Iraqi Journal of Veterinary Sciences

Abstract


Introduction
Heavy metals are considered a major class of water pollutants that may disturb the equilibrium in aqueous environments, impacting the variation of living organisms, including fish (1).Commonly, organisms require some heavy metals in trace amounts, even as co-factors in biochemical pathways or as essential molecules in cell construction.Unfortunately, non-illegible accumulation of heavy metals in the aqueous environment may result from human activities (2).The extent of accumulation in living bodies may depend on the metal's concentration in the surrounding environment, exposure time, temperature, salinity, feeding habits, and physiological state according to sex, age, and health status (2).The increasing elevation in heavy metals may lead to dire results since these metals have long half-lives, are difficult to disintegrate, could be spread away from the source of their emanation, and can accumulate in the tissues of living organisms at a toxic level (3).Monitoring the levels of these metals, especially in aquatic environments, is necessary.Fish are distinguished members of the aqueous communities in their tolerance to heavy metals; they either get them directly from the ingested food or indirectly through their gills.As the next predator eats the fish, the concentration of heavy metals would biomagnify through the food chain (4).Thus, fish has been used as bioindicators to estimate water pollution with heavy metals and other organic compounds (5).The muscle of C. carpio fish tissue was not safe for human consumption and that the groundwater in the Khor al-Zubair area in Basra governorate/South Iraq is possibly contaminated with the heavy metals: Cr, Ni, Hg, Pb, and Cd, mainly owing to industrial activity (6).Manganese (Mn): Mn is found naturally in water and soil, almost accompanied by ferrous.Mn is an essential element living organisms use as an enzyme activator and aids in bone hardening in vertebrate animals.Elevated amounts of Mn in the environment may result from the manufacture of batteries, plant fertilizers, and the preparation of alloys (3).The Nickeil (Ni): Nickeil does not exist freely in nature and is combined with other metals.It is needed in trace amounts to activate some enzymes in living organisms, added to its role in ferrous absorption and, thus, hemoglobin formation.The abnormality of Ni's existence in an aqueous environment may relate to fuel and industrial wastes (7).The Cobalt (Co): In living organisms, trace amounts of Co are necessary for metabolic activity, like carbohydrate and protein metabolism, and work as activating factor in several enzymatic and immunological cellular reactions, in addition to being included in the composition of vitamins B12 (8).Some workers set their sights on the accumulation of heavy metals in fish and their parasites, the impact of these metals on the parasite distribution, and host physiology (9)(10)(11)(12)(13).
The present work was aimed to investigate the bioaccumulation of three heavy metals (Mn, Ni, Co), those expected to be found in Tigris water in concentrations over the permissible limit, since they are exchanged into the river, especially with dairy and soft drinks factories.Organisms of two levels in the food chain were Chosen, Cyprinus carpio fish, and its intestinal Cestoda, Bothriocephalus acheilognathi.The two living organisms are setter populations in the water of Mosul`s Tigris (Within Nineveh Governorate).

Ethical approve
University of Mosul, College of Veterinary Medicine, the Institutional Animal Care and Use Committee, give the final acceptance to conduct experiment on animals numbered UM.VET.2023.015,sated 18 march 2023.

Study location
The study was performed in two locations in the Tigris River, In Al Rashedia and Sherikhan villages (about 6 km Northwest of Mosel City/ Nineveh Province) between June 2022 to October 2022.Both sites are densely populated, adding farms, poultry, and livestock fields to hospitals and healthcare centers.The two locations also contain power stations, dairy, and soft drink factories.As well as being residential areas.It seems that wastewater disposal is irregular there and discards directly to the Tigris River.

Sample collection
A 52 C. carpio fish were hunted using fishnet, and their weight ranged from 1-1.5 Kg.The fish then desiccated according to Dybem (13), locking for the intestinal cestode Bothriocephalus acheilognathi in the small intestine.Furthermore, four body organs were isolated from each hunted fish after killing (Gill, liver, intestinal muscles, skeletal muscles) either the fish was infected or uninfected with the intestinal cestodes.

Sample preparation
The isolated tissues and the cached cestodes were washed with Distilled water and put on filter paper to eliminate excess water.Each sample's Fresh weight was recorded, then frozen at -20ºC in a locked container until the next steps of the research work.

Estimation of heavy metals concentration
The concentration of the three chosen metals: Mn, Ni, and Co, was estimated in Fish and helminth tissues.The targeted cestoda Bothriocephalus acheilognathi was collected from fish after making longitudinal fissures along the fish intestine, put in a Petri dish, and washed three times with PBS at pH 7.3.After insurance of the Cestoda identity under a dissecting microscope, the fresh weight of each collected helminth was recorded.The isolated fish tissues and the helminths were digested according to (14).A 1 ml of concentrated HNO3 65% was added to 0.1 gm of each sample.Homogenizer was used to disintegrate the samples, and then the sample was put in a glass test tube with a tide lid and incubated at 70°C in the water bath for 24hrs; the tubes were then left for 72 hrs. to complete tissue digestion.To determine the heavy metal concentration in each sample, a colorimetric method was performed using Atomic Absorption Spectrophotometer (Perkin Elmer-4000 USA.).The concentration of each metal under study (µg of the metal/gm.fresh weight) was estimated depending on a standard curve of the metal (14).

Statistical analysis
Complete randomized design, Duncan multiple range tests, and T-tests were used to compare compatibility and differences between values mean (15).All differences were considered significant at P≤0.05.

Results
Bioaccumulation of some heavy metals commonly discharged to the Tigris River in Mosul City was estimated in the present work.C. carpio fish, and its intestinal cestoda, B. acheilognathi, were chosen as they are two setter populations in an ecosystem.
Table 1 illustrates the mean concentration of Mn in uninfected and infected C. carpio fish.The highest concentration of Mn was observed in gills 21.773 and 14.597 µg/gm fresh weight, followed by the liver 16.157 and 12.123 µg/gm fresh weight, intestine 12.607 and 9.11 µg/gm fresh weight, and skeletal muscles 5.597 and 2.8 µg/gm fresh weight, both in uninfected and infected fish; respectively.The different between values were significant at P≤0.05.In table 2 mean concentration of Ni both in uninfected and infected C. carpio fish was listed.The highest concentration of Ni was observed in the liver 8.10 and 4.44 µg/gm fresh weight, followed by the gills 5.393 and 3.41 µg/gm fresh weight, intestine 4.517 and 2.793 µg/gm fresh weight, and skeletal muscles 1.90 and 2.20 µg/gm fresh weight, both in uninfected and infected fish; respectively.3 illustrates that Co was highly accumulated in the intestine 7.467 and 4.790 µg/gm fresh weight of Carpio fish, and to a less extent in the gills 4.507 and 1.663 µg/gm fresh weight, then liver 2.133 and 1.823 µg/gm fresh weight and skeletal muscles 1.007 and 0.217 µg/gm fresh weight, both in uninfected and infected fish; respectively.and 2.067 µg/gm fresh weight, respectively.It is observed that the concentration of Mn and Ni was higher in helminthic tissues than in tissues of the host fish (Figure 1).It is worth knowing that the concentration of both Mn and Co was higher in fish muscles of uninfected fish 5.597 and 1.007 µg/gm fresh weight, respectively than in muscles of infected fish 2.80 and 0.217 µg/gm fresh weight, respectively.The concentration ion of Ni was higher the in the muscles of infected fish 2.20 µg/gm fresh weight tin than in the uninfected fish 1.90 µg/gm fresh weight.

Discussion
The present study aimed to evaluate the biomagnification possibility of several heavy metals commonly discharged into the Tigris River dairy.Two locations in Mosul distinct were chosen, Al Rashedia and Sherikhan villages.These villages are known to be important regions for industrial, agricultural, and other relhumanan activities.The study was conducted on two living organisms interconnected by a parasitic relationship.B. acheilognathi is a cestode recorded as an intestinal parasite for the common carp C. carpio living there (16).The parasitic cestode has a distinct location in the food chain as an internal consumer.It could be a unique model for estimating the bioaccumulation of a particular pollutant like heavy metals.
The concentration of the three chosen metals was first estimated in four metabolically active tissues in the fish, then estimated in tissues of the intestinal cestode as illustrated in up worded results.
The results agreed with that of Al-Weher (17) those that recorded the highest concentration of some metals in the gills of C. carpio uninfected fish and the lowest concentration in skeletal muscles.Elwasify et al. (18) also recorded a higher concentration of Mn in the Gills of Tilapia zillii fish (Settled in Qarun Lake in Egypt) and the lowest in skeletal muscles.On the other hand, recent results do not agree with that of Tekin-Özan and Barlas (19), which showed that the highest accumulation of Mn in Tinca tinca fish (Settled in Beysehir Lake in Turkey) was observed in the liver, then gills and skeletal muscles.
Gills are directly exposed to pollutants found in water because of their structure and physiology; thus, the highest concentration of heavy metals offset with the highest accumulation of that metal in fish gills (20).Karadede et al. (21) were referred to that the heavy metals that can combine with mucous materials in gills forming hardly removable complexes.Yousafzai et al. (22) combined the attraction between the positively charged heavy metals and the negatively charged phospholipids found in the mucous lining epithelium of fish gills.
On the other hand, Hantoush et al. (23), which illustrated that Ni was highly concentrated in the liver of silver carp fish (collected from the farms of Middle Iraq).Bhuvaneshwari et al. (24) was referred to the high accumulation of Ni in the liver of Oreochromis mass amicus fish 25.67 µg/gm fresh weight, then in gills 12.5 µg/gm and muscles 4.5 µg/gm in Kaveri River in India.Arantes et al. (25) estimated the accumulation of heavy metals in Pseudoplatystoma correct fish in (Paraopebia River in Brazil), and so Rajeshkumar Xiaoyu (26) those referred to the high accumulation of heavy metals in the fish liver in Taihu Lake in China.
The higher concentration of Ni in liver tissues is reliable because the liver is a metabolically active organ in which several biochemical pathways are performed (27).It is the first organ that receives metabolites from the intestine through blood and lymphatic circulation.Heavy metals also arrive in liver tissue in bloodstream, then either removed with bile salts, discharged to the small intestine and then to the outer environment, or bound with some side groups available in the liver like carboxyl, amino, sulfate, nitro, and mercapto groups, added to metallothionein (28).Some of these metalorganic complexes are reabsorbed in the small intestine and stored in the liver (29).
Yousafzai et al. (22), which concluded that the concentrations of heavy metals were higher in the intestine of C. carpio fish than in other body organs.They excluded that Co metal inter fish body during nutrition rather than respiration (through gills).Elsenhans et al. (30) referred to the tide combination of ingested metals with the mucous membrane in the fish intestine that make it hard to remove these metals.Furthermore, some heavy metals attached to mucous membranes could be absorbed by intestinal villi after a while; other heavy metals could be discharged with fecal materials (30).
The present work revealed that the body organs of fish differ in their deterioration to heavy metals.Mn, Ni, and Co were found in the lowest concentration in muscles.This result agreed with that of Akinsanya and Kuton (12), Tekin-Özan and Barlas (31), Yilmaz and Aldhamin et al. (32), who concluded that heavy metals accumulated in skeletal muscles to at less extent than in other fish organs.This status result is fortunate since the skeletal muscles of fish are put on the main food lists for humans and the mean concentrations of the studied metals, Mn, Ni, and Co, did not reach the lower permitted levels of such metals in fish tissues by FAO and FDA 4.4-7.9µg/gm of fish muscles for Mn, 17.8-20 µg/gm for Ni and 2.6-5.39µg/gm for Co (33).Besides, we must consider that the over threshold accumulation of heavy metals in fish muscles may hazard human health (22,34).The relatively low concentration of heavy metals in fish muscles may indicate the weak or disability of muscle proteins to combine with these heavy metals (35).Furthermore, Perimusculer connective tissue around skeletal muscle bundles contains small amounts of lipids, the favorable binding site for heavy metals (36).
It is worth knowing that the concentration of both Mn and Co was higher in fish muscles of uninfected fish 5.597 and 1.007 µg/gm fresh weight, respectively than in muscles of infected fish 2.80 and 0.217 µg/gm fresh weight; respectively.The concentration of Ni was higher in the muscles of infected fish 2.20 µg/gm than in the uninfected fish 1.90 µg/gm.The total concentration mean of Mn 11.846 µg/gm was higher than that of Ni 4.094 µg/gm and Co 2.616 µg/gm, respectively.The variation in concentration of metals may relate to the fact that body tissues and organs differ in their reactions toward each metal, either discharging or accumulating, or interacting with the heavy metal (34).Duration and extent of exposure to the metal added to the portal of entry to fish tissues, climate changes (like pH, salinity, and temperature) in the aqueous environment also affect factors (37)(38)(39).
The result came to agree with that of Hassan, et al. (1), Kirin and Kuzmanova (11), Akinsanya and Kuton (12), Eira et al. (40), and Oyoo-Okoth et al. (41), those who concluded that the concentration of heavy metals in parasitic helminths is almost higher than in tissues of their host fish.As for Co, its concentration was approximately the same in the host fish and the parasitic helminths.The higher concentration of heavy metals in parasitic helminths compared to its host fish may be referred to the biomagnification of these metals through the food chain, concerning that the parasite is consumed by the fish internally.This is evident especially in metals that enter the body with engulfed water and food since the worm is an intestinal parasite.This interpretation agrees with the opinion of Chowdhury et al. (42).
Cestodes that inhabit fish intestines have no digestive system and thus absorb ready nutrients from fish intestinal contents, including the swallowed heavy metals, throughout the helminth tegument.The concentration of heavy metals would be higher in helminth tissues than the fish tissues, especially if the fish ingest nutrients rich with fatty materials (43).Furthermore, intestinal cestodes were found to be able to accumulate different heavy metals in their tissues at a variable level.This may relate to host species, type of host food, age, and absorptive surface area of the parasitic cestoda (44).

Conclusion
Mn hit the highest accumulation both in the body of C. carpioo fish and B. acheilognathi cestoda, followed by Ni and Co.There was selectivity in the accumulation of the three metals in different body organs of common carp.The highest concentration of Mn was found in gills, Ni was more concentrated in the liver, and Co was relatively more accumulated in the small intestine.Less accumulation of the three metals was observed in skeletal muscles.The concentration of the three metals was less in the tissues of the infected fish than those uninfected.The concentration of both Mn and Ni was duplicated in cestoda tissues compared with the host fish tissues, but not Co, which had an approximate concentration in fish and cestoda tissues.

Table 4 :
Bioaccumulation of manganese, nickel, and cobalt µg/gm fresh weight of Bothriocephalus acheilognathi tissues * ±0.057 * Refer to the presence of significant differences between treatments at P≤0.05, according to T-test.

Figure 1 :
Figure 1: Variation in concentrations (µg/gm fresh weight) of the three heavy metals (Mn, Ni, and Co) in B. acheilognathi cestoda and uninfected and infected C. carpio fish, respectively.

Table 1 :
Bioaccumulation of Manganese in fresh weight of Cyprinus carpio fish internal organs

Table 2 :
Bioaccumulation of Nickel in fresh weight of Cyprinus carpio fish internal organs

Table 3 :
Bioaccumulation of cobalt in fresh weight of Cyprinus carpio fish internal organs

Table 4
displays the concentration of the metals; Mn and Ni, and Co in Bothriocephalus acheilognathi tissues 22.53, 10.45,