Accepted Manuscript

Title: Toxicity Effects of Profenofos on Embryonic and Larval

Development of Zebrafish (Danio rerio)

Author: Rajesh Pamanji B. Yashwanth M.S. Bethu S.

Leelavathi K. Ravinder J. VenkateswaraRao

PII: S1382-6689(15)00062-9
DOI: http://dx.doi.org/doi:10.1016/j.etap.2015.02.020
Reference: ENVTOX 2215

To appear in: Environmental Toxicology and Pharmacology

Received date: 23-12-2014

Revised date: 24-2-2015
Accepted date: 27-2-2015

Please cite this article as: Pamanji, R., Yashwanth, B., Bethu, M.S., Leelavathi,
S., Ravinder, K., VenkateswaraRao, J.,Toxicity Effects of Profenofos on Embryonic
and Larval Development of Zebrafish (Danio rerio), Environmental Toxicology and
Pharmacology (2015), http://dx.doi.org/10.1016/j.etap.2015.02.020

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Highlights

Profenofos is highly toxic to zebrafish embryos with 96h LC50 value of 1.56mg/L.

It induces several morphological abnormalities in a concentration dependent manner.

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The percent hatchability was significantly lower than that of the control.

Abnormal development of heart leads to brachycardia.

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Whole-body AChE inhibition resulted altered locomotor (swimming) behaviour.

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 Toxicity Effects of Profenofos on Embryonic and
Larval Development of Zebrafish (Danio rerio)

Rajesh Pamanjia, B. Yashwantha, M.S. Bethua, S. Leelavathia, K. Ravinderb,

J. VenkateswaraRaoa*

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a
Biology Division, CSIR-Indian Institute of Chemical Technology,

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Hyderabad 500 007, India
b
Zebrafish Laboratory, Centre for Cellular and Molecular Biology,

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Hyderabad 500 007, India

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________________
*Corresponding author
J. VenkateswaraRao
e -mail: jviict@gmail.com
Office: +91 (40) 2719 3191
Res.: +91 (40) 2720 5440
Mobile: +91 994 846 9470

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Abstract

The aim of the present study was to evaluate the developmental toxicity of profenofos to early

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developing Zebrafish (Danio rerio) embryos (4 h post fertilization) in a static system at 1.0 to

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2.25 mg/L. Median lethal concentrations (LC50) of profenofos at 24-h, 48-h, 72-h and 96-h were

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determined as 2.04, 1.58, 1.57 and 1.56 mg/L, respectively. The hatching of embryos were

recorded at every 12 h interval and the median hatching time (HT50) was also calculated for each

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concentration. In a separate set of experiments, 96-h LC10 (0.74 mg/L) and LC50 (1.56 mg/L)

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concentrations were used to assess the developmental toxicity in relation to behavior,

morphology, and interactions with the targeted enzyme acetylcholinesterase. Live video-
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microscopy revealed that the profenofos exposed embryos exhibited an abnormal development,

skeletal defects and altered heart morphology in a concentration-dependent manner, which leads
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to alterations in the swimming behavior of hatchlings at 144-h,which indicate that developing

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zebrafish are sensitive to profenofos.

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Key words: Profenofos, Zebrafish, Developmental toxicity, Swimming behavior.

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1. Introduction

Pesticides have now become an integral part of our modern life and are used to protect

agricultural land, stored grain, flower gardens as well as to eradicate the pests transmitting

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dangerous infectious diseases (Gill and Garg, 2014). Unfortunately, most of the pesticides are

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non-specific and may kill the organisms that are harmless or useful to the ecosystem. In general,

it has been estimated that less than 0.1% of the pesticide applied to crops actually reaches the

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target pest; the rest enters the surrounding environment, where it can adversely affect non-target

organisms (Carriger et al. 2006). Pesticides easily contaminate the air, ground and water when

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they run off from fields, escape storage tanks, and especially when they are sprayed aerially
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(Larson et al. 2010; Singh and Mandal, 2013). The impact of pesticides within an aquatic

environment is influenced by their water solubility and uptake ability within an organism
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(Pereira et al. 2013). Pesticides in natural water within the acceptable concentration range can
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still pose harmful effects, which may not enough to kill the fish, but associated with subtle

changes in behavior and physiology that impair both survival and development of fish (Nazia
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and Rita, 2014). Toxicomorphomics in test organisms (morphological alterations caused by

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toxicant) always associated with test concentration and length of exposure, and it should be
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considered as baseline data for identifying the developmental toxicity of a chemical. This

morphomics requires a systemic analysis, which often involves the complete organism rather

than specific tissue or cell specific targets (Raldua and Pina, 2014).

The zebrafish embryo is considered a promising alternative model for in vivo mammalian

predictive developmental toxicity testing. Zebrafish have high developmental similarity to

mammals in most aspects of embryo development, including early embryonic processes, and on

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cardiovascular, somite, muscular, skeletal, and neuronal systems (McCollum et al. 2011).

Embryos of zebrafish have been employed as a model organism for toxicological studies because

of their sensitivity to environmental changes, quick development (Cook et al. 2005), sharing of

many cellular, anatomical, and physiological characteristics with other vertebrates (Haendel et al.

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2004), wealth of knowledge available on its molecular genetics and developmental biology

(Linney et al. 2004; Hill et al. 2005). Profenofos, [O-(4-Bromo-2-chlorophenyl) O-ethyl S-

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propyl phosphorothioate] a well known organophosphate insecticide has been in agricultural use

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over the last two decades for controlling Lepidopteron pests of cotton, tobacco and vegetable

crops. This extensive use of profenofos has resulted in wide-spread distribution in aquatic and

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terrestrial ecosystems (Jabbar et al. 1993; USEPA, 2006). Furthermore, there are several

instances of fish mortality, which have occurred in the U.S due to aerial spray of profenofos
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(USEPA, 1998).
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Acetylcholinesterase (AChE) enzyme is critical to the normal development of the zebrafish

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nervous system (Nobonita and Suchismita, 2013; Pamanji et al. 2015), therefore AChE inhibitors

like profenofos is particularly relevant for studying vertebrate development. Nonetheless,

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extensive studies on the toxic effects of profenofos on embryonic development of species

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representing the aquatic system are lacking. Therefore, a complete systematic evaluation was
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carried out in the present study to determine the toxic effects of profenofos on 4 hours post

fertilized (hpf) zebrafish embryos with special emphasis on morphological aberrations, delay of

hatching, whole-body AChE activity and swimming behavior pattern of hatchlings.

2. Materials and Methods

2.1 Test chemical and zebrafish maintenance

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The test compound used, profenofos was synthesized at the Indian Institute of Chemical

Technology and was of 99% purity. The zebrafish species, Danio rerio (order: Cypriniformes,

family: Cyprinidae) were obtained from a local pet store and maintained in glass aquariums

(60x30x30 cm) of 40L water capacity at laboratory conditions for more than one month. The

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average values for the culture conditions in aquariums were quantified using the approved

methods for the sampling and analysis of water (APHA, 1998), i.e., temperature 28±1 °C, pH 7.2

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and dissolved oxygen 8.15±0.06 mg/L. The water was aerated continuously with a Jumbo-Jet

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aquarium air pump (Super-8300, made in India) with natural photoperiod of 14:10 light: dark

hours were maintained and the fish were fed with dry flakes twice per day and ad libitum with

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nauplii of brine shrimp (Artemia salina) once a day.
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2.2 Egg production

Fertilized eggs were obtained from induced spawning of an equal number of males and females
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from a glass aquarium containing a breeding trap. Briefly, before the collection of eggs the well
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fed male and females (separated by a divider) were transferred to breeding tanks containing
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marbles at the bottom. On the day of the experiment, the divider was removed just before the
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light cycle to initiate the breeding activity (starts within 10-30 minutes) and fertilized eggs were

collected from the bottom of the tank with glass pipette. The eggs were cleaned 2-3 washes with
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distilled water.

2.3 Embryo exposure

Fertilized eggs were separated from the non-fertilized ones with a pipette using digital video

microscope (HiROXco. Ltd., Japan, Model KH2200 MD2) connected to a computer-assisted

video image analysis system, Ethovision-version 2.3 (Noldus Information Technology,

Netherlands). Acute developmental toxicity studies of profenofos on zebrafish embryos (4 h

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post fertilization,4hpf) were determined in a static method for 96-h following OECD TG 236 test

guidelines (OECD, 2013). The test concentrations were chosen based on the initial experiments

to determine the median lethal concentration (LC50). Test solutions of pre-determined

concentrations (1.0, 1.25, 1.5, 1.75, 2.0 and 2.25 mg/L) were maintained in 20 ml of water in

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80x15-mm diameter glass petri dishes (Borosil Glass Works Ltd., India) by adding the toxicant

dissolved in acetone (carrier solvent). Two hundred fertilized embryos divided into 10 batches,

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(n=10x20=200) were exposed to each concentration separately. Control experiments were also

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performed by the addition of carrier solvent alone. Percent mortality of the embryos exposed to

different concentrations was recorded and dead embryos were removed in each concentration of

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the toxicant after 24, 48, 72 and 96-h. The data were used to estimate the median lethal

concentrations (LC50) for 24-h, 48-h, 72-h and 96-h by means of probit analysis (Finney, 1971).
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Secondly, the percent hatching rate of embryos in each test concentration was monitored during
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the exposure tenure of 48-96h, and in the recuperation period up to 144-h. The median hatching
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time (HT50) for each test concentration was calculated individually.

In order to assess lethality and gross developmental changes, embryos (4 hpf; n=500 with five
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replicates of 100 each) were exposed to LC10 (0.74 mg/L) and LC50 (1.56 mg/L) for 96-h

concentrations of profenofos. After 96-h of exposure, the survived embryos and larvae were
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transferred to distilled water to study the further development up to 144-h (6 days). At 96-h, a

minimum of 50 hatchlings from each control and treated groups were analyzed to measure the

heart rate per minute and also assessed their body lengths using a digital video microscope.

Likewise, digital images were used to determine the angle of curvature in the effected larvae in

comparison with the linear spinal axis of controls. The spinal curvature was measured using a

protractor (n=100 hatchlings from each group), and classified into low, medium, maximum and

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severely (i.e., 0-450, 46-900, 91-1350 & 136-1800) effected. After 96-h of exposure, the survived

embryos of each test concentration were transferred to toxicant free water and then, carefully

monitored their hatching success at every 12-h interval until 144-h.

2.4 Acetylcholinesterase (AChE EC 3.1.1.7) activity

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The whole body AChE activity of control and exposed embryos/larvae minimum of 50 numbers

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each were collected randomly and washed twice with ice cold PBS (pH-7.5). The embryos/larvae

were homogenized in ice cold 0.1M PBS (pH-7.5) containing 0.2M NaCl, 1% (v/v) Triton-X 100

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using Potter-Elvehjam homogenizer fitted with a Teflon pestle. The homogenates were

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centrifuged at 5000xg for 10 minutes and the supernatant was further centrifuged at 15000xg for

10 minutes at Kubota (Model 6930) refrigerated centrifuge. The resultant supernatant was
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collected and protein was estimated by the method of Bradford (1976), which further used as the

enzyme source for the estimation of AChE activity at regular intervals of 24-h to 144-h.
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The acetylcholinesterase (AChE, EC 3.1.1.7) activity was estimated by the modified method of
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Ellman et al.1976. Briefly, the AChE experiments were performed in a 96 well-plate consisting
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75 μl of 0.1 M phosphate buffer (pH 7.5), 25 μl of 2.4 mM DTNB (5,5-dithio-bis (-nitrobenzoic

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acid)) and 25 μl of homogenate (0.3 mg) for each well. The reaction was initiated by adding

25 μl of the substrate, 1.2 mM ATChI (Acetylthiocholine iodide) at 28 ± 1 °C, and color

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development was recorded continuously for 5 minutes at 412 nm in a spectrophotometer

(Molecular Devices, USA; supported by the software, Spectro-max Plus). AChE activity was

calculated as nanomoles of acetylcholine hydrolyzed minute per mg protein using Origin 6.0

statistical software.

2.5 Measurement of swimming behavior

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In a separate set of experiments, the effect of profenofos on the locomotor behavioral response

of 6 days (144-h) old zebrafish hatchlings was monitored in comparison to controls by using

small petridish with a diameter of 50x12mm with 2.5 ml of water. The petridish was placed

under a CCD camera (Sony CCD IRIS, Model No: SSC-M370CE) for continuous monitoring of

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the locomotor behavior of test organisms for 5 minutes with an interval of 10 seconds each. For

each concentration a minimum of 10 replicates were used to monitor the swimming behavior.

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Distance travelled in 5 minutes by the individual zebrafish larva was observed using the analysis

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module of Ethovision-2.3 package software (Noldus Information Technology, The Netherlands).

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2.6 Data analysis

The median lethal concentration (LC50) and median hatching time (HT50) was calculated using
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‘probit’ analysis (Finney, 1971) as recommended by the OECD guidelines as an appropriate

statistical method for toxicity data analysis. After linearization of the response curve by
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logarithmic transformation of concentrations, 95% confidence limits and slope function were
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calculated to provide a consistent presentation of the toxicity data. Furthermore, the

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morphological and behavioral alterations were analyzed in the embryos exposed to LC10 and
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LC50 concentrations of profenofos and compared with controls. All the experiments were

repeated three times each. The magnification of the digital images was calibrated with the aid of
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stage micrometer (ERMA, Tokyo, Japan). Mean and standard errors for all experimental

parameters were calculated using BioStat 2008 statistical software. One-way analysis of

variance (ANOVA) and the Tukey’s test (HSD) were carried out to determine whether

treatments were significantly different from control group.

3. Results

3.1 Embryo toxicity

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The effect of profenofos on zebrafish embryos, Danio rerio was concentration dependent and the

percentage survival decreased with increasing concentration of pesticide. Mortality was

identified by lack of embryonic development and coagulation of nuclear material. The median

lethal concentration (LC50) was determined up to 96-h at an interval of 24-h and the values are

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2.04, 1.58, 1.57 and 1.56 mg/L, respectively (Table 1). Mortality occurred within 24-h to 48-h of

exposure in all the test concentrations and no further significant mortality was noticed after 48-h.

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Probit analysis revealed that the LC50 values of 48-h, 72-h and 96-h are not significantly different

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from each other.

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3.2 Morphological abnormalities

The results indicated that profenofos induces morphological abnormalities during the
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early embryonic development of zebrafish in a concentration dependent manner, which includes

yolk sac edema, tail and head malformations with microphthalmia (Fig. 1). About 98% of
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control embryos were fully developed within 48±6 hours with well-developed notochord, caudal
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fin, head, eyes, and pigment extend to the whole body (1A). In contrast, the embryos exposed to
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1.0 and 1.25 mg/L were displayed malformed yolk sac with accumulation of body fluid that
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distorts the normal shape of yolk mass (1B). The majority of embryos exposed to 1.5 mg/L,

spontaneously developed adipose tissue masses (tumors) on the surface of yolk sac (1C-E).
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After 48-h the embryos from 2.0 mg/L concentration showed underdeveloped head with different

degrees of tail truncation. These embryos exhibited either partial or complete failure of eye

development and scattered hemorrhages in the yolk sac edema (1F-H). Consecutively, treatment

with 2.25 mg/L concentration leads to enhanced inhibition of development with no head or tail

formation (1I).

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Experiments were further extended to study the persisting morphological aberrations in larvae

with yolk sac and compared with controls (Fig. 2). Control larvae of zebrafish had transparent

bodies with straight tail, darkly pigmented eyes, possessing mean length of 3.43±0.2 mm (n=50).

The anterior part of the yolk sac in controls was bulbous, and its posterior part was cylindrical

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(2A). The embryos exposed to different concentrations of profenofos exhibited several

morphomics during development in a concentration dependent manner. Most of the embryos

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exposed to 1.0 and 1.25 mg/L displayed bulged yolk sac tube with pericardial edema, and about

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35-40% of hatched larvae (75-h to 96-h) exhibited spinal, tail and caudal fin malformations (2B-

D). The larvae hatched from 1.5 and 1.75 mg/L concentrations at 108 to120-h exhibited similar

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extended characteristics with notochord-related lesions and a combination of muscle weakness

leads to kinking in the trunk/tail region (2E) and severe kyphosis (2F, G). Nonetheless, about
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40% of hatched larvae from 2.0 mg/L exposure at 120-h have shown degenerative lordosis,
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bulged pericardial edema and partial microphthalmia (2H). Some of the larvae exhibited
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hydrocephalus, pear shaped body with fusion of swim bladder, yolk sac and pericardial regions

(2I). These malformations were further intensified in 2.25 mg/L exposed embryos with severe
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spinal curvature, edema formation, totally deformed eyes, accumulation of red blood cells in the
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yolk sac (black lined box) with hook-like tail (2J) at 144-h and majority of them have a lateral
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curvature of spine, scoliosis (2K) and few of them possessed stunted development (2L).

3.3 Hatchability

Control embryos started hatching from 48-h onwards and about 96% of embryos hatched within

72-h. However, the percent hatchability of embryos treated with profenofos was significantly

lower than that of the control (Fig. 3). After 96-h of exposure, all the embryos of each test

concentration was transferred to distilled water free from toxicant. The hatching percentage of

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embryos to different concentrations of profenofos was recorded (based on number of embryos

exposed for each concentration, n= 200) until 144-h and median hatching time (HT50) for each

concentration was calculated (Table 2). It is evident from the results that the time taken for

hatching of 50% embryos at highest concentrations of 2.0 and 2.25 mg/L was almost double in

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comparison to the time required for controls.

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3.4 Heart development and Body Length

In a separate set of experiment, a total number of 500 healthy embryos (4 hpf) exposed to lethal

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and sub-lethal concentrations (LC50 & LC10) for 96 hours. The development of heart and its

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function along with body lengths were measured using randomly selected 50 individuals from

the lots and their mean values were compared with controls (Fig. 4). The morphological
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alterations were more prominent in heart development, when the exposure progresses from 48-h

to 96-h. Typically, in control organisms the both ventricle and atrium placed side by side after
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completion of the looping process, and largely overlap each other at 60-h to 90-h. It is clearly
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visible (‘S-shaped’ loop) in the lateral view of control embryos. In contrast, elongated and
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string-like heart development without any overlap was observed in LC10 and LC50 exposed
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embryos in 96-h. Moreover, the atria were thin and elongated; additionally ventricle appeared

more compact and smaller in all the LC10 treated embryos. The process of looping of the heart
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tube of embryonic stage was greatly affected by the LC50 concentration of profenofos.

The lethal and sub-lethal concentrations of profenofos showed altered heart rate significantly (p

< 0.001, p < 0.0001) when compared to controls. Embryos incubated for 96-h in distilled water

(control group) had the highest number of heartbeat with a mean of 141±14.1 per min.

However, heartbeat rates of embryos treated with LC10 and LC50 concentrations were 38.29%

and 59.57% lower as compared to embryos in the control. Similar with the percent hatchability,

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heartbeat rate was also showed to be concentration-dependent (Fig. 4, inset-A). A significant

percent reduction in body length of zebrafish hatchlings was observed in LC50 concentration (p <

0.01) when compared to control length. Similarly, between LC10 and LC50 concentrations the

significance level is p < 0.05 and there is no significance difference in length between control

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and LC10 concentrations (Fig. 4, inset-B).

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3.5 Spinal curvature

Profenofos caused different degrees of spinal curvature and exhibited a concentration-dependent

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relationship. The incidence of 46-900 angle of curvature was more prominent in LC50 exposed

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larvae. But in case of 0-450 angle of curvature, the incidence percentage is more or less equal in

both LC10 and LC50 concentrations (Fig. 5). In LC10 exposed, the percentage of unaffected
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larvae is about 46 % and in LC50 it is 11% (Fig. 5, inset-A). About 88% of the hatchlings from

LC50 exposed embryos possessed inward or outward and/or lateral curvature of the spine, which
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is 34% higher than the larvae exposed to LC10 concentration (Fig. 5, inset-B).
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3.6 AChE inhibition and Swimming behavior

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The effect of profenofos at LC10 and LC50 concentrations on whole body AChE activity of
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hatchlings was estimated during and after exposure tenures of 24-h, 48-h, 72-h, 96-h, 120-h and

144-h and is presented in Fig. 6. The inhibitory pattern for whole body AChE activity was
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similar in both the test concentrations (LC10 and LC50 for 96-h), which initially inhibited 17%

and 48% by 24-h and it gradually increased to reach the maximum reduction of 43% and 89% by

96-h (Fig. 6, inset-A). Nevertheless, a small positive recovery trend was observed with 20% and

25% by the end of 144-h exposure for LC10 and LC50 concentrations, respectively.

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A significant reduction in swimming behavior was observed in profenofos treated hatchlings

when compared to controls (Fig. 7). It is apparent from the inset-A that the mobility (distance

travelled) of hatchlings was gradually decreased significantly (p < 0.0001) by the action of LC10

and LC50 concentrations at 96-hwith 38.34% and 67.87%, respectively when compared to

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controls. The average swimming speed (mm/sec) of hatchlings in every 10 seconds for 5 minutes

was statistically analyzed and presented in graph.

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4. Discussion

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Understanding the mechanism of profenofos will requires much further study, but through our

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experiment we try to shed light on certain developmental abnormalities in the zebrafish embryos.

Results indicated that the embryos exposed to different concentrations of profenofos exhibited
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significant mortality up to 48-h and no significant mortality were observed thereafter (72-h and

96-h exposure). It may be due to developmental complexity of growth tissues formation and
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production of detoxifying enzymes after 48-h of embryonic development. From this study it
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was found that the profenofos is highly toxic to zebrafish embryos with an LC50 value of 1.56
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mg/L at 96-h. It is evident from the earlier reports that the yolk sac fry of Oreochromis niloticus
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(Nile Tilapia) required less concentrations of profenofos for causing 50% mortality, at 2.1, 0.87,

0.66 and 0.42 mg/L for 24-h, 48-h, 72-h and 96-h, respectively (Phommakone, 2004). This
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confirms the chorion of the zebrafish acted as a barrier to profenofos, however, at high

concentrations it may penetrates through the minute pores of chorion and caused deleterious

effects. Earlier studies indicated that zebrafish mutant types those arrived from knock down or

knock out technology provide considerable insight into mechanisms of developmental toxicity

via the comparison of these mutant phenotypes with those arising from exposure to contaminants

or other small molecules (Stehr et al. 2006).

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The low hatchability could be attributed to the delayed development of embryos as one of the

important sub-lethal effects of the toxicant. The hatching rate in zebrafish embryos exposed to

profenofos decreased significantly compared to control and is concentration dependent. It may

be due to the inhibition of Tetraspanin cd63 gene, which resulted in lack of secreted proteolytic

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enzymes required for chorion-softening (Michael et al. 2011). In contrast, some other OP

insecticides like monocrotophos (Pamanji et al. 2015), malathion (Cook et al. 2005) and a

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pyrethyroid, bifenthrin (Jin et al. 2009) induced hatching rate in zebrafish embryos at lower

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concentrations, it may be due to either weakening the chorionic membrane or inducing the

activity of chorionase enzyme.

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The edema formation arises in embryonic condition and is persisted after hatching also, which is
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concentration dependent. With the increase in concentration of profenofos from 1.0 to 2.25

mg/L, the size of edema also increased significantly in embryos. It may be due to failure of
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osmoregulatory system associated with pesticide accumulation (Cook et al. 2005) or could be
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due to inhibition of slc2a10/glut10 and wwox genes (Willaert et al. 2012; Tsuruwaka et al. 2015).
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The embryos exposed to LC10 (0.74 mg/L) and LC50 (1.56 mg/L) concentrations of profenofos
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displayed a significant reduction in their body lengths (10 to 25%) than the controls. It could be

due to an increased yolk sac area, which resulted concomitant decrease in the body lengths of
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hatchlings, which is common in metal toxicity tests (Johnson et al. 2007). Similarly, Cook et al.

(2005) reported in zebrafish embryos exposed to 3 mg/L concentration of malathion that also

reduced its body length by 83% than control body length.

Spinal curvature is another malformation, which is quite commonly seen in zebrafish

embryos/larvae exposed to toxicants. Depending on the bending of spine these are three types

which include lordosis, kyphosis and scoliosis. These three types of spinal curvatures may

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depend on various factors, including differential accumulation of toxicant, inhibition of AChE

activity and lack of neuromuscular coordination. The literature further indicated that the spinal

curvature may be due to decreasing amounts of collagen in the spinal column, changing amino

acid composition (Ekrem et al. 2012) or due the inhibition/down regulation of pkt7 gene, a

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critical regulator of Wnt signaling (Hayes et al. 2014).

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Hydrocephalus, a condition in which swelling of head region with the accumulation of water was

observed in the embryos exposed to LC50 and higher concentrations of profenofos. Earlier

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reports indicated that these symptoms might appear due to inhibition/down regulation of

CCP1/CCP5 (Lyons et al. 2013) or lgi1b gene (Yong et al. 2011) expressions in the zebrafish.

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It appears that the profenofos might have been interrupted the expression of these genes. It is
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evident from our results that the profenofos induced microphthalmia or diminished eyes in the

exposed embryos at higher concentrations that may be due to the reduced levels of retinoic acid
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or inhibition of Alx1 gene in the developing zebrafish embryos (Le et al. 2012; Dee et al. 2013).
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As the heart formation and its function is the key target for profenofos-induced developmental
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toxicity, we examined the embryonic heart development and function in the LC10 and LC50
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exposed embryos from 48-h onwards. Cardiac function could be affected by the malformations

of the heart, which could result in cardiac arrhythmia and blood circulation failure (Pamanji et al.
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2015). Generally, the presence of AChE inhibitors increases the acetylcholine concentration in

the synaptic cleft, leading to continuous signals from the acetylcholine receptor leads to

tachycardia, but in this study, structural malformations make it bradycardia. It is apparent from

our results that profenofos exposure leads to structural malformations, altered looping, and

decreased size of the heart (Fig. 4), which resulted in a significant reduction in their heart beat by

38% and 59%, in the LC10 and LC50 exposed hatchlings, respectively.

Page 16 of 33
Tail fin deformities were observed in most of the larvae exposed to different concentrations of

profenofos, which is common with most of the toxicants. The reason could be the

inhibition/down regulation of Hoxc13a or Hoxc13b genes (Thummel et al. 2007) which are

critical in the development of tail fin. Similarly, lower jaw impairment was observed at lower

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concentrations of profenofos exposed larvae, which could be due to the Ahr2 mediated down

regulation of Hh signaling pathway leads to failure of cell proliferation (Teraoka et al. 2006) or

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inhibition/down regulation of Wnt9, which is critical in craniofacial morphogenesis (Curtin et al.

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2011).

The inhibition of AChE leading to the accumulation of ACh at synaptic junctions might have

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been altered the locomotor behavior of exposed fish. The present results indicates that
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significant inhibition of AChE was observed during the exposure with LC10 and LC50

concentrations, in a concentration and time dependent manner, which resulted in 43 to 89%

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inhibition that leads to 38 to 68% reduction in swimming behavior when compared to controls.
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It is evident from earlier reports that the chlorpyrifos exposure to 6 and 9 days post fertilized

zebrafish hatchlings with 100 ng/ml concentration that significantly reduced their swimming
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activity (Levin et al. 2004).

5. Conclusions
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In conclusion, by analyzing the lethal and sub lethal endpoints, the zebrafish embryos were

found to be more sensitive, even at low concentration of profenofos (1.0 mg/L), which exhibited

morphological aberrations during the development of zebrafish. The data obtained from this

study will be helpful in assessing the potential risk of profenofos in the aquatic environment.

Further research is warranted to study the molecular mechanism underlying in inducing

developmental malformations in zebrafish by profenofos.

Page 17 of 33
Acknowledgments

The authors are thankful to the Director, IICT for providing the facilities and for the constant

encouragement throughout the study. Mr. Rajesh Pamanji is also thankful to Council of

Scientific and Industrial Research (CSIR), Govt. of India, New Delhi and Ms. Suddala

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Leelavathi is thankful to Indian Council of Medical Research, Govt. of India, New Delhi for the

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grant of senior and junior research fellowships, respectively.

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FIGURE-LEGENDS

FIG. 1. Malformation in embryos exposed to different concentrations of profenofos for 48h.

Arrows indicates abnormalities in the developing embryos. All the images were
photographed live through a digital video microscope. Well-developed control embryo
(A). Malformed yolk sac (embryos exposed to 1.0 and 1.25 mg/L) with accumulation

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of body fluid (B). Profenofos concentration of 1.5 mg/L stimulates tumor growth on the

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surface of yolk sac (C-E). Multiple morphological deformities developed in the
survived embryos that exposed to 2.0 mg/L (F-H). Embryos exposed to 2.5 mg/L

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exhibited stunted growth during embryonic development of zebrafish (I).

FIG. 2. Morphological changes in zebrafish larvae emerged from the embryos that exposed to

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different concentrations of profenofos and were photographed live through a digital
video microscope. Arrows and circles indicate abnormalities in the hatchlings.
Representative photograph of fully developed embryo in control (A). Larvae hatch out

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from 1.0 and 1.25 mg/L shown developed edema and accumulation of fluid cavities in
the yolk sac region (B-D). A typical kinking effect in the trunk/tail region (E) and
severe kyphosis was observed in the larvae hatch out from 1.5 and 1.75 mg/L (E-G).
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Developed lordosis and bulged pericardial edema in the larvae hatch out from 2.0 mg/L
(H) and few of them exhibited pear shaped body with fusion of swim bladder, yolk sac
and pericardial regions (2I). Larvae hatch out at 2.25 mg/L shown hook-like tail
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accumulation of red blood cells in the yolk sac (black lined box) (J) and stunted
development (K & L).
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FIG. 3. Percent hatchability of embryos during and after exposed to different concentrations of
profenofos in relation to 12 h time interval (from 48 to 144 h). Figures in the
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parenthesis indicate total percent hatchability. Different colors in each bar indicate
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different time intervals of hatching and the values represent percent hatching.

FIG. 4. Effects of profenofos on zebrafish heart morphology. Representative lateral view

images (100X) of control, LC10 and LC50 exposed hatchlings are shown.
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Abbreviations: (A) Atrium, (V) Ventricle. An average heart rate (beats per minute) of
control and exposed hatchlings, and reduction in their body length are shown in inset
(A) and (B) at 96 h, respectively. Data was reported as mean ± SE of fifty
representative larvae for each inset panel. In inset A graph *** indicates the values are
significantly different at p < 0.0001. In inset B graph **indicates the values are
significantly different at p < 0.05; and ***indicates the values are significantly
different at p < 0.01.

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FIG. 5. Effect of profenofos on body axis of zebrafish hatchlings. The percentage of different
degrees of spinal curvature (0-450, 46-900, 91-1350& 136-1800) measured in
hatchlings emerged from LC10 and LC50 concentrations at 96 h. Each value is the mean
± SE of 3 independent experiments (n=100 for each experiment). Inset (A): percent
number of hatchlings among the test groups. *** indicates the values are significantly
different at p < 0.0001. Inset (B): Percent number of affected larvae with body

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curvature in control and exposed groups. [Data derived from the Inset (A) panel].

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FIG. 6. Effect of Profenofos on in vivo AChE activity. The graph representing the whole body
AChE activity during exposure and recovery with regular intervals. Each value is the

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mean ± SE of five individual observations. Inset (A): showing the percent inhibition of
AChE activity treated with LC10 and LC50 concentrations of profenofos compared to

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control AChE activity.

FIG. 7. Locomotor behavior of zebrafish hatchlings at 144 h in control, LC10 and LC50
concentrations. The graph represents the movement of hatchlings over a period of 5

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minutes with an interval of 10 seconds. Inset (A): The tracks showing the path of
movement during 5 minutes of tracking. The mean distance travelled in cm with SE
values (n=10) are presented just below each track.
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Table-1 Median lethal concentrations of Profenofos against zebrafish embryos, Danio rerio

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95% confidence
Regression equation
Exposure LC50
Limits
Y = (Ybar - bxbar)+b

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time (mg/ L)
log10 Conc.
LCL* UCL**

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24 hrs Y=3.77+3.97x 2.04±0.06 1.93 2.17

48 hrs Y=4.20+3.99x 1.58±0.03 1.52 1.65

72 hrs

96 hrs
Y=4.22+3.98x

Y=4.23+3.97x an
1.57±0.03

1.56±0.03
1.51

1.49
1.64

1.63
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*
LCL-Lower Control Level; **UCL-Upper Control Level
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Table-2 Median hatching time (HT50) of zebrafish embryos at different concentrations of
profenofos

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Test Regression equation 95% confidence limits
a
Concentrations Y = (Ybar - bxbar)+b HT50

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(mg/L) log10Time. LCL* UCL**

Control Y=-19.58+14.09x 55.61±0.62 54.29 56.71

1.0 Y=-6.67+6.41x
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66.26±1.65 62.81 69.26
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1.25 Y=-1.79+3.58x 78.31±2.98 72.17 83.80

1.5 Y=-3.25+4.21x 90.41±6.58 70.62 112.19

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1.75 Y=-5.43+5.30x 92.51±2.56 87.61 97.63

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2.0 Y=-4.69+4.83x 101.38±3.48 95.01 108.72

2.25 Y=-5.07+4.94x 109.16±4.94 100.69 120.23

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aHT50 stands for time required for 50% hatching of embryos.

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*
LCL-Lower Control Level; **UCL-Upper Control Level
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Figure-1

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Figure-2

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Figure-3

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Figure-4

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Figure-5

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Figure-6

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Figure-7

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