Levels of some heavy metals in the tissues of cestode Schyzocotyle acheilognathi (^{) and its definitive host fish}

Abstract

Background:

In recent years, parasitic helminths and their fish hosts have been used as a biomonitoring tool to indicate the status of ecosystems and their relationship with human health.

Aim:

This work aims to evaluate and use the potential ability of the cestode Schyzocotyle acheilognathi (Yamaguti, 1934) as a bioaccumulative indicator of zinc and iron, and compare it with the tissues of its final host, Common carp Cyprinus carpio Linnaeus, 1758, and to see if this cestode provides substantial evidence of the aquatic ecosystem pollution.

Methods:

Twenty-eight live fish were caught from the Tigris River passing through Rashidiya and Sharikhan sites in Mosul city, Iraq, during March 2024. The concentrations of Zn and Fe in the cestode tissues, gills, liver, intestines, and muscles of infected and uninfected fish were determined using an atomic absorption spectrometer.

Results:

The results showed that the ratio of accumulation of Fe and Zn in the cestode tissues to their host tissues was 1:0.622 and 1:0.616, respectively. In both uninfected and infected fish, the Zn levels were in the following order: gills > liver > intestine > muscles, while Fe levels were in the order liver > gills > intestine > muscles. Although the Fe concentrations were higher than the Zn in all samples that were analyzed, the values of Average Pollution Load Index were < 1, and they were within the permissible limits according to Food and Agriculture Organization and WHO.

Conclusion:

Thecestode S. acheilognathi shows enormous promise as a sensitive bioindicator for heavy metal pollution in aquatic habitats, as does its fish host, Common carp C. carpio. Frequent observation of these species can yield early indicators in environmental monitoring programs.

Introduction

Many heavy metals, including zinc and iron, are of great importance in vital processes and their regulation in living organisms, as they are essential for immune, reproductive, and growth activities, and play a key role as cofactors for some enzymes in metabolic pathways (Agashe et al., 2024). Although they are required in limited concentrations, their deficiency negatively affects these pathways, and at the same time, increasing their concentration to toxic levels causes various damages to the organisms (Al-Mayahi et al., 2021) and can affect their diversity (Van Der Spuy et al., 2023).

These elements are common pollutants as a result of the rapid development in agricultural and industrial fields around the world, such as the manufacture of fertilizers, pesticides, dry batteries, mining operations, the automotive industry, and central heating systems (Liu et al., 2021).

Heavy metals are dangerous pollutants that can enter aquatic food chains, including fish, in two ways: either directly through food or indirectly through gills and skin (Panda et al., 2023), and their toxic effect is transmitted through the food chain, and the highest level of toxicity is reached in organisms at the top of the food chain (Tahity et al., 2022).

Parasites help scientists understand the bioavailability of pollutants in the environment and the host’s chemical conditions, which has piqued their interest in studying the relationship between parasites and pollution (Tramboo et al., 2022). Both fish and their internal parasites, including helminths, have been used as important bioindicators in assessing the health of aquatic ecosystems (Sedaghat et al., 2022). The cestode Schyzocotyle acheilognathi (Yamaguti, 1934) Brabec et al. (2015) (syn. Bothriocephalus acheilognathi Yamaguti, 1934) belongs to the family Bothriocephalidae, order Bothriocephalidea, and class Cestoda. It can accumulate heavy metals in its body (Assis and Pinto, 2024). Also, fish are good bioindicators for detecting pollutants and good accumulators of them, as they can store these pollutants in larger amounts than in water and sediments because they feed on aquatic microorganisms, algae, and organic materials present in the water (Öktener and Bănăduc, 2023).

This field has drawn researchers from all over the world as it is becoming more important in monitoring environmental pollution and guaranteeing the health of fish and their safety for human consumption (Habib et al., 2024; Mostafa et al., 2023). Numerous scientific research institutions and international organizations have set acceptable limits for the concentration of these metals in certain ecosystems are based on in-depth studies and experiments (Rakib et al., 2021).

Given the detrimental effects of heavy metals on human health and the paucity of research, in Iraqi environments, on host-parasite systems as sensitive biomarkers for monitoring heavy metal pollution are still scarce, therefore, the current study intends to evaluate the potential ability of the cestode Schyzocotyle acheilognathi (Yamaguti, 1934) to accumulate iron and zinc in comparison to the tissues of its final host, Common carp Cyprinus carpio (Linnaeus, 1758; Cyprinidae), and regard them as bioaccumulative indicators of pollution and to assess the health hazards that consumers of these fish might be exposed to, as well as to evaluate the pollution of the Tigris River in Mosul City with these two metals.

Materials and Methods

Sample collection

During March 2024, 28 live C. carpio (Linnaeus, 1758), identified according to FAO (2009) and were collected from the Tigris river passing through two locations: Rashidiya and Sharikhan (about 5 km north of Mosul city, Iraq) (Fig. 1). These regions are crowded with poultry and livestock farms, and are characterized by the presence of a power station, a water supply system, gas stations, beverages and dairy factories, and health hospitals, as well as a large channel for sewage water draining into the river. The alive fish were placed in a thermo-insulated plastic container “esky” containing crushed ice and immediately carried to the research laboratory in the Biology Department, Science College, Mosul University.

Fig. 1. The current study area is in Rashidiya and Sharikhan, Nineveh Governorate, at 43.1333°E and 36.3333°N. Since Al-Sharikhan is located within the Mosul area, it falls within approximately the same general area. https://humanitarianatlas.org/iraq/atlasmaps/ninewa.pdf.

Sample preparation

The live fish placed in crushed ice were dissected immediately after arrival at the laboratory according to the method of Dybem (1983). Gills, liver, intestines, and muscle were removed from infected and uninfected fish. Cestode S. acheilognathi (Yamaguti, 1934) was isolated from the intestines of infected fish. Then, these fresh organs and the cestodes were washed with triple-distilled water several times to eliminate any possible contamination, then dried using blotting papers, labeled, and placed separately in clean, dry polyethylene bags and stored in a freezer at (−20°C) until digestion.

Sample digestion

Sample digestion is made according to the method of Adebayo (2017) with modifications. Fish and cestode samples were oven-dried at 105°C for 24 hours, and then they were milled using a mortar and pestle. Then, 0.5 g of the dry weight for each sample was put separately in glass bottles in triplicate, and 5 mL of 65% nitric acid 14.3 M (Fisher Chemical, USA) was added to each. The bottles were heated on a hot plate at 100°C for one hour to speed up the digestion process. Then the bottles were left for 24 hours, and 2 mL of 70% perchloric acid, 11.7 M (P.C.Chem, India) was added to each bottle and left for 45 minutes to complete the digestion. Finally, the samples were filtered by a 0.8 µm cellulose ester membrane filter and transferred to newly cleaned bottles for the determination of their heavy metal content.

Heavy metal estimation

The concentrations of heavy metals (zinc and iron) were determined in triplicate for each sample using an atomic absorption spectrophotometer (AAS) (Perkin-Elmer-2380, USA). The controls were a mixture of nitric acid and perchloric acid without samples. The Zn was estimated at a 213.9 nm wavelength and a 0.5 nm slit width according to the standards of 2, 5, 10, 20, and 30 mg/l of the mixture of nitric acid and perchloric acid. The Fe was estimated at a 248.3 nm wavelength and a 0.5 nm slit width, depending on the standards of 5, 10, 20, 30, and 40 mg/l of the mixture of nitric acid and perchloric acids.

Bioconcentration factor (BCF)

The BCFs of Zn and Fe were calculated as the following equation: (Xiong et al., 2024).

Pollution load evaluation

The average pollution load index (APLI) method, which can be at various levels, was used to assess the metal contamination of fish samples. APLI = 0.7–1.0: heavily polluted; 0.5– < 0.7: moderately polluted; 0.2– < 0.5: lightly polluted; 0.1– < 0.2: micro-pollution; < 0.1: unpolluted (Jahan et al., 2025). The equation below calculates the APLI (Adetutu et al., 2023):

where,

APLI: Average pollution load index

CS: The mean concentration of metal in the fish sample.

CM: The maximum allowable concentration of metal is 130.43 µg/g dry weight for Zn and 434.78 µg/g dry weight for Fe (FAO, 1983; Rakib et al., 2021).

n: number of metals in fish sample.

Statistical analysis

The obtained results were analyzed statistically using software SPSS v. 20. An independent t-test was used to compare the data between the infected and uninfected fish. Duncan’s test was applied to compare the data among fish organs and also among the infected, uninfected fish, and the cestode. The values were expressed as mean ± standard deviation.

Ethical approval

Not needed for this study.

Results

The fish weighed between 1256 and 1884 g (mean ± SD = 1570 ± 309.3) and measured 30.9–37.8 cm in length (mean ± SD = 34.4 ± 3.3). The prevalence and the mean intensity were 17.85% and 8.4, respectively.

The highest concentration of zinc was in the gills, followed by the liver and then the intestines, while the muscles showed the lowest concentration in uninfected and infected fish, C. carpio (Linnaeus, 1758), with the cestode Schyzocotyle acheilognathi (Yamaguti, 1934) (Table 1).

Table 1. Zinc levels (µg/g dry weight) in the organs of uninfected and infected Cyprinus carpio with the cestode Schyzocotyle acheilognathi.

Organ	Infected fish	Mean ± SD	Uninfected fish	Mean ± SD	Mean
Gills	16.883 ± 0.074	A	24.227 ± 0.025 *	a	20.555
Liver	12.377 ± 0.099	B	19.070 ± 0.044*	b	15.724
Intestine	9.523 ± 0.012	C	18.893 ± 0.015*	b	14.208
Muscle	5.903 ± 0.015	D	11.967 ± 0.015*	c	8.935

*Refers to the significant differences (p ≤ 0.01) according to the t-test. Different letters vertically refer to the significant differences (p ≤ 0.01) according to the Duncan test.

The differences in all results were considered statistically significant at p ≤ 0.01.

The highest iron concentration was found in the liver, followed by the gills and intestines and, finally, muscles, in both uninfected and infected fish (Table 2).

Table 2. Iron levels (µg/g dry weight) in the organs of uninfected and infected Cyprinus carpio with the cestode Schyzocotyle acheilognathi.

Organ	Infected fish	Mean ± SD	Uninfected fish	Mean ± SD	Mean
Gills	16.160 ± 0.053	B	27.033 ± 0.015*	b	21.597
Liver	19.220 ± 0.010	A	34.633 ± 0.032*	a	26.927
Intestine	11.793 ± 0.015	C	18.213 ± 0.015*	c	15.003
Muscle	7.597 ± 0.012	D	10.413 ± 0.012*	d	9.005

*Refers to the significant differences (p ≤ 0.01) according to the t-test.Different letters vertically refer to the significant differences (p ≤ 0.01) according to the Duncan test.

The concentration of iron was higher than that of zinc in all samples. The zinc and iron concentrations in the organs of uninfected fish were accumulated in higher amounts than those in infected ones. Also, the levels of the two metals in the tissues of the cestode Schyzocotyle acheilognathi (Yamaguti, 1934) were higher than the levels found in all organs of the infected fish (Table 3).

Table 3. Zinc and iron levels (µg/g dry weight) in the uninfected, infected Cyprinus carpio and its parasite, the cestode Schyzocotyle acheilognathi.

Heavy metal	Infected fish	Mean ± SD	Uninfected fish	Mean ± SD	Cestode	Mean ± SD
Zinc	11.172 ± 4.01	B	18.539 ± 4.35	a	18.133 ± 0.035	a
Iron	13.693 ± 4.39	B	22.573 ± 9.11	a	22.013 ± 0.035	a

Different letters horizontally refer to the significant differences (p ≤ 0.01) according to the Duncan test.

The BCF of the studied heavy metals was calculated to evaluate the ability of the cestode to accumulate these metals compared to the organs of the infected fish. The results showed that the ability of the cestode to accumulate zinc and iron was close to the ability of the gills and liver of host fish, respectively (Table 4).

Table 4. Bioconcentration factors of cestode Schyzocotyle acheilognathi for zinc and iron accumulations.

Fish organ	Bioconcentration factors C[parasite]/C[fish organ]
	Zinc		Iron
Gills	1:0.931	1:0.734
Liver	1:0.680	1:0.873
Intestine	1:0.525	1:0.536
Muscle	1:0.326	1:0.345
Average	1:0.616	1:0.622

Regarding the level of fish muscle contamination with heavy metals and its risk to consumers’ health, the values of average pollution load index (APLI) were lower than one (Table 5).

Table 5. Values of APLI in infected and uninfected fish muscles.

Pollution indicator	Infected fish	Uninfected fish
APLI	0.0313	0.0578

APLI = average pollution load index.

Discussion

The current result regarding zinc agreed with the result of Kareem et al. (2022) and Ouda et al. (2023), who indicated that the highest concentration of zinc was in the gills and the lowest concentration in the muscles of C. carpio (Linnaeus, 1758) caught from Lake Dukan and Shatt al-Arab in Iraq, respectively. Also, Tahity et al. (2022) found that many elements, including zinc, accumulated in high concentrations in the gills, than the liver and muscles of farmed and wild Barramundi fish from the Bengal Coast, while the present result did not agree with the findings of Huang et al. (2022) and Zaghloul et al. (2024) who indicated that the highest concentration of zinc was in the liver, followed by the intestine or gills, then the muscles with the lowest concentration.

The presence of zinc in high concentrations in the gills is attributed to the fact that the gills are one of the main sites exposed to environmental pollutants in fish, as they are areas for breathing and regulating ionic and osmotic exchange between the environment and the living organism. Also, their anatomical and tissue structure gives them a large surface area that allows pollutants to enter more (Chen et al., 2023). Also, the presence of the mucous substance containing phospholipids in the gills helps to bind elements to them, forming a complex that cannot be removed from the gill plates before the tissue decomposes. In addition, the surface of the gills is negatively charged, which provides a potential site for positively charged heavy metals to attach to it (Choudhary et al., 2023).

The high iron content in the fish liver was consistent with that of Al-Niaeemi et al. (2020), who found that the livers of Silurus glanis (Linnaeus, 1758; Siluridae) fish infected and uninfected with the cestode Postgangesia armata accumulated a significant amount of iron. The liver is the organ that stores the most iron and zinc throughout the year, followed by the gills, kidneys, and muscles of the fish Tilapia zilli ( Gervais, 1848), according to Elwasify et al. (2021). However, our findings contradicted those of Mahboob et al. (2016), who found that the gills had the highest iron concentration in C. carpio (Linnaeus, 1758), followed by the liver, while the muscles had the lowest level. The high content of iron in the liver is due to its status as one of the active organs of numerous metabolic pathways of crucial molecules that require enzymes and cofactors; it possesses a preventive function in eliminating toxins and is essential for the storage of minerals and vitamins (Gashkina, 2024). The liver is also responsible for the synthesis of metallothionein proteins, which can interact with heavy metals and expel them from the body (Setiyowati et al., 2020).

Additionally, our findings demonstrated that both infected and uninfected fish had medium levels of iron and zinc in their intestines. This can be shown by the fact that food in the digestive tract is a major source of metal accumulation. The existence of mucus that can bind to metals is another factor (Jamil Emon et al., 2023). The study of heavy metals in muscles is crucial because they make up a large portion of fish for human food, and the high concentration of metals in them has a detrimental impact on the fish and consumer health. The results showed lowest accumulation of zinc and iron in muscles among the organs of infected and uninfected fish, and this was consistent with Zaghloul et al. (2024), who observed that the concentrations of certain elements, including zinc, in the muscles of seven species of fish in Bardawil Lake, Egypt, were lower than their concentrations in the rest of the organs. This finding is in line with those of Al-Niaeemi and Dawood (2023) and Chatha et al. (2023), who found that muscles had the lowest metal concentrations. These findings may be aligned with the fact that fish muscles have less fat. These results can be interpreted by the fact that fish muscles have less fat than other organs, as metals easily penetrate fatty tissue and are difficult to remove, or due to the lack of binding of metals with muscle proteins, so muscles are inactive sites for metal accumulation (Huang et al., 2022).

The content of iron was higher than the zinc in all organs of infected and uninfected fish. This result is in line with that of Afifi et al. (2024), who reported that iron was the element with the highest concentration of Zn, Pb, Cu, As, and Cd in two fish: Clarias gariepinus (Burchell, 1822 ) and Oreochromis niloticus (Linnaeus, 1758; Cichlidae). This is explained by the fact that fish have distinct metabolic processes for different metals, the food that contains these metals, the length of exposure, and the fact that fish have varied ways of getting rid of these metals (Serviere-Zaragoza et al., 2021). The number of environmental factors, such as ionic strength, acidity, temperature, and the level of metal pollution in the water, also contribute to this (Mijošek et al., 2024).

The variations in heavy metal concentrations within fish organs can be cleared by their functional and anatomical differences (Younis et al., 2024). Our results reveal that the organs of uninfected fish contained higher levels of zinc and iron than those of infected fish. Compared to the all organs of the infected fish, the tissues of the cestode Schyzocotyle acheilognathi (Yamaguti, 1934) had higher concentrations of the two metals. This result is consistent with the findings of Al-Niaeemi et al. (2020) and Al-Niaeemi and Dawood (2023). The reason for this is due to the cestodes inhabiting the host’s intestines and competing with it for food and minerals, which depends on the parasite’s developmental stage, length of stay inside the host, and the extent of the parasite’s ability to absorb and accumulate metals in it (Goutte and Molbert, 2022). Furthermore, because these parasites cannot produce fat, so they take the minerals from their host’s intestines along with the fats in diet (Najm et al., 2022).

The cestode Schyzocotyle acheilognathi (Yamaguti, 1934) has undoubtedly contributed to decrease the bioaccumulation of zinc and iron in the tissues of its hosts, C. carpio (Linnaeus, 1758), and this was confirmed by Mijošek et al. (2024) and Mostafa et al. (2023). Fish consumers’ health is not at risk because the levels of APLI for zinc and iron were within the Food and Agriculture Organization’s permissible limits (Rakib et al., 2021).

Conclusion

The results of the study show that the liver and gills of the fish C. carpio (Linnaeus, 1758) are essential organs for accumulating zinc and iron. The results emphasize that the cestode S. acheilognathi (Yamaguti, 1934), which lives in the intestines of the C. carpio (Linnaeus, 1758), reduces the levels of heavy metals (biodilution process) in the fish’s organs, demonstrating the beneficial effects of the cestode and preventing the accumulation of these heavy metals in the fish’s tissues. Thus, Schyzocotyle acheilognathi (Yamaguti, 1934) can be regarded as sensitive bio-indicators for zinc and iron contaminations and assessing how it affects fish consumers’ health. Although the levels of Zn and Fe were within the globally permissible limits and do not pose any health risks to humans, it is highly recommended to regularly monitor heavy metal levels in aquatic ecosystems. This study emphasizes how crucial it is to investigate the host-parasite system as one of the future directions requested in our lives.

Limitations

The fish samples should be obtained in a live state. These live fish should be infected with only one type of worm. Obtaining two fish, one infected and the other uninfected, both should have approximately the same weight and length.

Suggestions for future work

Study many other heavy metals in other helminthes, like nematodes, and compare them to know which one is more efficient in accumulating metals in their hosts. Study other fish species or other aquatic organisms such as crustaceans and mollusks. Conduct representative studies in different environments and different seasons.

Conflict of interest

The authors declare that there is no conflict of interest.

Funding

None.

Authors’ contributions

Mohammad Hussein Mikael: Designed the study; carried out field sampling and laboratory analysis; interpreted the data; wrote the first draft of the manuscript. Wahda A. Kharofa: Participated in the study design, supervised sample dissection and anatomical analysis; critically revised the manuscript for important intellectual content. Bushra H. Al-Niaeemi: Participated in doing lab work and data collection; did statistical analysis; shared in drafting results and discussion. Muhammed S.A.F. Muhammed: Contributed in data validation and literature review; Reviewed and edited the final manuscript; Gave academic supervision and technical support for the whole research. All authors have read and approved the final version of the manuscript.

Data availability

All data were provided in the manuscript.