SavedAdd your journal

Journals / Turkish Journal of Analytical Chemistry / 2025 / Cilt: 7 Sayı: 3

Biocompatibility Assessment of Chlorhexidine Gluconate Versus a Natural Mouthwash Containing Olea europaea and Opuntia ficus-indica in Zebrafish Embryos

Zebra Balığı Embriyolarında Klorheksidin Glukonat ile Olea europaea ve Opuntia ficus-indica İçeren Doğal Ağız Gargarasının Biyouyumluluklarının Karşılaştırmalı Olarak Değerlendirilmesi

Ezgi Cahide Aydaş Bayramov(Marmara University, Faculty of Dentistry.)0000-0002-9894-8750
Selma Yaltkaya(Marmara University, Faculty of Dentistry.)0009-0008-3075-6796
Merih Beler(Marmara University, Institute of Health Sciences.)0000-0002-3828-4630
Gizem Eğilmezer(Marmara University, Institute of Health Sciences.)0000-0002-1231-5232
Efruz İrem Akkuş Yaylamış(Istanbul University-Cerrahpaşa, Cerrahpaşa Faculty of Medicine.)0000-0001-8504-2602
Armağan Begüm Özel Korlu(Istanbul Health and Technology University, Faculty of Medicine.)0000-0002-4197-3103
Zülal Mızrak(Marmara University, Institute of Health Sciences.)0009-0004-7647-7267
Semanur Işıkoğlu(Marmara University, Institute of Health Sciences.)0009-0003-9779-2867
Atakan Karagöz(Fenerbahce University, Vocational School of Health Services.)0000-0002-6310-1752
İsmail Ünal(Istanbul Medipol University, Faculty of Medicine.)0000-0002-8664-3298
Derya Cansız(Istanbul Medipol University, Faculty of Medicine.)0000-0002-6274-801X
Şebnem Erçalık Yalçınkaya(Marmara University, Faculty of Dentistry.)0000-0003-2924-1935
Ebru Emekli Alturfan(Marmara University, Faculty of Dentistry.)0000-0003-2419-8587
Pages
310–320
Publication Date
2025-09-30
🏛️Bibliographic metadata and full text access for this article are provided via DergiPark.
DergiPark Record ↗
✨

AI Summary & Key Takeaways

AI Insight
⚡ TL;DR(Too Long; Didn't Read)

These findings provide evidence regarding the differential biocompatibility of synthetic and plant-based mouthwashes in early developmental models.

📌 3 Key Takeaways

🎯
Objective & Hypothesis:Various chemical solutions are available for oral hygiene and care, but these chemical solutions also cause various side effects in living systems.
📊
Key Findings & Data:Therefore, the trend towards the production of highly biocompatible, natural-based oral care products has increased.
💡
Conclusion & Impact:These findings provide evidence regarding the differential biocompatibility of synthetic and plant-based mouthwashes in early developmental models.
🔬Design:Modeling & Simulation
📍Scope:Comprehensive Sample
⚙️Variables:Olea europaea · Opuntia ficus-indica · klorheksidin

Abstract

Various chemical solutions are available for oral hygiene and care, but these chemical solutions also cause various side effects in living systems. Therefore, the trend towards the production of highly biocompatible, natural-based oral care products has increased. This study aimed to compare the biocompatibility profiles of a conventional chlorhexidine gluconate-based mouthwash and a natural formulation containing Olea europaea leaf and Opuntia ficus-indica extracts using the zebrafish embryo model. Zebrafish embryos were exposed to two concentrations (100 ppm and 1000 ppm) of each mouthwash for 72 hours post-fertilization. Developmental properties such as mortality, hatching rate, pericardial edema, and body length were evaluated. Biochemical analyses included oxidative stress parameters and acetylcholinesterase (AChE) activity. Chlorhexidine exposure resulted in increased embryonic mortality, pericardial edema, and reduced body length. Biochemically, chlorhexidine increased lipid peroxidation and glutathione S-transferase (GST) activity while decreasing superoxide dismutase (SOD) and AChE activities. In contrast, embryos exposed to the natural formulation showed no significant developmental abnormalities and exhibited increased SOD and AChE activities without changes in lipid peroxidation. These findings provide evidence regarding the differential biocompatibility of synthetic and plant-based mouthwashes in early developmental models.

Özet

Çeşitli kimyasal solüsyonlar ağız hijyeni ve bakımı amacıyla kullanılmaktadır; ancak bu kimyasal solüsyonların canlılarda çeşitli yan etkilere sebep olduğu bilinmektedir. Dolayısıyla, yüksek biyouyumluluğa sahip, doğal içerikli ağız bakım ürünlerinin üretimine yönelim artmaktadır. Bu çalışma, klorheksidin glukonat içeren geleneksel bir ağız gargarası ile Olea europaea (zeytin) yaprağı ve Opuntia ficus-indica (hint inciri) özütlerini içeren doğal bir formülasyonun biyouyumluluklarını zebra balığı embriyo modeli kullanarak karşılaştırmayı amaçlamaktadır. Zebra balığı embriyoları, her bir gargaranın 100 ppm ve 1000 ppm konsantrasyonlarına döllenmeden sonraki 72 saat boyunca maruz bırakılmıştır. Mortalite, koryondan çıkış oranı, perikardiyal ödem ve vücut uzunluğu gibi gelişimsel özellikler değerlendirilmiştir. Biyokimyasal analizlerde ise oksidatif stres parametreleri ve asetilkolinesteraz (AChE) aktivitesi incelenmiştir. Klorheksidin maruziyeti, embriyonik mortalite, perikardiyal ödem ve vücut uzunluğunda azalma ile sonuçlanmıştır. Biyokimyasal olarak, klorheksidin lipid peroksidasyonunu (LPO) ve glutatyon S-transferaz (GST) aktivitesini artırırken, süperoksit dismutaz (SOD) ve AChE aktivitelerini azaltmıştır. Buna karşılık, doğal formülasyona maruz kalan embriyolarda anlamlı bir gelişimsel anomali gözlenmemiş öte yandan SOD ve AChE aktivitelerinde artış izlenmiş, lipid peroksidasyonunda ise bir değişiklik gözlenmemiştir. Bu bulgular, sentetik ve bitki bazlı ağız gargaralarının erken gelişim modellerindeki biyouyumlulukları arasındaki farklılıklara dair kanıt sunmaktadır.

🔖

Cite This Article

Bayramov, E. C. A., Yaltkaya, S., Beler, M., Eğilmezer, G., Yaylamış, E. İ. A., Korlu, A. B. Ö., Mızrak, Z., Işıkoğlu, S., Karagöz, A., Ünal, İ., Cansız, D., Yalçınkaya, Ş. E., Alturfan, E. E. (2025). Biocompatibility Assessment of Chlorhexidine Gluconate Versus a Natural Mouthwash Containing Olea europaea and Opuntia ficus-indica in Zebrafish Embryos. Turkish Journal of Analytical Chemistry, Cilt: 7 Sayı: 3, 310–320. https://doi.org/10.51435/turkjac.1757238

📄 Full Text PDF Viewer

📄

PDF görüntüleyici hazırlanıyor...

References (72)

  1. P. Axelsson, B. Nyström, J. Lindhe, The long-term effect of a plaque control program on tooth mortality, caries, and periodontal disease in adults, J Clin Periodontol, 31(9), 2004, 749–757.
  2. C.G. Jones, Chlorhexidine: Is it still the gold standard?, Periodontol 2000, 15(1), 1997, 55–62.
  3. Y.L. Hsieh, J.C. Yao, S.C. Hsieh, N.C. Teng, Y.T. Chu, W.X. Yu, et al., The in vivo toxicity and antimicrobial properties for electrolyzed oxidizing (EO) water-based mouthwashes, Materials, 13(19), 2020, 4299.
  4. A. Santos, Evidence-based control of plaque and gingivitis, J Clin Periodontol, 30, 2003, 13–16.
  5. C.A. Gürgan, E. Zaim, I. Bakirsoy, E. Soykan, Short-term side effects of 0.2% alcohol-free chlorhexidine mouthrinse used as an adjunct to non-surgical periodontal treatment: A double-blind clinical study, J Periodontol, 77, 2006, 370–384.
  6. J.A. Helms, M.A. Della-Fera, A.E. Mott, M.E. Frank, Effects of chlorhexidine on human taste perception, Arch Oral Biol, 40, 1995, 913–920.
  7. P. John, D. Cunha, Peridex (Chlorhexidine Gluconate 0.12% Oral Rinse), RxList, 2020.
  8. G. Pizzo, R. Guiglia, M. Imburgia, I. Pizzo, M. D’Angelo, G. Giuliana, The effects of antimicrobial sprays and mouthrinses on supragingival plaque regrowth: A comparative study, J Periodontol, 77, 2006, 248–256.
  9. L.S. Hasan, I. Ibrahim Latif, S.H. Mohammed, Efficacy of olive leaf extract mouthwash on clinical and inflammatory parameters of gingival inflammation in relation to chlorhexidine in acute gingivitis patients, Diyala J Med, 22(2), 2022.
  10. N.F.R. Mohamed, R.V. Geetha, P.S. Ganesh, In vitro evaluation of antimicrobial activity of Opuntia ficus-indica seed oil, J Pharm Res Int, 33(47B), 2021, 490–496.
  11. Y. Kwon, Y. Liao, B. Koo, H. Bae, J. Zhang, E.H. Han, et al., Ethanolic extract of Opuntia ficus-indica var. saboten ameliorates cognitive dysfunction induced by cholinergic blockade in mice, J Med Food, 21(10), 2018, 971–978.
  12. R. Zaman, E.S.S. Tan, N.A. Bustami, et al., Assessment of Opuntia ficus-indica supplementation on enhancing antioxidant levels, Sci Rep, 15, 2025, 3507.
  13. R. Gucci, L. Lombardini, M. Tattini, Analysis of leaf water relations in leaves of two olive (Olea europaea) cultivars differing in tolerance to salinity, Tree Physiol, 17, 1997, 13–21.
  14. N.E. Sedef, S. Karakaya, Olive tree (Olea europaea) leaves: potential beneficial effects on human health, 67(11), 2009, 632–638.
  15. O. Benavente-García, J. Castillo, J. Lorente, A. Ortuño, J.A. Del-Rio, Antioxidant activity of phenolics from Olea europaea L. leaves, Food Chem, 49, 2000, 2480–2485.
  16. K.M. Ahmed, The effect of olive leaf extract in decreasing the expression of two pro-inflammatory cytokines in patients receiving chemotherapy for cancer: A randomized clinical trial, Saudi Dent J, 25(4), 2013, 141–147.
  17. Z. Atai, M. Ansari, N. Torabi, Efficacy of olive leaf extract in the treatment of minor oral aphthous ulcers, Am J Infect Dis, 3(1), 2007, 24–26.
  18. N. Chougui, A. Tamendjari, W. Hamidj, S. Hallal, A. Barras, T. Richard, R. Larbat, Oil composition and characterisation of phenolic compounds of Opuntia ficus-indica seeds, Food Chem, 139(1–4), 2013, 796–803.
  19. H. Chahdoura, P. Morales, J.C.M. Barreira, L. Barros, V. Fernández-Ruiz, I.C.F. Ferreira, L. Achour, Dietary fiber, mineral elements profile and macronutrients composition in different edible parts of Opuntia microdasys (Lehm.) Pfeiff and Opuntia macrorhiza (Engelm.), LWT Food Sci Technol, 64(1), 2015, 446–451.
  20. V. Saxena, G. Mishra, A. Saxena, K. Vishwakarma, A comparative study on quantitative estimation of tannins in Terminalia chebula, Terminalia belerica, Terminalia arjuna and Saraca indica using spectrophotometer, Asian J Pharm Clin Res, 6(3), 2013, 148–149.
  21. S. Ali, D.L. Champagne, H.P. Spaink, MK. Richardson, Zebrafish embryos and larvae: a new generation of disease models and drug screens, Birth Defects Res C Embryo Today, 93(2), 2011, 115–133.
  22. G.E. Karaman, E. Emekli-Alturfan, S. Akyüz, Zebrafish: an emerging model organism for studying toxicity and biocompatibility of dental materials, Cell Mol Biol, 66(8), 2020, 41–46.
  23. P. Stachurski, W. Świątkowski, A. Ciszewski, K. Sarna-Boś, A. Michalak, A short review of the toxicity of dentifrices—zebrafish model as a useful tool in ecotoxicological studies, Int J Mol Sci, 24(18), 2023, 14339.
  24. S. Meşeli, G. Kaplan, D. Cansız, Ü.V. Üstündağ, İ. Ünal, E. Emekli -Alturfan, F. Yanıkoğlu, D. Tağtekin, The biocompatibility of sodium lauryl sulphate on developing zebrafish embryos, Experimed, 11(2), 2021, 67–72.
  25. G.E. Karaman, İ. Ünal, M. Beler, F.D. Üstündağ, D. Cansız, Ü.V. Üstündağ, E. Emekli-Alturfan, S. Akyüz, Toothpastes for children and their detergent contents affect molecular mechanisms of odontogenesis in zebrafish embryos, Drug Chem Toxicol, 47(1), 2022, 15–25.
  26. R. Oliveira, I. Domingues, C. Koppe Grisolia, A.M. Soares, Effects of triclosan on zebrafish early-life stages and adults, Environ Sci Pollut Res, 16(6), 2009, 679–688.
  27. H. Gaur, S. Purushothaman, N. Pullaguri, Y. Bhargava, A. Bhargava, Sodium benzoate induced developmental defects, oxidative stress and anxiety-like behaviour in zebrafish larva, Biochem Biophys Res Commun, 502(3), 2018, 364–369.
  28. M. d’Amora, F. Liendo, F.A. Deorsola, S. Bensaid, S. Giordani, Toxicological profile of calcium carbonate nanoparticles for industrial applications, Colloids Surf B Biointerfaces, 190, 2020, 110947.
  29. H. Yi, Z. Wang, X. Li, M. Yin, L. Wang, A. Aldalbahi, N.N. El-Sayed, H. Wang, N. Chen, C. Fan, Silica nanoparticles target a Wnt signal transducer for degradation and impair embryonic development in zebrafish, Theranostics, 6(11), 2016, 1810–1820.
  30. S. Meşeli, Ü.V. Üstündağ, P.S. Ateş, İ. Ünal, E. Işık Alturfan, D. Tağtekin, F. Yanikoglu, The biocompatibility of a ginger-containing herbal toothpaste on developing zebrafish embryos, J Dent Indones, 30(3), 2023, 150–156.
  31. A.G. Kramer, J. Vuthiganon, C.S. Lassiter, Bis-GMA affects craniofacial development in zebrafish embryos (Danio rerio), Environ Toxicol Pharmacol, 43, 2016, 159–165.
  32. H. Makkar, S.K. Verma, P.K. Panda, E. Jha, B. Das, K. Mukherjee, M. Suar, In vivo molecular toxicity profile of dental bioceramics in embryonic zebrafish (Danio rerio), Chem Res Toxicol, 31(9), 2018, 914–923.
  33. L. Zhao, J. Si, Y. Wei, S. Li, Y. Jiang, R. Zhou, B. Liu, H. Zhang, Toxicity of porcelain-fused-to-metal substrate to zebrafish (Danio rerio) embryos and larvae, Life Sci, 203, 2018, 66–71.
  34. A. Karagöz, M. Beler, B.D. Altun, İ. Ünal, D. Cansız, H. Gündüz, A.A. Alturfan, E. Emekli-Alturfan, Ş.E. Yalçınkaya, Panoramic dental X-ray exposure leads to oxidative stress, inflammation and apoptosis-mediated developmental defects in zebrafish embryos, J Stomatol Oral Maxillofac Surg, 124(6), 2023, 101661.
  35. B.Y. Kollayan, et al., Effects of low-dose ionizing radiation on the molecular pathways linking neurogenesis and autism spectrum disorders in zebrafish embryos, Drug Chem Toxicol, 2024, 1–10.
  36. G. Gündüz, M. Beler, İ. İsmail, D. Cansız, E.E. Alturfan, K.N. Kose, Gingipain injection affects intestinal oxidant–antioxidant status and alkaline phosphatase in overfed zebrafish, Experimed, 13(2), 2023, 80–85.
  37. S. Meşeli, D. Tağtekin, E. Emekli Alturfan, Zebrafish, A Model Organism For Research in Dentistry, Selcuk Dent J, 2025;12(1):144-8.
  38. M. Westerfield, The Zebrafish Book: A Guide for the Laboratory Use of Zebrafish, University of Oregon Press, Eugene, OR, 1995.
  39. E.K. Choi, B.M. Choi, Y. Cho, S. Kim, Myelin toxicity of chlorhexidine in zebrafish larvae, Pediatr Res, 93(4), 2023, 845–851.
  40. A.S. Valan, J. Krithikadatta, R. Eswaramoorthy, A. Guru, Comparative Analysis of the Cytotoxic Effects of Modified Triple Antibiotic Hydrogel: Insights From Experimental Models, Cureus, 16(6), 2024, e62662.
  41. S. Louhimies, Directive 86/609/EEC on the protection of animals used for experimental and other scientific purposes, Altern Lab Anim, 30(Suppl 2), 2002, 217–219.
  42. O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, Protein measurement with the Folin phenol reagent, J Biol Chem, 193, 1951, 265–275.
  43. K. Yagi, Assay for blood plasma or serum, Methods Enzymol, 105, 1984, 328–331.
  44. W.H. Habig, M.J. Pabst, W.B. Jakoby, Glutathione S-transferases: the first enzymatic step in mercapturic acid formation, J Biol Chem, 249, 1974, 7130–7139.
  45. A.A. Mylroie, H. Collins, C. Umbles, J. Kyle, Erythrocyte superoxide dismutase activity and other parameters of copper status in rats ingesting lead acetate, Toxicol Appl Pharmacol, 82, 1986, 512–520.
  46. G.L. Ellman, K.D. Courtney, V. Andres Jr, R.M. Featherstone, A new and rapid colorimetric determination of acetylcholinesterase activity, Biochem Pharmacol, 7, 1961, 88–95.
  47. M.H. Tabatabaei, F.S. Mahounak, N. Asgari, Z. Moradi, Cytotoxicity of the ingredients of commonly used toothpastes and mouthwashes on human gingival fibroblasts, Front Dent, 16(6), 2019, 450–457.
  48. Y. Ha, Y. Kim, J. Choi, I. Hwang, J.-Y. Ko, H. K. Jeon, Y.-J. Kim, Evaluation of cytotoxicity, genotoxicity, and zebrafish embryo toxicity of mixtures containing Hyssopus officinalis, Morus alba, Engraulis japonicus, and 27 other extracts for cosmetic safety assessment, Mol Cell Toxicol, 17, 2021, 221-232.
  49. U.S. Food and Drug Administration (FDA), Peridex (chlorhexidine gluconate) oral rinse – prescribing information, 2013.
  50. NHS, Pregnancy, breastfeeding and fertility while using chlorhexidine, 2024.
  51. E. Benramdane, N. Chougui, P.A.B. Ramos, N. Makhloufi, A. Tamendjari, A.J.D. Silvestre, et al., Lipophilic compounds and antibacterial activity of Opuntia ficus-indica root extracts from Algeria, Int J Mol Sci, 23(19), 2022, 11161.
  52. A.P.N. Newton, L.L. Cogo, C.M.R. de Oliveira, C.C. Kanunfre, J. Freitas-Astúa, New data on biological effects of chlorhexidine: Fe²⁺ induced lipid peroxidation and mitochondrial permeability transition, Toxicol Lett, 151(3), 2004, 407–416.
  53. D. Biswas, M. Tiwari, V. Tiwari, Molecular mechanism of antimicrobial activity of chlorhexidine against carbapenem-resistant Acinetobacter baumannii, PLOS ONE, 14(10), 2019, e0224107.
  54. F. Abdellah, K. Hamden, T. Silarbi, N. Ayad, R. Benaraba, Antioxidant activity and protective effect of olive leaf extracts (Olea europaea L.) against lipid peroxidation, Phytothérapie, 21(6), 2023, 259–267.
  55. F. Saputra, M. Kishida, S.Y. Hu, Oxidative stress induced by hydrogen peroxide disrupts zebrafish visual development by altering apoptosis, antioxidant and estrogen related genes, Sci Rep, 14(1), 2024, 14454.
  56. K. Ukaegbu, D. Foyle, X. Luan, E. Schneiderman, E.P. Allen, J. Plemons, et al., The effect of an antioxidant gel compared to chlorhexidine during the soft tissue healing process: An animal study, J Periodontol, 95(11), 2024, 1086–1096.
  57. Y.C. Li, Y.H. Kuan, T.H. Lee, F.M. Huang, Y.C. Chang, Assessment of the cytotoxicity of chlorhexidine by employing an in vitro mammalian test system, J Dent Sci, 9(2), 2014, 130–135.
  58. R. Pennisi, I. Ben Amor, B. Gargouri, H. Attia, R. Zaabi, A. Ben Chira, M. Saoudi, A. Piperno, P. Trischitta, M. P. Tamburello, M. T. Sciortino, Analysis of antioxidant and antiviral effects of olive (Olea europaea L.) leaf extracts and pure compound using cancer cell model, Biomolecules, 13, 2023, 238.
  59. I. Besné-Eseverri, M. A. Martín, G. Lobo, M. P. Cano, M. P. Portillo, J. Trepiana, Antioxidant and anti-inflammatory effects of Opuntia extracts on a model of diet-induced steatosis, Antioxidants, 13, 2024, 1416.
  60. S.Z. Moussa, S.A. El Meadawy, H.A. Ahmed, M. Refat, Efficacy of Chelidonium majus and propolis against cytotoxicity induced by chlorhexidine in rats, 2007.
  61. H.J. Forman, H. Zhang, A. Rinna, Glutathione: Overview of its protective roles, measurement, and biosynthesis, Mol Aspects Med, 30(1–2), 2009, 1–12.
  62. M. Valko, D. Leibfritz, J. Moncol, M.T.D. Cronin, M. Mazur, J. Telser, Free radicals and antioxidants in normal physiological functions and human disease, Int J Biochem Cell Biol, 39(1), 2006, 44–84.
  63. J. Çoban, S. Öztezcan, S. Doğru-Abbasoğlu, I. Bingül, K. Yeşil-Mizrak, M. Uysal, Olive leaf extract decreases age-induced oxidative stress in major organs of aged rats, Geriatr Gerontol Int, 14(4), 2014, 996–1002.
  64. H. Jemai, A. Mahmoudi, A. Feryeni, I. Fki, Z. Bouallagui, S. Choura, M. Chamkha, S. Sayadi,, Hepatoprotective effect of oleuropein-rich extract from olive leaves against cadmium-induced toxicity in mice, Biomed Res Int, 2020, 4398924.
  65. N. Hfaiedh, M.S. Allagui, M. Hfaiedh, A.E. Feki, L. Zourgui, F. Croute, Protective effect of cactus (Opuntia ficus indica) cladode extract upon nickel-induced toxicity in rats. Food Chem Toxicol. 2008, 46(12),3759–3763.
  66. E. Hanneman, M. Westerfield, Early expression of acetylcholinesterase activity in functionally distinct neurons of the zebrafish, J Comp Neurol, 276(2), 1988, 244–255.
  67. D. Yang, H. Lauridsen, K. Buels, L.H. Chi, J. La Du, R.L. Tanguay, Chlorpyrifos-oxon disrupts zebrafish axonal growth and motor behavior, Toxicol Sci, 121(1), 2011, 146–159.
  68. C. Gravato, F.R. Abe, D.P. de Oliveira, A.M.V.M. Soares, I. Domingues, Acetylcholinesterase (AChE) activity in embryos of zebrafish, Methods Mol Biol, 2240, 2021, 119–124.
  69. J.M. Romero-Márquez, M.D. Navarro-Hortal, T.Y. Forbes-Hernández, A. Varela-López, J.G. Puentes, C. Sánchez-González, et al., Effect of olive leaf phytochemicals on the anti-acetylcholinesterase, anti-cyclooxygenase-2 and ferric reducing antioxidant capacity, Food Chem, 444, 2024, 138516.
  70. S.S. Khizrieva, S.N. Borisenko, E.V. Maksimenko, N.I. Borisenko, V.I. Minkin, Study of the composition and anti-acetylcholinesterase activity of olive leaf (Olea europaea L.) extracts obtained in subcritical water, Russ J Phys Chem B, 15(8), 2021, 1286–1290.
  71. D. Butera, L. Tesoriere, F. Di Gaudio, A. Bongiorno, M. Allegra, A.M. Pintaudi, et al., Antioxidant activities of Sicilian prickly pear (Opuntia ficus-indica) fruit extracts and reducing properties of its betalains: Betanin and indicaxanthin, J Agric Food Chem, 50(23), 2002, 6895–6901.
  72. E. Teixidó, E. Piqué, J. Gómez-Catalán, J.M. Llobet, Assessment of developmental delay in the zebrafish embryo teratogenicity assay, Toxicol In Vitro, 27(2), 2013, 469–478.