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Toxicity effects of profenofos on embryonic and larval development of Zebrafish (Danio rerio) Rajesh Pamanji a , B. Yashwanth a , M.S. Bethu a , S. Leelavathi a , K. Ravinder b , J. Venkateswara Rao a,∗ a b
Biology Division, CSIR-Indian Institute of Chemical Technology, Hyderabad 500 007, India Zebrafish Laboratory, Centre for Cellular and Molecular Biology, Hyderabad 500 007, India
a r t i c l e
i n f o
a b s t r a c t
Article history:
The aim of the present study was to evaluate the developmental toxicity of profenofos to
Received 23 December 2014
early developing Zebrafish (Danio rerio) embryos (4 h post fertilization) in a static system
Received in revised form
at 1.0 to 2.25 mg/L. Median lethal concentrations (LC50 ) of profenofos at 24-h, 48-h, 72-h
24 February 2015
and 96-h were determined as 2.04, 1.58, 1.57 and 1.56 mg/L, respectively. The hatching of
Accepted 27 February 2015
embryos were recorded at every 12 h interval and the median hatching time (HT50 ) was also
Available online 7 March 2015
calculated for each concentration. In a separate set of experiments, 96-h LC10 (0.74 mg/L) and LC50 (1.56 mg/L) concentrations were used to assess the developmental toxicity in relation
Keywords:
to behavior, morphology, and interactions with the targeted enzyme acetylcholinesterase.
Profenofos
Live video-microscopy revealed that the profenofos exposed embryos exhibited an abnormal
Zebrafish
development, skeletal defects and altered heart morphology in a concentration-dependent
Developmental toxicity
manner, which leads to alterations in the swimming behavior of hatchlings at 144-h, which
Swimming behavior
indicate that developing zebrafish are sensitive to profenofos. © 2015 Elsevier B.V. All rights reserved.
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 dangerous infectious diseases (Gill and Garg, 2014). Unfortunately, most of the pesticides are 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 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 they run
∗
off from fields, escape storage tanks, and especially when they are sprayed aerially (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 (Pereira et al., 2013). Pesticides in natural water within the acceptable concentration range can 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 and Rita, 2014). Toxicomorphomics in test organisms (morphological alterations caused by toxicant) always associated with test concentration and length of exposure, and it should be considered as baseline data for identifying the developmental toxicity of a chemical. This morphomics requires a systemic
Corresponding author. Tel.: +91 40 2719 3191/2720 5440. E-mail addresses: [email protected], [email protected], [email protected] (J. Venkateswara Rao).
http://dx.doi.org/10.1016/j.etap.2015.02.020 1382-6689/© 2015 Elsevier B.V. All rights reserved.
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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 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., 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-propyl phosphorothioate] a well known organophosphate insecticide has been in agricultural use 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 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 (USEPA, 1998). Acetylcholinesterase (AChE) enzyme is critical to the normal development of the zebrafish nervous system (Nobonita and Suchismita, 2013; Pamanji et al., 2015), therefore AChE inhibitors like profenofos is particularly relevant for studying vertebrate development. Nonetheless, extensive studies on the toxic effects of profenofos on embryonic development of species representing the aquatic system are lacking. Therefore, a complete systematic evaluation was carried out in the present study to determine the toxic effects of profenofos on 4 h 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
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 (60 × 30 × 30 cm) of 40 L water capacity at laboratory conditions for more than one month. The 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 and dissolved oxygen 8.15 ± 0.06 mg/L. The water was aerated continuously with a Jumbo-Jet 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 nauplii of brine shrimp (Artemia salina) once a day.
2.2.
Egg production
Fertilized eggs were obtained from induced spawning of an equal number of males and females from a glass aquarium containing a breeding trap. Briefly, before the collection of eggs the well fed male and females (separated by a divider) were transferred to breeding tanks containing marbles at the bottom. On the day of the experiment, the divider was removed just before the light cycle to initiate the breeding activity (starts within 10–30 min) and fertilized eggs were collected from the bottom of the tank with glass pipette. The eggs were cleaned 2–3 washes with distilled water.
2.3.
Embryo exposure
Fertilized eggs were separated from the non-fertilized ones with a pipette using digital video microscope (HiROX Co Ltd., Japan, Model KH-2200 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 post fertilization, 4hpf) were determined in a static method for 96h 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 80 × 15-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, (n = 10 × 20 = 200) were exposed to each concentration separately. Control experiments were also 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 the toxicant after 24, 48, 72 and 96-h. The data were used to estimate the median lethal concentrations (LC50 ) for 24h, 48-h, 72-h and 96-h by means of probit analysis (Finney, 1971). Secondly, the percent hatching rate of embryos in each test concentration was monitored during the exposure tenure of 48-96 h, and in the recuperation period up to 144-h. The median hatching 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 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 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 severely (i.e., 0–45◦ , 46–90◦ , 91–135◦ & 136–180◦ ) effected. After 96-h of exposure, the survived embryos of each test concentration were transferred to toxicant free water and then,
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Table 1 – Median lethal concentrations of Profenofos against zebrafish embryos, Danio rerio. Exposure time
24 h 48 h 72 h 96 h a b
Regression equation Y = (Ybar − bxbar ) + b log10 Conc.
LC50 (mg/L)
Y = 3.77 + 3.97x Y = 4.20 + 3.99x Y = 4.22 + 3.98x Y = 4.23 + 3.97x
2.04 ± 0.06 1.58 ± 0.03 1.57 ± 0.03 1.56 ± 0.03
95% confidence Limits LCLa 1.93 1.52 1.51 1.49
UCLb 2.17 1.65 1.64 1.63
LCL-Lower Control Level. UCL-Upper Control Level.
carefully monitored their hatching success at every 12-h interval until 144-h.
2.4.
Acetylcholinesterase (AChE EC 3.1.1.7) activity
The whole body AChE activity of control and exposed embryos/larvae minimum of 50 numbers each were collected randomly and washed twice with ice cold PBS (pH-7.5). The embryos/larvae were homogenized in ice cold 0.1 M PBS (pH-7.5) containing 0.2 M NaCl, 1% (v/v) Triton-X 100 using Potter-Elvehjam homogenizer fitted with a Teflon pestle. The homogenates were centrifuged at 5,000 × g for 10 min and the supernatant was further centrifuged at 15,000 ×g for 10 min at Kubota (Model 6930) refrigerated centrifuge. The resultant supernatant was 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. The acetylcholinesterase (AChE, EC 3.1.1.7) activity was estimated by the modified method of Ellman et al., 1976. Briefly, the AChE experiments were performed in a 96 wellplate consisting 75 l of 0.1 M phosphate buffer (pH 7.5), 25 l of 2.4 mM DTNB (5,5-dithio-bis (-nitrobenzoic acid)) and 25 l of homogenate (0.3 mg) for each well. The reaction was initiated by adding 25 l of the substrate, 1.2 mM ATChI (Acetylthiocholine iodide) at 28 ± 1 ◦ C, and color development was recorded continuously for 5 min 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
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 50 × 12 mm 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 the locomotor behavior of test organisms for 5 min with an interval of 10 s each. For each concentration a minimum of 10 replicates were used to monitor the swimming behavior. Distance travelled in 5 min by the individual zebrafish larva was observed using the analysis module of Ethovision-2.3 package software (Noldus Information Technology, The Netherlands).
2.6.
Data analysis
The median lethal concentration (LC50 ) and median hatching time (HT50 ) was calculated using ‘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 logarithmic transformation of concentrations, 95% confidence limits and slope function were calculated to provide a consistent presentation of the toxicity data. Furthermore, the morphological and behavioral alterations were analyzed in the embryos exposed to LC10 and 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 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
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 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. Probit analysis revealed that the LC50 values of 48-h, 72-h and 96-h are not significantly different from each other.
3.2.
Morphological abnormalities
The results indicated that profenofos induces morphological abnormalities during the 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 control embryos were fully developed within 48 ± 6 h with well-developed
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Fig. 1 – Malformation in embryos exposed to different concentrations of profenofos for 48 h. 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 of body fluid (B). Profenofos concentration of 1.5 mg/L stimulates tumor growth on the 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 exhibited stunted growth during embryonic development of zebrafish (I).
notochord, caudal fin, head, eyes, and pigment extend to the whole body (1A). In contrast, the embryos exposed to 1.0 and 1.25 mg/L were displayed malformed yolk sac with accumulation of body fluid that 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). 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). 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 (2A). The embryos exposed to different concentrations of profenofos exhibited several morphomics during development in a concentration dependent manner. Most of the embryos exposed to 1.0 and 1.25 mg/L displayed bulged yolk sac tube with pericardial edema, and about 35–40% of hatched larvae (75-h–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 to 120-h exhibited similar 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 40% of hatched larvae from 2.0 mg/L exposure at 120-h have shown degenerative lordosis, bulged pericardial edema and partial microphthalmia (2H). Some of the larvae exhibited 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 spinal curvature, edema formation, totally deformed eyes, accumulation of red blood cells in the yolk sac (black lined box) with hook-like tail (2J) at 144-h and majority of them
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Fig. 2 – Morphological changes in zebrafish larvae emerged from the embryos that exposed to 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 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). 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 accumulation of red blood cells in the yolk sac (black lined box) (J) and stunted development (K & L).
have a lateral curvature of spine, scoliosis (2K) and few of them possessed stunted development (2L).
was almost double in comparison to the time required for controls.
3.3.
3.4.
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 were transferred to distilled water free from toxicant. The hatching percentage of 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
Heart development and Body Length
In a separate set of experiment, a total number of 500 healthy embryos (4 hpf) exposed to lethal and sub-lethal concentrations (LC50 & LC10 ) for 96 h. The development of heart and its 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 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 completion of the looping process, and largely overlap each other at 60-h to 90-h. It is clearly visible (‘S-shaped’ loop) in the lateral view of
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Table 2 – Median hatching time (HT50 ) of zebrafish embryos at different concentrations of profenofos. Test Concentrations (mg/L)
Control 1.0 1.25 1.5 1.75 2.0 2.25 a b c
Regression equation Y = (Ybar − bxbar ) + b log10 Time.
Y = −19.58 + 14.09x Y = −6.67 + 6.41x Y = −1.79 + 3.58x Y = −3.25 + 4.21x Y = −5.43 + 5.30x Y = −4.69 + 4.83x Y = −5.07 + 4.94x
a HT 50
55.61 ± 0.62 66.26 ± 1.65 78.31 ± 2.98 90.41 ± 6.58 92.51 ± 2.56 101.38 ± 3.48 109.16 ± 4.94
95% confidence limits LCLb
UCLc
54.29 62.81 72.17 70.62 87.61 95.01 100.69
56.71 69.26 83.80 112.19 97.63 108.72 120.23
HT50 stands for time required for 50% hatching of embryos. LCL-Lower Control Level. UCL-Upper Control Level.
control embryos. In contrast, elongated and string-like heart development without any overlap was observed in LC10 and LC50 exposed 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 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 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, 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 and LC10 concentrations (Fig. 4, inset-B).
3.5.
Profenofos caused different degrees of spinal curvature and exhibited a concentration-dependent relationship. The incidence of 46–90◦ angle of curvature was more prominent in LC50 exposed larvae. But in case of 0–45◦ 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 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 is 34% higher than the larvae exposed to LC10 concentration (Fig. 5, inset-B).
3.6.
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 parenthesis indicate total percent hatchability. Different colors in each bar indicate different time intervals of hatching and the values represent percent hatching.
Spinal curvature
AChE inhibition and Swimming behavior
The effect of profenofos at LC10 and LC50 concentrations on whole body AChE activity of 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 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. 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-h with 38.34% and 67.87%, respectively when compared to controls. The average swimming speed (mm/sec) of hatchlings in every 10 s for 5 min was statistically analyzed and presented in graph.
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Fig. 4 – Effects of profenofos on zebrafish heart morphology. Representative lateral view images (100X) of control, LC10 and LC50 exposed hatchlings are shown. 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.
4.
Discussion
Understanding the mechanism of profenofos will requires much further study, but through our 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 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 production of detoxifying enzymes after 48-h of embryonic development. From this study it was found that the profenofos is highly toxic to zebrafish embryos with an LC50 value of 1.56 mg/L at 96-h. It is evident from the earlier reports that the yolk sac fry of Oreochromis niloticus (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, 72h and 96-h, respectively (Phommakone, 2004). This 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). 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 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 pyrethyroid, bifenthrin (Jin et al., 2009) induced hatching rate in zebrafish embryos at lower concentrations, it may be due to either weakening the chorionic membrane or inducing the activity of chorionase enzyme. The edema formation arises in embryonic condition and is persisted after hatching also, which is 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 osmoregulatory system associated with pesticide accumulation (Cook et al., 2005) or could be due to inhibition of slc2a10/glut10 and wwox genes (Willaert et al., 2012; Tsuruwaka et al., 2015).
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Fig. 5 – Effect of profenofos on body axis of zebrafish hatchlings. The percentage of different degrees of spinal curvature (0–45◦ , 46–90◦ , 91–135◦ & 136–180◦ ) 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 curvature in control and exposed groups. [Data derived from the Inset (A) panel].
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 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 control AChE activity.
The embryos exposed to LC10 (0.74 mg/L) and LC50 (1.56 mg/L) concentrations of profenofos displayed a significant reduction in their body lengths (10–25%) than the controls. It could be due to an increased yolk sac area, which resulted concomitant decrease in the body lengths of 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 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 critical regulator of Wnt signaling (Hayes et al., 2014). 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 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. It appears that the profenofos might have been interrupted the expression of these genes. It is evident from our results that the profenofos induced microphthalmia or diminished eyes in the exposed embryos
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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 min with an interval of 10 s. Inset (A): The tracks showing the path of movement during 5 min of tracking. The mean distance travelled in cm with SE values (n = 10) are presented just below each track.
at higher concentrations that may be due to the reduced levels of retinoic acid or inhibition of Alx1 gene in the developing zebrafish embryos (Le et al., 2012; Dee et al., 2013). As the heart formation and its function is the key target for profenofos-induced developmental toxicity, we examined the embryonic heart development and function in the LC10 and LC50 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., 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. 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 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 inhibition/down regulation of Wnt9, which is critical in craniofacial morphogenesis (Curtin et al., 2011). The inhibition of AChE leading to the accumulation of ACh at synaptic junctions might have been altered the locomotor behavior of exposed fish. The present results indicates that significant inhibition of AChE was observed during the exposure with LC10 and LC50 concentrations, in a concentration and time dependent manner, which resulted in 43–89% inhibition that leads to 38–68% reduction in swimming behavior when compared to controls. 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 activity (Levin et al., 2004).
5.
Conclusions
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.
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Conflict of Interest I (we) certify that there is no conflict of interest with any financial organization regarding the material discussed in the manuscript.
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 Leelavathi is thankful to Indian Council of Medical Research (ICMR), Govt. of India, New Delhi for the grant of senior and junior research fellowships, respectively.
references
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Hill, A.J., Teraoka, H., Heideman, W., Peterson, R.E., 2005. Zebrafish as a model vertebrate for investigating chemical toxicity. Toxicol. Sci. 86, 6–19. Jabbar, A., Masood, S.Z., Parveen, Z., Ali, M., 1993. Pesticide residues in cropland soils and shallow groundwater in Punjab Pakistan. Bull. Environ. Contam. Toxicol. 51 (2), 268–273. Jin, M., Zhang, X., Wang, L., Huang, C., Zhang, Y., Zhao, M., 2009. Developmental toxicity of bifenthrin in embryo-larval stages of zebrafish. Aquat. Toxicol. 95, 347–354. Johnson, A., Carew, E., Sloman, K.A., 2007. The effects of copper on the morphological and functional development of zebrafish. Aquat. Toxicol. 84, 431–438. Larson, S.J., Capel, P.D., Majewski, M., 2010. Pesticides in surface waters: Distribution, trends, and governing factors (No. 3). CRC Press. Le, H.G., Dowling, J.E., Cameron, D.J., 2012. Early retinoic acid deprivation in developing zebrafish results in microphthalmia. Vis. Neurosci. 29 (4–5), 219–228. Levin, E.D., Swain, H.A., Donerly, S., Linney, E., 2004. Developmental chlorpyrifos effects on hatchling zebrafish swimming behavior. Neurotoxicol. Teratol. 26 (6), 719–723. Linney, E., Upchurch, L., Donerly, S., 2004. Zebrafish as a neurotoxicological model. Neurotoxicol. Teratol. 26, 709–718. Lyons, P.J., Sapio, M.R., Fricker, L.D., 2013. Zebrafish cytosolic carboxypeptidases 1 and 5 are essential for embryonic development. J. Biol. Chem. 288, 30454–30462. McCollum, C.W., Ducharme, N.A., Bondesson, M., Gustafsson, J.A., 2011. Developmental Toxicity Screening in Zebrafish. Birth Defects Res. (Part C) 93, 67–114. Michael, Z.T., Pete, M., Henry, R., Lynda, J.P., 2011. Regulation of Zebrafish Hatching by Tetraspanin cd63. PLoS One 6 (5), e19683. Nazia, K., Rita, M., 2014. A Study on the Effect of Chlorpyrifos (20% EC) on thyroid hormones in freshwater Fish, Heteropneustes fossilis (Bloch.) by using EIA Technique. Sci. Probe. 2 (2), 8–16. Nobonita, D., Suchismita, D., 2013. Chlorpyrifos Toxicity in Fish: A Review. Current World Environ. 8 (1), 77–84. OECD, 2013. OECD TG 236 test guidelines for testing of chemicals. Fish embryos acute toxicity (FET) test. Pamanji, R., Bethu, M.S., Yashwanth, B., Leelavathi, S., Venkateswara Rao, J., 2015. Developmental toxic effects of monocrotophos, an organophosphorous pesticide, on zebrafish (Danio rerio) embryos. Environ. Sci. Pollut. Res., http://dx.doi.org/10.1007/s11356-015-4120-8. Pereira, L., Fernandes, M.N., Martinez, C.B., 2013. Hematological and biochemical alterations inthe fish Prochiloduslineatuscaused by the herbicide clomazone. Environ. Toxicol. Pharmacol. 36 (1), 1–8. Phommakone, S., 2004. The toxic effects of pesticides dimethoate and profenofos on nile tilapia fry (Oreochromis niolticus) and water flea (Moina macrocopa). Asian Institute of Technology, School of Environment and Resources Development, Pathumthani, Thailand, Ph. D. thesis. Raldua, D., Pina, B., 2014. In vivo zebrafish assays for analyzing drug toxicity. Expert. Opin. Drug Metab. Toxicol. 10 (5), 685–697. Singh, B., Mandal, K., 2013. Environmental impact of pesticides belonging to newer chemistry. In: Dhawan, A.K., Singh, B., Brar-Bhullar, M., Arora, R. (Eds.), Integrated Pest Management. Scientific Publishers, Jodhpur, India, pp. 152–190. Stehr, C.M., Linbo, T.L., Incardona, J.P., Scholz, N.L., 2006. The developmental neurotoxicity of fipronil: notochord degeneration and locomotor defects in zebrafish embryos and larvae. Toxicol. Sci. 92 (1), 270–278.
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Teraoka, H., Dong, W., Okuhara, Y., Iwasa, H., Shindo, A., Hill, A.J., Kawakami, A., Hiraga, T., 2006. Impairment of lower jaw growth in developing zebrafish exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin and reduced hedgehog expression. Aquat. Toxicol. 78 (2), 103–113. Thummel, R., Ju, M., Sarras Jr., M.P., Godwin, A.R., 2007. Both Hoxc13 orthologs are functionally important for zebrafish tail fin regeneration. Dev. Genes Evol. 217 (6), 413–420. Tsuruwaka, Y., Konishi, M., Shimada, E., 2015. Loss of wwox expression in zebrafish embryos causes edema and alters Ca2+ dynamics. Peer J. 3, e727, http://dx.doi.org/10.7717/peerj.727. USEPA, 1998. Revised EFED Environmental Risk Assessment for Profenofos. United States Environmental Protection Agency, Office of Prevention, Pesticides and Toxic substances, Washington DC.
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Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.elsevier.com/locate/etap
Toxicity effects of profenofos on embryonic and larval development of Zebrafish (Danio rerio) Rajesh Pamanji a , B. Yashwanth a , M.S. Bethu a , S. Leelavathi a , K. Ravinder b , J. Venkateswara Rao a,∗ a b
Biology Division, CSIR-Indian Institute of Chemical Technology, Hyderabad 500 007, India Zebrafish Laboratory, Centre for Cellular and Molecular Biology, Hyderabad 500 007, India
a r t i c l e
i n f o
a b s t r a c t
Article history:
The aim of the present study was to evaluate the developmental toxicity of profenofos to
Received 23 December 2014
early developing Zebrafish (Danio rerio) embryos (4 h post fertilization) in a static system
Received in revised form
at 1.0 to 2.25 mg/L. Median lethal concentrations (LC50 ) of profenofos at 24-h, 48-h, 72-h
24 February 2015
and 96-h were determined as 2.04, 1.58, 1.57 and 1.56 mg/L, respectively. The hatching of
Accepted 27 February 2015
embryos were recorded at every 12 h interval and the median hatching time (HT50 ) was also
Available online 7 March 2015
calculated for each concentration. In a separate set of experiments, 96-h LC10 (0.74 mg/L) and LC50 (1.56 mg/L) concentrations were used to assess the developmental toxicity in relation
Keywords:
to behavior, morphology, and interactions with the targeted enzyme acetylcholinesterase.
Profenofos
Live video-microscopy revealed that the profenofos exposed embryos exhibited an abnormal
Zebrafish
development, skeletal defects and altered heart morphology in a concentration-dependent
Developmental toxicity
manner, which leads to alterations in the swimming behavior of hatchlings at 144-h, which
Swimming behavior
indicate that developing zebrafish are sensitive to profenofos. © 2015 Elsevier B.V. All rights reserved.
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 dangerous infectious diseases (Gill and Garg, 2014). Unfortunately, most of the pesticides are 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 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 they run
∗
off from fields, escape storage tanks, and especially when they are sprayed aerially (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 (Pereira et al., 2013). Pesticides in natural water within the acceptable concentration range can 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 and Rita, 2014). Toxicomorphomics in test organisms (morphological alterations caused by toxicant) always associated with test concentration and length of exposure, and it should be considered as baseline data for identifying the developmental toxicity of a chemical. This morphomics requires a systemic
Corresponding author. Tel.: +91 40 2719 3191/2720 5440. E-mail addresses: [email protected], [email protected], [email protected] (J. Venkateswara Rao).
http://dx.doi.org/10.1016/j.etap.2015.02.020 1382-6689/© 2015 Elsevier B.V. All rights reserved.
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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 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., 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-propyl phosphorothioate] a well known organophosphate insecticide has been in agricultural use 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 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 (USEPA, 1998). Acetylcholinesterase (AChE) enzyme is critical to the normal development of the zebrafish nervous system (Nobonita and Suchismita, 2013; Pamanji et al., 2015), therefore AChE inhibitors like profenofos is particularly relevant for studying vertebrate development. Nonetheless, extensive studies on the toxic effects of profenofos on embryonic development of species representing the aquatic system are lacking. Therefore, a complete systematic evaluation was carried out in the present study to determine the toxic effects of profenofos on 4 h 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
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 (60 × 30 × 30 cm) of 40 L water capacity at laboratory conditions for more than one month. The 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 and dissolved oxygen 8.15 ± 0.06 mg/L. The water was aerated continuously with a Jumbo-Jet 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 nauplii of brine shrimp (Artemia salina) once a day.
2.2.
Egg production
Fertilized eggs were obtained from induced spawning of an equal number of males and females from a glass aquarium containing a breeding trap. Briefly, before the collection of eggs the well fed male and females (separated by a divider) were transferred to breeding tanks containing marbles at the bottom. On the day of the experiment, the divider was removed just before the light cycle to initiate the breeding activity (starts within 10–30 min) and fertilized eggs were collected from the bottom of the tank with glass pipette. The eggs were cleaned 2–3 washes with distilled water.
2.3.
Embryo exposure
Fertilized eggs were separated from the non-fertilized ones with a pipette using digital video microscope (HiROX Co Ltd., Japan, Model KH-2200 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 post fertilization, 4hpf) were determined in a static method for 96h 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 80 × 15-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, (n = 10 × 20 = 200) were exposed to each concentration separately. Control experiments were also 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 the toxicant after 24, 48, 72 and 96-h. The data were used to estimate the median lethal concentrations (LC50 ) for 24h, 48-h, 72-h and 96-h by means of probit analysis (Finney, 1971). Secondly, the percent hatching rate of embryos in each test concentration was monitored during the exposure tenure of 48-96 h, and in the recuperation period up to 144-h. The median hatching 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 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 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 severely (i.e., 0–45◦ , 46–90◦ , 91–135◦ & 136–180◦ ) effected. After 96-h of exposure, the survived embryos of each test concentration were transferred to toxicant free water and then,
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Table 1 – Median lethal concentrations of Profenofos against zebrafish embryos, Danio rerio. Exposure time
24 h 48 h 72 h 96 h a b
Regression equation Y = (Ybar − bxbar ) + b log10 Conc.
LC50 (mg/L)
Y = 3.77 + 3.97x Y = 4.20 + 3.99x Y = 4.22 + 3.98x Y = 4.23 + 3.97x
2.04 ± 0.06 1.58 ± 0.03 1.57 ± 0.03 1.56 ± 0.03
95% confidence Limits LCLa 1.93 1.52 1.51 1.49
UCLb 2.17 1.65 1.64 1.63
LCL-Lower Control Level. UCL-Upper Control Level.
carefully monitored their hatching success at every 12-h interval until 144-h.
2.4.
Acetylcholinesterase (AChE EC 3.1.1.7) activity
The whole body AChE activity of control and exposed embryos/larvae minimum of 50 numbers each were collected randomly and washed twice with ice cold PBS (pH-7.5). The embryos/larvae were homogenized in ice cold 0.1 M PBS (pH-7.5) containing 0.2 M NaCl, 1% (v/v) Triton-X 100 using Potter-Elvehjam homogenizer fitted with a Teflon pestle. The homogenates were centrifuged at 5,000 × g for 10 min and the supernatant was further centrifuged at 15,000 ×g for 10 min at Kubota (Model 6930) refrigerated centrifuge. The resultant supernatant was 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. The acetylcholinesterase (AChE, EC 3.1.1.7) activity was estimated by the modified method of Ellman et al., 1976. Briefly, the AChE experiments were performed in a 96 wellplate consisting 75 l of 0.1 M phosphate buffer (pH 7.5), 25 l of 2.4 mM DTNB (5,5-dithio-bis (-nitrobenzoic acid)) and 25 l of homogenate (0.3 mg) for each well. The reaction was initiated by adding 25 l of the substrate, 1.2 mM ATChI (Acetylthiocholine iodide) at 28 ± 1 ◦ C, and color development was recorded continuously for 5 min 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
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 50 × 12 mm 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 the locomotor behavior of test organisms for 5 min with an interval of 10 s each. For each concentration a minimum of 10 replicates were used to monitor the swimming behavior. Distance travelled in 5 min by the individual zebrafish larva was observed using the analysis module of Ethovision-2.3 package software (Noldus Information Technology, The Netherlands).
2.6.
Data analysis
The median lethal concentration (LC50 ) and median hatching time (HT50 ) was calculated using ‘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 logarithmic transformation of concentrations, 95% confidence limits and slope function were calculated to provide a consistent presentation of the toxicity data. Furthermore, the morphological and behavioral alterations were analyzed in the embryos exposed to LC10 and 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 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
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 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. Probit analysis revealed that the LC50 values of 48-h, 72-h and 96-h are not significantly different from each other.
3.2.
Morphological abnormalities
The results indicated that profenofos induces morphological abnormalities during the 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 control embryos were fully developed within 48 ± 6 h with well-developed
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Fig. 1 – Malformation in embryos exposed to different concentrations of profenofos for 48 h. 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 of body fluid (B). Profenofos concentration of 1.5 mg/L stimulates tumor growth on the 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 exhibited stunted growth during embryonic development of zebrafish (I).
notochord, caudal fin, head, eyes, and pigment extend to the whole body (1A). In contrast, the embryos exposed to 1.0 and 1.25 mg/L were displayed malformed yolk sac with accumulation of body fluid that 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). 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). 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 (2A). The embryos exposed to different concentrations of profenofos exhibited several morphomics during development in a concentration dependent manner. Most of the embryos exposed to 1.0 and 1.25 mg/L displayed bulged yolk sac tube with pericardial edema, and about 35–40% of hatched larvae (75-h–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 to 120-h exhibited similar 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 40% of hatched larvae from 2.0 mg/L exposure at 120-h have shown degenerative lordosis, bulged pericardial edema and partial microphthalmia (2H). Some of the larvae exhibited 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 spinal curvature, edema formation, totally deformed eyes, accumulation of red blood cells in the yolk sac (black lined box) with hook-like tail (2J) at 144-h and majority of them
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Fig. 2 – Morphological changes in zebrafish larvae emerged from the embryos that exposed to 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 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). 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 accumulation of red blood cells in the yolk sac (black lined box) (J) and stunted development (K & L).
have a lateral curvature of spine, scoliosis (2K) and few of them possessed stunted development (2L).
was almost double in comparison to the time required for controls.
3.3.
3.4.
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 were transferred to distilled water free from toxicant. The hatching percentage of 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
Heart development and Body Length
In a separate set of experiment, a total number of 500 healthy embryos (4 hpf) exposed to lethal and sub-lethal concentrations (LC50 & LC10 ) for 96 h. The development of heart and its 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 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 completion of the looping process, and largely overlap each other at 60-h to 90-h. It is clearly visible (‘S-shaped’ loop) in the lateral view of
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Table 2 – Median hatching time (HT50 ) of zebrafish embryos at different concentrations of profenofos. Test Concentrations (mg/L)
Control 1.0 1.25 1.5 1.75 2.0 2.25 a b c
Regression equation Y = (Ybar − bxbar ) + b log10 Time.
Y = −19.58 + 14.09x Y = −6.67 + 6.41x Y = −1.79 + 3.58x Y = −3.25 + 4.21x Y = −5.43 + 5.30x Y = −4.69 + 4.83x Y = −5.07 + 4.94x
a HT 50
55.61 ± 0.62 66.26 ± 1.65 78.31 ± 2.98 90.41 ± 6.58 92.51 ± 2.56 101.38 ± 3.48 109.16 ± 4.94
95% confidence limits LCLb
UCLc
54.29 62.81 72.17 70.62 87.61 95.01 100.69
56.71 69.26 83.80 112.19 97.63 108.72 120.23
HT50 stands for time required for 50% hatching of embryos. LCL-Lower Control Level. UCL-Upper Control Level.
control embryos. In contrast, elongated and string-like heart development without any overlap was observed in LC10 and LC50 exposed 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 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 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, 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 and LC10 concentrations (Fig. 4, inset-B).
3.5.
Profenofos caused different degrees of spinal curvature and exhibited a concentration-dependent relationship. The incidence of 46–90◦ angle of curvature was more prominent in LC50 exposed larvae. But in case of 0–45◦ 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 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 is 34% higher than the larvae exposed to LC10 concentration (Fig. 5, inset-B).
3.6.
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 parenthesis indicate total percent hatchability. Different colors in each bar indicate different time intervals of hatching and the values represent percent hatching.
Spinal curvature
AChE inhibition and Swimming behavior
The effect of profenofos at LC10 and LC50 concentrations on whole body AChE activity of 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 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. 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-h with 38.34% and 67.87%, respectively when compared to controls. The average swimming speed (mm/sec) of hatchlings in every 10 s for 5 min was statistically analyzed and presented in graph.
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Fig. 4 – Effects of profenofos on zebrafish heart morphology. Representative lateral view images (100X) of control, LC10 and LC50 exposed hatchlings are shown. 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.
4.
Discussion
Understanding the mechanism of profenofos will requires much further study, but through our 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 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 production of detoxifying enzymes after 48-h of embryonic development. From this study it was found that the profenofos is highly toxic to zebrafish embryos with an LC50 value of 1.56 mg/L at 96-h. It is evident from the earlier reports that the yolk sac fry of Oreochromis niloticus (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, 72h and 96-h, respectively (Phommakone, 2004). This 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). 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 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 pyrethyroid, bifenthrin (Jin et al., 2009) induced hatching rate in zebrafish embryos at lower concentrations, it may be due to either weakening the chorionic membrane or inducing the activity of chorionase enzyme. The edema formation arises in embryonic condition and is persisted after hatching also, which is 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 osmoregulatory system associated with pesticide accumulation (Cook et al., 2005) or could be due to inhibition of slc2a10/glut10 and wwox genes (Willaert et al., 2012; Tsuruwaka et al., 2015).
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Fig. 5 – Effect of profenofos on body axis of zebrafish hatchlings. The percentage of different degrees of spinal curvature (0–45◦ , 46–90◦ , 91–135◦ & 136–180◦ ) 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 curvature in control and exposed groups. [Data derived from the Inset (A) panel].
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 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 control AChE activity.
The embryos exposed to LC10 (0.74 mg/L) and LC50 (1.56 mg/L) concentrations of profenofos displayed a significant reduction in their body lengths (10–25%) than the controls. It could be due to an increased yolk sac area, which resulted concomitant decrease in the body lengths of 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 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 critical regulator of Wnt signaling (Hayes et al., 2014). 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 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. It appears that the profenofos might have been interrupted the expression of these genes. It is evident from our results that the profenofos induced microphthalmia or diminished eyes in the exposed embryos
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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 min with an interval of 10 s. Inset (A): The tracks showing the path of movement during 5 min of tracking. The mean distance travelled in cm with SE values (n = 10) are presented just below each track.
at higher concentrations that may be due to the reduced levels of retinoic acid or inhibition of Alx1 gene in the developing zebrafish embryos (Le et al., 2012; Dee et al., 2013). As the heart formation and its function is the key target for profenofos-induced developmental toxicity, we examined the embryonic heart development and function in the LC10 and LC50 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., 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. 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 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 inhibition/down regulation of Wnt9, which is critical in craniofacial morphogenesis (Curtin et al., 2011). The inhibition of AChE leading to the accumulation of ACh at synaptic junctions might have been altered the locomotor behavior of exposed fish. The present results indicates that significant inhibition of AChE was observed during the exposure with LC10 and LC50 concentrations, in a concentration and time dependent manner, which resulted in 43–89% inhibition that leads to 38–68% reduction in swimming behavior when compared to controls. 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 activity (Levin et al., 2004).
5.
Conclusions
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.
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Conflict of Interest I (we) certify that there is no conflict of interest with any financial organization regarding the material discussed in the manuscript.
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 Leelavathi is thankful to Indian Council of Medical Research (ICMR), Govt. of India, New Delhi for the grant of senior and junior research fellowships, respectively.
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