Studying embryotoxicity of petroleum products for carp fish family

  • Lina Lebedeva
  • Karakoz Mukhambetiyar
  • Mariya Suvorova
Original Article
  • 6 Downloads

Abstract

To study the embryotoxicity of the oil fraction Karakuduk locality (Kazakhstan, oil deposit) for zebrafish Danio rerio (family Cyprinidae), in the conditions of the experiment. The method of breeding fishes of Danio rerio, receiving, and incubating zebrafish in vitro. Experiments have been carried out on the development of Danio rerio embryos in the oil-bearing fraction (Karakuduk locality) in various in vitro concentrations. The paper presents the results of experiments on exposure zebrafish embryos (Danio rerio) oil fractions in different concentrations and shows data on mortality of embryos and embryonic development violations. The originality of studying embryotoxicity of petroleum products for Cyprinidae fish family is that the object of our experiment is the most vulnerable age group in the population-fish larvae. Due to investigations, number of eggs could exactly determine the vitality of the fish population in general and can counter the number of the population through a certain period. Following the epidemic of mass mortality of caviar and larvae from oil pollution, the toxicant acts in the role of the immunosuppressive agent. Thus, it leads to the reduction of resistance to various infections with the reduction of the variability of even slight temperature and salinity fluctuations.

Keywords

Oil Water-soluble fractions of oil Embryotoxicity Carp Danio rerio Embryos 

Introduction

According to the data received by experts, nowadays, there are no water reservoirs that are not contaminated by oil or oil products. Over the last 20 years, human beings have been shocked by several ecologic catastrophes, related to the tanker crushes, explosions at drilling rigs, and oil spills within a radius of 10 km (de Mora et al. 2004). It is obviously seen that water ecosystem, including animals and plants, struggles because of oil spills. Thus, evaluation of the effects of native oil on the processes of embryonic development of live animals is essential. In the world practice, OECD (Organization for Economic Co-operation and Development) has accepted the use of Danio rerio in embryotoxicological studies to evaluate the toxicity of fish embryos (Fish Embryo Toxicity Assays 2006; DOE/EIA-0383 2006) February 2006 Annual Energy Outlook). One of the good points of these fish is that it is convenient for breeding, has fast rate of growth, large cavity, and a full-fledged genotype of this species. In addition, the studies on fish could be adapted to study the reactions of other animals to this or other toxicant.

The goal of this work is to study the embryotoxicity of the oil fraction Karakuduk locality (Kazakhstan, oil deposit) for zebrafish Danio rerio (family Cyprinidae), in the conditions of the experiment. To achieve this goal, the following tasks were set:
  1. 1.

    To establish experiment for checking the influence of the oil fraction on the embryos of Danio rerio.

     
  2. 2.

    To investigate the mortality factors of Danio rerio under 1–5% concentration of oil.

     
  3. 3.

    Identify changes in Danio rerio embryo, which leaded under the effect of oil fraction.

     

Research area

People regularly meet with technogenic catastrophes that have aftermaths such as penetration of huge amount of toxic solutions into environment. World’s most serious oil spill related to Torrey Canyon oil spill in 1967 until now followed by other new catastrophes (DOE/EIA-0383(2006) February 2006 Annual Energy Outlook). In other words, many endemic and rare species that are not adapted to the stresses are closer to extinction. Moreover, such ecological issue might lead to death of biotypes, biocenosis, or even ecosystems (Braginsky and Shlikhter 2002). In total, there are more than 6500 of drilling platforms and more than 3000 tankers involved in transshipment of oil and oil products (Mabro 2006). Every year, more than two billion tons of crude oil are obtained, while the loss and emissions to the environment due to accidents at drilling wells and tankers is 0.23% of total output or more than five million tons (SA Cherkashin 2005). In sum, pollution area is reached 5,000,000 km3. Taking into account that oil almost immediately begins to change its density, not only surface water is contaminated, but also the pelagic, as well as the bottom and coastal zones (Mikhaylova LV 1991).

The issue of saving the ecologic state of a unique natural object, the Caspian Sea, has current interest. The reason is that Caspian Sea is the largest in the world unique inland waterway, with an area, approximately, equal 398,000 km2. Its hydrocarbon resources and biologic wealth do not have analogues in the world. The biocenosis of the Caspian Sea includes 500 species of plants and 850 species of animals. The Caspian sea is the main migratory route and the habitat of inland and coastal birds. However, the Caspian Sea, as a unique system, is strictly supported by the antropogenic activity. The main pollutant of the sea, unconditionally, is oil. The drilling mud, drilling and oil-bearing waters, oil and oil products, surfactants, and petroleum products are next potential pollutants, because they take part in such stages as drilling, production of wells, transportation, production, and storage of oil and gas. Thus, within several days after the spill, crude oil transforms to aggregate fractions, such as the surface films, which do not entrain acid and sunlight. This causes overheating of smaller reservoirs, however, the fastest forms of intermittent oil films and emulsions. The fastest transformation appears in the form of waterproof oil films and emulsions.

Despite the large number of studies conducted by scientists, there is still no clear data for oil concentration, with which the first irreversible changes occur. Additionally, have not yet been fully explored all the available oil products and their wagging on living organisms, the period of their effect on hydrobiotics, finally, the maximum permissible concentration. According to the above results of scientific research, it has been shown that, although one type of oil concentration and its derivatives in the water cannot cause any patency in one species, it could be lethal for other species.

Materials and methods

Fishes

Mature Danio rerio contained in the aquarium with the volume of 60 l, a landing rate of 2 l per fish with a natural change of days and nights, and an average temperature of 22–24 °C (Fig. 1). The aquarium for the content of mature fishes is situated near the window, as these fish are very fond of the sun. They were equipped with an internal filter, to maintain azotist balance and saturation of the water with oxygen. Weekly, 30% of the amount of water was replaced by a clean, unoccupied tap water. The same water was used for spawning and incubation.
Fig. 1

The condition of the content of the producers of Danio rerio in the aquarium

The nest of fishes ready to breeding (the ratio of males and females 3:2) was breeded in a spawning aquarium (20 l), disinfected previously, also separator mesh was placed at the bottom. The stimuli to spawning are a fresh water in the spawning, a bright morning sun, and an increase in the temperature of the water. Firstly, spawned fishes were removed from the aquarium. Secondly, the separating grid was shaken and removed. Finally, the eggs were collected and selected for the next experiment (Westerfield M 2000).

The content Karakuduk crude oil

According to official classification, all types of oil might be grouped in dependence on their chemical characteristics. For the experiment, it has been selected oil from Karakuduk oil deposit. This oil can be described as light, low-resinousness, sweet because of its low sulfur percentage (0.03–0.49%), with high paraffin concentration (approximately 4.2–22.3%) and low acidity. Many grades of oil such as LLS, Saharan Bl, Tapis, Bonny Lt, Qua Lbo and others, i.e., WTI (West Texas Intermediate) and Brent Crude, serving as a marker standard for all global companies, producing petroleum, also belong to light oil. In comparison with the oil from Karakuduk, Brent Crude contains 0.40% of sulfur and WTI 0.24% of sulfur. Thus, it is adequately suppose, that the results of current investigation can be predicted to be the same not only for the Karakuduk, but for other oil deposits all over the world, being that composition of Kazakhstani oil is almost completely equal to the main samples.

Water-soluble oil fraction

For the preparation of the water-soluble fraction, the proprietary oil from Karakuduk locality was mixed with distilled water at a ratio of 1:10, and stirred on a magnetic stirrer for a day. The mixture was then left to fully separation of the fractions; the contiguous fraction was carefully filtered and stored at 4 °C before use.

Embryos were divided into groups below:
  • group 1—control

  • group 2—addition of the oil fraction at the rate of 1% of the volume of incubation medium

  • group 3—addition of the oil fraction at the rate of 5% of the volume of the incubation medium

  • group 4—addition of the oil fraction at the rate of 10% of the volume of incubation medium

  • group 5—addition of the oil fraction at the rate of 30% of the volume of the incubation medium

  • group 6—addition of the oil fraction at the rate of 50% of the volume of the incubation medium.

Eggs were placed in a sterile 12-holed incubation plate, 10 eggs per hole; each of the four groups had a triplicate sequence. The eggs were incubated at 27 °C for 24 h; the oil fractions were dispensed for 1 h, after the medium was fully replenished for incubation. In order to prevent fungal infection, in the water for incubation, methylene blue was added. Moreover, the dead eggs get a dye, which allows them to easily recognize them in the analysis. Dead and unprocessed eggs were removed as far as the experiment was concerned. Some of the surviving embryos were fixed in 10% neutral buffered formalin before further analysis. The medium for incubation was updated daily, saturated it with oxygen by using an oxygenator and adding methylene blue for disinfection previously. To fix the embryos, we have prepared a solution of buffered formalin:

For solution A
$$ 2.76\ g\ \mathrm{Na}{\mathrm{H}}_2\mathrm{P}{\mathrm{O}}_4\times {\mathrm{H}}_2\mathrm{O} or\ 3.12\ g\ \mathrm{Na}{\mathrm{H}}_2{\mathrm{PO}}_4\times 2{\mathrm{H}}_2\mathrm{O} $$
(1)
for 100 ml of distilled water.
For solution B
$$ 2.84\ g\ {\mathrm{NaH}}_2\mathrm{P}{\mathrm{O}}_4\times {\mathrm{H}}_2\mathrm{O} or\ 5.36\ g\ \mathrm{Na}{\mathrm{H}}_2{\mathrm{PO}}_4\times {\mathrm{H}}_2\mathrm{O} or\ 7.17\ g\ \mathrm{Na}{\mathrm{H}}_2\mathrm{P}{\mathrm{O}}_4\times 12{\mathrm{H}}_2\mathrm{O} $$
(2)
for 100 ml of distilled water

For the preparation of the total drug, we sequentially put fixed embryos in 98, 90, 80, and 70% alcohol, then in distilled water, hematoxylin, water, eosin, alcohols, and xylol and, finally, filled with balsam. The duration of each staining stage was 30 s. After washing, embryos were painted with eosin and removed from water (from weak alcohol to strong), put in xylol, and then in balm. Continuity of dyeing held was 1 min for each stage.

The analysis of embryos was carried out in vivo, using the digital stereomicrophone Motic DM 143 with a built-in camera and the output of the image on the PC screen. The analysis of the eggs was continued after 24, 72, 98, and 120 h after reconstitution in the following scheme: coagulation (death) of the embryo, the development of the spinal column and the tail (length, curvature), development of the eye, development of the head, pigmentation, blood circulation, and edema. Analysis and photogravure of live and fixed embryos were carried out using the program Motic Images Plus 2.0 (Fig. 2).
Fig. 2

Analysis of images in the on-line mode with the help of the program Motic Images Plus 2.0

Statistical analysis of the data was conducted using the statistical application Microsoft Office Exel; the data expressed as the average and the error of the medium. Differences in the data were considered statistically unreliable for p ≤ 0.05.

Results

In our research, we used D. rerio to study the toxic effects of the oil fractions. The experiment was started approximately 5 h after the reconstitution. In the control group, the mortality of embryos is reached for about 10%, with the greatest death occurring at 12 and 24 h after fertilization. Within 24 h after fertilization, the feces are in the pharyngula stage, and the notochord is clearly discernible.

Exposition of embryos of striped zebrafish to the oil fractions in large concentrations (30–50%) during the hour leads to an increased mortality of the feces. The greatest mortality was observed after 12 and 24 h after fertilization. In other words, almost 90% of the embryos died before the 24-h p/o stage (Fig. 3).
Fig. 3

Embryos D. rerio, 10 h after fertilization, after the use of oil fractions in 30% of the concentration, × 2

The cause of death of the respiratory organs is very difficult to determine, but patologic changes in development are observed, in the form of a strong hypophyseal edema. Changes were recorded after 3 h next to the procedure, while the development of caudal bud (10 h/o), and at the end of the fouling. All embryos with fixed phenomena of the edema died during the next few instants. Data on the mortality of Danio rerio embryos when exposed to a healthy fraction of oil in various concentrations are presented in Fig. 4.
Fig. 4

Mortality of embryos of Danio rerio in experimental conditions during the operation of the oil fraction (Karakuduk locality)

The exposition of the inorganic oil fraction started from about 5 h after the fertilization, which can be described as a stage of 30% epiboly (Fig. 5).
Fig. 5

Embryos of the Danio rerio, 5 h after the fertilization, the beginning of the process. Fixation with 10% neutral buffered formalin, × 4

The period from 24 to 28 h after the treatment is considered as pharyngula. During 24 h after fertilization, clearly distinguished notochord, somites, the developing brain, in which, with a constant increase in magnification, it is possible to distinguish five parts, and the eyes (Fig. 6). This period is very important as long as the development of the rudiments of the visceral arcs occurs. At this stage, the zodiac contains 30 somites, 13 of them in the hive department.
Fig. 6

Embryos of Danio rerio, 24 h after the fertilization, control. Fixation with 10% neutral buffered formalin, × 4

The rate of discharge of 1 and 5% of the oil fraction in the oil phase already at these stages leads to the appearance of feces with a curved monochord (Figs. 7 and 8).
Fig. 7

Embryos of Danio rerio, 24 h after the fertilization, the exposition with 1% concentrated oil fraction. Fixation of 10% neutral buffered formalin, × 4

Fig. 8

Embryos of Danio rerio, 24 h after the fertilization, increase of concentration to 5% oil fraction. Fixation of 10% neutral buffered formalin, × 4

At this stage, pigmentation of retinal cells begins, and then the skin melanocytes becomes dorsolateral. The heart differs in the form of a tube enclosed in the pericardial fringe at the anterior end of the yolk. By 26 o’clock, the heart tube lengthens; its pulsation is weakly discernible.

By 30 o’clock after fertilization, the yolk height is longer than the diameter of the yolk. The yolk occupies about twice as much as the halo of the fetus. Additionally, rudiments of pectoral fins are revealed in the form of cupola-shaped protrusions.

In our experiment, the impact of the fractional oil fractions in the Karakuduk field, even at a concentration of 10% of the volume of the medium, leads to a delay and disruption of the development of embryos, almost to 100% at the 26 h p/o. It is necessary to continue the experiments on the development of embryos D. rerio in the appropriate fractions of oil in smaller concentrations, to identify the causes of death of embryos. It is clear that the severe pollution of the reservoirs by the oil and oil product fractions with a high degree of faith will lead to the death of pelagic fish eggs. All the key peculiarities in our experiment was collected and demonstrated in the Table 1.
Table 1

The main changes, which were observed by comparison control and experimental group of Danio rerio

Hours after fertilization

Growth peculiarities in control groups

Growth peculiarities among the groups exposed to water-soluble oil fraction

24 h

Somites appear. Tail well extended and yolk extension is prominent. In developing brain with a constant increase in magnification, it is possible to distinguish five divisions; lens and oitc placodes are clearly visible.

Heartbeat may be detected. This period is very important as it promotes the development of the rudiments of the visceral arches.

Axial malformations as notochord and end-tail curving

30 h

The yolk height is longer than the diameter of the yolk.

The yolk occupies about twice as much as the halo of the fetus. The rudiments of the pectoral fins are revealed in the form of cupola-shaped protrusions. Pigmentation in retina and skin. Spontaneous movement and weak circulation are detected.

No visible (important) changes

72 h

Protruding-mouth stage. Yellow pigmentation due to xanthophore development is spreading dorsally; melanophores fill lateral stripe. Circulation is clearly visible but blood is colorless. Active spontaneous movement of an embryo.

Pericardial edema may be detected in many groups. The majority of effects refer to axial malformations as end-tail and tail malformations and scoliosis. This changes embarrass hatching.

98 h

The morphogenesis is almost complete and larva continues to growth rapidly. Early larva begins to swim about actively, moves jaw, pectoral fins, and eyes.

Axial malformations are now very prominent and sometimes embarrass swimming. In laboratory conditions with excessive food sources, these may be considered as non-lethal ones, but in natural habitation may affect larvae survival.

Discussion

In the exposition with water-soluble oil fractions under concentration of 1% of the medium, the greatest mortality among Danio rerio burdens is also observed for 24 h after fertilization. Before the pecking, mortality of the eggs in this group is even lower than in the control. Nevertheless, the mortality rate of the larvae by 120 h after fertilization in this group is significantly higher than the control group and the group with the rate of 5% of the oil fraction. In this last group, the greatest mortality is observed for 72 h after the fertilization. This may be due to the fact that the oil fractions at low concentrations act as a nonspecific adaptagen, mobilizing for a certain time a protective mechanism.

The next 48 to 72 h are characterized as a period preceding hatching. In our experiment, the hatching was observed during the period from 70 to 75 h in different embryos (Fig. 9). This asynchronicity in principle is not a pathological process and is characteristic of all groups under investigation, both in the control group and in the experimental groups. When the larva leaves the chorion, the development does not stop, and the course of individual development does not differ in the hatched and left behind in the harpoons. The most dramatic changes occur in the area of glitches - in the early stages of the hatching begins, initially primordially far from the end of the head. In the next 12 h of embryogenesis, the mouth moves forward, toward the end of the head.
Fig. 9

Embryos of Danio rerio, 72 h after fertilization, control. Fixation with 10% by neutral buffered formalin, × 4

At 72-h stage with the concentration such as 1 and 5% of the oil fraction, it is possible to distinguish puffiness in the area of the heart tube and the curvature of the notochord in the tail section. There are diseases with a strongly curved body, but they, like the majority of embryos with developmental disorders, fixed at earlier stages, are practically high-potential candidates for death. This assumption is confirmed by the fact that as the experiment is going on, the dead bodies are eliminated and remain more appropriately adapted, with the least developmental disabilities (Figs. 10, 11, and 12).
Fig. 10

The embryos of Danio rerio, 72 h/o, the 1% production of the water-soluble oil fraction. Fixation with 10% neutral buffered formalin, × 4

Fig. 11

Embryos of Danio rerio, 72 h after fertilization, increase of concentration to 5% oil fraction. Fixation with 10% neutral buffered formalin, × 4

Fig. 12

Embryos of Danio rerio, 72 h after fertilization, increase of concentration to 5% oil fraction. Fixation with 10% neutral buffered formalin, × 4

The next control point of the experiment was 98 h after the fertilization (Fig. 13). By this time, the yolk sack is already empty, a larva is very small and the fish go over to active diet. They actively move, therefore, starting from this stage, we moved the larva from the cells or petri dishes into sterile glasses to which live dust was added, so that the larvae did not die of the starvation.
Fig. 13

Embryos of Danio rerio, 98 h after fertilization, control. Fixation with 10% by neutral buffered formalin, × 4

In the active, actively moving and feeding larvae, the ribs of the curvature of the piercing column are quite clear, since by this time, the overwhelming majority of the processes of morphogenesis and organogenesis have come to an end. As with the action of the oil fractions in the concentration of 1 and at 5%, the disturbances in the larvae are found quite often (Figs. 14 and 15). Sleekly, we did not have the need to trust the fish tissues separately, because the mutations were very obvious and are of the type of non-life-threatening ones. In natural conditions, these fish will not survive.
Fig. 14

The embryos of Danio rerio, 98 h after fertilization, the 1% production of a water-soluble oil fraction. Fixation of 10% neutral buffered formalin, × 4

Fig. 15

Embryos of Danio rerio, 98 h after fertilization, increase of concentration to 5% oil fraction. Fixation of 10% neutral buffered formalin, × 4

The long-term effect of oil on adult blood D. rerio causes minor changes in the anatomy of the heart and a decrease in the minute volume of the heart (Westerfield 2000).

In our experiment, the impact of the fractional oil fractions in the Karakuduk field, even at a concentration of 10% of the volume of the medium, leads to a delay and disturbance in the development of embryos, almost to 100% mortality at hatching period. It is necessary to continue the experiments on the development of embryos D. rerio in the appropriate fractions of oil in smaller concentrations, to identify the causes of death of embryos (Figs. 16, 17, and 18). It is no doubt that the severe pollution of the reservoirs by the oil and oil product fractions with a high degree of faith will lead to the death of the pelagic fish eggs.
Fig. 16

Embryos of Danio rerio, 120 h after reconstitution, control. Fixation of 10% neutral buffered formalin, × 4

Fig. 17

Embryos of Danio rerio, 120 h after fertilization, the 1% exposition of the oil fraction in the oil. Fixation of 10% neutral buffered formalin, × 4

Fig. 18

Embryos of Danio rerio, 120 h after fertilization, the 5% increase in the oil fraction. Fixation of 10% neutral buffered formalin, × 4

Conclusion

The toxic properties of the oil fraction of the Karakuduk field for embryos Danio rerio of the Ciprinidae family have been investigated. Based on the results of the study, the effect of oil fractions at concentration of 10, 30, and 50% v/v leads to the death of embryos up to 48 h after fertilization, while concentration of 1 and 5% v/v leads to the developmental disorders such as axial malformations—scoliosis and tail curvatures.

Notes

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. All procedures performed in studies involving animals were in accordance with the ethical standards of the institution or practice at which the studies were conducted.

Informed consent

Informed consent was obtained from all individual participants included in the study. Additional informed consent was obtained from all individual participants for whom identifying information is included in this article.

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Copyright information

© Springer-Verlag London Ltd., part of Springer Nature 2018

Authors and Affiliations

  1. 1.Molecular biology and genetics departmentAl-Farabi Kazakh National UniversityAlmatyRepublic of Kazakhstan

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