Evaluation of the developmental toxicity of 2 phenoxyethanol and clove oil anaesthetics using the Frog Embryo Teratogenesis Assay: Xenopus (FETAX)

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(1)Veterinarni Medicina, 57, 2012 (5): 245–250. Original Paper. Evaluation of the developmental toxicity of 2-phenoxyethanol and clove oil anaesthetics using the Frog Embryo Teratogenesis Assay: Xenopus (FETAX) D. Vrskova, H. Modra University of Veterinary and Pharmaceutical Sciences, Brno, Czech Republic ABSTRACT: The developmental toxicity of two anaesthetics, 2-phenoxyethanol and clove oil, used in aquaculture was evaluated using the Frog Embryo Teratogenesis Assay: Xenopus (FETAX) and the results were compared to outcomes in fish. Xenopus laevis embryos were exposed to 50, 100, 300, 500, 700 and 1000 mg/l of 2-phenoxyethanol or 1, 5, 10, 20, 30 and 40 mg/l of clove oil. Values of 96 h LC50, 96 h EC50 (malformation) and teratogenic index (ratio of 96 h LC50 and 96 h EC50) were determined and the types and severities of the induced malformations and minimal concentration inhibiting the growth of embryos were estimated. Teratogenic index values for 2-phenoxyethanol and clove oil were estimated at 1.69 and 0.61 respectively. The most frequently observed malformations produced by 2-phenoxyethanol were axial flexure and oedema and for clove oil, axial flexure, gut malformation, microphthalmia and oedema. 2-phenoxyethanol was found to induce growth inhibition of frog embryos at concentrations above 300 mg/l and clove oil at concentrations above 20 mg/l. In summary, both 2-phenoxyethanol and clove oil affected the growth of Xenopus embryos, while only 2-phenoxyethanol represented a teratogenic risk. Keywords: eugenol; amphibian; anaesthesia; teratogenic index; malformation; African clawed frog. Anaesthetics such as 2-phenoxyethanol or clove oil are used in aquaculture to prevent stress or mechanical damage in fish during manipulation, veterinary intervention, artificial spawning or measuring (Ross and Ross 1999). The right use of anaesthesia prevents stress-induced complications in fish such as decreased immune functions or food intake (Ross and Ross 1999). With the growing popularity of amphibians in research and as pets a need for high-quality anaesthesia that could be used to manage amphibians for diagnostic and surgical procedures without sideeffects, arose. A large number of anaesthetics for parenteral administration have been evaluated in amphibians; tricaine methanesulfonate, isoflurane or ketamine rank among the most frequently used (Whitaker and Wright 2001). In consideration of the small size of some amphibians and difficulty with injectable administration, the possibility of. anaesthetic immersion baths for water amphibians represents an ideal route of anaesthetic administration. Clove oil, tricaine methanesulfonate (MS 222) and propofol rank among the most efficacious compounds for immersion bath anaesthesia in amphibian medicine (Mitchell 2009). Clove oil is a naturally occurring compound obtained from clove plants Eugenia aromatica or Eugenia caryophylatta. The active substance, eugenol, comprises from 80% to 90% of clove oil. Clove oil’s advantages include low price, relatively little adverse reactions for both fish and amphibians and safety for staff (Svobodova et al. 2007). 2-phenoxyethanol (ethylene glycol monophenyl ether) has been suggested as a good anaesthetic for short term immobilisation of fish (Ortuno et al. 2002; Tsantilas et al. 2006). The advantages of 2-phenoxyethanol include short anaesthesia induction phase, rapid recovery and low price (Weyl. Supported by the Ministry of Education, Youth and Sports of the Czech Republic (Grant No. MSM 6215712402).. 245.

(2) Original Paper et al. 1996). Compared to clove oil, 2-phenoxyethanol is less safe for staff; in closed rooms it can cause skin and eye irritation in people (Svobodova et al. 2007). The African clawed frog Xenopus laevis is a popular worldwide amphibian model and is one of the amphibian species most commonly used in toxicity testing. Embryos of X. laevis are sensitive indicators for evaluation of the developmental toxicity of substances in the water environment. The purpose of this study was (1) to evaluate the teratogenic risk of 2-phenoxyethanol and clove oil to Xenopus laevis embryos and (2) to estimate the risk for amphibian embryos in the field when subjected to an anaesthetic immersion bath for fish containing 2-phenoxyethanol or clove oil.. MATERIAL AND METHODS In this study, clove oil supplied by the Kulich Company (Hradec Kralove/Ricany, Czech Republic) and 2-phenoxyethanol supplied by MERCK (Hohenbrunn, Germany) were used. The substances were dissolved in standard FETAX solution (625.0 NaCl, 96.0 NaHCO3, 30.0 KCl, 15.0 CaCl2, 60.0 CaSO4.H2O and 70.0 MgSO4 all in mg/l dissolved in distilled water) (ASTM 1998). The South African clawed frog (Xenopus laevis) embryos were obtained from an adult pair injected with human chorionic gonadotropin (Pregnyl 1500, N.V. Organon, Oss, The Netherlands) into the dorsal lymph sac (females 600 IU, males 300 IU). Embryonal tests were conducted using the Standard guide for the frog embryo teratogenesis assay-Xenopus (FETAX) (ASTM 1998). Normally developed embryos in mid-blastula (stage 8) to early gastrula (stage 11) were selected for testing (Nieuwkoop and Faber 1994). Groups of 25 embryos were randomly placed in plastic Petri dishes (60 mm) with 10.0 ml of the tested solution and incubated in Zanussi ZT 155 BR at 24 ± 1 °C. A photoperiod of 12 : 12 LD was maintained throughout the test. The control groups of embryos exposed to FETAX solution were tested four times and 2-phenoxyethanol-treated groups (50, 100, 300, 500, 700, 1000 mg/l) and clove oil-treated groups (1, 5, 10, 20, 30, 40 mg/l) were tested twice. Subsequently a series of three tests were performed using embryos produced by different parental pairs. All 2-phenoxyethanol and clove oil concentrations (stock solutions) were prepared 246. Veterinarni Medicina, 57, 2012 (5): 245–250 immediately before changing the bath. The test medium was changed daily and dead embryos were removed. At the end of the experiment (after 96 h), surviving embryos were euthanized with carbon dioxide-saturated water and fixed in 3.0% formaldehyde. Head-tail length and an assessment of the morphological abnormalities of fixed embryos were determined under a dissecting microscope according to the Atlas of Abnormalities (Bantle et al. 1991). The results of the FETAX assay were analysed using the statistical package Unistat® v. 5.1 (Unistat Ltd., Great Britain). Concentrations causing 50% lethality (96 h LC50) and concentrations eliciting malformations in 50% of surviving embryos (96 h EC50) were estimated using a probit model with 95% confidence. The homogeneity of variances prior to ANOVA was assessed using Levene’s test. The teratogenic index (TI) was calculated as a ratio of 96 h LC50 and 96 h EC50 for tested samples. Differences among head-tail lengths were evaluated using one-way ANOVA and Fischer LSD test. P-values less than 0.05 were considered statistically significant.. RESULTS Controls for the FETAX assay met the criteria for the test acceptance – mortality and malformation rates were lower than 10% (ASTM 1998). In 2-phenoxyethanol, statistically significant increases in mortality were observed at concentrations of 500, 700 and 1000 mg/l and increases in malformation at concentrations of 300, 500 and 700 mg/l (Table 1). 96 h LC 50 and 96 h EC50 were estimated at 588 mg/l and 348 mg/l, respectively. Severe oedema and axial abnormality were the most frequent malformations observed. The calculated teratogenic index (TI; ratio of 96 h LC50 and 96 h EC50) for 2-phenoxyethanol was 1.69. Statistically significant decreases in growth were found in embryos exposed to 2-phenoxyethanol at concentrations of 300, 500 and 700 mg/l compared to the control (Figure 1). The minimal concentration that inhibited growth (MCIG) was estimated to be 300 mg/l. In clove oil, statistically significant increases in mortality were observed at concentrations of 20, 30 and 40 mg/l, and increases in malformation were seen at concentrations of 10, 20 and 30 mg/l (Table 2). 96 h LC50 and 96 h EC50 values were esti-.

(3) Veterinarni Medicina, 57, 2012 (5): 245–250. Original Paper. Table 1. Effects of 2-phenoxyethanol on the survival and malformation of Xenopus laevis embryos after 96 h FETAX (mean of three tests) Mortality (%). Concentration (mg/l) . Malformation (%). mean. SD. P. mean. SD. P. 50. 5.25. 2.55. > 0.05. 1.25. 2.12. > 0.05. 100. 4.63. 5.66. > 0.05. 2.50. 3.04. > 0.05. 300. 6.14. 2.83. > 0.05. 40.00*. 1.91. < 0.001. 500. 26.22*. 2.83. < 0.001. 83.00*. 5.87. < 0.001. 700. 80.36*. 5.66. < 0.001. 86.50*. 4.95. < 0.001. 1000. 100.00*. 0.00. < 0.001. 5.00. 3.83.  . Control.   8.00. 2.33.  . *indicate statistically significant difference from the control (ANOVA plus LSD post-test). mated to be 21.60 mg/l and 35.71 mg/l, respectively. Axial flexure, gut malformation, microphthalmia and oedema were the most frequent malformations observed. The TI for clove oil was 0.61. Statistically. 10. *. Head-tail lenght (mm). 9. significant decreases in growth were found in embryos exposed to clove oil at concentrations of 20 and 30 mg/l compared to the control (Figure 2). The MCIG was estimated to be 20 mg/l.. *. 8. *. 7 6 5 4. Figure 1. Effects of 2-phenoxyethanol on the growth of Xenopus laevis embryos in 96  h FETAX (mean of three tests). 3 2 1 0. Control. 50. 100. 300. 500. 700. Head-tail lenght (mm). Concentration of 2-phenoxyethanol (mg/l) 11 10 9 8 7 6 5 4 3 2 1 0. *. *indicate a statistically significant difference from the control (ANOVA plus LSD post-test). *. Figure 2. Effects of clove oil on the growth of Xenopus laevis embryos in 96 h FETAX (mean of three tests) Control. 1. 5. 10. Concentration of clove oil (mg/l). 20. 30. *indicate a statistically significant difference from the control (ANOVA plus LSD post-test). 247.

(4) Original Paper. Veterinarni Medicina, 57, 2012 (5): 245–250. Table 2. Effects of clove oil on the survival and malformation rate of Xenopus laevis embryos in 96 h FETAX (mean of three tests) Concentration (mg/l). Mortality (%). Malformation (%). mean. SD. P. mean. SD. P. 1. 2.36. 1.54. > 0.05. 0.50. 1.21. > 0.05. 5. 12.50. 2.83. > 0.05. 11.25. 6.24. > 0.05. 10. 26.75. 6.36. > 0.05. 20.41*. 2.12. < 0.05. 20. 34.69*. 3.54. > 0.05. 24.62*. 4.49. < 0.01. 30. 50.00*. 3.87. < 0.001. 64.57*. 3.65. < 0.001. 40. 100.00*. 0.00. < 0.001. 6.50. 5.72. 2.25. 1.38. Control. *indicate statistically significant difference from the control (ANOVA plus LSD post-test). DISCUSSION There exists only a small number of reports dealing with the toxic effects of xenobiotics on amphibians in different developmental stages (Pauli et al. 2000; Richards and Cole 2006; Gungordu et al. 2010) and according to our knowledge, there are no data on the embryotoxicity and teratogenity of 2-phenoxyethanol or clove oil. According to the ASTM (1998), three separate criteria have been considered for the identification of teratogens using the FETAX assay: a TI higher than 1.5, the occurrence of severe malformations at concentrations near the 96 h LC50 and growth inhibition (the growth should be significantly affected at concentrations below 30% of the 96 h LC50). A substance presents a teratogenic risk when any one of the three criteria is met. FETAX assay with 2-phenoxyethanol gained TI value 1.69, and the occurrence of malformations in 96 h LC50 reached over 80%, so our study met two criteria for estimating teratogenic hazard. The MCIG of 2-phenoxyethanol was estimated at 300 mg/l, the 30% value of 96 h LC50 was 177 mg/l, so this criterion for teratogenic risk was not met. Acute toxicity of 2-phenoxyethanol for juvenile fish is higher than for embryonal stages of fish. Values 96 h LC50 for juvenile fish range from 188 mg/l (0.17 ml/l) (Cyprinus carpio) to 338 mg/l (Danio rerio) (Velisek and Svobodova 2004a). Embryotoxicity for 2-phenoxyethanol in zebrafish embryo according OECD No. 212 method yield 168 h LC50 values 486 mg/l (Macova et al. 2008). Compared to these results embryos of X. laevis are less sensitive to effects of 2-phenoxyethanol than Danio rerio embryos. 248. According to our knowledge, there is only one experimental paper reporting the effect of 2-phenoxyethanol on the adult amphibians, because 2-phenoxyethanol is not used in amphibian clinical practice. Pitkin and Pettyjohn (1992) described sinus bradycardia in the red-spotted newt (Notophthalmus viridescens) after the bath with 2-phenoxyethanol at concentration of 5000 mg/l. The generally recommended concentration of 2-phenoxyethanol for fish anaesthetic bath varies depending on genus from 167 mg/l to 442 mg/l (Barton and Helfrich 1981; Velisek and Svobodova 2004a,b) and anaesthetic effect differs according fish age, weight, gender and physical condition of water (Weyl et al. 1996). In our experiment 96 h EC50 was estimated at 348 mg/l, TI at 1.69 and MCIG at 300 mg/l, so we concluded, that direct affecting of amphibian embryos by 2-phenoxyethanol immersion bath solution at commonly used concentration would cause increase in malformation rate and it would also affect the larval growth. The FETAX assay with clove oil gave a TI value of 0.61 and the occurrence of severe malformations at concentrations near the 96 h LC50 reached about 25% (this value was statistically significant). The MCIG of clove oil was estimated to be 20.0 mg/l while the 30% value of the 96 h LC50 was 6.5 mg/l. Our results did not meet the criteria for the identification of a teratogenic hazard and it is thus proven that clove oil does not represent a teratogenic risk for Xenopus laevis embryos. The acute toxicity of clove oil for fish according to the OECD No. 203 method in juvenile common carp (Cyprinus carpio), rainbow trout (Oncorhynchus mykiss) and zebrafish (Danio rerio) gave 96 h LC50 values of 18.1 mg/l, 14.1 mg/l and.

(5) Veterinarni Medicina, 57, 2012 (5): 245–250 18.8 mg/l (Velisek et al. 2005). The embryotoxicity of clove oil for zebrafish embryos according to the OECD No. 212 method yielded a 168 h LC50 value of 15.6 mg/l (Macova et al. 2008). The 96 h LC50 value for clove oil obtained using FETAX (21.6 mg/l) indicated that X. laevis embryos are less sensitive than fish embryos and juvenile fish. Although the recommended concentrations of clove oil for the short-term immobilization of fish range from 40 up to 100 mg/l (Keene et al. 1998; Waterstrat 1999), convenient clove oil concentrations for immersion anaesthesia for amphibians differ according to the genus and size of the animal and range from 300 to 450 mg/l (Lafortune et al. 2001; Guenette et al. 2007; Mitchell et al. 2009). Interspecies differences in anaesthetic effects, induction time and analgesia in amphibians are attributed to differences in metabolisms of individual species, the route of administration and in the case of immersion or topical administration, to the rate of absorption of the drug across the skin (Mitchell et al. 2009). Clove oil is generally considered a safe anaesthetic agent in amphibians (Guenette et al. 2007), but Goulet et al. (2011) reported side effects in adult X. laevis, where a single 10 minute anaesthetic bath containing clove oil at a concentration of 350 mg/l caused renal distal tubular apoptosis, hepatic necrosis, formation of lung hyaline membranes and adipose tissue haemorrhages. Cutaneous necrosis after topical administration of clove oil at concentrations of 60 and 100 mg/l were reported by Ross et al. (2006). Mitchel et al. (2009) described other side effects of baths with clove oil at a concentration of 450 mg/l that included gastric prolapse and respiratory depression in leopard frogs (Rana pipiens) and bradycardia in leopard frogs and tiger salamander (Ambystoma tigrinum). It is suggested that when used in fish, clove oil be administered in anaesthesia baths at concentrations between 25–100 mg/l, according to the genus and size of the fish (Hikasa et al. 1986; Walsh and Pease 2002; Hamackova et al. 2004). When we compare our experimental results (21.60 mg/l for 96 h LC50, 35.71 mg/l for 96 h EC50, 20.0 mg/l for MCIG) and the recommended concentrations for fish anaesthesia, it is clear that although the TI of clove oil is low (0.61), the effect on amphibian embryos of a clove oil immersion bath solution could result in higher mortality, malformation and growth inhibition. In conclusion, our study showed a lower sensitivity of X. laevis embryos to both tested anaesthetic. Original Paper agents compared to fish embryos. Furthermore, our study proved that, in contrast to clove oil, 2-phenoxyethanol represents a teratogenic risk for amphibian embryos. An extended period of exposure could increase the toxicity of anaesthetics. Our results indicate that the risk to amphibian embryos is high when they are exposed to anaesthetic concentrations of 2-phenoxyethanol and clove oil for 96 hours.. REFERENCES ASTM (1998): ASTM E1439-98: Standard guide for conducting fhe Frog Embryo Teratogenesis Assay – Xenopus (FETAX). ASTM, Philadelphia. 16 pp. Bantle JA, Dumont JN, Finch RA, Linder G (1991): Atlas of Abnormalities: A Guide for Performance of FETAX. Oklahoma State University Publications, Stillwater. 72 pp. Barton BA, Helfrich H (1981): Time-dose responses of juvenile rainbow-trout to 2-phenoxyethanol. Progressive Fish-Culturist 43, 223–223. Goulet F, Vachon P, Helie P (2011): Evaluation of the toxicity of eugenol at anesthetic doses in African clawed frogs (Xenopus laevis). Toxicologic Pathology 39, 471–477. Guenette SA, Helie P, Beaudry F, Vachon P (2007): Eugenol for anesthesia of African clawed frogs (Xenopus laevis). Veterinary Anaesthesia and Analgesia 34, 164–170. Gungordu A, Birhanli A, Ozmen M (2010): Assessment of embryotoxic effects of cadmium, lead and copper on Xenopus laevis. Fresenius Environmental Bulletin 19, 2528–2535. Hamackova J, Lepicova A, Kozak P, Stupka Z, Kouril J, Lepic P (2004): The efficacy of various anaesthetics in tench (Tinca tinca L.) related to water temperature. Veterinarni Medicina 49, 467–472. Hikasa Y, Takase K, Ogasawara T, Ogasawara S (1986): Anesthesia and recovery with tricaine methanesulfonate, eugenol and thiopental sodium in the carp, Cyprinus-carpio. Japanese Journal of Veterinary Science 48, 341–351. Keene JL, Noakes DLG, Moccia RD, Soto CG (1998): The efficacy of clove oil as an anaesthetic for rainbow trout, Oncorhynchus mykiss (Walbaum). Aquaculture Research 29, 89–101. Lafortune M, Mitchell MA, Smith JA (2001): Evaluation of medetomidine, clove oil, and propofol for anesthesia in leopard frogs (Rana pipiens). Journal of Herpetological Medicine and Surgery 11, 13–18.. 249.

(6) Original Paper Macova S, Dolezelova P, Pistekova V, Svobodova Z, Bedanova I, Voslarova E (2008): Comparison of acute toxicity of 2-phenoxyethanol and clove oil to juvenile and embryonic stages of Danio rerio. Neuroendocrinology Letters 29, 680–684. Mitchell MA (2009): Anesthetic considerations for amphibians. Journal of Exotic Pet Medicine 18, 40–49. Mitchell MA, Riggs SM, Singleton CB, Diaz-Figueroa O, Hale LK (2009): Evaluating the clinical and cardiopulmonary effects of clove oil and propofol in tiger salamanders (Ambystoma tigrinum). Journal of Exotic Pet Medicine 18, 50–56. Nieuwkoop PD, Faber J (1994): Normal Table of Xenopus laevis (Daudin). Garland Publishing, New York. 252 pp. Ortuno J, Esteban MA, Meseguer J (2002): Effects of phenoxyethanol on the innate immune system of gilthead seabream (Sparus aurata L.) exposed to crowding stress. Veterinary Immunology and Immunopathology 89, 29–36. Pauli BD, Perrault JA, Money SL (2000): Ratl: A Database of Reptile and Amphibian Toxicology Literature. Canadian Wildlife Service, Quebec. 494 pp. Pitkin RB, Pettyjohn A (1992): 2-phenoxyethanol effects on the heart rate of the adult, red-spotted newt. Journal of the Pennsylvania Academy of Science 65, 195. Richards SM, Cole SE (2006): A toxicity and hazard assessment of fourteen pharmaceuticals to Xenopus laevis larvae. Ecotoxicology 15, 647–656. Ross LG, Ross B (1999): Anaesthetic and Sedative Techniques for Aquatic Animals. Blackwell Science Ltd., Oxford. 159 pp. Ross A, Guenette SA, Helie P, Vachon P (2006): Case of cutaneous necrosis in african clawed frogs Xenopus laevis after the topical application of eugenol. Canadian Veterinary Journal-Revue Veterinaire Canadienne 47, 1115–1117.. Veterinarni Medicina, 57, 2012 (5): 245–250 Svobodova Z, Kolarova J, Navratil S, Vesely T, Chloupek P, Tesarcik J (2007): Diseases of freshwater and aquarium fish. Informatorium Prague, Prague. 264 pp. Tsantilas H, Galatos AD, Athanassopoulou F, Prassinos NN, Kousoulaki K (2006): Efficacy of 2-phenoxyethanol as an anaesthetic for two size classes of white sea bream, Diplodus sargus L., and sharp snout sea bream, Diplodus puntazzo C. Aquaculture 253, 64–70. Velisek J, Svobodova Z (2004a): Anaesthesia of common carp (Cyprinus carpio L) with 2-phenoxyethanol: Acute toxicity and effects on biochemical blood profile. Acta Veterinaria Brno 73, 247–252. Velisek J, Svobodova Z (2004b): Anaesthesia of rainbow trout (Oncorhynchus mykiss) with 2-phenoxyethanol: Acute toxicity and biochemical blood profile. Acta Veterinaria Brno 73, 379–384. Velisek J, Svobodova Z, Piackova V (2005): Effects of clove oil anaesthesia on rainbow trout (Oncorhynchus mykiss). Acta Veterinaria Brno 74, 139–146. Walsh CT, Pease BC (2002): The use of clove oil as an anaesthetic for the longfinned eel, Anguilla reinhardtii (Steindachner). Aquaculture Research 33, 627–635. Waterstrat PR (1999): Induction and recovery from anesthesia in channel catfish Ictalurus punctatus fingerlings exposed to clove oil. Journal of the World Aquaculture Society 30, 250–255. Weyl O, Kaiser H, Hecht T (1996): On the efficacy and mode of action of 2-phenoxyethanol as an anaesthetic for goldfish, Carassius auratus (L.), at different temperatures and concentrations. Aquaculture Research 27, 757–764. Whitaker BR, Wright KN (2001): Amphibian medicine and captive husbandry. Krieger Publishing Company, Malabar, USA. 570 pp. Received: 2012–05–14 Accepted after corrections: 2012–05–28. Corresponding Author: Dagmar Vrskova, DVM., Ph.D., Veterinary and Pharmaceutical Sciences Brno, Faculty of Veterinary Medicine, Department of Pharmacology and Pharmacy, Palackeho 1–3, 612 42 Brno, Czech Republic Tel. +420 541 562 559, E-mail: vrskovad@vfu.cz. 250.

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