Bioaccumulation of Cadmium, Copper, Lead, and Zinc in Water Buffaloes (Bubalusbubalis) Infected With Liver Flukes (Fasciolagigantica)

Introduction

Heavy metals are found in ample amounts in the environment due to its abundant use in industrial factories. Exposure of humans and animals via inhalation or ingestion to these heavy metals can have detrimental consequences to health. The toxicities of these heavy metals vary depending on the dose, route of exposure, and chemical species, as well as the age, gender, genetics, and nutritional status of exposed individuals¹. However, some studies indicated that certain organisms accumulate toxins at certain levels without any harmful health effects, whereas for Fasciolagigantica(F. gigantica), high exposure to lead results to tegumental deformities such as crumpling and blebbing².

Many livestock animals such as sheep and ruminants, particularly cattle, are prone to parasitic infections. Due to the quality of a parasite’s ability to bioaccumulate heavy metals in its system, these parasites may serve as potential indicators of environmental quality³. Parasitic flatworms belonging to Phylum Platyhelminthes, Class Trematoda require intermediate and definitive hosts to complete its life cycle⁴. Examples of common parasites infecting ruminants belonging to Class Trematoda are Fasciola hepatica and Fasciolagiganticacommonly known as liver flukes. These liver flukes cause fascioliasis or fasciolosis, a condition wherein the liver is infected by either or both Fasciola making them economically significant⁵. Moreover, fascioliasis is a zoonotic condition where parasites infecting animals can be transmitted to humans. Recent studies show that the two species are increasing in Europe, United States of America, Oceania, Africa, and Asia⁶. Other examples of these parasites are the intestinal acanthocephalans, nematodes, and cestodes which can accumulate heavy metals present in the environment.

Bioaccumulation of heavy metals in a trophic level may be passed on to higher trophic levels in significantly increasing amounts and can be introduced into new environments⁷. A trophic level is a part of a functioning biological community that is composed of various organisms such as primary producers, preys, and predators⁸.In the case of organisms at a higher trophic level that consume its prey having accumulated heavy metal, the predator can break down the heavy metal leading to absorption in its body. Thus, this makes the present study relevant in determining the presence of heavy metals in the liver flukes as well as in the liver tissues of water buffaloes as these serve as bioindicators. Since humans consume water buffalo products, liver tissues may be studied as bioindicators. Due to these problems involving heavy metals and liver flukes, this study determined and compared the concentrations of heavy metals, namely cadmium, copper, lead, and zinc in both water buffalo liver tissues and liver flukes, and determined the potential of the liver flukes as bioaccumulator and bioindicator.

Materials and Methods

Collection of Samples and Species Identification

Fourteen infected livers were collected from slaughtered water buffaloes in the Veterinary Inspection Board located in Tondo, Manila, Philippines. The liver samples were immediately transported to the Zoology and Parasitology Laboratory, Science and Technology Research Center, De La Salle University. Liver samples were then processed for extraction of the liver flukes and the collected flukes were placed in petri dishes for further analysis. Species identification of liver flukes was based on morphological characteristics⁶.

Heavy Metal Concentration Determination

The collected liver tissue and liver fluke samples were digested in 6.0 mL of 30% hydrogen peroxide and 12.0 mL of 60% nitric acid, and subsequently heated at around 35-40 °C until no observable material is left⁹. The samples were then filtered and the filtrate obtained was diluted to 100 mL distilled water. The diluted samples were then kept in amber bottles until atomic absorption spectrophotometric analysis was conducted.

Standards of 0.125, 0.250, 0.500, and 1.000 ppm were separately prepared for cadmium, copper, lead, and zinc. Spectrophotometric analysis of each metal was carried out using Shimadzu-6700 spectrophotometer. In each run, triplicate measurements of the metal concentration were obtained for each of the heavy metals in both the liver tissue and liver fluke samples.

Data and Statistical Analysis

Heavy metal concentrations (µg/g) were reported as median ± interquartile range. Nonparametric tests were employed to analyze the obtained experimental data. Comparisons on the heavy metal concentrations between liver tissue and liver fluke samples were performed using median test and two-sample Wilcoxon rank-sum (Mann-Whitney) test. Assessment on the differences in the bioindicator level and bioconcentration factor was done using Kruskal-Wallis equality of population rank test. All statistical analyses were performed at 1% level of significance using STATA® V12.0.

Results and Discussion

A total of 1,329 liver flukes were extracted from 14 water buffalo livers and all were identified as F. gigantica. These liver flukes were prevalent in the tropical areas and Southeast Asia including the Philippines¹⁰. Only F.gigantica was identified to infect water buffaloes¹¹, but can also coexist with Fasciola hepatica¹².

Spectrophotometric studies on liver tissue and liver fluke samples revealed presence of varying concentrations of cadmium, copper, lead, and zinc (Table 1). These heavy metal concentrations were found to be consistently higher in the liver fluke samples compared to the liver tissue samples where these flukes were extracted (Table 1). Zinc has the highest concentration, in both the liver tissue and liver fluke samples, among the four heavy metals identified in this study (Zn > Cu >Pb> Cd). The liver tissue and liver fluke samples differ with respect to their levels of heavy metal concentrations (p < 0.001, Table 2). Consequently, the significant differences in concentrations of each of the heavy metals in the liver tissue and liver fluke samples resulted to variations in the bioconcentration factor levels of these heavy metals (p=0.0026, χ²=14.253, df=3, Table 3).

Table 1: Heavy metal concentrations (µg/g) in the liver tissue and liver fluke samples

	Heavy metal concentration, median ± interquartile range (µg/g)
	Cadmium	Copper	Lead	Zinc
Samples
Liver Tissues	0.9300±0.4040	9.1290±9.6160	4.7480±3.6940	48.9540±5.1360
Fasciolagigantica	3.3158±1.1754	72.2588±44.1052	20.8246±26.0351	159.3684±69.1140

 

Table 2: Median and Mann-Whitney tests on heavy metal concentrations between the liver tissue and liver fluke samples

	Heavy Metal
	Cadmium	Copper	Lead	Zinc
Median Test
Pearson χ2	28.000	20.5714	20.5714	20.5714
p-value	0.0000	0.0000	0.0000	0.0000
Mann-Whitney Test
Rank sum
Liver Tissues	105	109	111	117
Fasciolagigantica	301	297	295	289
z-statistic	-4.505	-4.319	-4.228	-3.952
p-value	0.0000	0.0000	0.0000	0.0001

 

Table 3: Kruskal-Wallis equality of heavy metal concentration rank test on bioconcentration factor

	Heavy Metal
	Cadmium	Copper	Lead	Zinc
Bioconcentration factor
Median ± IQR	3.6484±1.201	7.9332±5.7436	4.3860±1.8657	3.3914±1.330
Rank sum	309	559	456	272

IQR: interquartile range

 

The bioconcentration factor (BCF) reflects the transfer of a heavy metal from the liver tissue to liver flukes, and was obtained by dividing the concentration of a heavy metal measured in the parasite’s tissue with the concentration of the same heavy metal in the liver tissue where the parasite was extracted¹³. All the heavy metals identified in this study had BCF values greater than 1.0 (Table 3). Copper has the highest BCF value compared to other heavy metals (Cu >Pb> Cd > Zn). With these results, the authors identified that liver flukes accumulated higher concentrations of these heavy metals than their respective hosts. This indicates the indirect impacts of the biotic effects of pollutants¹⁴. Hence, it can be ascertained that these liver flukes can serve as potential bioindicators in assessing the current conditions of an ecosystem.

Conclusion

Fasciolagigantica accumulated higher concentrations of heavy metals when compared to liver tissues where these liver flukes were extracted. These liver flukes can serve as bioaccumulators and bioindicators of environmental pollution. Considering how these concentrations of heavy metals in the flukes and tissues can be associated with the area where the water buffaloes feed, certain mitigation measures particularly rehabilitation of the area in case of heavy metal contamination should be in place. Likewise, it is recommended that the pastureland where the water buffaloes graze should be tested for the presence of other heavy metals. Furthermore, knowledge about heavy metal contamination in the adjacent trophic levels such as grass or plants which serve as food for the water buffaloes is significant due to possible adverse effects on human health.

References

1. Tchounwou, P.; Yedjou, C.; Patlolla, A.; Sutton, D. Heavy metal toxicity and the environment. A. Luch (ed.), Molecular, Clinical and Environmental Toxicology, ExperientiaSupplementum. 2012, 101,133-164.
2. Chang, A.C.G.; Flores, M.J.C. Tegumental studies of adult Fasciolagigantica (giant liver fluke) from Philippine Carabaos (Bubalusbubalis) using scanning electron microscopy for lead bio-indicator analysis. Manila Journal of Science. 2015, 11.
3. Lotfy, W.; Ezz, A.; Hassan, A. Bioaccumulation of some heavy metals in the liver flukes Fasciola hepatica and F. gigantica. Iranian Journal of Parasitology. 2013, 8, 552–558.
4. Fried, B.; Graczyck, T.K. Advances in trematode biology. Boca Raton: CRC Press, 1997.
5. Phalee, A.; Wongsawad, C.; Rojanapaibul, A.; Chai, J.Y. Experimental life history and biological characteristics of Fasciolagigantica(Digenea: Fasciolidae). The Korean Journal of Parasitology. 2015, 53(1), 59–64.
   CrossRef
6. World Health Organization (WHO). Fascioliasis. Retrieved 2016 Oct 17 from http://www.who.int/ foodborne trematode _infections/fascioliasis/en/.
7. Mitchell, K. The transfer of heavy metals through trophic levels and their toxicity effects on organisms including humans. Nottingham Trent University’s Institutional Repository.2007.
8. Branca, B.; Focazio, P.; Dunn, K. Heavy metal in the food chain.2011.Retrieved 2017 June 1 from http://www.seagrant.sunysb.edu/images/uploads/pdfs/cl-spr11.pdf.
9. Furukawa, J. Nitric acid digestion protocol. University of Tsukuba. Retrieved from https://www.plantcellwall.jp/protocol/pdf/protocol_10.pdf,2013.
10. Saleha, A.A. Liver fluke disease (fascioliasis): Epidemiology, economic impact, and public health significance. Southeast Asian J Trop Med Public Health.1991, 22, 361-364.
11. Chang, A.C.G.; Flores, M.J.C. Morphology and viability of adult Fasciolagigantica (giant liver flukes) from Philippine carabaos (Bubalusbubalis) upon in vitro exposure to lead. Asian Pacific Journal of Tropical Biomedicine.2015, 5(6), 493–496.
    CrossRef
12. Aguila, J.A.; Saldonido, G.B. Trace metal analysis of copper (Cu) and lead (Pb) in water buffaloes (Bubalusbubalis) infected with liver flukes. [undergraduate thesis], De La Salle University.2015.
13. Satpathy, D.; Reddy, M.V.; Dhal, S.P. Risk assessment of heavy metals contamination in paddy soil, plants, and grains (Oryza sativa L.) at the East Coast of India. BioMed Research International. 2014, 1–11.
    CrossRef
14. Holt, E.A.; Miller, S.W.Bioindicators: using organisms to measure environmental impacts. Nature Education Knowledge. 2010, 3(10), 8.

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