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Article

Accumulation of Astaxanthin and Canthaxanthin in Liver and Gonads of Rainbow Trout (Oncorhynchus mykiss (Walbaum, 1792)) Reared in Water Containing the Fungicide Mancozeb in Concentration Level Permitted by European Legislation

by
Milena Tzanova
1,*,
Zvezdelina Yaneva
2,
Elica Valkova
1,
Donika Ivanova
2 and
Vasil Atanasov
1
1
Department of Biochemistry, Microbiology and Physics, Faculty of Agriculture, Trakia University, 6000 Stara Zagora, Bulgaria
2
Department of Pharmacology, Animal Physiology, Biochemistry and Chemistry, Faculty of Veterinary Medicine, Trakia University, 6000 Stara Zagora, Bulgaria
*
Author to whom correspondence should be addressed.
Appl. Sci. 2022, 12(13), 6646; https://doi.org/10.3390/app12136646
Submission received: 25 May 2022 / Revised: 16 June 2022 / Accepted: 28 June 2022 / Published: 30 June 2022
(This article belongs to the Section Environmental Sciences)
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Abstract

:
In this study, we studied the levels of both of the main pigments in Salmonidae—astaxanthin (Ax) and canthaxanthin (Cx)—accumulated in the liver, female gonads, and male gonads of rainbow trout (Oncorhynchus mykiss) reared in water containing the fungicide mancozeb (MZ) in concentration levels permitted by European legislation. Experimental fish were divided into three groups: the first was a control group, the second was fed with market feed (containing Ax and Cx), and the third was fed with market feed (containing Ax and Cx) and reared in environmental water containing permissible MZ levels. The diet preparation followed the manufacturer’s recommendations. The accumulated pigment quantities were measured using an HPLC-PDA method after selective extraction: Ax ranged from 2.490 ± 0.247 mg/kg (female gonads, second group) to 0.176 ± 0.007 mg/kg (liver, control group), and Cx—from 2.406 ± 0.166 mg/kg (female gonads, second group) to 0.103 ± 0.010 mg/kg (liver, control group). The pattern of the accumulation of both pigments in the three organs in the specimens of the three groups was sustainable: the amount of Ax was always greater than that of Cx, and the correlation between their concentrations was very high. The pigments were accumulated most intensively in the female gonads, followed by the male gonads and the liver. This trend was confirmed for all three experimental groups. However, the differences in the last third group were very small, and the levels of the xanthophylls accumulated were the lowest. A particular cause of the latter findings was the ongoing detoxification reactions and the disposal of MZ, in which Ax and Cx were involved as antioxidants.

1. Introduction

The pollution of water bodies with plant protection products is a huge problem worldwide. They often cannot be removed and can be absorbed by aquatic organisms. The pesticide level accumulated is often multiplied thousands of times higher than its concentration in the environment [1]. Pesticides can indirectly influence fish health through their feeding, even at low amounts [1]. Salmonids are very sensitive to xenobiotics, including pesticides, in their early life cycle [2]. Environmental pollutants are the reason for the early mortality of salmonids, well known as Syndrom M74 [3]; they reduce disease resistance and cause the long-term decrease in the humoral immune capacity [4,5].
The dithiocarbamate mancozeb (MZ) is a polymeric mixture of Zn- and Mn-ethylene dithiocarbamates. It was used in European Union (EU) countries as a fungicide [6]. The Commission Implementing Regulation (EU) 2020/2087 restricted the use of crop protection products based on MZ in the EU since 4 January 2022 [7]. However, worldwide, the amount used in the agriculture has increased significantly in recent years [8,9]. Dithiocarbamates are strong chelating agents and can directly interact with signaling proteins and enzymes. In this way, they change the cellular metabolism [10]. Bojarski and Witeska summarized the toxicity of various fungicides for different fish species: 7-day exposure in water containing 1.1 mg/L MZ caused adverse hematological changes in rainbow trout, e.g., hemoglobin and mean corpuscular hemoglobin levels were decreased [11].
Rainbow trout ((Oncorhynchus mykiss (Walbaum, 1792)) is a member of the Salmonidae family, which are distributed in the word fish market. These fish products are safe, nutritious, and of high quality for the consumer [3]. Because of their high sensitivity to the environment, these fish species are used as an indicator of water pollution [12]. In an earlier study, our research team established that the concentration limit of MZ, permitted by the European legislation, does not affect the reproduction of rainbow trout. In particular, its toxic metabolite—ethylenethiourea (ETU)—does not accumulate in fish eggs and meat, it and does not make them harmful to consumers [8].
Harmful environmental effects can be neutralized by using nutritional supplements that exhibit strong antioxidant capacities [3,8,13]. In the aquaculture industry, xanthophylls such as astaxanthin (Ax) and canthaxanthin (Cx) have been included in the diets of salmonids to provide a desirable coloration to these cultured organisms and to protect them from various diseases [14,15,16,17]. Salmonids are able to absorb them from their diet [18]. A combination of both pigments in their diet gives a higher total xanthophyll accumulation than either one alone. Moreover, the rainbow trout O. mykiss and brook trout Salvelinus fontinalis (Mitchill, 1814) are able to absorb Ax and Cx in different muscle tissues from the nutritional base of the environment, which contains them in very low concentrations [19].
The aim of the present study was to determine the accumulation of Ax and Cx in the liver and the gonads of rainbow trout (O. mykiss) reared in water containing the fungicide MZ in a concentration level permitted by the European legislation (0.5 μg/L) and to evaluate its impact on their accumulation in different organs, with high significance placed on fish reproduction and safety. A diet poor in antioxidants or increased metabolic consumption in the females can lead to reproductive disorders [20].

2. Materials and Methods

2.1. Experimental Design

The object of this research was rainbow trout (O. mykiss). This study is part of a larger experiment to explore xanthophyll deposition in different tissues of males and females in various physiological states. The new condition in this work is the environment: the fish were reared in water containing a fungicide (MZ) in a concentration level permitted by European legislation (0.5 μg/L).
The fish were bred on a Bulgarian fish farm near Tundzha River with the location of 42°36′16″ N and 25°59′49″ E. This is an intensive fish breeding farm. The water is taken from the river and includes rain and groundwater. Its quality parameters meet the legal requirements of the country [21] (Table 1).
The trial started with 36-month-old male and female fishes with body weights of 912 ± 45 g. The fish species were reared for 60 days (from 15 August to 15 October 2019) in three tanks as two experimental groups and one control group, each of them comprising 6 male and 6 female specimens.
The fish were fed with specialized, slowly sinking, extruded feed delivered from a certified European producer, the content of which, according to the manufacturer’s specifications, is: 42% raw proteins, 20% raw fats, 1.5% raw fiber, 1.3% phosphorus, 10,000 I.U. Vit A, 1500 I.U. Vit D3, and 200 mg of Vit E. This feed was selected due to the following advantages: it is suitable for rearing fish; it has high nutritious quality; it has high digestibility; it causes low water pollution and has no genetically modified organism (GMO) content according to the EU requirements [22]. Two types of fish feed were used: feed without and feed containing ensured quantities of both xanthophylls: 40 mg/kg Ax and 25 mg/kg Cx. The diet plan was recommended by the manufacturer and was consistent with the size of the fish and the water temperature: three times a day with 6 h intervals, at a feed ratio of 1.0 kg per 100 kg of fish.
The 1st group (group 1) was the control. The diet was conventional feed without pigments.
The fish in the 2nd trial group (group 2) were fed with feed containing 40 mg/kg Ax and 25 mg/kg Cx.
The fish in the 3rd trial group (group 3) were fed with feed containing ensured quantities of the pigments (40 mg/kg Ax and 25 mg/kg Cx), and MZ was added in the water during feeding in quantities which led to the final concentration of 0.5 μg/L, meeting the requirements of Bulgarian [23] and European legislation [6].
No mortality was recorded in all of the observed fish groups during the experimental period.
The biological material was provided by a fish farm, where fish products are constantly produced, and none of the fish were killed especially for this study. After sampling the biological material, it was immediately frozen and stored for a maximum of 7 days at −12 °C prior to the analyses. The xanthophyll extraction followed the procedure of the method developed and patented by Schweigert [24].
The fish number analyzed in each sampling point (experimental group and fish organs) was 6 (n = 6).

2.2. Xanthophyll Quantification

The content of Ax and Cx in the livers and male and female gonads of rainbow trout O. mykiss was measured using the HPLC-PDA method, described by Tzanova et al. [25]. Analytical HPLC measurements were performed with a C−18 column Hypersil Gold (5 µm; 150 mm × 4.6 mm) on a Thermo system combined with a Surveyor LC Pump Plus, Surveyor Plus, and Surveyor photodiode array detector PDA Plus. The mobile phase was methanol: water (97:3 v/v) which was previously filtered through a 0.45 μm membrane filter and degassed. Under isocratic conditions, the analysis was carried out at a flow rate of 1.0 mL/min at room temperature for 6 min for a single run. The chromatograms were recorded at a wavelength of 474 nm.
The reference materials, Ax (purity of min 97%, for HPLC analysis) from Haematococcus pluvailis (Flotow 1844) and Cx OEKANAL® (purity of min 97%, for HPLC analysis), were provided by Sigma-Aldrich (St. Louis, MO, USA, and Seelze, Germany, respectively). All solvents—methanol and chloroform CHROMASOLV® HPLC grade, ethanol p.a., n-hexane p.a., and i-propanol p.a. were also purchased from Sigma-Aldrich. Deionized water (σ ≤ 0.4 µS/cm) was used thoroughly.
The standard solutions used for the calibration were at five concentration levels in the range from 0.10 mg/L to 10.00 mg/L of both Ax and Cx. Each of the calibration standards was run in triplicate. The correlation coefficients (r2) of 0.9998 for Ax and of 0.9997 for Cx were obtained. They demonstrated an excellent relationship between the peak area and concentration according to the International Conference on Harmonization guidelines [26]. Figure 1 is a typical chromatogram of the standard solution. Here, the retention times of Ax (ca 2.7 min) and Cx (ca 4.2 min) can be seen.
The results in mg/kg were calculated using the following formula:
Xanthophylls   = Xanthophylls   in   mg / L ∗ V M
where V is the volume of the methanol solution in mL and M is the mass of the sample in g.

2.3. Statistical Analysis

All analytical measurements were carried out in triplicate. The results were expressed as mean values ± standard deviation (SD). Fisher’s test was used for mean comparison. A value of p ≤ 0.05 was considered statistically significant. The statistical analyses were performed using Statistica 10, StatSoft Inc., Tulsa, OK, USA.

3. Results and Discussion

Figure 2, Figure 3 and Figure 4 illustrate typical chromatograms of the sample solutions.
The Ax and Cx amounts accumulated in the liver and female and male gonads for all of the fish groups are presented in Table 2.
Many research teams explored the influence of dithiocarbamates including MZ on fish and their health. An investigation of the effect of MZ on biochemical and enzymatic parameters in Clarius batrachus (Linnaeus, 1758) in different fish life stages was carried out, and the authors concluded they were dose- and time-dependent [27,28]. MZ can cause genotoxic effects [29] and changes in the physiology of the liver and muscle tissues of catfish [30]. The authors concluded that fingerlings were more sensitive to MZ than adults. The dose of 1.1 mg/L (1/2 LC50 of MZ) caused important changes in the hematological properties of rainbow trout [31]. However, data concerning long-term exposure to MZ and the resulting impact on fish health are scarce. According to the official EPA information, the 72 h exposure of rainbow trout (O. mykiss) to MZ at concentration levels higher than 0.91 mg/L is categorized as highly toxic [32]. In this study, a 60-day trial with rainbow trout O. mykiss reared in water containing the fungicide MZ in the concentration level permitted by the European legislation was carried out. In a 60-day-trial-period experiment, a Ginko-biloba-extract-supplemented diet was applied to neutralize the negative effect of organophosphorus pesticide on rainbow trout [33]. The biochemical parameters measured were very encouraging, but specific amounts of the antioxidant supplement were included. In our study, to neutralize the negative effect of this long-term exposure, the fish were fed with a xanthophyll-supplemented diet. Ax and Cx were selected due to their known antioxidant activity [34]. Salmonids are able to adsorb them from their diet, and a carotenoid-supplemented diet is used in the fish farming industry. So, many researchers studied the accumulation rate of the pigment in fish products. An experiment on the effect of carotenoids, including liposomes as a diet supplement, on their accumulation in the flesh of rainbow trout was carried out during a trial period of three months [35]. Our team conducted a similar experiment and concluded that the accumulation of Ax and Cx in the muscle tissues of rainbow trout was most intensive up to the 60th day of the xanthophyll-supplemented diet [36].
Because of the great importance of the age and size of the fish specimens for optimal pigment accumulation [37], 36-month-old males and females with body weights of 912 ± 45 g were selected.
In all of the experimental fish groups, the pattern of accumulation of Ax and Cx was constant: the amount of Ax was always greater than that of Cx (Table 2), and the correlation between their concentrations was very high. The coefficients of the Pearson correlation were higher than 0.99 for all of the tested organs (gonads and liver) of fishes from the groups bred under no-Mz-containing environmental conditions (Figure 5 and Figure 6) and 0.9738 for all of the measured samples in the third fish group (Figure 7), where the specimens were bred in an environment containing permitted levels of MZ during the 60 d period.
The accumulation of Ax and Cx in the control fish group was the most significant in the female gonads, followed by the accumulation in the male gonads, and it was the lowest in the liver (Table 2). The fish were fed with a conventional diet. These fish species are able to absorb and cumulate Ax and Cx regardless of xanthophyll content in concentrations under the detection limit [19]. Similar results obtained in an earlier study by our team were collected: 0.176 and 0.141 mg/kg were the mean contents of Ax and Cx in Salmonidae eggs, respectively [25]. Due to their antioxidant potential and reproductive syndrome M74 prevention, other authors determined the content of both pigments in Salmonidae eggs [3]. In addition, Petterson and Lignell suggested a concentration limit of 2.22 mg/kg in order to prevent or overcome this early mortality syndrome [38]. The determination of the main carotenoids with such prevention potential in different organs of Salmonidae can assist the clarification of the bio-ability, some physiological processes during feeding, and their health status.
During sexual maturation and spawning migration, Ax is mobilized from the muscle to the skin and gonads [39,40]. This confirms the xanthophyll quantities accumulated and determined in the present study: the fish species were in the breeding season, and the highest concentration levels of Ax and Cx were determined in the gonads. The female gonads contained higher concentrations of Ax and Cx than the male gonads. The females accumulate these pigments in the eggs, avoiding the early mortality of their offspring. This natural phenomenon is explained by the fact that each species primarily provides resources for its reproduction and therefore for the survival of the species. The stockpiling of xanthophylls in trout eggs is an effective instrument for naturally overcoming the reproductive syndrome M74 and the early mortality of their offspring [3,20]. These powerful antioxidants have been shown to counteract thiamine deficiency and protect post hatch embryos and fry from a range of negative effects of this reproductive disorder until the yolk sac has been completely resorbed. Low concentration levels of thiamine and xanthophylls in fish eggs result from an insufficient dietary intake of the females [20].
Analogous results were obtained for the second fish group. The specimens were bred under no-Mz-containing environmental conditions with a xanthophyll-supplemented diet. The highest Ax and Cx concentration levels were determined in the order of female gonads, male gonads, and liver (Table 2).
A xanthophyll accumulation rate for the third experimental group was established. The specimens were bred in an environment containing MZ at permitted levels. These conditions led to low accumulation levels of Ax and Cx in the gonads and liver, regardless of the xanthophyll-supplemented diet (Table 2).
Another tendency was confirmed: the accumulation rate of Ax and Cx in the gonads was highest in the second experimental group. However, the highest xanthophyll levels in the liver were found in the samples of the third experimental group. Under the conditions of the static renewal test, exposure to maneb (uncoordinated with zinc mangozeb) mainly affected the gills, trunk kidney, and liver [41]. Under the conditions of contamination, detoxification reactions take place in the liver, which explains the higher rate of antioxidant accumulation there.
The xanthophyll levels accumulated in the organs of fishes from the last group were the lowest. The differences between the measured quantities in the gonads and liver were small. A certain reason for this was the ongoing detoxification reactions and degradation of MZ, in which Ax and Cx were involved as antioxidants.

4. Conclusions

The patterns of accumulation of both pigments in the three organs in the specimens of the three groups did not change: the amount of Ax was always greater than that of Cx, and the correlation between their concentrations was very high. The pigments were accumulated most intensively in the female gonads, then in the male gonads, and the least in the liver. This trend was confirmed for all three experimental groups. However, the differences in the last third group were very small, and the levels of the xanthophyll accumulated were the lowest. A certain reason for this was the ongoing detoxification reactions and the disposal of MZ, in which Ax and Cx were involved as antioxidants.

Author Contributions

Conceptualization, M.T. and V.A.; methodology, M.T., Z.Y. and V.A.; software, M.T. and Z.Y.; validation, M.T. and V.A.; formal analysis, M.T., E.V., Z.Y. and D.I.; investigation, M.T. and E.V.; resources, V.A.; data curation, M.T. and D.I.; writing—original draft preparation, M.T; writing—review and editing, V.A., Z.Y. and D.I.; supervision, V.A. and Z.Y.; project administration, V.A. and Z.Y.; funding acquisition, M.T., Z.Y. and V.A. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported financially by the Bulgarian Ministry of Education and Science under the National Research Program “Healthy Foods for a Strong Bio-Economy and Quality of Life” approved by DCM # 577/17.08.2018, and by Scientific Project No. 06/2021 FVM, Trakia University.

Institutional Review Board Statement

Not applicable. For this study are not involving humans or animals. The biological material was actually the production waste of a fish farm.

Informed Consent Statement

Not applicable.

Data Availability Statement

All data are contained within the article.

Acknowledgments

The authors would like to thank the fish farm “Happy Fish” Ltd., Bulgaria, for technical support and for providing biological material for this study.

Conflicts of Interest

The authors declare no conflict of interest.

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Applsci 12 06646 g001 550
Figure 1. Typical chromatogram of standard solution.
Figure 1. Typical chromatogram of standard solution.
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Figure 2. Typical chromatogram of sample solution of female gonads.
Figure 2. Typical chromatogram of sample solution of female gonads.
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Figure 3. Typical chromatogram of sample solution of male gonads.
Figure 3. Typical chromatogram of sample solution of male gonads.
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Figure 4. Typical chromatogram of sample solution of liver.
Figure 4. Typical chromatogram of sample solution of liver.
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Figure 5. Pearson correlation between Ax and Cx accumulation in the female gonads of rainbow trout, O. mykiss.
Figure 5. Pearson correlation between Ax and Cx accumulation in the female gonads of rainbow trout, O. mykiss.
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Figure 6. Pearson correlation between Ax and Cx accumulation in the male gonads of rainbow trout, O. mykiss.
Figure 6. Pearson correlation between Ax and Cx accumulation in the male gonads of rainbow trout, O. mykiss.
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Figure 7. Pearson correlation between Ax and Cx accumulation in the liver of rainbow trout, O. mykiss.
Figure 7. Pearson correlation between Ax and Cx accumulation in the liver of rainbow trout, O. mykiss.
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Table
Table 1. Water quality self-monitoring (mean ± SD).
Table 1. Water quality self-monitoring (mean ± SD).
Monitoring PeriodOptimal [21]
August SeptemberOctober
Temperature, °C18.9 ± 1.617.9 ± 1.816.1 ± 2.4not more than 20
Dissolved oxygen, mg/L9.4 ± 0.29.6 ± 0.39.8 ± 0.3not less than 9.0
Nitrates, mg/L0.13 ± 0.040.15 ± 0.030.15 ± 0.05not more than 2.00
Nitrites, mg/L0.05 ± 0.010.06 ± 0.010.06 ± 0.01not more than 0.01
Amonium, mg N/Lnonenonenonenone
pH7.83 ± 0.127.80 ± 0.157.67 ± 0.217–8
Harndess, mmo1/L6.24 ± 0.086.12 ± 0.056.18 ± 0.046–14
Table
Table 2. Ax and Cx amounts in different organs of rainbow trout, O. mykiss (n = 6, mean ± SD).
Table 2. Ax and Cx amounts in different organs of rainbow trout, O. mykiss (n = 6, mean ± SD).
Group NoFemale GonadsMale GonadsLiver
AxCxAxCxAxCx
10.319 ± 0.036 a *0.207 ± 0.015 a0.211 ± 0.009 a0.099 ± 0.006 a0.176 ± 0.007 c0.103 ± 0.010 c
22.490 ± 0.247 b2.206 ± 0.166 b1.215 ± 0.010 a0.888 ± 0.010 b0.257 ± 0.007 c0.156 ± 0.006 c
30.518 ± 0.020 a0.335 ± 0.019 a0.395 ± 0.012 a0.210 ± 0.010 a0.351 ± 0.008 c0.308 ± 0.009 c
* The content of Ax and Cx is expressed as mg/kg; different letters in the table denote significant differences between the experimental groups and control values according to LSD test (p ≤ 0.05).
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Tzanova, M.; Yaneva, Z.; Valkova, E.; Ivanova, D.; Atanasov, V. Accumulation of Astaxanthin and Canthaxanthin in Liver and Gonads of Rainbow Trout (Oncorhynchus mykiss (Walbaum, 1792)) Reared in Water Containing the Fungicide Mancozeb in Concentration Level Permitted by European Legislation. Appl. Sci. 2022, 12, 6646. https://doi.org/10.3390/app12136646

AMA Style

Tzanova M, Yaneva Z, Valkova E, Ivanova D, Atanasov V. Accumulation of Astaxanthin and Canthaxanthin in Liver and Gonads of Rainbow Trout (Oncorhynchus mykiss (Walbaum, 1792)) Reared in Water Containing the Fungicide Mancozeb in Concentration Level Permitted by European Legislation. Applied Sciences. 2022; 12(13):6646. https://doi.org/10.3390/app12136646

Chicago/Turabian Style

Tzanova, Milena, Zvezdelina Yaneva, Elica Valkova, Donika Ivanova, and Vasil Atanasov. 2022. "Accumulation of Astaxanthin and Canthaxanthin in Liver and Gonads of Rainbow Trout (Oncorhynchus mykiss (Walbaum, 1792)) Reared in Water Containing the Fungicide Mancozeb in Concentration Level Permitted by European Legislation" Applied Sciences 12, no. 13: 6646. https://doi.org/10.3390/app12136646

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