Bull 623 Al-Warid et al. Bull. Iraq nat. Hist. Mus. (2021) 16 (4): 623-634. https://doi.org/10.26842/binhm.7.2021.16.4.0623 ACCUMULATION OF SOME HEAVY METALS IN LARVAE OF CONTRACAECUM SP. AND THEIR HOST TIGRIS CATFISH SILURUS TRIOSTEGUS HECKEL, 1843 IN BAGHDAD, IRAQ Harith Saeed Al-Warid * Ahmed Saad Aldhamin *♦ and Azhar Ahmed Al-Moussawi ** * Department of Biology, College of Science, University of Baghdad, Baghdad, Iraq. ** Iraq Natural History Research Center and Museum, University of Baghdad, Baghdad, Iraq. ♦Corresponding author: e-mail: ahmed.aldhamin@sc.uobaghdad.edu.iq Received Date: 06 Nov. 2021, Accepted Date: 08 December 2021, Published Date: 20 December 2021 This work is licensed under a Creative Commons Attribution 4.0 International License ABSTRACT This study was achieved to investigate the accumulation of some heavy metals included: Cadmium, Lead and Nickel in the tissues (gill, intestine, liver, muscles and skin) of Silurus triostegus Heckel, 1843 (Siluriformes, Siluridae) and its larval stage of the nematode Contracaecum sp. (Rhabditida, Anisakidae). As well as to assess the infection patterns of Contracaecum among S. triostegus specimens which were purchased fresh from the local market in Baghdad. One hundred and nine nematodes specimens in larval stage were recovered from the fish host; the overall prevalence of Contracaecum sp. was 38.6%. The sex of the host was not significantly (P ˃ 0.05) associated with the infection of this nematode. Results showed that the overall mean intensity of Contracaecum sp. was 6.41; mean intensity did not differ significantly (P ˃ 0.05) between the fish sexes.The lead (Pb) was the only element detected in all fish tissues investigated as well as in the parasite, while the cadmium and nickel elements were not detected in all specimens. Skin and muscles of the fish, as well the parasite Contracaecum sp. contained the lowest lead levels compared to other fish tissues (gill, intestine and liver), although no significant differences were noticed among all investigated tissues and the parasite regarding the concentration of Pb. Keywords: Accumulation, Contracaecum, Heavy metals, Iraq, Silurus triostegus. INTRODUCTION Extreme action in the industrial and agricultural has undoubtedly boosted heavy element concentrations in natural water streams; as a result, determining the amounts of heavy metals https://doi.org/10.26842/binhm.7.2021.16.4.0623 https://creativecommons.org/licenses/by/4.0/ 624 Accumulation of some heavy metals in larvae in fish, especially those which are widely consumed by human, has lately got some attentions. It is critical to determine the potential harm of fish consumption to human health (Djedjibegovic et al., 2020). Many researches on heavy metal levels in the aquatic ecosystem have been published (Pandey, 2006). A wide range of creatures have been studied to see if they can be used as biological markers of various types of pollution in the aquatic environment (El-Shafei, 2015). Certain species have been discovered as being very sensitive to aquatic contaminants, either in terms of their functional response or their propensity to accumulate certain poisons in a dose-time dependent manner (Vinodhini and Narayanan, 2008). Tigris catfish, Silurus triostegus (Siluriformes, Siluridae) has been used to identify heavy metal pollution in freshwater environments and can be considered a reliable bioindicator for biomonitoring (Karadede et al., 2004; Rasheed, 2012; Jawad et al., 2020). As seen by various reviews, there has recently been growing attention to the interaction between parasitism and pollution in the aquatic ecosystem (MacKenzie et al., 1995; Aldhamin et al., 2021). Heavy metal concentrations in fish parasites have been discovered to be quite high, primarily in adult acanthocephalan worms, but also to a lesser extent in the adult cestodes (Aldhamin et al., 2021). Limited investigations were achieved regarding the accumulation of heavy metals in parasites and their fish hosts in Iraq. The current study was conducted to compare the accumulation of some haevy elements in the different tissues of S. triostegus and in its nematode Contracaecum sp., as well as to investigate the infection patterns of nematode in Tigris catfish. MATERIALS AND METHODS Specimens' collection Tigris catfish, Silurus triostegus (n=44) were purchased fresh from some local markets in Baghdad during the period between July to December 2020. Using a cooling box, the fish specimens were transferred to the laboratory in Iraq Natural History Research Center and Museum within 24 hours. The fish was identified following Coad (2010); Weight and total length were recorded for all obtained fish. The fish body weights were between 0.664 and 1.680 Kg and the total lengths were between 43 to 63 cm. The sex was identified for each fish (female, n=25; male, n=19). All fish carcasses were dissected. Approximately 5 g of the skin, gills, muscles, intestine and liver of the fish were dissected, washed by double deionized distilled water, dried with filter paper, weighed, and kept at – 20 o C until analysis (El-Shafei, 2015). Identification of nematodes The mesenteries and intestine tract of each fish were isolated, then opened and checked for nematodes. The isolated larvae of the nematodes were collected separately from body cavity and intestine of each fish, washed with saline solution (0.9% NaCl), fixed in 70% ethyl alcohol, cleared with lactophenol, and identified microscopically to a genus level according to 625 Al-Warid et al. the morphological description of Anderson (2000). All nematodes were weighted, washed, dried and preserved frozen until subsequent analysis. Heavy metal analysis The analyses of the heavy metals (Nickel, Cadmium and Lead) were done according to the procedure described by Lazarus et al. (2013). After thawing, 4 to 5 g wet weight of homogenized fish tissues and up to 1.1 g of nematodes were weighed into reaction vessels, followed by 2.5 ml hydrogen peroxide (30% H2O2) and 1.3 ml nitric acid (65% HNO3). Using the microwave digestion method, the vessels were cooked for 90 minutes at around 175°C. After digestion, the clear sample solution was transported to a 10 ml volume with deionized distilled water in a glass container. Subsequently, the concentrations of Nickel, Cadmium and Lead were analyzed using spectrometry (CARBOLITE, England). Data analysis Infection prevalence was calculated as a percentage of infected/examined hosts (Bush et al., 1997). Using Fisher's exact tests and the chi-square test in quantitative parasitology 3.0 (Rózsa et al., 2000), differences in prevalence between male and female hosts were investigated. Bush et al. (1997) defined infection intensity as the number of parasites in an infected host divided by the number of infected hosts, with 95 percent CIs of mean intensity calculated using bootstrap testing (Rózsa et al., 2000). Using a Mood's median test, mean infection intensities were statistically compared between males and females. The variance/mean ratios (s 2 /m) were used to determine parasite aggregation among hosts. QP 3.0 was used to conduct all statistical evaluations of intensity and aggregation. The variations in heavy metals accumulation among different host tissues and Contracaecum sp. were compared using a one-way ANOVA. Statistical Package for the Social Sciences was used to conduct the analysis (SPSS Inc, Chicago IL, USA). P value of less than 0.05 was considered statistically significant in all statistical analyses. RESULTS AND DISCUSSION The examination of the body cavity and intestine of the fish revealed that Contracaecum Railliet & Henry, 1912 (Rhabditida, Anisakidae) was the only genus identified. Seventeen out of 44 fish specimens (38.6%) were found parasitized with the larval stage of Contracaecum sp. (Pl. 1). This nematode taxa has been previously reported from the Tigris catfish, Silurus triostegus in different parts of Iraq (Abdulkarim and Abdullah, 2010; Al-Moussawi et al., 2018; Mhaisen and Abdul-Ameer, 2021). Mhaisen and Abdul-Ameer (2021) showed that S. triostegus was detected as a host of Contracaecum species larvae in 22 local studies, as well as, Contracaecum species has been recorded from other fish species such as Planiliza abu (Heckel, 1843), Acanthobrama marmid Heckel, 1843, Arabibarbus grypus (Heckel, 1843), Capoeta trutta (Heckel, 1843), Carasobarbus luteus (Heckel, 1843), and Chondrostoma regium (Heckel, 1843) (Mhaisen et al., 1988; Abdullah et al., 2021). Adults of different species of the genus Contracaecum were also reported from some bird species in Iraq such as Ardea alba Linnaeus, 1758, Ardea purpurea Linnaeus, 1766, Ardeola ralloides (Scopoli, 1769), Botaurus stellaris (Linnaeus, 1758), Ceryle rudis (Linnaeus, 1758), Egretta garzetta 626 Accumulation of some heavy metals in larvae (Linnaeus, 1766), and Phalacrocorax carbo (Linnaeus, 1758) (Al-Awadi et al., 2010; Al- Moussawi and Mohammad, 2011; Alqaisi et al., 2020). Sex of the host was not significantly associated with the occurrence of Contracaecum sp. (X 2 =2.14; df=1; p=0.143), although the occurrence of Contracaecum sp. among the females was higher (48%) than of the males (26.3%) (Tab.1). This result was likely due to slight differences in exposure or resistance between the sexes to some infective stages of parasites, and no differences in the diet and what containing of the possible intermediate hosts of both sexes. One hundred and nine nematodes larvae were recovered from fish specimens (range= 2-13 nematode larvae/fish). The overall mean intensity of Contracaecum sp. was 6.41. For subsets of hosts (Tab. 2, Diag. 1), the mean intensity did not change significantly between sexes (t = 0.4; bootstrap p = 0.711), although the intensity of Contracaecum sp. was slightly higher in males than females. Similarities in host susceptibility or host contact with the parasite in the same group are likely to generate such a pattern of intensity (Moravec and Jirků, 2014).Variance /mean for the larval stage of Contracaecum sp. showed an aggregated distribution of the parasite across examined fish (Tab. 2, Diag. 2). The aggregation of Contracaecum sp. showed to be high (5.64) across the fish. Moreover, the aggregation was higher in males (7.62) than females (4.72). Parasites were aggregated across the host population, with the majority (74/109, 67.88%) of the parasite population concentrated into a minority (7/44, 15.9%) of the host population. The distribution of Contracaecum sp. across the fish host in the present study is similar to the distribution of most macro parasites among their hosts (Wilson et al., 2002). The lead was the only element detected in all fish tissues investigated as well as in the parasites, while other elements (Cd and Ni) were not detected in all samples which is likely due to their low concentrations. This result agreed with Karadede and Unlu (2000), who did not detect the levels of three heavy metals in addition to Cd and Pb in the muscles of six studied cyprinids collected from the Atatürk Dam Lake on Euphrates River in Turkey, Acanthobrama marmid Heckel, 1843, Capoeta trutta (Heckel, 1843) , Carasobarbus luteus (Heckel, 1843), Chalcalburnus mossulensis (Heckel, 1843) (synonym of Alburnus mossulensis Heckel, 1843, Chondrostoma regium (Heckel, 1843) and Cyprinus carpio Linnaeus, 1758. Current result was also agreed with Suhendan et al. (2010) who showed Cadmium was not detected in Silurus triostegus and other six cyprinid fish species: Acanthobrama marmid, Aspius vorax, Capoeta trutta, Carasobarbus luteus, Chalcalburnus mossulens and Cyprinus carpio. The lead concentrations in Tigris catfish tissue samples and Contracaecum larvae specimens are illustrated in Table (3). It is evident that the skin, muscles and the nematode Contracaecum sp. biomass contained the lowest lead levels (1.16 ± 1.4) ppm, (1.4 ± 1.3) ppm and (1.4 ± 0.99) ppm respectively, compared to other tissues (intestine, liver and gills), although no significant differences (F-test= 1.32, P value= 0.26) were noticed among all 627 Al-Warid et al. investigated tissues and the parasites regarding the concentration of Pb. Of all the tissues investigated, the mean value of lead accumulation was maximum in the intestine followed by gills and liver. Lower accumulations were seen in Contracaecum sp. This result agreed with some other investigators who showed that the parasitic nematode Anguillicola crassus has less accumulated lead concentrations than their fish hosts (Sures et al., 1994; Zimmermann et al., 1999). Tenora et al. (1999, 2000) found higher amounts of metals in nematodes than in their fish hosts in their experiments. Additional influences that may affect metal accumulation include the parasite's developmental stage and the length of time it has been present in a particular host (Bergey et al., 2002). The properties of the parasite, its stage, the heavy element, the parasite's position in the host, and the host species are likely to cause differences in accumulation levels. Finally, it is concluded that Contracecum sp. is highly aggregated in Silurus triostegus as well as this worm has the ability to accumulate Pb in low levels comparing to the tissues of fish. Plate (1): Anterior (A) and Posterior (B) extremity of Contracaecum larvae recovered from S. triostegus (100X). Table (1): The infection rate of Contracaecum sp. among the males and females of S. triostegus. Population Number of examined fish Number of infected fish (%) All 44 17 (38.6%) Female 25 12 (48%) Male 19 5 (26.3%) X 2 =2.14; df =1; p=0.143 Table (2): The mean intensity and Variance/mean ratio of Contracaecum sp. among males than females of S. triostegus. Population Number of fish examined Mean intensity Var/mean ratio All 44 6.41 5.64 Female 25 6.17 4.72 Male 19 7 7.62 t = 0.4; bootstrap p = 0.711 628 Accumulation of some heavy metals in larvae Diagram (1): The intensity of Contracaecum sp. (larval stage) collected from S. triostegus. Diagram (2): Distribution of Contracaecum sp. abundance (number of parasites per host, including uninfected hosts) among n = 44 S. triostegus examined. 629 Al-Warid et al. Table (3): The mean ± SD of Pb concentrations in different tissues of S. triostegus and of Contracaecum sp. Tissue Pb concentration ± SD (ppm) Skin 1.16 ± 1.4 Gills 1.9 ± 1.16 Muscles 1.4 ± 1.3 Intestine 2.08 ± 1.2 Liver 1.7 ± 1.2 Nematodes 1.4 ± 0.99 F-test= 1.32, P value= 0.26 CONFLICT OF INTEREST STATMENT "The authors have no conflicts of interest to declare". LITERATURE CITED Abdulkarim, A. S. and Abdullah, S. M. 2010. Endoparasites of the Asian catfish Silurus triostegus (Heckel, 1843) from greater zab river-Kurdistan Region-Iraq. Journal of Dohuk University, 13(1):172-179. Abdullah, Y. S., Abdullah, S. M. A. and Hussein, R. H. 2021. Ultramorphology and molecular studies of Contracaecum larvae (Nematoda: Anisakidae) collected in five cyprinid fish species from Sulaimani Province, Kurdistan Region- Iraq. Helminthologia, 58(1): 41-58. Al-Awadi, H. M., Mhaisen, F. T. and Al-Joborae, F. F. 2010. Helminth parasitic fauna of aquatic birds in Bahr Al-Najaf depression, mid Iraq. Bulletin of the Iraq Natural History Museum, 11(2): 7-15. Aldhamin, A. S., Al-Warid, H. S. and Al-Moussawi, A. A. 2021. Helminths and their fish hosts as bioindicators of heavy metal pollution: A review. International Journal of Aquatic Science, 12(2): 3401-3408. Al-Moussawi, A. A. and Mohammad, M. K. 2011. Contracaecum rudolphii Hartwich, 1964 (Nematoda: Anisakidae) in the cormorant Phalacrocorax carbo (Linnaeus, 1758) wintering in Baghdad area: a new host record in Iraq. Bulletin of the Iraq Natural History Museum, 11(3): 7-16. Al-Moussawi, A. A., Hadi, A. M. and Macawi, Z. A. 2018. Diagnosis of some parasites of Asian catfish Silurus triostegus (Heckel, 1843). Advance in Bioresearch, 9: 86-90. Alqaisi, A. Q., Al-Warid, H. S. and Al-Moussawi, A. A. 2020. Molecular characterization of Contracaecum rudolphii Hartwich, 1964 (Nematoda: Anisakidae) from the cormorant Phalacrocorax carbo in Iraq. Bulletin of the Iraq Natural History Museum, 16(2): 135-150. 630 Accumulation of some heavy metals in larvae Anderson, R. C. 2000. Nematode parasites of vertebrates (2nd edition): Their development and transmission. CABI Publishing, Wallingford, 672 pp. Bergey, L., Weis, J. S. and Weis, P. 2002. Mercury uptake by the estuarine species Palaemonetes pugio and Fundulus heteroclitus compared with their parasites, Probopyrus pandalicola and Eustrongylides sp. Marine Pollution Bulletin, 44(10): 1046-1050. Bush, A. O., Lafferty, K. D., Lotz, J. M. and Shostak, A. W. 1997. Parasitology meets ecology on its own terms: Margolis et al. revisited. The Journal of Parasitology, 83(4): 575-583. Coad, B. W. 2010. Freshwater fishes of Iraq. Sofia-Moscow, 274pp. Djedjibegovic, J., Marjanovic, A., Tahirovic, D., Caklovica, K., Turalic, A., Lugusic, A., Omeragic, E., Sober, M. and Caklovica, F. 2020. Heavy metals in commercial fish and seafood products and risk assessment in adult population in Bosnia and Herzegovina. Scientific Reports, 10(1):1-8. El-Shafei, H. M. 2015. Some heavy metals concentration in water, muscles and gills of Tilapia niloticus as biological indicator of Manzala Lake pollution. Journal of Aquaculture Research and Development, 6(9): 1-5. Jawad, L. A., Al-Janabi, M. I. and Rutkayová, J. 2020. Directional fluctuating asymmetry in certain morphological characters as a pollution indicator: Tigris catfish (Silurus triostegus) collected from the Euphrates, Tigris, and Shatt al-Arab Rivers in Iraq. Fisheries and Aquatic Life, 28(1):18-32. Karadede, H. and Unlu, E. 2000. Concentrations of some heavy metals in water, sediment and fish species from The Ataturk Dam Lake (Euphrates), Turkey. Chemosphere, 41: 1371-1376. Karadede, H., Oymak, S. A. and Ünlü, E. 2004. Heavy metals in mullet, Liza abu, and catfish, Silurus triostegus, from the Atatürk Dam Lake (Euphrates), Turkey. Environment International, 30(2): 183-188. MacKenzie, K., Williams, H. H., Williams, B., McVicar, A. H. and Siddall, R. 1995. Parasites as indicators of water quality and the potential use of helminth transmission in marine pollution studies. Advances in Parasitology, 35: 85-144. Mhaisen, F. T. and Abdul-Ameer, K. N. 2021. Checklist of fish hosts of species of Contracaecum Railliet & Henry, 1912 (Nematoda: Ascaridida: Anisakidae) in Iraq. Biological and Applied Environmental Research, 5(2): 281-306. 631 Al-Warid et al. Mhaisen, F. T., Al‐Salim, N. K. and Khamees, N. R. 1988. Occurrence of parasites of the freshwater mugilid fish Liza abu (Heckel) from Basrah, Southern Iraq. Journal of Fish Biology, 32(4): 525-532. Moravec, F. and Jirků, M. 2014. Rhabdochona spp. (Nematoda: Rhabdochonidae) from fishes in the Central African Republic, including three new species. Folia Parasitologica, 61(2): 157-72. Rasheed, R. O. 2012. Assessment of some heavy metals in muscle tissue of Silurus triostegus from Derbendikhan reservoir, Kurdistan Region–Iraq. Rafidain Journal of Science, 23(1): 11-18. Pandey, S. 2006. Water pollution and health. Kathmandu University Medical Journal, 4(1): 128-134. Rózsa, L., Reiczigel, J. and Majoros, G. 2000. Quantifying parasites in samples of hosts. The Journal of Parasitology, 86(2): 228-32. Suhendan, M. O. L., Özden, Ö. and Oymak, S. A. 2010. Trace metal contents in fish species from Ataturk Dam Lake (Euphrates, Turkey). Turkish Journal of Fisheries and Aquatic Sciences, 10: 209-213. Sures, B., Taraschewsk, H. and Jackwerth, E. 1994. Lead content of Paratenuisentis ambiguus (Acanthocephala), Anguillicola crassus (Nematodes) and their host Anguilla anguilla. Diseases of Aquatic Organisms, 19: 105-107. Tenora, F., Baruš, V., Kráčmar, S. and Dvořáček, J. 2000. Concentrations of some heavy metals in Ligula intestinalis plerocercoids (Cestoda) and Philometra ovata (Nematoda) compared to some of their hosts (Osteichthyes). Helminthologia, 37(1): 15-18. Tenora, F., Baruš, V., Kraćmar, S., Dvořáćek, J. and Srnková, J. 1999. Parallel analysis of some heavy metals concentrations in the Anguillicola crassus (Nematoda) and the European eel Anguilla anguilla (Osteichthyes). Helminthology, 36: 79-81. Lazarus, M. V., Sekovanić, A., Kljaković-Gašpić1, Z., Orct, T., Jurasović, J., Kusak, J., Reljić, S. and Huber, D. 2013. Cadmium and lead in grey wolf liver samples: Optimisation of a microwave-assisted digestion method. Archives of Industrial Hygiene and Toxicology, 64(3): 395-402. Vinodhini, R. and Narayanan, M. 2008. Bioaccumulation of heavy metals in organs of fresh water fish Cyprinus carpio (Common carp). International Journal of Environmental Science and Technology, 5(2): 179-182. 632 Accumulation of some heavy metals in larvae Wilson, K., Bjørnstad, O. N., Dobson, A. P., Merler, S., Poglayen, G., Randolph, S. E. and Skorping, A. 2002. Heterogeneities in macroparasite infections: patterns and processes. The ecology of Wildlife Diseases, 44: 6-44. Zimmermann, S., Sures, B. and Taraschewski, H. 1999. Experimental studies on lead accumulation in the eel-specific endoparasites Anguillicola crassus (Nematoda) and Paratenuisentis ambiguus (Acanthocephala) as compared with their host, Anguilla anguilla. Archives of Environmental Contamination and Toxicology, 37(2): 190-195. 633 Al-Warid et al. Bull. Iraq nat. Hist. Mus. (2021) 16 (4): 623-634. .Contracaecum spتراكم بعض العناصر الثقيلة في يرقة الدودة الخيطية Silurus triostegus Heckel, 1843 ومضيفها سمكة جري دجلة بغداد، العراق في **و أزهار احمد املوسوي *احمد سعد الضامن ،حارث سعيد جعفر الورد* .عراققسم علوم الحياة، كلية العلوم، جامعة بغداد، بغداد، ال* .مركز بحوث و متحف التاريخ الطبيعي، جامعة بغداد، بغداد، العراق** 2021/ 12/ 20، تاريخ النشر: 2021/ 12/ 08، تاريخ القبول: 2021/ 11/ 06تاريخ االستالم: الخالصة أجريت هذه الدراسة للتعرف على تراكم بعض املعادن الثقيلة )الكادميوم انسجة )الخياشيم واألمعاء والكبد والعضالت والجلد( والرصاص والنيكل( في بعض Silurus triostegus Heckel ,1843 (Siluriformes, Siluridae)سمكة جري دجلة املعزولة Contracaecum sp. (Rhabditida, Anisakidae)والطوراليرقي للدودة الخيطية االصابة بالدودة من التجويف الجسمي واالمعاء لهذه االسماك. تم تقييم أنماط والتي تم شراؤها من السوق املحلي في S. triostegus من اسماك عينة( 44الخيطية في ) من هذه .Contracaecum spيرقات الدودة الخيطية عينة ل( 109) و عزل بغداد ظهر ، و ٪38.6 الدودة الخيطية االسماك. بلغت النسبة املئوية لالصابة بيرقات ُ لم ت بين جنس االسماك و االصابة بيرقة الدودة الخيطية ) ً معنوية ً (.P ˃ 0.05النتائج عالقة . لم6.41كما أوضحت النتائج أن متوسط كثافة االصابة بيرقة الدودة الخيطية بلغ ( ً ( بين الجنسين. كان الرصاص هو P 0 ˃.05يختلف متوسط شدة االصابة معنويا 634 Accumulation of some heavy metals in larvae شافه في جميع أنسجة األسماك التي خضعت للفحص العنصر الوحيد الذي تم اكت وكذلك في يرقة الدودة ، بينما لم يتم اكتشاف العناصر األخرى في جميع العينات. على أقل مستويات .Contracaecum spاحتوى جلد وعضالت األسماك ، وكذلك الرغم الرصاص مقارنة بأالنسجة األخرى لالسماك )الخياشيم واألمعاء والكبد( ، على ( بين جميع األنسجة التي تم فحصها واليرقة P ˃ 0.05من عدم وجود فروق معنوية ) فيما يتعلق بتركيز الرصاص.