVA, E., NATHANAILIDES, C., SKOUFOS, I., PASCHOS, I., ATHANASSOPOULOU, F., and PAPPAS, I. S., 2020b. Reduced metabolic rate and increased heartbeat as early signs of sub lethal copper toxicity in developing zebrafish. Asian Journal of Agriculture and Biology, 8,119–128. https://doi.org/10.35495/ajab.2019.09.415
GÜLDEN, M., and SEIBERT, H., 2005. Impact of bioavailability on the correlation between in vitro cytotoxic and in vivo acute fish toxic concentrations of chemicals. Aquatic Toxicology, 72(4), 327–337. https://doi.org/10.1016/j.aquatox.2005.02.002
GRIFFITT, R. J., WEIL, R., HYNDMAN, K. A., DENSLOW, N. D., POWERS, K., TAYLOR, D., and BARBER, D. S., 2007. Exposure to copper nanoparticles causes gill injury and acute lethality in zebrafish (Danio rerio). Environmental Science and Technology, 41, 8178–8186. https://doi.org/10.1021/es071235e
EL-SHERIF, A. A., SHABAAN, A. M., ABDO, M. H., ABDEL LATIF, A. K. M., and GOHER, M. E., 2025. Water feature and heavy metal pollution indices in Lake Qarun, Fayoum, Egypt. Egyptian Journal of Aquatic Biology and Fisheries, 29(3), 119–149.
EMENIKE, E. C., IWUOZOR, K. O., and ANIDIOBI, S. U., 2022. Heavy metal pollution in aquaculture: Sources, impacts and mitigation techniques. Biological Trace Element Research, 200(10), 4476–4492. https://doi.org/10.1007/s12011-021-03037-x
HAQUE, S., PAL, S., MUKHERJEE, A. K., and GHOSH, A. R., 2012. Histopathological and ultramicroscopic changes induced by fluoride in soft tissue organs of the air-breathing teleost, Channa punctatus (Bloch). Fluoride, 45, 263–273.
HARDISTY, M. W. 1976. Cysts and tumour-like lesions in the endocrine pancreas of the lamprey (Lampetra fluviatilis). Journal of Zoology, 178, 305–317. https://doi.org/10.1111/j.1469-7998.1976.tb02272.x
JANBAKHSH, S., HOSSEINI SHEKARABI, S. P., and SHAMSAIE MERGAN, M., 2018. Nutritional value and heavy metal content of fishmeal from the Southwest Caspian Sea. Caspian Journal of Environmental Sciences, 16, 307–317. https://doi.org/10.22124/cjes.2018.3200
KAMUNDE, C., CLAYTON, C., and WOOD, C. M., 2002. Waterborne vs. dietary copper uptake in rainbow trout and the effects of previous waterborne copper exposure. American Journal of Physiology-Regulatory, Integrative and Comparative Physiology, 283(1), R69–R78. https://doi.org/10.1152/ajpregu.00016.2002
KARLSSON, L. 1983. Gill morphology in the zebrafish, Brachydanio rerio (Hamilton-Buchanan). Journal of Fish Biology, 23, 511–524. https://doi.org/10.1111/j.1095-8649.1983.tb02931.x
KARLSSON-NORRGREN, L., RUNN, P., HAUX, C., and FÖRLIN, L., 1985. Cadmium-induced changes in gill morphology of zebrafish, Brachydanio rerio (Hamilton-Buchanan), and rainbow trout, Salmo gairdneri Richardson. Journal of Fish Biology, 27, 81–95. https://doi.org/10.1111/j.1095-8649.1985.tb04011.x
KOVRIŽNYCH, J. A., SOTNÍKOVÁ, R., ZELJENKOVÁ, D., ROLLEROVÁ, E., and SZABOVÁ, E., 2013. Acute toxicity of 31 different nanoparticles to zebrafish tested in adulthood and early life stages—comparative study. Interdisciplinary Toxicology, 6, 67–73. https://doi.org/10.2478/intox-2013-0012
LIU, Y., DOU, Z., JI, C., ZHOU, Q., ZHAO, J., WANG, K., CHEN, C., and LIU, Q., 2025. Effects of dietary ferric EDTA levels on vegetables and mirror carp (Cyprinus carpio var. specularis) in aquaponics system. Animals, 15, Article 792. https://doi.org/10.3390/ani15060792
LU, K., QIAO, R., AN, H., and ZHANG, Y., 2018. Influence of microplastics on the accumulation and chronic toxic effects of cadmium in zebrafish (Danio rerio). Chemosphere, 202, 514–520. https://doi.org/10.1016/j.chemosphere.2018.03.145
MACIRELLA, R., and BRUNELLI, E., 2017. Morphofunctional alterations in zebrafish (Danio rerio) gills after exposure to mercury chloride. International Journal of Molecular Sciences, 18, Article 824. https://doi.org/10.3390/ijms18040824
MARASCUILO, L. A. 1966. Large-sample multiple comparisons. Psychological Bulletin, 65, 280–282. https://doi.org/10.1037/h0023189 https://doi.org/10.1037/h0023189
MAJID, M., JANMOHAMMAD, M., ENAYAT, B., and AKBARTABAR, T. M., 2019. Heavy metal content in farmed rainbow trout in relation to aquaculture area and feed pellets. Foods and Raw Materials, 7(2), 329–338. http://doi.org/10.21603/2308-4057-2019-2-329-338
MERRIFIELD, D. L., SHAW, B. J., HARPER, G. M., SAOUD, I. P., DAVIES, S. J., and HENRY, T. B., 2013. Ingestion of metal-nanoparticle contaminated food disrupts endogenous microbiota in zebrafish. Environmental Pollution, 174, 157–163. https://doi.org/10.1016/j.envpol.2012.11.017
MORCILLO, P., CORDERO, H., MESEGUER, J., ESTEBAN, M. Á., and CUESTA, A., 2015. Toxicological in vitro effects of heavy metals on gilthead seabream (Sparus aurata L.) head-kidney leucocytes. Toxicology in Vitro, 30, 412–420. https://doi.org/10.1016/j.tiv.2015.09.021
NAZ, S., HUSSAIN, R., ULLAH, Q., CHATHA, A. M. M., SHAHEEN, A., and KHAN, R. U., 2021. Toxic effect of some heavy metals on hematology and histopathology of major carp (Catla catla). Environmental Science and Pollution Research, 28(6), 6533–6539. https://doi.org/10.1007/s11356-020-10980-0
PIEPHO, H. P. 2004. An algorithm for a letter-based representation of all-pairwise comparisons. Journal of Computational and Graphical Statistics, 13, 456–466. https://doi.org/10.1198/1061860043515
PÉREZ-RUZAFA, A., PÉREZ-MARCOS, M., and MARCOS, C., 2018. From fish physiology to ecosystems management: Keys for moving through biological levels of organization in detecting environmental changes and anticipating their consequences. Ecological Indicators, 90, 334–345. https://doi.org/10.1016/j.ecolind.2018.03.019
RAEESZADEH, M., KHOEI, A. J., PARHIZKAR, S., RAD, F. T., and SALIMI, B., 2023. Assessment of some heavy metals and their relationship with oxidative stress and immunological parameters in aquatic animal species. Biological Trace Element Research, 201, 4547–4557. https://doi.org/10.1007/s12011-022-03507-w
RAJESHKUMAR, S., LIU, Y., MA, J., DUAN, H. Y., and LI, X., 2017. Effects of exposure to multiple heavy metals on biochemical and histopathological alterations in common carp, Cyprinus carpio L. Fish and Shellfish Immunology, 70, 461–472. https://doi.org/10.1016/j.fsi.2017.08.013
RODRIGUES, G. Z. P., DE SOUZA, M. S., DA SILVA, A. H., ZWETSCH, B. G., and GEHLEN, G., 2018. Evaluation of intestinal histological damage in zebrafish exposed to environmentally relevant concentrations of manganese. Ciência e Natura, 40, 52. https://doi.org/10.5902/2179460X31681 https://doi.org/10.5902/2179460X31681
SHAW, P., MONDAL, P., BANDYOPADHYAY, A., and CHATTOPADHYAY, A., 2019. Environmentally relevant concentration of chromium activates Nrf2 and alters transcription of related XME genes in liver of zebrafish. Chemosphere, 214, 35–46. https://doi.org/10.1016/j.chemosphere.2018.09.104
SMITHSON, M., and VERKUILEN, J., 2006. A better lemon squeezer? Maximum-likelihood regression with beta-distributed dependent variables. Psychological Methods, 11, 54–71. https://doi.org/10.1080/00049530600940006
SÖNMEZ, V. Z., AKARSU, C., ALTAY, M. C., ERCAN, N., and SIVRI, N., 2025. Can the combination index be used as a new perspective in characterizing the toxicity of metal mixtures? International Journal of Environmental Science and Technology, 1–16. https://doi.org/10.1007/s13762-025-06517-y
TSAI, J. W., JU, Y. R., HUANG, Y. H., DENG, Y. S., CHEN, W. Y., WU, C. C., and LIAO, C. M., 2013. Toxicokinetics of tilapia following high exposure to waterborne and dietary copper and implications for coping mechanisms. Environmental Science and Pollution Research, 20, 3771–3780. https://doi.org/10.1007/s11356-012-1304-3
WARD, J. H., 1963. Hierarchical grouping to optimize an objective function. Journal of the American Statistical Association, 58, 236–244.
WU, Y., LIU, W., LI, L., TAI, Z., and LIU, J. X., 2023. Transcriptional profiles in zebrafish atp7a mutants and responses of atp7a mutants to Cu stress. Water Biology and Security, 2, Article 100186. https://doi.org/10.1016/j.watbs.2023.100186
YACOUB, A. M., MAHMOUD, S. A., and ABDEL-SATAR, A. M., 2021. Accumulation of heavy metals in tilapia fish species and related histopathological changes in muscles, gills and liver of Oreochromis niloticus occurring in the area of Qahr El-Bahr, Lake Al-Manzalah, Egypt. Oceanological and Hydrobiological Studies, 50(1), 1–15. https://doi.org/10.2478/oandhs-2021-0001
YIN, J., WANG, A. P., LI, W. F., SHI, R., JIN, H. T., and WEI, J. F., 2018. Time-response characteristic and potential biomarker identification of heavy metal-induced toxicity in zebrafish. Fish and Shellfish Immunology, 72, 309–317. https://doi.org/10.1016/j.fsi.2017.10.047
YOUNIS, N. A., LABAN, S. E., AL-MOKADDEM, A. K., and ATTIA, M. M., 2020. Immunological status and histopathological appraisal of farmed Oreochromis niloticus exposed to parasitic infections and heavy metal toxicity. Aquaculture International, 28(6), 2247–2262. https://doi.org/10.1007/s10499-020-00589-y | ||
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