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ORIGINAL ARTICLE
Year : 2017  |  Volume : 7  |  Issue : 2  |  Page : 39-43

Induced Developmental Toxicity Studies With Mercuric Chloride and Polychlorinated Biphenyls on Danio Rerio (Zebrafish) Embryo


PG and Research Department of Zoology, ADM College for Women, Nagapattinam, Tamil Nadu, India

Date of Web Publication27-Apr-2017

Correspondence Address:
Vijayalakshmi Venkatarajulu
PG and Research Department of Zoology, ADM College for Women, Nagapattinam 611001, Tamil Nadu
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijnpnd.ijnpnd_2_17

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   Abstract 

Introduction: Fish population is generally considered very sensitive to all kinds of environmental changes to which it is exposed, as they are exclusively aquatic with an external mode of fertilization. The aim of this study is to highlight the effects of some of the most powerful environmental toxicants such as mercuric chloride (HgCl2) and polychlorinated biphenyls (PCBs), which alter the behavioral response, embryo survival rate, embryo hatching ratio, mortality rate, and heart rates of Danio rerio (zebrafish) embryo during its developmental stages. Materials and Methods: Zebrafish (D. rerio) were exposed to progressive concentrations of HgCl2 and PCBs. The water quality parameters were analyzed, and the acute toxicity test was conducted using eight fish for different concentrations; the results were compared with the control group. In addition, behavioral changes at each concentration were observed for the individual fish. Results and Discussion: The fish were exposed to various concentrations of 5, 10, 20, 30, 40, 50, and 60 μg/L of HgCl2 and PCB for a period of up to 2 weeks. The toxicity was concentration as well as time dependent. Toxicity tests revealed that the larvae of zebrafish were more sensitive to these toxicants as compared to the embryos.

Keywords: Days post fertilization, environmental toxicants, exposure, fish embryo, larvae, zebrafish


How to cite this article:
Venkatarajulu V, Sundaram K. Induced Developmental Toxicity Studies With Mercuric Chloride and Polychlorinated Biphenyls on Danio Rerio (Zebrafish) Embryo. Int J Nutr Pharmacol Neurol Dis 2017;7:39-43

How to cite this URL:
Venkatarajulu V, Sundaram K. Induced Developmental Toxicity Studies With Mercuric Chloride and Polychlorinated Biphenyls on Danio Rerio (Zebrafish) Embryo. Int J Nutr Pharmacol Neurol Dis [serial online] 2017 [cited 2017 Nov 18];7:39-43. Available from: http://www.ijnpnd.com/text.asp?2017/7/2/39/205286


   Introduction Top


Fish are relatively sensitive to the changes in their surrounding environment. These changes are reflected on the health of the fish, which may be a good indication regarding the status of a specific ecosystem.[1] Generally, fish have been used to evaluate the quality of aquatic systems by serving as bioindicators for environmental pollutants.[2] Water-soluble toxicants from industrial and municipal wastes as well as those leaked out from the soil and the atmosphere hastily enter into aquatic systems, which act as the essential collectors of all these end products of anthropogenic activities. In this manner, the water bodies become overloaded with several pollutants. While some of the pollutants putrefy or volatilize, the others form insoluble salts, which get precipitated and integrated into the sediment.[3] The uptake of such toxicants by the aquatic organisms is habitually followed by the metabolism of the toxicants into more toxic derivatives.[4]

Mercury is classified as a heavy metal, and it is considered as a poisonous metal.[5],[6] In recent years, it is a key source of environmental pollution, generally because of the anthropogenic emissions not only due to the usage of brown coal, hard coal, and other fossil fuels, but also due to uncontrolled waste combustion.[7],[8] Fish contaminated with mercury undergo severe pathological changes, including inhibition of metabolic processes and reduced fertility and survival.[9] Because of the stability of polychlorinated biphenyls (PCBs), unfortunately, they too do not break down in the environment and bioaccumulate in animals and humans. This might pose significant health risks to various organisms including humans. Once the PCBs enter the body, they are absorbed by our fat cells and stored. Because the PCBs are not water-soluble, they are not excreted from the body and accumulate over a person’s lifetime, increasing that person’s body burden of PCBs. Exposure of a human to PCBs causes severe acne, rash, eye irritation, liver damage, weakened immune system, fatigue, certain cancers, developmental disorders, and allergies.[10] In addition, embryonic exposure to PCB can lead to yolk sac edema, pericardial sac edema, and retardation of growth in the embryos of zebrafish.[11]

Zebrafish are particularly sensitive to a chemical exposure, especially during its early development. This has been widely studied and defined, as well as has become a prevalent model for toxicology, developmental biology, and recently for the discovery of new drugs.[12],[13] These fish were used to assess toxicity, lethality, and malformation during embryonic development. Zebrafish spawns every 1–6 days during the spawning season. The female releases 5–20 eggs at a time. Zebrafish have been widely used for the detection of heavy metal contamination, making this animal a convenient biological water contaminant sensor.[14],[15] There are several reports regarding the effects of heavy metals on the embryo and the larvae of fish living in polluted water bodies.[16] Animal embryos, in particular, had been recognized as a valuable and cost effective tool for monitoring water quality, because this life stage generally responds quickly even at lower toxicant concentration.[17] Zebrafish embryos have some important characteristics such as in vitro fertilization, rapid embryonic development, and optical transparency, which make it easy to detect morphological endpoints or observe the development process in its early life stages.[4] Thus, the environmental toxicants find their way into a human either by direct absorption via air or drinking water or via the food chain. Therefore, any negative effects on the fish would also be detrimental to the human system.


   Materials and Methods Top


Collection and maintenance

Zebrafish (Danio rerio) was recorded for the first time from Uttar Pradesh, where it was collected from local ponds. They were acclimatized under laboratory conditions in a 35 L glass aquaria containing dechlorinated water, aerated continuously through stone diffusers connected to a mechanical air compressor. The zebrafish is a hardy fish and can withstand a pH ranging between 7.2 and 7.5. The temperature of the water was maintained at 26 ± 1°C throughout the study. During the acclimation period, the adult fish was fed twice per day with brine shrimp and egg albumin. The care and husbandry of the zebrafish used in this study was in conformity with the guidelines[1] that regulate the care and use of laboratory animals by humans for research purposes.

The zebrafish we used as spawners had a length of 3.62 ± 0.04 cm and a weight of 1.00 ± 0.48 g. Prior to spawning, the male and female fish were housed separately for a minimum of 5 days. Care was taken in collecting the eggs. A nylon net was placed between the fish and the bottom of the aquarium; the spawned eggs fell through the net, thus, preventing the adult fish from eating the eggs. The day before breeding, male and female fish were taken in the ratio of 2:1 (male:female) to get the maximum number of embryos. Two hours after oviposition, the eggs were transferred to a  Petri dish More Details and checked for fertilization under a microscope.

Determination of median lethal concentration (96 h LC50)

The acute toxicity of mercuric chloride (HgCl2) to D. rerio was determined using a standard, 24 h, static renewal technique.[18] Desired concentrations of HgCl2 and PCB were prepared by adding 1% HgCl2 and PCB stock solution to a known volume of water. The initial experiment was conducted on a minimum of two random concentrations (0.03 and 0.10 mg L−1) to determine the mortality of the fish within the range of 5–95%. The tests were repeated thrice to check for the reproducibility of the results.

Subsequent tests were conducted using six concentrations of HgCl2 and PCBs (i.e., 5, 10, and 20–60 mg L−1). The mortality in these different concentrations ranged from 5 to 95%. For embryo toxicity, 300 fertilized eggs were separated in 500 mL beakers with 300 mL dechlorinated water. These eggs were exposed to different concentrations of HgCl2 and PCBs (5, 10, 20, 30, 40, and 50 μg/L), for up to 3 days. Because the fertilized egg takes almost 72 h to form a complete embryo, opaque, nonfertilized eggs and inactive embryos were removed immediately. Controls without toxicant were also run simultaneously. Behavioral manifestations and the condition of the fish were noted every 24 h up to 96 h.

To determine larvae toxicity, the eleuthero embryonic stage (i.e., the 5-day-old free-swimming larvae) of D. rerio was used. Thirty larvae were used to evaluate the toxic effect of HgCl2 and PCB. The mortalities of larvae were recorded after 24-, 48-, 72-, and 96-h exposure periods. The larvae that failed to respond even to strong tactile stimuli were considered dead and removed immediately. The number of dead larvae was recorded for each concentration of the toxicant, and the data were used to determine the median lethal concentration (LC50) by means of probit analysis.[19] The corresponding results were generated with a computer program. In these, the aquaria water was replaced daily with fresh treatment of pesticide so as to maintain a constant concentration of the toxicant.

Mortality and hatching rates

The embryos were observed daily under a dissection microscope for observing mortality, hatching, and delayed growth. The delayed growth included skeletal malformations, pericardial edema, decreased pigmentation of eyes, and swim bladder inflation or embryos that did not hatch by 10 days post fertilization (dpf) were considered dead. The morphology, mortality, and hatching rates were determined. To evaluate the possible toxicity of HgCl2 and PCB (12.5, 25, 50, 100, and 200 µg/mL) to zebrafish embryos, we performed an acute toxicity test through 120 hours post fertilization to characterize the general response of the developing zebrafish embryos and the larvae to HgCl2 and PCB exposure, with specific endpoints of survival and hatching rates. The percentage of hatching (% of hatch success) was defined as (the number of larvae/the initial number of embryos) × 100. The water quality parameters were maintained the same as for the acute toxicity test. The behavioral changes of the fish were recorded after every 12 h.


   Results and Discussion Top


Survival rate

The observed percentages of survival of D. rerio for HgCl2 and PCBs for different concentrations are shown in [Figure 1] and [Figure 2], respectively. The acute toxicity test determined the mortality in zebrafish embryos exposed to a wide range of PCB and HgCl2 concentrations for 96 hpf and monitored for severe malformations and death. No significant effect on the survival rate was observed for the 50 and 60 µg/L treatment groups. However, the treatment of groups of zebrafish larvae (n = 6–8 in each group) with 50 and 60 μg/L of HgCl2 and PCB for 3 days resulted in 60 and 68% survival rates, respectively.
Figure 1: Survival rate (%) of Danio rerio (larvae) exposed to HgCl2

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Figure 2: Survival rate (%) of Danio rerio (larvae) exposed to PCB

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The survival rate decreased with the increasing concentration of the toxicants. None of the zebrafish survived when the exposure periods continued beyond 9 days at concentrations of 60 μg/L. With shorter incubation times, such as within 24 h, significant mortality was observed even at higher concentration treatments. The survival rate was analyzed for 9 days, and it was found that the survival rate percentage was low at the concentrations of 50 and 60 μg/L. The survival rate was presented as the percentage (mean ± SD) of embryos surviving at each time point, determined using three replicates of eight embryos each/group. Similarly, treatments exhibited significant effects on the growth of the body’s length of zebrafish after 2 full days of incubation of one day post fertilized embryo. At the same time, obvious morphological abnormalities or changes in the body length were observed for those fish that survived these mortality studies. According to[17] the developing fish embryos/larvae are generally considered to have the highest susceptibility in the fish cycle.

Mortality and hatchability of zebrafish

In this study, mortality of the groups treated with 40, 50, and 60 µg/mL concentrations increased significantly compared to that of the control group. The normal embryos had a hatching period from 48 to 72 hpf. However, treatment with HgCl2 and PCB showed that the delayed hatching rate of the groups treated with 40, 50, and 60 µg/L concentrations was significantly different when compared to the controls during the 72 h exposure period. The hatching ratio (%) is shown in [Figure 3] and [Figure 4] for both the toxicants. The embryos treated with the highest concentrations had a survival percentage that began to differ from the control (P ≤ 0.05) at 96 hpf, respectively, and reached near or complete mortality before the end of the experiment. There was no significant difference in the mortality at the low concentrations (5 and 10 µg/L). Our data showed that exposure to the both HgCl2 and PCB caused a dose-dependent embryonic toxicity. Overall, these results suggested that the treatment with HgCl2 and PCB exhibited certain toxic effects on both embryonic and larval stages (e.g., hatchability and mortality) of the zebrafish. The reduced fitness and growth of fish occurs at sublethal levels depending on the exposure time, toxicity, and concentration of the chemical substances involved. In this study, the 96 h LC50 for both the toxicants was found to be 40 µg/L [Table 1]; [Figure 5],[Figure 6],[Figure 7]. According to[20] the reproductive ability and early life stages of fish, such as eggs and larvae, are particularly sensitive to contaminants. Thus, this study clearly indicates that the number of dead larvae increased with increase in the concentration and also by increasing the duration (time) of exposure of both HgCl2 and PCBs.
Figure 3: Hatching ratio (%) of Danio rerio (embryo) exposed to HgCl2

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Figure 4: Hatching ratio (%) of Danio rerio (embryo) exposed to PCB

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Figure 5: Concentration of HgCl2 and PCB (μg/L) versus probit mortality rate (%)

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Figure 6: Concentration of HgCl2 and PCB (μg/L) versus probit mortality rate (%)

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Figure 7: Median LC50 value of HgCl2 and PCB

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Table 1: LC50 value of Danio rerio exposed to different concentrations of HgCl2 and PCB for 96 h

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Behavioral response

The test fish, D. rerio, exposed to various lethal concentrations of HgCl2 and PCB during 24 h exposure, displayed altered behavioral responses. During the exposure time, at all tested concentrations, the fish showed rapid movement, faster opercular activities, and hyperexcitability. It was observed that in median and above median lethal concentrations, hyperbehavioral activities were relatively increased initially (up to 6 h of exposure) and subsequently reduced, expressing signs of distress. These behavioral effects increased in a dose-dependent and time-dependent manner in both the treatment groups.It is clear from this study that zebrafish and its early life stages are sensitive to low levels of HgCl2 and PCBs and significantly affect its populations. It may be presumed that the exposure to sublethal doses of metals might have caused acetylcholinesterase inhibition, which could drastically affect growth, hatchability, survival, feeding, and reproductive success of fish as reported by other workers.[21] Thus, fish are not the exception, and they may be highly polluted with heavy metals leading to serious problems and ill effects. These data would be useful for further studies regarding these toxicant effects on fish or for determining the water quality guidelines for the protection of aquatic biota as well as for solving the problems regarding aquatic toxicity. Toxicity tests using early life stages of fish are of great importance in assessing the risks to growth, reproduction, and survival in polluted environments and are important tools for good environmental monitoring. Therefore, the pollution-free environment plays a key role to perish each compartment of the ecosystem.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]
 
 
    Tables

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