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Abstract

In this study, the toxicity of thymol, the essential oil (EO) of thyme plant, cumin seeds, and caraway seeds on rainbow trout (Oncorhynchus mykiss) was investigated and compared through a static method. The acute and short-term exposure study were conducted at a pH of 7.4 and a temperature of 15°C. In Acute toxicity test, concentrations of agents that killed 50% of rainbow trout (LC50) within 96-h for EOs of thyme, thymol, cumin, and caraway were 6.6, 2.6, 35, and 14 mg L⁻¹, respectively. Changes in fish behavior were restless, aimless swimming, and imbalances that were the same for all agents in acute toxicity test. In short-term study, histopathological changes consisted of hyperemia and edema in most organs. But most of the changes were observed in gill and brain of fish that included cellular hyperplasia and fusion of lamellae in the gills and necrosis and inflammation in the brain in addition to hyperemia and edema. The results showed that EOs used in this study were likely to exert their effects through competition with oxygen insolubility in the water and, for this reason, most of the changes were seen in the gills and brain that are more sensitive to the amounts of oxygen. This study concluded that the acute toxicity of these EOs is significant and the use of these substances in the aquatic industry should be cautious.

Keywords

Essential oil toxicity LC50 rainbow trout pathological assessment

Introduction

Today, the use of chemical agents is necessary for aquaculture. Compounds such as drugs, herbal drugs, and detergent are used in aquaculture industries.¹ Aquatic wildlife exposed to contaminants in their natural environments, and toxic agents may induce toxicity in sensitive target tissues and cells.² The widespread use of antibiotics to control diseases in aquaculture industry not only suppresses the growth of aquaculture but also leads to antibiotic residues and chemical resistance in the body of human as consumers.³

Recently, availability of natural products, fewer side toxicity, as well as better biodegradability, in comparison with available antibiotics and preservatives, encourage people to use these products more. Natural herbal products with antimicrobial, antifungal, and antioxidant properties have an important role in diseases control. The plant extracts contain essential oils (EOs) with active and effective ingredients.⁴ EOs with terpenes, phenolics, and alcoholic components are obtained by various procedures like cold-pressing, hydro-distillation, or steam distillation of botanicals.⁵ In addition to the mentioned properties, plant products stimulate appetite and promote weight gain. They exhibit all their desired properties due to active compounds such as alkaloids, terpenoids, tannins, saponins, glycosides, flavonoids, phenolics, steroids, or EOs.⁶ Especially in aquaculture, the potential of phytogenic substances in fish diets is not only depleted by their usage as alternatives for antibiotic growth promoters but also may be extended to other areas of interest, such as disease control, immune response and resistance of fish, or storage quality improvement and antioxidant properties of fish fillet.⁷

Along with the health benefits, using high doses or exposure for a long time with these substances may be harmful. As we know, the use of medicinal plants takes longer time compared to antibiotics (7 days) to resolve infection as tested in laboratory trials.^8,9 Like any other agents, overuse and misuse of EOs can also lead to detrimental effects.⁹

Immunostimulatory effects of black cumin (caraway) on fish¹⁰ and antibacterial and anthelmintic impacts of that in human and animals have been reported.¹¹ Dorucu et al. showed that black cumin seed stimulates specific defense mechanisms of rainbow trout (Oncorhynchus mykiss), and it is recommended to be used for farmed fish to decrease mortalities caused by pathogens.¹⁰ Antioxidant effects are very important, because free radicals produced in physiological metabolisms in the body are frequent and act on the same pattern. They have been implicated in more than 100 diseases such as tumors and autoimmune diseases. It will take many years to identify the role of radicals and the true importance of antioxidants.¹² Some adverse effects of Carum carvi have been reported such as allergies and contact dermatitis in human especially under sunlight.¹³ But there is no report for toxicity effects of these herbal extracts on fish. However, the toxicity of these natural and innocuous compounds must be evaluated in aquaculture.¹⁴ Before some organizations registered chemicals (drugs), they must undergo laboratory testing for short-term (acute) and long-term (chronic) health effects. Laboratory animals are purposely given high enough doses to cause toxic effects. These tests help scientists judge how these chemicals might affect humans, domestic animals, and wildlife in cases of overexposure.

Thyme (Thymus vulgaris) is one of the most applicable herbs since thousands of years. The antibacterial, antiviral, anti-inflammatory, and antioxidant effects of this plant were approved.^15
–17 The EO is obtained from its leaves and flowers. Its most important component is thymol. This component is responsible for many activities of this plant.¹⁸

To date, the toxicity of these materials has not been established for rainbow trout. The objective of this study was to evaluate the acute toxicity of EOs of thyme plant (T vulgaris), cumin (Cuminum cyminum), and caraway seeds and to determine the histopathological changes in some organs of rainbow trout in short-term exposure to caraway. Among organs, the gill plates, which are directly exposed to the ambient water, may be more sensitive to waterborne inducers than other organs. Because not only pure concentrations of toxicants are in direct contact to the gills but also most toxicant may be degraded in the first-pass metabolism in the gill and then reach to the internal metabolism organs such as liver and kidney. Therefore, their purity and toxicity are reduced.² According to many studies, usage of medicinal plants for treatment of illness in animals and human is developing and we should prepare ourselves for continuous exposure with these kinds of treatments.

Materials and methods

Thymol was obtained from Sigma-Aldrich (Darmstadt, Germany). The EO was obtained from C carvi seeds, C cyminum seeds, and thyme by direct steam distillation (water and vapor method) using an analytical scale Clevenger-type apparatus (Agilent Technology, Wilmington, USA). Three hundred grams of materials were immersed in 350 mL aqueous solution in a 1000-mL round-bottomed flask and crushed using a high-speed dispersing tool. The flask was attached to the Clevenger apparatus and kept at boiling point for 12 h. No organic solvent was used in the receiver vessel. After cooling, the isolated oil was lowered into the graduated capillary, measured, and transferred to an amber vial.

The extracted compounds were analyzed on a 6890N Agilent gas chromatograph (Agilent Technology, Wilmington, USA) coupled to a 5975 C Agilent mass-selective detector¹⁹ (Agilent Technology, Wilmington, USA) with a 7683 Agilent autosampler (Agilent Technology, Wilmington, USA), and 1.0 µL of the sample was injected in the splitless mode at 250°C into a 30 m × 0.25 mm × 0.5 µm DB-5 MS capillary column and operated by MSD ChemStation Software (Agilent Technologies, California, USA). The temperature program used for the chromatographic separation was as follows: 50°C for 2 min, the temperature increases from 25°C min⁻¹ to 100°C and held for 2 min, and then the temperature increases at 5°C min⁻¹ to 290°C, where it was finally held for 5 min. The carrier gas was helium (99.999%) and was kept at a constant flux of 1.0 mL min⁻¹. The MS device was administered in the electron impact ionization mode, and the energy of the electrons was maintained at 70 eV. After injection of a sample to gas chromatography–mass spectrometry (GC/MS), several unknown peaks were observed. The impact mass spectra of these obtained peaks were searched in the computer of our library.

Sixty-five rainbow trout with an average weight of 3.5 g and 30 days old were selected and bought from a fish farm in Varamin (a city in the southeast of Tehran) and transported to Faculty of Veterinary Medicine in Tehran University (Toxicology Research Center). All fish were held in a 500-L polyvinyl chloride (PVC) tank outfitted with a refrigerator and one powerful air compressor for aeration of water. Fish were maintained for 2 weeks before the start of the experiments in aerated urban drinking water with continuous dechlorination and recirculation. This study was conducted indoors using eight 9-L polyethylene (PET) tanks. Each tank was cleaned carefully before and after each experiment, whereas the tank was covered with netting to prevent fish escapement. The light period was 8 h lightness and 16 h darkness, and the fish were fed twice a day with a commercial trout diet. At least 24 h before the test starts, because of fish adaptation, they were introduced to the eight PET tanks (containing five fish in PET tank hanging in a 500-L PVC tank), while continuously aerated. The fish in 9 L were not fed during the experiment. The experiments were performed in static water. Each test concentration (of each seed EO 0.5, 15, 30, 40, and 60 mg L⁻¹) was replicated three times and the doses selected (in acute test) based on the geometric progression.²⁰ Dissolved oxygen, pH, and temperature measurements were determined for each test vessel before fish introduction and at the end of the 96-h test period.

Acute toxicity test for EOs

Five fish were gently placed in each test vessel. Cumulative fish mortality was recorded at time intervals 24, 48, 72, and 96 h. Test temperatures were approximately 15°C and it was stable. The pH was 7.4 and dissolved oxygen was 6.9 ± 0.5 mg L⁻¹. The entire experiment was under ethical approval, and fish were subject to independent health checks during the work.

Short-term toxicity test for EOs

The fish in PET tanks were not fed during the experiment. The experiments were performed in static water. Each test concentration of EO (0 and 25 mg L⁻¹) was replicated three times. Five fish were gently placed in each test vessel. Test solutions were not renewed, and mortalities were monitored daily. Tests were considered invalid if control survival was <90%. Among EOs in this study, caraway was selected for the short-term test because the seeds of caraway, commonly known in Iran as “Zireh Siah,” have been used extensively in Iranian foods and traditional medicine to treat several disorders. In addition to that, there was ethical prevention to use more fish for other substances. Fourteen days after EOs exposure,²¹ fish were sacrificed according to animal care guidelines. Some organs such as liver, brain, gill, skin, and kidney were removed from fish bodies. The organs were quickly rinsed with cold phosphate buffered saline (PBS) and then immersed in 10% neutral-buffered formalin, embedded in paraffin, and sliced into 5-µm thickness sections. The sliced sections were stained with hematoxylin and eosin (H&E)²² and examined under light microscopy.

Data analysis

Determining the relative toxicity of chemicals to living organisms is normally executed by probit analysis. Probit analysis assumes that the relationship between number responding (not percent response) and concentration is normally distributed. We can use logit analysis if data are not normally distributed. In this study, the acute toxicities of EOs on rainbow trout (LC50) were determined by the use of logit analysis of Statistics 19 SPSS IBM (it changes the dose–response curve to a straight line that can then be assessed by regression either through least squares or through maximum likelihood).

Results and discussion

Acute toxicity tests

The concentration values causing 50% mortality at the end of the 96-h period were analyzed. For example, LC50 for caraway EO was found only in the concentrations of 35 mg L⁻¹ that is displayed in Table 1. The statistical analysis of LC50 of caraway is shown in Table 2.

Table 1.

The cumulative mortalities, acute 96 h LC50 of caraway essential oil in rainbow trout (weight of fish = 3.5 g) according to logit analysis.

Caraway essential oil dose (mg/L)	Number of exposed fish	Number of dead fish				Overall deaths within 96 h
Caraway essential oil dose (mg/L)	Number of exposed fish	Day 1	Day 2	Day 3	Day 4	Overall deaths within 96 h
0	5	0	0	0	0	0
5	5	0	0	0	0	0
15	5	0	0	0	0	0
30	5	0	0	0	0	0
40	5	3	2	0	0	5
60	5	4	1	0	0	5

96 – h LC50 = 35 mg/L.

Table 2.

Acute 96-h LC50 of studied compounds in rainbow trout, according to logit analysis.

Compound	Weight of fish (g)	Number of fish	LC50 (mg L⁻¹)
Cuminum cyminum seeds essential oil	3.5	5	35
Carum carvi seeds essential oil	16	5	14
Thyme essential oil	13	5	6.6
Thymol	22	5	2.6
Surfactant I	2.5	5	5.5
Surfactant II	2.5	5	7.5

The same method was used for the determination of LC50 of cumin and thyme EOs and thymol. The calculated LC50s are given in Table 2. As shown, the toxicity order of studied compounds is as follows: thymol > thyme EO > C carvi seeds EO > C cyminum seeds EO.

The aromatic and medicinal properties of the genus Thymus have made it one of the most popular plants all over the world. The basic components of the studied thyme oil were as follows: thymol (40%), p-cymene (20%), and γ-terpinene (10%). It is important to be known that thyme EO contains 40% thymol and thus we can conclude that the toxicity of thyme EO is related to that of thymol. This means that the toxicity of thyme EO is 2.5 times lower than that of pure thymol. According to Table 2, LC50s of these two agents confirm this truth (2.6 vs 6.6). Also, GC-MS analyses of the caraway EO led to the identification of 21 different compounds representing 93.95%. Some unknown peaks were identified as following compounds: carvone (31.88%), p-cymene (14.88%), γ terpinene (13.05%), p-cymene–a-ol (6.98%), limonene (2.89%), 3-isoterpinelene (3.7%), and 2-beta-pinene (1.32%) (Table 3).

Table 3.

Chemical composition of caraway essential oil.

Order	Compounds	Area%
1	Carvone	31.88
2	p-Cymene	14.88
3	γ-Terpinene	13.05
4	p-Cymene–a-ol	6.98
5	Isoterpinolene	3.7
6	Tricyclene	3.38
7	dl-Limonene	2.89
8	1-Phenyl-1-butanol	2.12
9	1-Phenylethanol	1.59
10	3-Cyclopentylcyclopentan-1-one	1.45
11	Benzenemethanol, 4-(1-methylethyl)-, acetate	1.35
12	Benzoic acid, p-isopropyl	1.34
13	Phenol, 2-(1,1-dimethylethyl)-4,5-dimethyl	0.99
14	β-Pinene	0.92
15	4-Nitroso-N, N-dimethylaniline	0.84
16	Cyclobutane (1-methylethylidene)	0.8
17	4-Terpineol	0.67
18	α Terpinolene	0.54
19	3,5-Lutidine	0.52
20	α-Pinene	0.49
21	α Bisabolol	0.46

EOs extracted from spices are natural compounds that are added to fish food as appetizer and growth stimulator. These agents also have antibacterial, antiviral, anti-inflammatory, antioxidant, and immune-enhancer properties.²³ A little research on the toxicity or potentially harmful effects of these oils is done, but according to the study of Lewis in 1977, spices can be toxic in high doses and prolonged exposure.⁸ In 2015, Haghiroalsadat et al. studied the EO of black caraway of Yazd province. They found that the main components of this kind of cumin are γ-terpinene and cuminic aldehyde with amounts of 21.8% and 17.2%, respectively.²⁴ Abdel- Hadi et al. evaluated LC50 of garlic EO (3 g L⁻¹) and its toxic effects on hatchability of common carp.²⁵ Sharif Rohani et al. measured the LC50 of a kind of thyme in rainbow trout that was 13.6 ppm.²⁰

Identifying the most active toxicant compounds of EOs is cumbersome because the EOs are complex mixtures of different constituents, and the composition of particular EO may vary depending on the season of harvest and the methods used to extract the oil. According to the research by Ultee et al, the toxicity of EO increases with increasing hydroxyl group.²⁶

Effects of agents on behavior in acute toxicity test

A checklist has been accompanied by guidelines and definitions of terminology and has been divided into major categories covering equilibrium, locomotor activities, fish dispersion, body movements, coloration, pathology, and unique behaviors not previously defined.²⁷

In our study, no fish died before agent exposure, and no control fish died during toxicity tests. When the fish were exposed to concentrations equal to or higher than LC50 of agents, the fish exhibited signs of restlessness, erratic swimming, dark pigmentation, flashing, convulsions, increased respiratory rates, and loss of balance. The fish also had excessive mucus. These findings correspond with the results of Sharif Rohani et al.²⁰ Therefore, we can conclude that the transportation of oxygen through the gills must be perturbed. Gills are vital organs in aquatic organisms because of transport of respiratory gases and regulation of osmotic and ionic balances. According to this theory, toxicity increases with increasing hydrophilic properties of agent in water and can affect the gills more than other organs. Thymol has more solubility than thyme and other compounds used in this study. Thus, it has the most toxicity within them. EOs of C cyminum and C carvi seeds have lower solubility and lower toxicity.

Short-term toxicity test and histopathological changes

The acute toxicity of these EOs was more important than short-term toxicity because the rate of mortality in fish was very high in the early hours of administration. To verify impaired transport of oxygen from the water to the gills, histopathological examination was performed. Selected tissues including gill, brain, liver, and kidney were histopathologically investigated. H&E-stained tissue sections revealed tissue-specific pathological changes in fish treated with EO of C carvi seeds.

Our reasons for selecting these organs were that the skin and the gills are more vulnerable due to direct contact with the environment. The brain is the most sensitive organ to hypoxia, and the liver and the kidney are the main sites of metabolism of most toxicants. The concentration of the toxicant and the period of exposure determine the severity of the damage to these organs.

In this study, fish gills showed shortening of lamellae, edema at the secondary lamellae, and swelling of the epithelial cells. There was fusion at adjacent secondary lamellae as a result of hyperplasia after a 2-week exposure to EO of C carvi seeds (Figure 1). And their skin showed inflammation in dermis, hypodermis, and subcutaneous muscles. There was a severe edematous change characterized by epithelial detachment (Figure 2). These findings can be in line with the results of previous studies on the occurrence of dermatitis in the exposure to EO of C carvi seeds.¹⁴ Almost all brains had neurodegenerative changes. Hyperemia and edema in the surround of neurons and vessels were notable. Infiltration of mononuclear inflammatory cells into the brain parenchyma was also significant. In some cases, neuronal necrosis was observed (Figure 3(a) and (b)). Thus, necrotic encephalitis and edema occurred due to this toxicity. The results of these studies clearly indicate that sublethal concentrations of EO of caraway (or other agents) have diverse effects on fish skin and gills. The liver and kidney showed only minimal changes such as hyperemia and edema. Changes of the liver have been shown in Figure 4.

Figure 1.

Histopathological images of gill of the fish in chronic exposure with Carum carvi seed essential oil; cellular proliferation (long arrow), shortening of primary lamellae (short arrow), and fusion of secondary lamellae (arrow head). H&E: ×100. H&E: hematoxylin and eosin.

Figure 2.

Histopathological images of skin of the fish in chronic exposure with Carum carvi seed essential oil: edema and tissue fracture in derma and hypoderm (long arrows), edema in subcutaneous muscles (short arrow), and infiltration of mononuclear inflammatory cells (arrow head). H&E: ×100. H&E: hematoxylin and eosin.

Figure 3.

(a, b) Histopathological images of brain of the fish in chronic exposure with Carum carvi seed essential oil; preneuronal (long arrows) and prevascular (short arrow) edema, and neuronal necrosis (arrow head). H&E: ×100. H&E: hematoxylin and eosin.

Figure 4.

Histopathological image of liver of the fish in chronic exposure with Carum carvi seed essential oil; hyperemia (long arrow) and necrosis in some hepatocytes (short arrow). H&E: ×100. H&E: hematoxylin and eosin.

As we mentioned previously, there are few studies on the adverse effects and possible toxicity of EOs, and correspondingly, lower histopathological studies have been conducted in this area. According to the authors’ knowledge, histopathological reports are only about the protective effect of natural materials and plants. Nouh et al. studied the pathological evaluations of some probiotics on the health and immune status of Nile Tilapia,²⁸ and Fuat Gulhan et al. investigated the therapeutic effects of propolis for the toxicity of cypermethrin on histopathological changes in tissues of rainbow trout.²⁹

In the present study, gills, brain, and skin were found to be more severely affected organs compared to liver and kidney. These EOs decrease the solubility of oxygen in the water, and so they affect the most oxygen-sensitive organs (gills and brain).

Conclusion

This study shows that the acute toxicity of these chemical compounds in fish is related to the solubility of them to water: The acute toxicity increases with increasing the solubility of agents. On the other hand, these materials exert their effects by changing the physical properties of water and reducing the solubility of oxygen in the water. And fish die due to hypoxia. In histopathology, most lesions were observed in the gills and brain that are most sensitive organs to hypoxia.

In this study, acute toxicity of EOs was more significant rather than short-term toxicity, because the mortality rates were very high in the early hours of EOs exposure. Fish died quickly in the acute toxicity test of thyme, cumin, and caraway. According to our results, cumin is toxic, caraway is more toxic than cumin, and thyme is the most toxic.

Time–dose relationship was studied and reported by Elias and El Ghany on tilapia fed with diets mixed with garlic and black seed.³⁰ Exact dose and duration of herbs and plant extracts still have not yet been established in aquaculture and animal production sectors, in general.⁹ We suggest that compounds have a principal effect on physicochemical properties of water and perturb oxygen transformation through the gill of fish.

Finally, it is recommended that more studies be done on acute and short-term toxicity of EOs and histopathological changes resulting from them. The assessment of biochemical and hematological factors also can be added to these studies. Specific staining and immunohistochemistry can be used for future studies.

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

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Determination of LD50 of some essential oils and histopathological changes in short-term exposure to one of them in rainbow trout ( Oncorhynchus mykiss )