Developmental Toxicity of Nonylphenol in Zebrafish (Danio Rerio) Embryos
Indian Journal of Marine Sciences Vol. 40(4), August 2011, pp. 509-515
Developmental toxicity of nonylphenol in zebrafish (Danio rerio) embryos
Tamer El-Sayed Ali1* & Juliette Legler2 1Oceanography Department, Faculty of Science, Alexandria University, Alexandria, Egypt. 2 Institute for Environmental Studies, VU University, 1081 HV Amsterdam, The Netherlands *[E.mail: [email protected]] Received 5 January 2010; revised 7 December 2010
Present study reports the response of zebrafish embryos exposed in vivo to graded concentrations of Nonylphenol (NP), ranging from high lethal levels to lower, more environmentally relevant levels. Embryos treated with NP showing developmental abnormalities and morphological alterations in a dose-dependent pattern. Results suggest that early exposure to NP can cause direct and delayed mortalities or even non-lethal malformations at doses more than 0.3 µM, while EC50 was shown to be 1 µM after 6 days of exposure. [Keywords: Nonylphenol, Zebrafish, Toxicity, Non-estrogenic action, Developmental abnormalities].
Introduction Materials and Methods Nonylphenol (NP) is a widely used industrial Chemicals organic compound that enters the environment as a Seven stock solutions (0.1, 0.3, 1, 3, 10, 30 and 100 product of microbial degradation of nonylphenol mM) of NP (a mixtures of isomers, CAS Number: polyethoxylates (NPEs). Majority of research on NP 84852-15-3, Sigma-Aldrich, Netherlands) were has concentrated on the estrogenic effects of NPs. The dissolved in dimethyl sulfoxide (DMSO, 0.01%) endocrine disrupting chemicals on several fish immediately prior to use and then directly diluted species, where plasma vitellogenine (VTG) gene 10000 times in Dutch standard water (nominal expression has been used as a biomarker for fish concentrations: 0.01, 0.03, 0.1, 0.3, 1, 3 and 10 µM). exposure to oestrogens1. Kinetics of hepatic VTG Solvent (DMSO, 0.01%) and negative controls were mRNA expression, plasma VTG accumulation and incorporated in the experiment. VTG clearance have been determined after exposure to NP in zebrafish (Danio rerio)2,3, rainbow trout Fish maintenance (Onchorynchus mykiss)4,5, Atlantic salmon (Salmo Zebrafish were raised and kept under standard salar)6, Japanese medaka (Oryzias latipes)7, Fathead laboratory conditions at about 28°C and a photoperiod minnows (Pimephales promelas)8, common carp of 14:10 h. light:dark12. Fish were fed with dry fish (Cyprinus carpio)9. However, to our knowledge, feed, Tetra-Pro Flakes (Tetra GmbH, Germany) in the reporting the non-estrogenic action of such morning and hatched brine shrimp (Artemia cysts compounds is poorly studied. NP can cause from INVE, Grantsvillle, UT, USA) in the afternoon. developmental toxicity in aquatic organisms and it Fish were acclimated in glass aquaria containing was demonstrated in killifish (Fundulus heteroclitus) copper free water. Eggs were spawned synchronously and zebrafish (Danio rerio) causing both lethal and at dawn of the next morning. One hour later, eggs sublethal developmental abnormalities after 96 h, 48 h quality has been checked under the microscope (Leica of exposure, respectively10,11, respectively. MZ 75), being sure to select the healthy, fertilized This study aimed to determine concentration- eggs for the experiment. Fish breeding and embryo dependent effects of graded series of NP on the manipulation were conducted according to development of zebrafish embryos according to gross Westerfield13. morphology. It also examine the extent to which the magnitude of the effects is dependent on the Zebrafish embryo test concentration of NP with which the embryos are Selected eggs (1 hour post fertilization, hpf) were treated. placed in 24-well cell culture sterilized plates (one 510 INDIAN J. MAR. SCI., VOL. 40, NO. 4, AUGUST 2011
embryo/well). Embryos were exposed to these were depicted at all treatment levels to complete the concentrations of NP at the 4:8 – cell stage (1:1.25 picture of the morphological abnormalities in hour post fertilization, hpf). Ten embryos/ different organs. concentration were used and incubated at 28°C. Embryos/larvae were screened daily - till 6 days - and Calculation of LC50 and EC50 scored for survival, alterations in morphology, The LC50 and EC50 were calculated at 6-days post developmental abnormalities and endpoints of fertilization from concentration-% lethality and toxicity14. Toxic/lethal end points (coagulation, concentration-% effect curves, respectively for all end missing heart beat, missing somites, missing tail points separately as well as for the sum of lethal detachment, missing spontaneous movement) and affected embryos. Logistic curves with binomially non-lethal malformations (pericardial or yolk sac distributed errors were used to describe the oedema, bent notochord, fin malformation, no relationships. From these, LC50 and EC50 values and pigmentation, incomplete head and eye development) their 95% confidence intervals were calculated using were reported separately. For the later stages, GraphPad Prism 5.01. (6 days), larvae from each replicate (n = 20 per concentration) were immobilized in 2% Results methylcellulose. The experiment was repeated twice. To gain more insight in the embryotoxic effects of Developed embryos/larvae were examined and NP, zebrafish embryos were exposed from 1 hour post photographed daily by a stereo microscope. Paintshop fertilization (hpf) for the first 6 days of development Pro. 8 image analysis software was utilized to control to follow up the developmental alterations caused by a Roper digital camera on the microscope. Images graded levels of NP (Figs.1-5).
Fig. 1―Morphological chang3es in zebrafish embryos exposed to different concentrations of NP and were photographed live in lateral orientation through a stereomicroscope at 24 h post fertilization (hpf). Embryos exposed to concentrations of 0.01, 0.03, 0.1, 0.3 µM, showing well developed embryo with yolk sac, tail, head, eyes and pigmentation similar to the control group embryos. Embryos exposed to 1 µM showing yolk sac oedema (arrow). Embryos exposed to 3 µM, showing extended mal-formed yolk sac accompanied with oedema (×4). ALI & LEGLER: DEVELOPMENT TOXICITY OF NONYLPHENOL IN ZEBRAFISH (DANIO RERIO) EMBRYOS 511
Fig. 2―Morphological changes in zebrafish embryos exposed to different concentrations of NP and were photographed live through a stereomicroscope at 48 h post fertilization (hpf). Embryos exposed to concentrations of 0.01, 0.03, 0.1, 0.3 µM, showing embryos with well developed notochord with muscles, otolith, caudal fin, head, eyes and pigmentation similar to the control group embryos, 1 µM group, showing biffer oedema and a slightly unstraight notochord. 3 µM group, (a) line oedema around yolk sac (arrow), growth retardation (small head and eyes), (b) mal formed tail (curved, short, no tail fin), blood clotting around yolk sac (arrow, visualized as tissue discoloration), simple scoliosis was shown (×4).
For the groups treated with 0.01, 0.03 and 0.1 µM, stage, while at 0.1 µM, 20% of the exposed embryos NP has no effect during all the experimental period. died (Fig. 6). The developmental effects of NP were Otherwise, NP leads to lethal and non-lethal dose dependent with an EC50 value of 0.8 µM for all malformations in embryos varied according to the endpoints (Fig. 7). concentration and duration of exposure. Respecting to 1 and 3 µM, the NP started its toxic non-lethal action 24 hpf 24 hpf with simple oedema which increased stepwise The concentrations of 0.01, 0.03, 0.1 and 0.3 µM leading to severe head, yolk sac and heart oedemas have not caused morphological alterations in embryos after 6 days of exposure at the first concentration level compared with those of the control group during the and death at the second one. Concentration 10 µM first 24 h of development, showing well developed was toxic within the first three hours of exposure, all healthy embryos with somites, yolk sac, tail, head, embryos stopped their development in the epiboly eyes, prominently sculptured brain and few pigment 512 INDIAN J. MAR. SCI., VOL. 40, NO. 4, AUGUST 2011
Fig. 3―Morphological changes in zebrafish embryos exposed to different concentrations of NP and were photographed live Fig. 4―Morphological changes in zebrafish embryos exposed to through a stereomicroscope at 72 h post fertilization (hpf). different concentrations of NP and were photographed through a Embryos exposed to concentrations of 0.01, 0.03, 0.1, 0.3 µM, stereomicroscope at 72 h post fertilization (hpf). (×2, except when showing well developed hatched larvae similar to the control mentioned). Embryos exposed to concentrations of 0.01, 0.03, 0.1 group larvae (×4). 1 µM trated group, showing delayed growth µM, showing well developed activbe swimming larvae- lateral “un hatched” with oedema all around the body “eye, peri9cardial view- with caudal fin, swim bladder, notochord similar to the and yolk sac oedema” (a, arrows, ×4), o4r hatched with a ruved control group embryos, (0.1 µM plate showing an inset-ventral notochord (b, ×2), 3 µM group, necrosis in body tissue and brain view- with an arrow referring to the swim bladder; control plate (white arrow, detected by condensed spots of pigments), oedema with an inset (a) showing notochord with swim bladder (arrow), with blood clotting in yolk (arrow) and heart (head arrow), coiled inset (b) showing the caudal fin, (×5). Embryos exposed to 0.3 tail, but the heart still beating (×4). µM showing scoliosis (notochord (a) and tail (b)). Embryos exposed to 1 µM showing a slightly curved notochord, hypoactive cells are present along the axis dorsal to the yolk heart beating and late developed fins. Embryos exposed to 3 µM showing 50% hatched larvae with so curved notochord, thinner extension and on the dorsal part of the yolk ball, caudal fin than the control group (a) and 50% unhatched embryos similar to the control ones. While, embryos exposed with head, heart and yolk sac oedema (arrows, 1, 2 and 3, to 1 and 3 µM showed yolk sac oedema and extended respectively, ×4). mal-formed yolk sac in the second case only (Fig. 1). and pigment extends the whole length of the body, 48 hpf similar to the control group embryos. The 1 µM Embryos exposed to concentrations of 0.01, 0.03, treated-group showing bigger oedema and a slightly 0.1 and 0.3 µM showing embryos with well- unstraight notochord. Embryos exposed to 3 µM developed notochord, otolith, caudal fin, head, eyes group seems more affected with the time, showing ALI & LEGLER: DEVELOPMENT TOXICITY OF NONYLPHENOL IN ZEBRAFISH (DANIO RERIO) EMBRYOS 513
line oedema and blood clotting around yolk sac accompanied with growth retardation (small head and eyes) and mal formed tail (curved, short, no tail fin). However, Blood circulates through a closed set of channels and clear heart beats were measured and ranged between 119-120 beats/min., as all other groups (Fig. 2).
72 hpf Hatched larvae with quite elongated pectoral fin buds and vigorous heart beats were observed in the control group and those treated with 0.01, 0.03, 0.1 and 0.3 µM of NP. Also, it was shown that the yolk sac started to be shrunk making the pericardial cavity more conspicuous. For the embryos treated with 1 and 3 µM, severe oedemas all around the body Fig. 6―Dose-effect curve of lethal malformation (% relative t6o control) of zebrafish embryos caused by different concentrations accompanied by growth retardation and curved of NP. notochord were shown, the embryos of these treatments still looked like those that were 48 h old and approximately no hatching was recorded in these group except 20% of those of the 1 µM treated group. Focusing on the 3 µM treated group, malformed- coiled tail, necrosis in brain and other body tissues, blood clotting in yolk sac and reduction in heart beats number (80 beats/min.) were also detected (Fig. 3).
96 hpf Further regular development was shown in 0.01, 0.03, 0.1 and 0.3 µM treated groups. Also those of 1 and 3 µM treated embryos have presented 100% and 50% hatching, respectively. However the hatched larvae of the later group were severely curved.
Fig. 7―Abnormal development (% relative to control) of zebrafish embryos caused by different concentrations of NP at 6 days post fertilization.
120 hpf For the groups treated with 0.01, 0.03 and 0.1 µM, hatched larvae have completed most of their morphogenesis, started to grow rapidly and swim about actively, showing inflated swim bladders and protruded mouths. The ventral yolk sac extending in both directions and nearly empty. For 0.3 µM treated
Fig. 5―Morphological changes in zebrafish embryos exposed to group, although the larvae were healthy and behaved different concentration of NP and were photographed live through as the control group from the developmental point of a stereomicroscope at 6 days post fertilization (×2, except when view, a slight scoliosis (curvature in notochord and mentioned). Control and 0.3 µM treated groups showing well- tail fin) was shown. Embryos exposed to 1 µM developed active larvae with an empty yolk sac. 1 µM group with severe oedemas around eyes, heart and yolk sac (arrows) and showing curved notochord, less active heart beating curved notochord. 3 µM group showing severe oedemas (arrows), and delayed development of caudal fin. Embryos development stop in 120 h stage and death (×2). exposed to 3 µM presenting only 60% survival, 50% 514 INDIAN J. MAR. SCI., VOL. 40, NO. 4, AUGUST 2011
of these living are so curved, with thinner caudal fin development, presenting EC50 for lethal endpoints of than the control group, so big yolk sac, not so far in 6.7 mg/L, after 48 hours of exposure16 demonstrated the development with small head and jaws are mal that exposure of zebrafish juveniles of 17dpf to 0.01-1 formed. The rest of living embryos are unhatched µM NP, strongly enhanced the expression of with head, heart and yolk sac oedema (Fig. 4). CYP19A2 gene in dose-dependent manner.
6 days post fertilization Conclusion For the groups treated with 0.01, 0.03 and 0.1 µM, The recorded abnormalities, lethal and non-lethal active swimming larvae have been demonstrated with malformations occurred at different concentrations an absorbed yolk sac and a more ventrally dropped levels may be due to the ability of NP to be gut tube. The larva also moves its jaws, opercular metabolized in the fish causing numerous direct and flaps, pectoral fin and eyes. Partial recovery of the tail indirect effects. It ranges from changes in gene and notochord curvature was shown in embryos expression17,18 through induction of estrogen treated with 0.3 µM and the development was parallel responsive genes19 and protein1 and effects on brain to those of the control and previously mentioned muscarinic receptor20 to increased apoptosis21, treated groups. Severe oedema was shown around expression of acute phase protein22 and changes in different body organs with a slight curved notochord phase II detoxication23. This study confirms the action was shown in the larvae of the 1 µM treated group. of NP as a toxic compound causing internal and For those treated with 3 µM, all larvae were dead with morphological malformation and mortality in no further growth than presented at 120 h pf stage zebrafish at dose rates approximately equal to the (Fig. 5). LC50 (1 µM ) at 6 days post fertilized larvae level. The range of responses of NP shows that exceeding a thresholds concentration of 0.3 µM would put the Discussion embryos in risk. Chronic and acute toxicities of NP on aquatic 15 organisms have been recently reviewed by Staple . References The degree of toxicity of NP varies according to the 1 Sumpter, J. P. & Jobling, S., Vitellogenesis as a biomarker dose and exposure period. Additionally, the nature of for estrogenic contamination of the aquatic environment, effects in the zebrafish embryo test differs according Environ. Health Perspect., 103 (1995) 173-178. to the embryonic/larval phase. According to11, acute 2 Holth, T.F., Nourizadeh-Lillabadi, R., Blaesbjerg, M., Grung, M., Holbech, H., Petersen, G.I., Aleström, P. & toxicity tests with zebrafish embryo can only be a Hylland, K., Differential gene expression and biomarkers in preliminary step for the assessment of the zebrafish (Danio rerio) following exposure to produced environmental risk of NPs. In the present study, NP water components, Aquatic Toxicology, 90 (2008) 277-291. caused abnormal development at nominal 3 Yang, F.X., Xu, Y. & Hui, Y., Reproductive effects of concentration of 1 µM at the beginning of the test, prenatal exposure to nonylphenol on zebrafish (Danio rerio), Comparative Biochemistry and Physiology, 142 (2006) reached to severe oedemas after 6 days of exposure, 77-84. whereas higher concentrations led to full development 4 Uguz, C., Iscan, M., Erguven, A. Isgor, B. & Togan, I., The arrest and mortality. The action of lethality varied bioaccumulation of nonyphenol and its adverse effect on the according to the concentration, meaning that, for the liver of rainbow trout (Onchorynchus mykiss), 92 (2003) 262-270. highest nominal concentration of 10 µM, the 5 Vetillard, A. & Bailhache, T., Effects of 4-n-Nonylphenol experiment was terminated at 24 h examination, and Tamoxifen on Salmon Gonadotropin-Releasing whereas for 3 µM the lethality was shown on the sixth Hormone, Estrogen Receptor, and Vitellogenin Gene day with a delayed-hatched larvae, explaining the Expression in Juvenile Rainbow Trout, Toxicological acute immediate toxicity of the first concentration and Sciences, 92 (2006) 537-544. 6 Yadetie, F. & Male, R., Effects of 4-nonylphenol on gene the non-lethal action (endpoints are inhibition of the expression of pituitary hormones in juvenile Atlantic salmon embryonal development and oedemas) of the second (Salmo salar), Aquatic Toxicology, 58 (2002) 113-129. one during the first 120 hpf. This study demonstrates 7 Balch, G. & Metcalfe, C., Developmental effects in Japanese that the developmental effect of NP is dose dependent medaka (Oryzias latipes) exposed to nonylphenol ethoxylates 11 and their degradation products, Chemosphere, 62 (2006) with a LC50 value of 1 µM. Very recently, 1214-1223. demonstrated that NP caused lethal as well as non- 8 Barber, L.B., Lee, K.E., Swackhamer, D.L., Heiko, L. & lethal malformation during zebrafish embryo Schoenfuss, H.L., Reproductive responses of male fathead ALI & LEGLER: DEVELOPMENT TOXICITY OF NONYLPHENOL IN ZEBRAFISH (DANIO RERIO) EMBRYOS 515
minnows exposed to wastewater treatment plant effluent, 17 Arukwe, A., Kullman, S.W., Berg, K., Goksoyr, A. & effluent treated with XAD8 resin, and an environmentally Hinton, D.E., Molecular cloning of rainbow trout relevant mixture of alkylphenol compounds, Aquatic (Oncorhynchus mykiss) eggshell zona radiata protein Toxicology, 82 (2007) 36-46. complementary DNA: mRNA expression in 17beta- 9 Barse, A.V., Chakrabarti, T., Ghosh, T.K., Pal, A.K. & estradioland nonylphenol-treated fish, Comp. Biochem. Jadhao, S.B., One-tenth dose of LC50 of 4-tert-butylphenol Physiol. B., 132 (2002) 315-326. causes endocrine disruption and metabolic changes in 18 Larkin, P., Knoebl, I. & Denslow, N.D., Differential gene Cyprinus carpio, Pesticide Biochemistry and Physiology, 86 expression analysis in fish exposed to endocrine disrupting (2006) 172-179. compounds, Comp. Biochem. Physiol. B., 136 (2003) 10 Kelly, S.A. & Di Giulio, R.T., Developmental toxicity of 149-161. estrogenic alkylphenols in killifish (Fundulus heteroclitus), 19 Arukwe, A., Kullman, S.W. & Hinton, D.E., Differential Environ. Toxicol. Chem., 19 (2000) 2564-2570. biomarker gene and protein expressions in nonylphenol and
11 Kammann, U., Vobach, M., Wosniok, W., Schäffer, A. & estradiol-17beta treated juvenile rainbow trout Telscher, A., Acute toxicity of 353-nonylphenol and its (Oncorhynchus mykiss), Comp. Biochem. Physiol. C., 129 metabolites for zebrafish embryos, Environ. Sci. Pollut. Res., (2001) 1-10. 16 (2009) 227-231. 20 Jones, S.B., King, L.B., Sappington, L.C., Dwyer, F.J., 12 Brand M, Granato M & Nüsslein-Volhard, C., Keeping and Ellersieck, M. & Buckler, D.R., Effects of carbaryl, raising zebrafish, in: Zebrafish: A Practical Approach, edited permethrin, 4-nonylphenol, and copper on muscarinic by C. Nüsslein-Volhard & R. Dahm, (Oxford- IRL Press, cholinergic receptors in brain of surrogate and listed fish UK) 2002 , pp 261. species, Comp. Biochem. Physiol. C., 120 (1998) 405-414. 13 Westerfield, M., Doerry, E., Kirkpatrick, A. E., Driever, W. & Douglas, S. A., An on-line database for zebrafish 21 Weber, L.P., Kiparissis, Y., Hwang, G.S., Niimi, A.J. & development and genetics research, Sem. Cell Dev. Biol., 8 Jantz, D.M., Increased cellular apoptosis after chronic (1997) 477-488. aqueous exposure to nonylphenol and quercetin in adult Oryzias latipes Comparative Biochemistry and 14 Nagel, R., DarT: the embryo test with the zebrafish Danio medaka ( ), Physiology, rerio—a general model in ecotoxicology and toxicology, 131 (2002) 51-59.
Altex-Altern Tierexp, 19 (2002) 38–48. 22 Baldwinm, W.S., Roling, J.A., Peterson, S. & Chapman, 15 Staples, C., Mihaich, E., Carbone, J., Woodbrun, K. & L.M., Effects of nonylphenol on hepatic testosterone Klecka, G., A weight of evidence analysis of the chronic metabolism and the expression of acute phase proteins in ecotoxicity of nonylphenol ethoxylates, nonylphenol ether winter flounder (Pleuronectes americanus): comparison to carboxylates, and nonylphenol, Human Ecol. Risk Assessm., the effects of Saint John's Wort, Comp. Biochem. Physiol. C., 10 (2004), 999-1017. 140 (2005) 87-96. 16 Kazeto Y., Place, A.R. & Trant, J.M., Effects of endocrine 23 Hughes, E.M. & Gallagher, E.P., Effects of 17-beta estradiol disrupting chemicals on the expression of CYP19 genes in and 4-nonylphenol on phase II electrophilic detoxification zebrafish (Danio rerio) juveniles, Aquatic Toxicology, 69 pathways in largemouth bass (Micropterus salmoides) liver, (2004) 25-34. Comp. Biochem. Physiol. C., 137 (2004) 237-247.