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Developmental Toxicity of Nonylphenol in Zebrafish (Danio Rerio) Embryos

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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).
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