- Email: [email protected]
Influence of parasitism on the use of small terrestrial rodents in environmental pollution monitoring
Environmental Pollution 157 (2009) 2584–2586
Contents lists available at ScienceDirect
Environmental Pollution journal homepage: www.elsevier.com/locate/envpol
Short communication
Influence of parasitism on the use of small terrestrial rodents in environmental pollution monitoring Ivana Jankovska´ a, *, Daniela Miholova´ b, Iva Langrova´ a, Vladimı´r Bejcˇek c, Jaroslav Vadlejch a, Dana Kolihova´ b, Miloslav Sˇulc b a b c
Department of Zoology and Fisheries, Faculty of Agrobiology, Food and Natural Resources, Czech University of Life Sciences, Kamycka 129, 165 21 Prague 6 – Suchdol, Czech Republic Department of Chemistry, Faculty of Agrobiology, Food and Natural Resources, Czech University of Life Sciences, Kamycka 129, 165 21 Prague 6 – Suchdol, Czech Republic Department of Ecology, Faculty of Environmental Sciences, Czech University of Life Sciences, Kamycka 129, 165 21 Prague 6 – Suchdol, Czech Republic
Liver tissue from voles infected by Paranoplocephala dentata was less suitable as a biomonitor for metal contamination than kidney tissue.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 October 2008 Received in revised form 2 April 2009 Accepted 11 April 2009
Bioaccumulation of cadmium, chromium, copper, manganese, nickel, lead and zinc in small terrestrial rodents – voles and their cestode parasite Paranoplocephala dentata was studied. Contents of Pb, Mn, Ni and Zn in the parasite were found to be higher than in the kidney and liver of the parasitized animals. Lead level in the cestode was 37 fold higher than in the liver of the infected rodents. Bioaccumulation factors of zinc, nickel and manganese in the cestode are mostly in the range from 2 to 4.5. Considering the different contents of manganese and zinc in livers of non-parasitized and parasitized rodents, kidney tissue was found to be more reliable than liver as an indicator of environmental pollution by manganese and zinc; the kidneys of parasitized animals showed no significant change in the concentrations of those elements that are accumulated in the cestode. Ó 2009 Elsevier Ltd. All rights reserved.
Keywords: Heavy metal Bioaccumulation Small terrestrial rodent Parasite AAS
1. Introduction Small terrestrial rodents have been used in the monitoring of environmental pollution by heavy metals for a long time (Anderson et al., 1982; Fendick et al., 1989; Korolova et al., 1999; Ieradi et al., 2003; Sa´nchez-Chardi and Nadal, 2007). Tissues of the animals are used as biomonitors regardless of the knowledge of possible infection by parasites, which is very frequent (Klimpel et al., 2007). Certain parasites can accumulate some trace elements at concentrations that are orders of magnitude higher than those in the host tissues (Sures et al., 1999, 2000a,b; Barusˇ et al., 2003; Torres et al., 2004, 2006; Eira et al., 2005). Therefore, the use of tissues from infected animals in pollution monitoring studies could produce biased concentrations of such elements. The aim of the present study was to assess the level of environmental pollution using small terrestrial rodents (voles) and their helminths as biomonitors for input of heavy metals in natural field conditions. Moreover, the impact of the parasite burden on bioaccumulation of heavy metals in some tissues of
* Corresponding author. Tel.: þ420 224382793. E-mail address: [email protected] (I. Jankovska´). 0269-7491/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.envpol.2009.04.008
voles was studied. This information can be important for the use of rodent tissues in the monitoring of environmental pollution, if no information about possible infection of rodents by parasites is available. 2. Material and methods 2.1. Study area and sampling The small terrestrial rodents (voles) were collected in June 2005 at the polluted area in Krusˇne´ hory (Northwest Bohemia), where pollutants from petrochemical industry and brown coal power plants are common. They were caught by snap traps and stored in plastic bags and kept frozen (20 C) until laboratory analyses were performed. The temperature of cadavers was increased to 0 C before they were dissected for removal of the liver, kidneys and digestive tract. The digestive tract was investigated according to standard helminthological procedures. The liver and kidneys were taken from each rodent and stored frozen for analysis. The collection was formed by 29 bank voles (Clethrionomys glareolus) and 31 field voles (Microtus agrestris). Among other things, these species differ in terms of habitat use and diet, which may account for a different exposition to pollution. In fact, bank voles (C. glareolus) feed on plants, insects and carrion, whereas field voles (M. agrestris) consume wetland plants only. Forty-one animals (68.3%) were infected by helminths. The number of animals infected by both cestode Paranoplocephala dentata and nematode Heligmosomoides polygyrus, or by H. polygyrus only was insufficient to allow for any statistical evaluation (n 5). Therefore, 24 voles infected by P. dentata and 19 without cestode were selected for this study, corresponding to a total of 43 voles (19 C. glareolus and 24 M. agrestris).
´ et al. / Environmental Pollution 157 (2009) 2584–2586 I. Jankovska Table 1 Detection limits (ng ml1) and results of the quality assessment of analytical data by simultaneous analysis of certified reference material CRM 12-02-01 (Bovine liver) (mg g1). Value Detection limit (mean 3SD of blanks) CRM 12-02-01 (Bovine liver) Certified Found
Cd
Cr
0.07
0.17
Mean CI (95%)
0.48 0.03
–
Mean SD
0.46 0.05
Cu
Mn
Ni
Pb
Zn
0.37
0.21
0.21
6.5
26.3 1.6
7.6 0.5
–
0.71 0.08
162 6
25.7 0.9
7.5 0.6
0.69 0.11
160 5
0.69
2585
3.1. Bioaccumulation of heavy metals in P. dentata As can be seen in Table 2, lead, manganese, nickel and zinc were accumulated more in P. dentata than in host tissues. P. dentata presented 7.3 and 37 times more lead than was determined in the kidney and liver of M. agrestris, and 4.7 and 36 times more than was detected in the kidney and liver of C. glareolus. Though the bioaccumulation of zinc, manganese and nickel in cestodes is not so high, levels of manganese in P. dentata in comparison with those in liver were around 2 times higher, and with those in kidney 4 times higher. Similarly as reported by Torres et al. (2004, 2006), no accumulation of cadmium was observed in the cestode in this study. 3.2. Differences in heavy metal contents in tissues of parasitized and non-parasitized rodents
2.2. Determination of trace elements in animal tissues and parasites The contents of cadmium, chromium, copper, lead, manganese, nickel and zinc were determined in digested samples of kidney and liver of rodents, and parasites by atomic absorption spectrometry. Frozen samples of tissues (0.2–5 g of wet material) were dried by lyophilisation using the LYOVAC GT 2 (LEYBOLD-HERAEUS, GmbH, FRG) – average content of dryness was 32/25% in liver/kidney – and decomposed by dry ashing procedure in the Dry Mineralizer Apion (Tessek, Ltd., CZ) in the presence of a mixture of gases – O2, O3, and NOx as described by Miholova´ et al. (1993). Off-white ash obtained was leached with 1.5% nitric acid prepared from HNO3 65%, p.a. ISO (Merck) and deionised water (Barnstead). The concentrations of analytes in the digests were measured by ET-AAS technique using Varian AA 280Z (Varian, Australia) with GTA 120 (Cd, Cr, Ni and Pb), and F-AAS using Varian SpectrAA 110 (Cu, Mn, and Zn). The analytical procedure was validated using certified reference material CRM 12-02-01 (Bovine liver). Analytical blanks prepared under the same conditions, but without samples, were used to determine the analytical detection limit. Eleven replicates of reference material representing 4% of the analyzed samples were used along with 44 blanks representing 17% of the samples analyzed in this study. The detection limits (mean 3SD of blanks) for determined elements and the results of analytical quality assessment are given in Table 1. Samples of animal tissues were analyzed in two replicates; the number of replicates in the case of parasites depended on the sample quantity. If overall mass of a sample was less than 5 mg dry weight, the sample was analyzed only once. Bioaccumulation factors (BFs) in the cestode were calculated as the ratio of the metal concentration in the parasite to those in different tissues of parasitized animal as proposed by Sures et al. (1999). 2.3. Statistical evaluation The data were evaluated in the Microsoft Excel program and in Statistica 7.0 (Mann–Whitney U-test). The results are expressed as median, minimum and maximum from total range within the groups.
3. Results and discussion All results for heavy metal contents determined in analyzed tissues of voles and their parasite are given in Table 2.
With respect to the differences in the feeding habits of both vole groups data of individual animal groups were evaluated separately (Table 2). The accumulation of Pb, Mn, Ni and Zn in P. dentata is accompanied by significant decrease of zinc content in liver of M. agrestris only; no significant decrease of lead and nickel concentrations was found in tissues of infected groups in comparison with non-infected animals. As bioaccumulation of cadmium in the cestode was not observed, significant differences between Cd contents found in the tissues of both animal groups cannot be connected with infection by P. dentata. Interesting results were found in the case of manganese. Along with accumulation of manganese in the cestode, a significant increase of Mn content in livers of parasitized M. agrestris was found. Although not significant, there was an increase in the liver manganese concentration of parasitized C. glareolus. Manganese is an essential nutrient required for proper growth and maintenance of many biological systems. However, at high concentration it is neurotoxic. The transport of Mn across the blood-brain barrier, predominantly Mn speciation, is the subject of recent studies (Fitsanakis et al., 2007; Michalke et al., 2007). This is the reason why the movement of Mn in the environment is to be monitored. The increase of manganese content in liver of parasitized animals may be caused by some defence reaction of the infected organism in which manganese compounds participate. The role of manganese as a part of enzymes participating in processes proceeding in the liver is very complex. Our study does not allow us to specify a type of reaction that might result in an increase of the Mn concentration in the liver of voles infected by
Table 2 Concentrations (mg g1 dry weight) of selected elements in cestode Paranoplocephala dentata and tissues of parasitized and non-parasitized rodents. Asterisks denote a significant difference (*p < 0.05, **p < 0.01) between non-parasitized and parasitized rodents calculated by Mann–Whitney test. BF (bioaccumulation factor) ¼ concentration in cestode (median)/concentration in tissue (median). Rodent
Element
Non-parasitized animals
Parasitized animals Liver
Kidney
P. dentata
BFliver
BFkidney
Microtus agrestris
Cd Cr Cu Mn Ni Pb Zn
0.056** 0.097 16.41 13.29** 0.095 0.223 98.91*
(0.051–0.226) (0.037–0.107) (15.59–19.79) (9.31–13.95) (0.072–0.363) (0.152–0.311) (92.49–110.0)
0.138** 0.034 21.96 8.02 0.094 0.975 89.05
(0.125–0.469) (0.027–0.037) (18.23–24.78) (5.74–8.93) (0.085–0.241) (0.858–1.349) (84.55–94.89)
0.186** (0.078–1.280) 0.077 (0.028–0.327) 13.90 (12.58–17.49) 20.11** (14.00–23.15) 0.085 (0.048–0.181) 0.173 (0.054–0.399) 87.67* (69.4–99.29))
0.593** 0.063 19.71 7.95 0.130 0.884 85.19
(0.276–7.641) (0.024–0.673) (16.30–23.96) (6.77–18.16) (0.075–0.345) (0.483–2.701) (75.84–98.16)
0.195 0.054 16.22 34.73 0.367 6.42 183.8
(0.102–0.360) (0.050–0.101) (6.33–45.71) (28.98–99.03) (0.350–0.506) (4.67–11.43) (156.0–199.7)
1.01 0.70 1.20 1.7 4.3 37.0 2.1
0.33 0.85 0.83 4.4 2.8 7.3 2.1
Clethrionomys glareolus
Cd Cr Cu Mn Ni Pb Zn
2.03* 0.128 16.44 10.49 0.235 0.142 105.5
(0.869–3.729) (0.053–0.196) (14.04–18.65 (9.35–12.20) (0.125–0.466) (0.038–1.570) (90.93–125.0)
12.56** 0.065 19.69 7.60 0.372 0.374 98.13
(8.11–19.24) (0.010–0.204) (18.52–20.84) (6.36–8.87) (0.272–1.056) (0.122–2.017) (87.68–114.0)
1.186* (0.686–1.277) 0.074 (0.003–0.080) 15.03 (13.93–19.44) 11.80 (9.06–12.71) 0.235 (0.192–0.292) 0.126 (0.036–0.276) 103.0 (97.89–115.6)
5.54** 0.082 19.91 7.97 0.532 0.966 84.40
(1.13–6.35) (0.012–0.088) (18.44–41.16) (4.21–12.09) (0.182–0.573) (0.235–1.105) (72.75–120.8)
0.349 0.045 11.18 26.15 0.481 4.54 182.4
(0.305–3.641) (0.038–0.107) (9.79–24.00) (22.69–76.23) (0.350–0.699) (1.60–6.83) (90.7–199.0)
0.29 0.58 0.74 2.2 2.0 36.0 1.8
0.06 0.53 0.56 3.3 0.9 4.7 2.2
Liver
Kidney
2586
I. Jankovska´ et al. / Environmental Pollution 157 (2009) 2584–2586
cestodes. However, it is known that manganese superoxide dismutase – Mn(SOD) active in antioxidative reactions is present in the human parasite Clonorchis sinenses (Li et al., 2005). Frisk et al. (2007) studied sequential trace element changes in serum and blood during a common viral infection in mice and found not only an expected increase in copper and zinc concentrations both in serum and blood after infection of mice by myocarditic CB3 virus, but the temporary increase of manganese concentration, as well. 4. Conclusion As mentioned by Sures (2004), more field and experimental studies are required to evaluate the relationship between bioaccumulation in cestode parasites of rodents and environmental metal exposure. We present M. agrestris/P. dentata and C. glareolus/ P. dentata as useful models for the evaluation of environmental lead exposure in terrestrial habitats, especially outside urban areas. These could be promising bioindication systems complementary to the models Rattus norvegicus/Hymenolepis diminuta (Sures et al., 2002, 2003), Apodemus sylvaticus/Gallegoides arfaai (Torres et al., 2004) and A. sylvaticus/Skrjabinotaenia lobata (Torres et al., 2006). The models evaluated in this study could also be promising bioindication systems for Mn, Ni and Zn, since higher concentrations were detected in P. dentata when comparing to kidney and liver tissues of the parasitized rodents. Since there are no significant differences in contents of lead and nickel in the liver and kidney of the non-parasitized voles and those parasitized by P. dentata, both tissues can be used in monitoring of environmental pollution by Pb and Ni as well as Cd, Cr and Cu. Because of the significant differences in the contents of manganese and zinc in livers of non-parasitized and parasitized M. agrestris, liver is less reliable as an indicator of environmental pollution by Mn and Zn than kidney tissue. Conversely, when comparing parasitized and non-parasitized animals, there were no significant differences in kidney concentrations of those elements that are accumulated in P. dentata (Pb, Mn, Ni and Zn), indicating that kidney tissue is more reliable to assess trace element pollution, thus avoiding the potential bias caused by cestode infection. Acknowledgements This study was supported by project MSM 6046070901 of the Ministry of Education, Youths and Sports, Czech Republic and by project No. 524/06/0687 of the Grant Agency of the Czech Republic. The authors declare that the experiments comply with the current laws of the Czech Republic, in which they were performed. References Anderson, T.J., Barret, G.W., Clark, C.S., Alia, V.J., 1982. Metal concentrations in tissues of meadow voles from sewage sludge treated fields. Journal of Environmental Quality 11, 272–277.
Barusˇ, V., Tenora, F., Sˇumbera, R., 2003. Relative concentrations of four heavy metals in the parasites Protospirura muricola (Nematoda) and Inermicapsifer arvicanthidis (Cestoda) in their definitive host silvery mole-rat (Heliophobius argenteocinereus: Rodentia). Helminthologia 40, 227–232. Eira, C., Torres, J., Vingada, J., Miquel, J., 2005. Concentration of some toxic elements in Oryctolagus cuniculus and in its intestinal cestode Mosgovoyia ctenoides, in Dunas de Mira (Portugal). Science of Total Environment 346, 81–86. Fendick, E.A., Stevens, G.L., Brown, R.J., Jordan, W.P., 1989. Element content in tissues of four rodent species sampled in the Geysers geothermal steamfield. Environmental Pollution 58, 155–178. Fitsanakis, V.A., Piccola, G., dos Santos, A.P., Aschner, J.L., Aschner, M., 2007. Putative proteins involved in manganese transport across the blood-brain barrier. Human Experimental Toxicology 26, 295–302. Frisk, P., Darnerud, P.O., Friman, G., Blomberg, J., Ilba¨ck, N.G., 2007. Sequential trace element changes in serum and blood during common viral infection in mice. Journal of Trace Elements in Medicine and Biology 21, 29–36. Ieradi, L.A., Zima, J., Allegra, F., Kotla´nova´, E., Campanella, L., Grossi, R., Cristaldi, M., 2003. Evaluation of genotoxic damage in wild rodents from a polluted area in the Czech Republic. Folia Zoologica 52 (1), 57–66. Klimpel, S., Forster, M., Schmabl, G., 2007. Parasites of two abundant sympatric rodent species in relation to host phylogeny and ecology. Parasitology Research 100, 867–875. Korolova, E., Miholova´, D., Mader, P., Cibulka, J., Sla´mova´, A., 1999. Small mammals as bioindicators for terrestrial ecosystems in Bohemia. In: Peakall (Ed.), Biomarkers: a Pragmatic Basis for Remediation of Severe Pollution in Eastern Europe. Kluwer Academic Publishers (NL), pp. 308–309. Li, A.H., Kong, Y., Cho, S.H., Na, B.K., Pak, J.K., Kim, T.S., 2005. Molecular cloning and characterization of the copper/zinc and manganese superoxide dismutase genes from the human parasite Clonorchis sinenses. Parasitology 130, 687–697. Michalke, B., Berthele, A., Mistriotis, P., Ochsenkiihn-Petropoulou, M., Halbach, S., 2007. Manganese species from human serum, cerebrospinal fluid analysed by size exclusion chromatography-, capillary electrophoresis coupled plasma mass spectrometry. Journal of Trace Elements in Medicine and Biology 21, 4–9. Miholova´, D., Mader, P., Sza´kova´, J., Sla´mova´, A., Svatosˇ, Z., 1993. Czechoslovakian biological certified reference materials and their use in the analytical quality assurance system in a trace element laboratory. Fresenius Journal of Analytical Chemistry 345, 256–260. Sa´nchez-Chardi, A., Nadal, J., 2007. Bioaccumulation of metals and effects of landfill pollution in small mammals. Part I. The greater white-toothed shrew, Crocidura russula. Chemosphere 68, 703–711. Sures, B., 2004. Environmental parasitology: relevancy of parasites in monitoring environmental pollution. Trends in Parasitology 20, 170–177. Sures, B., Siddall, R., Taraschewski, H., 1999. Parasites as accumulation indicators of heavy metal pollution. Parasitology Today 15, 16–21. Sures, B., Ju¨rges, G., Taraschewski, H., 2000a. Accumulation and distribution of lead in the acanthocephalan Moniliformis moniliformis from experimental infected rats. Parasitology 121, 427–433. Sures, B., Franken, M., Taraschewski, H., 2000b. Element concentrations in the archiacanthocephalan Macracanthorhynchus hirudinaceus compared with those in the porcine host from a slaughterhouse in La Paz, Bolivia. International Journal of Parasitology 30, 1071–1076. Sures, B., Grube, K., Taraschewski, H., 2002. Experimental studies on the lead accumulation in the cestode Hymenolepis diminuta and its final host Rattus norvegicus. Ecotoxicology 11, 365–368. Sures, B., Scheible, T., Bashtar, A.R., Taraschewski, H., 2003. Lead concentrations in Hymenolepis diminuta adults and Taenia taeniaeformis larvae compared to their rat hosts (Rattus norvegicus) sampled from the city of Cairo, Egypt. Parasitology 127, 483–487. Torres, J., de Lapuente, J., Eira, C., Nadal, J., 2004. Cadmium and lead concentrations in Gallegoides arfaai (Cestoda Anoplocephalidae) and Apodemus sylvaticus (Rodentia: Muridae) from Spain. Parasitology Research 94, 468–470. Torres, J., Peig, J., Eira, C., Borras, M., 2006. Cadmium and lead concentrations in Skrjabinotaenia lobata (Cestoda: Catenotaeniidae) and its host, Apodemus sylvaticus (Rodentia: Muridae), in the urban dumping side of Garraf (Spain). Environmental Pollution 143, 4–8.
Contents lists available at ScienceDirect
Environmental Pollution journal homepage: www.elsevier.com/locate/envpol
Short communication
Influence of parasitism on the use of small terrestrial rodents in environmental pollution monitoring Ivana Jankovska´ a, *, Daniela Miholova´ b, Iva Langrova´ a, Vladimı´r Bejcˇek c, Jaroslav Vadlejch a, Dana Kolihova´ b, Miloslav Sˇulc b a b c
Department of Zoology and Fisheries, Faculty of Agrobiology, Food and Natural Resources, Czech University of Life Sciences, Kamycka 129, 165 21 Prague 6 – Suchdol, Czech Republic Department of Chemistry, Faculty of Agrobiology, Food and Natural Resources, Czech University of Life Sciences, Kamycka 129, 165 21 Prague 6 – Suchdol, Czech Republic Department of Ecology, Faculty of Environmental Sciences, Czech University of Life Sciences, Kamycka 129, 165 21 Prague 6 – Suchdol, Czech Republic
Liver tissue from voles infected by Paranoplocephala dentata was less suitable as a biomonitor for metal contamination than kidney tissue.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 October 2008 Received in revised form 2 April 2009 Accepted 11 April 2009
Bioaccumulation of cadmium, chromium, copper, manganese, nickel, lead and zinc in small terrestrial rodents – voles and their cestode parasite Paranoplocephala dentata was studied. Contents of Pb, Mn, Ni and Zn in the parasite were found to be higher than in the kidney and liver of the parasitized animals. Lead level in the cestode was 37 fold higher than in the liver of the infected rodents. Bioaccumulation factors of zinc, nickel and manganese in the cestode are mostly in the range from 2 to 4.5. Considering the different contents of manganese and zinc in livers of non-parasitized and parasitized rodents, kidney tissue was found to be more reliable than liver as an indicator of environmental pollution by manganese and zinc; the kidneys of parasitized animals showed no significant change in the concentrations of those elements that are accumulated in the cestode. Ó 2009 Elsevier Ltd. All rights reserved.
Keywords: Heavy metal Bioaccumulation Small terrestrial rodent Parasite AAS
1. Introduction Small terrestrial rodents have been used in the monitoring of environmental pollution by heavy metals for a long time (Anderson et al., 1982; Fendick et al., 1989; Korolova et al., 1999; Ieradi et al., 2003; Sa´nchez-Chardi and Nadal, 2007). Tissues of the animals are used as biomonitors regardless of the knowledge of possible infection by parasites, which is very frequent (Klimpel et al., 2007). Certain parasites can accumulate some trace elements at concentrations that are orders of magnitude higher than those in the host tissues (Sures et al., 1999, 2000a,b; Barusˇ et al., 2003; Torres et al., 2004, 2006; Eira et al., 2005). Therefore, the use of tissues from infected animals in pollution monitoring studies could produce biased concentrations of such elements. The aim of the present study was to assess the level of environmental pollution using small terrestrial rodents (voles) and their helminths as biomonitors for input of heavy metals in natural field conditions. Moreover, the impact of the parasite burden on bioaccumulation of heavy metals in some tissues of
* Corresponding author. Tel.: þ420 224382793. E-mail address: [email protected] (I. Jankovska´). 0269-7491/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.envpol.2009.04.008
voles was studied. This information can be important for the use of rodent tissues in the monitoring of environmental pollution, if no information about possible infection of rodents by parasites is available. 2. Material and methods 2.1. Study area and sampling The small terrestrial rodents (voles) were collected in June 2005 at the polluted area in Krusˇne´ hory (Northwest Bohemia), where pollutants from petrochemical industry and brown coal power plants are common. They were caught by snap traps and stored in plastic bags and kept frozen (20 C) until laboratory analyses were performed. The temperature of cadavers was increased to 0 C before they were dissected for removal of the liver, kidneys and digestive tract. The digestive tract was investigated according to standard helminthological procedures. The liver and kidneys were taken from each rodent and stored frozen for analysis. The collection was formed by 29 bank voles (Clethrionomys glareolus) and 31 field voles (Microtus agrestris). Among other things, these species differ in terms of habitat use and diet, which may account for a different exposition to pollution. In fact, bank voles (C. glareolus) feed on plants, insects and carrion, whereas field voles (M. agrestris) consume wetland plants only. Forty-one animals (68.3%) were infected by helminths. The number of animals infected by both cestode Paranoplocephala dentata and nematode Heligmosomoides polygyrus, or by H. polygyrus only was insufficient to allow for any statistical evaluation (n 5). Therefore, 24 voles infected by P. dentata and 19 without cestode were selected for this study, corresponding to a total of 43 voles (19 C. glareolus and 24 M. agrestris).
´ et al. / Environmental Pollution 157 (2009) 2584–2586 I. Jankovska Table 1 Detection limits (ng ml1) and results of the quality assessment of analytical data by simultaneous analysis of certified reference material CRM 12-02-01 (Bovine liver) (mg g1). Value Detection limit (mean 3SD of blanks) CRM 12-02-01 (Bovine liver) Certified Found
Cd
Cr
0.07
0.17
Mean CI (95%)
0.48 0.03
–
Mean SD
0.46 0.05
Cu
Mn
Ni
Pb
Zn
0.37
0.21
0.21
6.5
26.3 1.6
7.6 0.5
–
0.71 0.08
162 6
25.7 0.9
7.5 0.6
0.69 0.11
160 5
0.69
2585
3.1. Bioaccumulation of heavy metals in P. dentata As can be seen in Table 2, lead, manganese, nickel and zinc were accumulated more in P. dentata than in host tissues. P. dentata presented 7.3 and 37 times more lead than was determined in the kidney and liver of M. agrestris, and 4.7 and 36 times more than was detected in the kidney and liver of C. glareolus. Though the bioaccumulation of zinc, manganese and nickel in cestodes is not so high, levels of manganese in P. dentata in comparison with those in liver were around 2 times higher, and with those in kidney 4 times higher. Similarly as reported by Torres et al. (2004, 2006), no accumulation of cadmium was observed in the cestode in this study. 3.2. Differences in heavy metal contents in tissues of parasitized and non-parasitized rodents
2.2. Determination of trace elements in animal tissues and parasites The contents of cadmium, chromium, copper, lead, manganese, nickel and zinc were determined in digested samples of kidney and liver of rodents, and parasites by atomic absorption spectrometry. Frozen samples of tissues (0.2–5 g of wet material) were dried by lyophilisation using the LYOVAC GT 2 (LEYBOLD-HERAEUS, GmbH, FRG) – average content of dryness was 32/25% in liver/kidney – and decomposed by dry ashing procedure in the Dry Mineralizer Apion (Tessek, Ltd., CZ) in the presence of a mixture of gases – O2, O3, and NOx as described by Miholova´ et al. (1993). Off-white ash obtained was leached with 1.5% nitric acid prepared from HNO3 65%, p.a. ISO (Merck) and deionised water (Barnstead). The concentrations of analytes in the digests were measured by ET-AAS technique using Varian AA 280Z (Varian, Australia) with GTA 120 (Cd, Cr, Ni and Pb), and F-AAS using Varian SpectrAA 110 (Cu, Mn, and Zn). The analytical procedure was validated using certified reference material CRM 12-02-01 (Bovine liver). Analytical blanks prepared under the same conditions, but without samples, were used to determine the analytical detection limit. Eleven replicates of reference material representing 4% of the analyzed samples were used along with 44 blanks representing 17% of the samples analyzed in this study. The detection limits (mean 3SD of blanks) for determined elements and the results of analytical quality assessment are given in Table 1. Samples of animal tissues were analyzed in two replicates; the number of replicates in the case of parasites depended on the sample quantity. If overall mass of a sample was less than 5 mg dry weight, the sample was analyzed only once. Bioaccumulation factors (BFs) in the cestode were calculated as the ratio of the metal concentration in the parasite to those in different tissues of parasitized animal as proposed by Sures et al. (1999). 2.3. Statistical evaluation The data were evaluated in the Microsoft Excel program and in Statistica 7.0 (Mann–Whitney U-test). The results are expressed as median, minimum and maximum from total range within the groups.
3. Results and discussion All results for heavy metal contents determined in analyzed tissues of voles and their parasite are given in Table 2.
With respect to the differences in the feeding habits of both vole groups data of individual animal groups were evaluated separately (Table 2). The accumulation of Pb, Mn, Ni and Zn in P. dentata is accompanied by significant decrease of zinc content in liver of M. agrestris only; no significant decrease of lead and nickel concentrations was found in tissues of infected groups in comparison with non-infected animals. As bioaccumulation of cadmium in the cestode was not observed, significant differences between Cd contents found in the tissues of both animal groups cannot be connected with infection by P. dentata. Interesting results were found in the case of manganese. Along with accumulation of manganese in the cestode, a significant increase of Mn content in livers of parasitized M. agrestris was found. Although not significant, there was an increase in the liver manganese concentration of parasitized C. glareolus. Manganese is an essential nutrient required for proper growth and maintenance of many biological systems. However, at high concentration it is neurotoxic. The transport of Mn across the blood-brain barrier, predominantly Mn speciation, is the subject of recent studies (Fitsanakis et al., 2007; Michalke et al., 2007). This is the reason why the movement of Mn in the environment is to be monitored. The increase of manganese content in liver of parasitized animals may be caused by some defence reaction of the infected organism in which manganese compounds participate. The role of manganese as a part of enzymes participating in processes proceeding in the liver is very complex. Our study does not allow us to specify a type of reaction that might result in an increase of the Mn concentration in the liver of voles infected by
Table 2 Concentrations (mg g1 dry weight) of selected elements in cestode Paranoplocephala dentata and tissues of parasitized and non-parasitized rodents. Asterisks denote a significant difference (*p < 0.05, **p < 0.01) between non-parasitized and parasitized rodents calculated by Mann–Whitney test. BF (bioaccumulation factor) ¼ concentration in cestode (median)/concentration in tissue (median). Rodent
Element
Non-parasitized animals
Parasitized animals Liver
Kidney
P. dentata
BFliver
BFkidney
Microtus agrestris
Cd Cr Cu Mn Ni Pb Zn
0.056** 0.097 16.41 13.29** 0.095 0.223 98.91*
(0.051–0.226) (0.037–0.107) (15.59–19.79) (9.31–13.95) (0.072–0.363) (0.152–0.311) (92.49–110.0)
0.138** 0.034 21.96 8.02 0.094 0.975 89.05
(0.125–0.469) (0.027–0.037) (18.23–24.78) (5.74–8.93) (0.085–0.241) (0.858–1.349) (84.55–94.89)
0.186** (0.078–1.280) 0.077 (0.028–0.327) 13.90 (12.58–17.49) 20.11** (14.00–23.15) 0.085 (0.048–0.181) 0.173 (0.054–0.399) 87.67* (69.4–99.29))
0.593** 0.063 19.71 7.95 0.130 0.884 85.19
(0.276–7.641) (0.024–0.673) (16.30–23.96) (6.77–18.16) (0.075–0.345) (0.483–2.701) (75.84–98.16)
0.195 0.054 16.22 34.73 0.367 6.42 183.8
(0.102–0.360) (0.050–0.101) (6.33–45.71) (28.98–99.03) (0.350–0.506) (4.67–11.43) (156.0–199.7)
1.01 0.70 1.20 1.7 4.3 37.0 2.1
0.33 0.85 0.83 4.4 2.8 7.3 2.1
Clethrionomys glareolus
Cd Cr Cu Mn Ni Pb Zn
2.03* 0.128 16.44 10.49 0.235 0.142 105.5
(0.869–3.729) (0.053–0.196) (14.04–18.65 (9.35–12.20) (0.125–0.466) (0.038–1.570) (90.93–125.0)
12.56** 0.065 19.69 7.60 0.372 0.374 98.13
(8.11–19.24) (0.010–0.204) (18.52–20.84) (6.36–8.87) (0.272–1.056) (0.122–2.017) (87.68–114.0)
1.186* (0.686–1.277) 0.074 (0.003–0.080) 15.03 (13.93–19.44) 11.80 (9.06–12.71) 0.235 (0.192–0.292) 0.126 (0.036–0.276) 103.0 (97.89–115.6)
5.54** 0.082 19.91 7.97 0.532 0.966 84.40
(1.13–6.35) (0.012–0.088) (18.44–41.16) (4.21–12.09) (0.182–0.573) (0.235–1.105) (72.75–120.8)
0.349 0.045 11.18 26.15 0.481 4.54 182.4
(0.305–3.641) (0.038–0.107) (9.79–24.00) (22.69–76.23) (0.350–0.699) (1.60–6.83) (90.7–199.0)
0.29 0.58 0.74 2.2 2.0 36.0 1.8
0.06 0.53 0.56 3.3 0.9 4.7 2.2
Liver
Kidney
2586
I. Jankovska´ et al. / Environmental Pollution 157 (2009) 2584–2586
cestodes. However, it is known that manganese superoxide dismutase – Mn(SOD) active in antioxidative reactions is present in the human parasite Clonorchis sinenses (Li et al., 2005). Frisk et al. (2007) studied sequential trace element changes in serum and blood during a common viral infection in mice and found not only an expected increase in copper and zinc concentrations both in serum and blood after infection of mice by myocarditic CB3 virus, but the temporary increase of manganese concentration, as well. 4. Conclusion As mentioned by Sures (2004), more field and experimental studies are required to evaluate the relationship between bioaccumulation in cestode parasites of rodents and environmental metal exposure. We present M. agrestris/P. dentata and C. glareolus/ P. dentata as useful models for the evaluation of environmental lead exposure in terrestrial habitats, especially outside urban areas. These could be promising bioindication systems complementary to the models Rattus norvegicus/Hymenolepis diminuta (Sures et al., 2002, 2003), Apodemus sylvaticus/Gallegoides arfaai (Torres et al., 2004) and A. sylvaticus/Skrjabinotaenia lobata (Torres et al., 2006). The models evaluated in this study could also be promising bioindication systems for Mn, Ni and Zn, since higher concentrations were detected in P. dentata when comparing to kidney and liver tissues of the parasitized rodents. Since there are no significant differences in contents of lead and nickel in the liver and kidney of the non-parasitized voles and those parasitized by P. dentata, both tissues can be used in monitoring of environmental pollution by Pb and Ni as well as Cd, Cr and Cu. Because of the significant differences in the contents of manganese and zinc in livers of non-parasitized and parasitized M. agrestris, liver is less reliable as an indicator of environmental pollution by Mn and Zn than kidney tissue. Conversely, when comparing parasitized and non-parasitized animals, there were no significant differences in kidney concentrations of those elements that are accumulated in P. dentata (Pb, Mn, Ni and Zn), indicating that kidney tissue is more reliable to assess trace element pollution, thus avoiding the potential bias caused by cestode infection. Acknowledgements This study was supported by project MSM 6046070901 of the Ministry of Education, Youths and Sports, Czech Republic and by project No. 524/06/0687 of the Grant Agency of the Czech Republic. The authors declare that the experiments comply with the current laws of the Czech Republic, in which they were performed. References Anderson, T.J., Barret, G.W., Clark, C.S., Alia, V.J., 1982. Metal concentrations in tissues of meadow voles from sewage sludge treated fields. Journal of Environmental Quality 11, 272–277.
Barusˇ, V., Tenora, F., Sˇumbera, R., 2003. Relative concentrations of four heavy metals in the parasites Protospirura muricola (Nematoda) and Inermicapsifer arvicanthidis (Cestoda) in their definitive host silvery mole-rat (Heliophobius argenteocinereus: Rodentia). Helminthologia 40, 227–232. Eira, C., Torres, J., Vingada, J., Miquel, J., 2005. Concentration of some toxic elements in Oryctolagus cuniculus and in its intestinal cestode Mosgovoyia ctenoides, in Dunas de Mira (Portugal). Science of Total Environment 346, 81–86. Fendick, E.A., Stevens, G.L., Brown, R.J., Jordan, W.P., 1989. Element content in tissues of four rodent species sampled in the Geysers geothermal steamfield. Environmental Pollution 58, 155–178. Fitsanakis, V.A., Piccola, G., dos Santos, A.P., Aschner, J.L., Aschner, M., 2007. Putative proteins involved in manganese transport across the blood-brain barrier. Human Experimental Toxicology 26, 295–302. Frisk, P., Darnerud, P.O., Friman, G., Blomberg, J., Ilba¨ck, N.G., 2007. Sequential trace element changes in serum and blood during common viral infection in mice. Journal of Trace Elements in Medicine and Biology 21, 29–36. Ieradi, L.A., Zima, J., Allegra, F., Kotla´nova´, E., Campanella, L., Grossi, R., Cristaldi, M., 2003. Evaluation of genotoxic damage in wild rodents from a polluted area in the Czech Republic. Folia Zoologica 52 (1), 57–66. Klimpel, S., Forster, M., Schmabl, G., 2007. Parasites of two abundant sympatric rodent species in relation to host phylogeny and ecology. Parasitology Research 100, 867–875. Korolova, E., Miholova´, D., Mader, P., Cibulka, J., Sla´mova´, A., 1999. Small mammals as bioindicators for terrestrial ecosystems in Bohemia. In: Peakall (Ed.), Biomarkers: a Pragmatic Basis for Remediation of Severe Pollution in Eastern Europe. Kluwer Academic Publishers (NL), pp. 308–309. Li, A.H., Kong, Y., Cho, S.H., Na, B.K., Pak, J.K., Kim, T.S., 2005. Molecular cloning and characterization of the copper/zinc and manganese superoxide dismutase genes from the human parasite Clonorchis sinenses. Parasitology 130, 687–697. Michalke, B., Berthele, A., Mistriotis, P., Ochsenkiihn-Petropoulou, M., Halbach, S., 2007. Manganese species from human serum, cerebrospinal fluid analysed by size exclusion chromatography-, capillary electrophoresis coupled plasma mass spectrometry. Journal of Trace Elements in Medicine and Biology 21, 4–9. Miholova´, D., Mader, P., Sza´kova´, J., Sla´mova´, A., Svatosˇ, Z., 1993. Czechoslovakian biological certified reference materials and their use in the analytical quality assurance system in a trace element laboratory. Fresenius Journal of Analytical Chemistry 345, 256–260. Sa´nchez-Chardi, A., Nadal, J., 2007. Bioaccumulation of metals and effects of landfill pollution in small mammals. Part I. The greater white-toothed shrew, Crocidura russula. Chemosphere 68, 703–711. Sures, B., 2004. Environmental parasitology: relevancy of parasites in monitoring environmental pollution. Trends in Parasitology 20, 170–177. Sures, B., Siddall, R., Taraschewski, H., 1999. Parasites as accumulation indicators of heavy metal pollution. Parasitology Today 15, 16–21. Sures, B., Ju¨rges, G., Taraschewski, H., 2000a. Accumulation and distribution of lead in the acanthocephalan Moniliformis moniliformis from experimental infected rats. Parasitology 121, 427–433. Sures, B., Franken, M., Taraschewski, H., 2000b. Element concentrations in the archiacanthocephalan Macracanthorhynchus hirudinaceus compared with those in the porcine host from a slaughterhouse in La Paz, Bolivia. International Journal of Parasitology 30, 1071–1076. Sures, B., Grube, K., Taraschewski, H., 2002. Experimental studies on the lead accumulation in the cestode Hymenolepis diminuta and its final host Rattus norvegicus. Ecotoxicology 11, 365–368. Sures, B., Scheible, T., Bashtar, A.R., Taraschewski, H., 2003. Lead concentrations in Hymenolepis diminuta adults and Taenia taeniaeformis larvae compared to their rat hosts (Rattus norvegicus) sampled from the city of Cairo, Egypt. Parasitology 127, 483–487. Torres, J., de Lapuente, J., Eira, C., Nadal, J., 2004. Cadmium and lead concentrations in Gallegoides arfaai (Cestoda Anoplocephalidae) and Apodemus sylvaticus (Rodentia: Muridae) from Spain. Parasitology Research 94, 468–470. Torres, J., Peig, J., Eira, C., Borras, M., 2006. Cadmium and lead concentrations in Skrjabinotaenia lobata (Cestoda: Catenotaeniidae) and its host, Apodemus sylvaticus (Rodentia: Muridae), in the urban dumping side of Garraf (Spain). Environmental Pollution 143, 4–8.