- Email: [email protected]
Effect of heavy metals on coelomocytes of the earthworm Allolobophora chlorotica
Pedobiologia 47, 640–645, 2003 © Urban & Fischer Verlag http://www.urbanfischer.de/journals/pedo
The 7th international symposium on earthworm ecology · Cardiff · Wales · 2002
Effect of heavy metals on coelomocytes of the earthworm Allolobophora chlorotica Joanna Homa1*, Maria Niklinska2 and Barbara Plytycz1 1 2
Department of Evolutionary Immunology, Institute of Zoology, Jagiellonian University, Ingardena 6, 30-060 Cracow, Poland Department of Ecotoxicology, Institute of Environmental Sciences, Jagiellonian University, Gronostajowa 3, 30-387 Cracow, Poland
Submitted September 6, 2002 · Accepted September 4, 2003
Summary Earthworms are sensitive bioindicators of soil pollution. The aim of present investigations was to study the effects of heavy metals on earthworms and on their coelomocytes involved in the defence reactions. Adult individuals of Allolobophora chlorotica collected in Krakow (K) soil were kept in the laboratory either in the K soil, or were transferred to unpolluted soil from the rural area Sierbowice (S) or to the heavily polluted (Zn>Pb>Cd>Cu) soil from the industrial area, Bukowno (B). They were kept there at 22 °C for up to 8 weeks. Cocoons and juveniles appeared in S and K soil samples. The number and activity of the coelomocytes of worms maintained in S and K soils were unaffected despite some accumulation of heavy metals in the earthworm tissues. In contrast, in the B soil samples, bioaccumulation of metals was strongest, high mortality of adults was recorded, body mass was reduced, and reproduction completely inhibited. Coelomocytes retrieved from the B soil survivors exhibited significant impairment of pinocytosis and plastic adherence. Perhaps impairment of immune functions contributed to the poor survival under conditions of heavily polluted B soil samples. Key words: Coelomocytes, copper, lead, zinc, cadmium, in vivo metal accumulation, in vitro
Introduction Earthworms play a major role in physical, chemical, and biological processes in the soil. They are keystone species within ecosystems and are used extensively as bioindicators of environmental contamination (Bunn et al. 1996; Fitzpatrick et al. 1996; Scott-Fordsmand & Weeks 2000). Soil organisms inhabiting contaminated habitats are usually exposed to various chemicals simultaneously (Weltje 1997). Earthworms may affect the chemical forms of pollutants (organic and inor-
*E-mail corresponding author: [email protected]
0031-4056/03/47/05–06–640 $15.00/0
ganic residues) during food consumption, metabolism, and excretion (Mariño et al. 1992; Morgan et al. 1992). Furthermore, metal contaminants usually interact with each other, and with an array of edaphic factors, such that the bioviabilities of the metals, and their potential toxicities, are modulated in ways that are difficult to predict from individual concentration values (Marinussen et al. 1996; Weltje 1997). Immunoactive coelomocytes and their viability is one of our most promis-
Effect of heavy metals on coelomocytes
ing surrogate assays to assess immunotoxic risks (Bunn et al. 1996; Burch et al. 1999; Fugère et al. 1996). The aim of present experiments was to check the effects of unpolluted and contaminated soil samples on the earthworm species Allolobophora chlorotica. We investigate the viability, reproduction, and accumulation of heavy metals (Zn, Pb, Cd, Cu) in the body and its effects on viability and activity of immunoactive coelomocytes.
Materials and Methods Animals
Sexually mature earthworms (with well-developed clitella) of Allolobophora chlorotica were field-collected from the soil in the garden of the Institute of Zoology, in Krakow (K). They were transferred to the laboratory and maintained for 2, 4 or 8 weeks (under a constant temperature of 22 °C, and a 12 h light/12 h dark lighting regime) in groups of 6 animals in plastic boxes with 0.4 l of the soil samples from 3 different localities. Soil samples
Soil samples were collected from the top 15 cm mineralised layers at three geographical locations: the urban area Krakow (K), the industrial area Bukowno (B), and the uncontaminated rural area Sierbowice (S). Prior to analysis, the soils were air-dried. Soil pH was measured by a glass-calomel electrode in suspension of 16 g soil with 40 ml distilled H2O, or 40 ml 1 M KCl equilibrated for 24 h. Organic carbon (OC) and organic matter (OM) were determined by Tiurin method (Oleksynowa et al. 1991). (a-b)f 0.0006 × 100 % O.C = ——————————— q (a-b)f 0.0006 × 1.724 × 100 % O.M = ——————————————— q a – amount of 0.2 n FeSO4 (ml) used to titration blank test b – amount of 0.2 n FeSO4 (ml) used to titration sample f – normality of 0.2 n FeSO4 solution (f = 0.98) q – amount of soil in grams
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Heavy metal accumulation in the soil and in earthworm tissues
Earthworms were placed on wet filter paper in Petri dishes for 48 h to allow the depuration of their gut contents (Helling et al. 1999). After this period the worms were washed in distilled water and dried in 70 °C. Heavy metal (Zn, Pb, Cd, and Cu) content in earthworm tissues and in soil samples was analysed according to the methods described by Hopkin (1989) using an Instrumentation Laboratory Model Analyst 800 spectrophotometer (Perkin Elmer). Three replicates were run for all samples. Metal concentration factors (CF) were estimated according to the formula: whole earthworm metal content / soil metal content. Earthworm viability, body weight and reproduction
Viability, body weight, and the presence of cocoons and juveniles in particular soil samples were checked every 2 weeks. Coelomocyte retrieval and viability
Extrusion fluid: Cells were extruded to 1 % NaCl solution supplemented with the mucolytic agent guiacol glyceryl ether (10mg.ml–1, Sigma Chemical Co.) and 2.5mg.ml–1 EDTA (Sigma Chemical Co.) to prevent cell aggregation as described previously (Cossarizza et al. 1996; Roch 1979; Stankiewicz & Plytycz 1998a). Incubation fluid: Cells were incubated in Hank’s balanced salt solution – HBSS (10 g.l–1 NaCl, 0,5 g.l–1 KCl, 1,25 g.l–1 glucose, 75 mg.l–1 KH2PO4, 475 mg.l–1 Na2HPO4 × 2H2O) with osmolarity adjusted with NaCl to 320mOsm (osmometer Trident) and pH 7.4 (1N NaOH, 1M HCl; CP-315 Elmetron) (Cossarizza et al. 1996; Roch 1979; Stankiewicz & Plytycz 1998b). Cell retrieval: The earthworms were gently massaged to remove an intestine contents, surface cleaned by dipping them in water and blotting them dry. Then they were placed into Petri dishes (60 mm diameter) containing 3 ml of extrusion fluid. Coelomocytes were extruded through dorsal pores after stimulation with 5V electric current for 1 min (Roch 1979). The fluid with the extruded cells was transferred into centrifuge tubes previously treated with Sigmacote (Sigma Chemical Co.) to avoid cell adhesion to glass. Cells were washed in HBSS (10 min, 2000 rpm), counted and adjusted to 106 cell.ml–1. The viability of cells was estimated by the trypan blue exclusion test. Plastic adherence: After 1 hour of incubation in 96-well flat-bottomed plates, the nonadherent cells were removed. Cells, which adhered to the bottom of plates, were fixed with 2 % paraformaldehyde and Pedobiologia (2003) 47, 640–645
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Table 1. Physico-chemical characteristics (mean±SD) of soil samples from Krakow (K), Sierbowice (S) and Bukowno (B) K
S
Table 2. Metal concentration (mean±SD) in tissues of Allolobophora chlorotica kept in Krakow soil samples (K), or transferred for 4 weeks to soil samples from Bukowno (B).
B
pH H2O 7.5 5.9 7.6 pH KCl 7.2 5.7 7.4 Metals (µg/g) Zn 331.2 ± 2.20 172.6 ± 14.50 9820.5 ± 345.8 Pb 94.1 ± 2.7 3.2 ± 2.9 1174.8 ± 126.8 Cd 1.4 ± 0.1 3.4 ± 0.2 72.2 ± 1.7 Cu 40.4 ± 0.7 7.6 ± 1.4 18.6 ± 2.6 O.C (%) 2.9 1.4 4.4 O.M (%) 5.0 2.5 7.7
Metals
Locality
Tissue [µg/g]
Zn
K B K B K B K B
478.8 ± 139.3 3250.0 ± 1178.1 7.2 ± 1.0 325.2 ± 109.2 3.2 ± 16.0 27.7 ± 3.1 9.4 ± 3.7 22.4 ± 2.3
Pb Cd Cu
O.C = organic carbon, O.M = organic matter
a)
S
K
B
earthworms viability
7,5 a
6
a
a
a
4,5
a
a ***
3 b
1,5 0
b) 0,5 body weight [g]
tested by crystal violet (CV; Sigma Chemical Co.) staining/extraction (Plytycz et al. 1992; Plytycz & Jozkowicz 1994; Stankiewicz & Plytycz 1998b). Pinocytosis ability: Cells were incubated for 1 hour in 96-well flat-bottomed plates in HBSS supplemented with 500 µg.ml–1 of neutral red (NR; Sigma Chemical Co.). Supernatant containing nonadherent cells and free NR was removed and wells with adherent cells were washed with HBSS. Then NR was extracted from adherent cell with acid alcohol (3 % HCl in 95 % ethanol) (Plytycz et al. 1992; Plytycz & Jozkowicz 1994; Stankiewicz & Plytycz 1998b). Absorbance measurement: The results were read on Uniskan II spectrophotometer (Labsystem, Helsinki) at 570nm (CV) and 540nm (NR). The relative values were compared using a Student’s t-test; the level of significance was established at P<0.05.
Results
b
0,4 0,3
a
0,2
* a * b
b a
*a
a
a
*
c
0,1 0
Soil characteristics
Heavy metal accumulation in Allolobophora chlorotica tissues
Heavy metal accumulation at 4 weeks after transfer of the animals from the K to B soil in tissues of A. chlorotica are given in Table 2. Zn, Pb, Cd and Cu concentration in the earthworms were highest in B soil but Pedobiologia (2003) 47, 640–645
cocoons number
c)
Standard soil properties (pH, organic carbon, organic matter), and soil heavy metal contamination are included in Table 1. Soil pH was similar in K and B soil, and lower in S soil. The content of Zn, Pb, and Cd was negligible in K and S but high in B soil (Zn>Pb>Cd), while Cu contamination was generally low and increased in the sequence S
30
c
c
24 ***
18
b
**
c
d
12 6
a
*b
***
***
***
0 0
2
4
6
8
time [weeks]
Fig. 1. Effects of soil pollution on Allolobophora chlorotica kept in the K, S and B soil samples for 0-8 weeks on (a) viability; (b) body weight; and (c) cocoons production. Different letters in given groups indicate values significantly different according to Sudent’s t-test *P<0,05, ** P<0,01, *** P<0,005. (n=8-24)
Effect of heavy metals on coelomocytes
S
K
c)
pinocytosis OD 540 nm
b)
cell number/body weight x 10 4 cell number/earthworm x 10 4
a)
50 0 010
B
175 140
a
105
c a
a ab
70
a
0
c * b * a
750 600 450 a 300
a
a
150
* b
*
a
a
a
a **
ab
bc
0
0,03 0,024
a
0,018 0,012
b
a a ** c
Coelomocyte number and ex vivo activity
The number of coelomocytes was constant from 0 to 8 weeks in animals from S soil. Total number was temporarily (at 2–4 weeks) reduced in K soil, and especially in animals from the B soil. When recalculated per body mass, the number of coelomocytes was constant in the S samples and increased between 4 and 8 weeks in K and B samples (Fig. 2a–b). At week eight, cell activity measured by plastic adherence and pinocytosis of neutral red was lower in B than in S and K samples. Activities in K and S samples were similar (Fig. 2c–d).
Discussion
0,006 0
d) plastic adherence OD 570 nm
Animal viability, body weight and reproduction
Animals were fully vital and reproduced (as evidenced by the presence of cocoons) in S and K soil, while high mortality and no reproduction were recorded in heavily polluted B soil (Fig. 1a–c). Earthworm body mass was reduced after 2 weeks in B soil, remained constant in the K soil, and was increased during 2–4 weeks in S soil (Fig. 1).
b
b
concentration factors for B were lower for Zn and Cd. Concentration factors for K soil were: 1.44, 0.08, 2.23, 0.23 for Zn, Pb, Cd and Cu respectively (Table 2). Respective factors for B soil were: 0.33, 0.28, 0.38 and 1.2.
a
*b
*
35
643
2 1,6
a
b * a
1,2 0,8
a
a
* b
a *** c
0,4 0 0
2
4
6
8
time[weeks] Fig. 2. Effects of soil pollution on coelomocytes of Allolobophora chlorotica retrieved after 0–8 weeks animal maintenance in K, S, or B soil samples; (a) number of coelomocytes; (b) number of coelomocytes per body weight; (c) in vitro pinocytosis of neutral red; (d) in vitro plastic adherence. OD – optical density. Different letters in given groups indicate values significantly different according to Sudent’s t-test. *P<0,05, **P<0,01, ***P<0,005. (n=8-24)
Organisms inhibiting contaminated habitats are almost always exposed to various chemicals simultaneously (Lock & Janssen 2002). Therefore, in some ecotoxicological tests the animals are collected in the field from various areas or animals reared in the knows soil samples are transferred to the more or less polluted natural soil samples (Mariño et al. 1998; Mariño & Morgan 1999; Marinussen et. al 1997). Mariño et al. (1998) showed significant interactions of Cu and Cd ions on Lumbricus rubellus. Another attempt is to use an artificial soil (OECD) deliberately contaminated with the known solution of one or several contaminants, in the latter case applied simultaneously or in sequence. The earthworms can accumulate the heavy metal ions (Maboeta et al. 1999; Maenpaa et al. 2002). For example, Maboeta et al. (1999) showed negative effects of lead nitrate on the growth of Asian earthworm Perionyx excavatus with no effects on maturation and cocoon production, while Posthuma et al. (1997) investigated the effects of copper and zinc applied singly or in mixtures in the artificial soil on reproduction of Enchytraeus crypticus. Pedobiologia (2003) 47, 640–645
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Mortality and cocoon production are the main endpoints in acute toxicity tests (Reinecke et al. 2001; Spurgeon & Hopkin 1999) and were used also in our present experiments. As expected, animals were fully vital and successfully reproduced in a relatively unpolluted K and S soil samples while their reproduction was inhibited and viability was significantly decreased in the B soil samples from the industrial area (Fig. 1a–c). The impairment of earthworm viability and reproduction in the B soil samples corresponded with a high bioaccumulation of Zn, Pb, Cd and Cu in the earthworm bodies leading to high metal concentration in their tissues (Table 2). Similar body concentrations of the investigated heavy metals were obtained for Dendrobaena veneta from a commercial supplier transferred to the K or B soil samples (Olchawa-Wieczorek et al. in press). In our experiments individuals of A. chlorotica were much more vital in the polluted B soil samples than D. veneta. Perhaps the field-collected individuals of A. chlorotica from the city area were genetically more resistant to some of contaminating heavy metals. Earthworms living in the field can develop a tolerance to metal ions (Fugère et al. 1996; Spurgeon & Hopkin 1999). The toxic effects of heavy metals are partly determined by soil pH and the content of organic matter, high values being protective (Peredney & Williams 2000). Perhaps this was in a case of B soil samples with pH around 7.5 and 7.7 % of organic matter (Table 1). These factors can explain the relatively good survival of animals despite high soil contamination with Zn and Pb ions. Perhaps also others factors, not investigated here, had a protective effects on earthworm viability as, for example, bioavailibity of Zn, Pb and Cd is limited in the presence of phosphorus compounds (Maenpaa et al. 2002). Several studies concerned the effects of environmental pollution, including heavy metals, on earthworm immune functions mediated by coelomocytes (Kurek et al. 2002; Scott-Fordsmand & Weeks 2000). For example, Fugère et al. (1996) recorded inhibition of phagocytic activity of coelomocytes exposed in vitro on heavy metal (Cd, Zn) solutions. Also, Burch et al. (1999) showed toxic effects of Cu ions (at 11×106 µg.l–1 Cu) on coelomocyte viability and phagocytosis. Therefore, it was not surprising that in our experiments coelomocyte viability and activity was significantly affected in A. chlorotica maintained in the B soil samples, being transiently decreased in number. However, in animals surviving in such a condition for a longer period the number of coelomocytes increased but their pinocytic activity and plastic adherence were significantly impaired. The parallel experiments performed by (Olchawa–Wieczorek et al. in press) on D. veneta revealed a better bacterial survival in the Pedobiologia (2003) 47, 640–645
coelomic cavity of earthworms kept in the polluted B soil samples. In conclusion, perhaps in both D. veneta and A. chlorotica, metal contamination of the soil caused their accumulation in the earthworm body including their coelomocytes which, in turn, affects their health and viability. Acknowledgements. This work was supported by KBN grant 6PO4G01318 and IZ/DS/ZIE. We would like to thank Professor Stefan Skiba and his co-workers the valuable help in soil characteristics.
References Bunn, K. E., Thompson, H. M., Tarrant, K. A. (1996) Effects of agrochemicals on the immune systems of earthworms. Bulletin of Environmental Contamination Toxicology 57, 632–639. Burch, S. W., Fitzpatrick, L. C., Goven, A. J., Venables, B. J., Giggleman, M. A. (1999) In vitro earthworm Lumbricus terrestris coelomocyte assay for use in terrestrial identification evaluation. Bulletin of Environmental Contamination Toxicology 62, 547–554. Cossarizza, A., Cooper, E. L., Suzuki, M. M., Salvioli, S., Capri, M., Gri, G., Quaglino, D., Franceschi, C. (1996) Earthworm leukocytes that are not phagocytic and crossreact with several human tumor cell lines. Environmental Cell Research 224, 174–182. Fitzpatrick, L. C., Muratti-Ortiz, J. F., Venables, B. J., Goven, A. J. (1996) Comparative toxicity in earthworms Eisenia fetida and Lumbricus terrestris exposed to cadmium nitrate using artifical soil and filter paper protocols. Bulletin of Environmental Contamination Toxicology 57, 63–68. Fugère, N., Brousseau, P., Krzystyniak, K., Coderre, D., Fournier, M. (1996) Heavy metal-specific inhibition of phagocytosis and different in vitro sensitivity of heterogenous coelomocytes from Lumbricus terrestris (Oligochaeta). Toxicology 109, 157–166. Helling, B., Reinecke, S. A., Reinecke, A. J. (1999) Effects of the fungicide cooper oxychloride on the growth and reproduction of Eisenia fetida (Oligochaeta). Ecotoxicology and Environmental Safety 46, 108–116. Hopkin, S. P. (1989) Ecophysiology of metals in terrestrial invertebrates. Elsevier, London. Kurek, A., Homa, J., Plytycz, B. (2002) Earthworm coelomocytes: convenient model for basic and applied sciences. In: Beschin, A., Bilej, M., Cooper, E. L. (eds) A New Model for Analyzing Antimicrobial Peptides with Biomedical Applications. IOS Press, Ohmsha, pp. 38–46. Lock, K., Janssen, C. R. (2002) Mixture toxicity of Zinc, Cadmium, Cooper, and Lead to the Potworm Enchytraeus albidus. Ecotoxicology and Environmental Safety 52, 1–7. Maboeta, M. S., Reinecke, A. J., Reinecke, S. A. (1999) Effects of low levels on growth and reproduction of Asian earthworm Perionyx excavatus (Oligochaeta). Ecotoxicology and Environmental Safety 44, 236–240.
Effect of heavy metals on coelomocytes Maenpaa, K. A., Kukkonen, J. V. K., Lydyn, M. J. (2002) Remediation of heavy metal-contaminated soils using phosphorus: evaluation of bioavailability using an earthworm bioassay. Archives of Environmental Contamination Toxicology 43, 389–398. Marinussen, M. P. J. C., Van der Zee, S. E. A. T. M., De Haan, F. A. M. (1996) Effect of Cd or Pb addition to Cu-contaminated soil on tissue Cu accumulation in the earthworm, Dendrobaena veneta. Ecotoxicology and Environmental Safety 38, 309–315. Marinussen, M. P. J. C., Van der Zee, S. E. A. T. M., De Haan, F. A. M. (1997) Ecotoxicology and Environmental Safety 36, 17–26. Mariño, F., Ligero, A., Cosin, D. J. (1992) Heavy metals and earthworms on the border of a road next to Santiago (Galicia, Northwest of Spain). Soil Biology and Biochemistry 24, 1705–1709. Mariño, F., Stürzenbaum, S. R., Kille, P., Morgan, A. J. (1998) Cu-Cd interactions in earthworms maintained in laboratory microcosms: the examination of a putative cooper paradox. Comparative Biochemistry and Physiology Part C 120, 217–223. Mariño, F., Morgan, A. J (1999) The time-course of metal (Ca, Cd, Cu, Pb, Zn) accumulation from a contaminated soil by three populations of the earthworm, Lumbricus rubellus. Applied Soil Ecology 12, 169–177. Morgan, J. E., Morgan, A. J., Corp, N. (1992) Assessing soil metals pollution with earthworms: indices derived from regression analyses. In: Grieg-Smith, P. W., Becker, H., Edwards, P. J., Heimbach, F. (eds) Ecotoxicology of earthworms. Intercept Press, Andover, UK 233–237. OECD (1984) Guidelines for the testing of chemicals. No. 207. Earthworm acute toxicity tests. Adopted April 4, 1984. Olchawa-Wieczorek, E., Niklin´ska, M., Miedzobrodzki, J., Plytycz, B. (2003) Effects of temperature and soil pollution on the presence of bacteria, coelomocytes and brown bodies in coelomic fluid of Dendrobaena veneta. Pedobiologia 47, 702–709. Oleksynowa, K., Tokaj, J., Jakubiec, J., Komornicki, T. (eds) (1991) Przewodnik do c´wiczen´ z gleboznawstwa i geologii. Agricultural Academy, Cracow (in Polish). Peredney, C. L., Williams, P. L. (2000) Utility of Caenorhab-
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ditis elegans for assessing heavy metal contamination in artifical soil. Archives of Environmental Contamination and Toxicology 39, 113–118. Plytycz, B., Róz·anowska, M., Seljelid, R. (1992) Quantification of Neutral Red pinocytosis by small numbers of adherent cells: comparative studies. Folia biologica (Kraków) 40, 3–9. Plytycz, B., Jozkowicz, A. (1994) Differential effects of temperature on macrophages of ectothermic vertebrates. Journal of Leukocytes Biology 56, 729–731. Posthuma, L., Baerselman, R., Van Veen, R. P. M., DirvenVan Breemen, E.M. (1997) Single and joint toxic effects of cooper and zinc on reproduction of Enchytraeus crypticus in relation to sorption of metals in soils. Ecotoxicology Environmental Safety 38, 108–121. Reinecke, A. J., Reinecke, S. A., Maboeta, M. S. (2001) Cocoon production and viability as endpoints in toxicity testing of heavy metals with three earthworms species. Pedobiologia 45, 61–68. Roch, P. (1979) Protein analysis of earthworm coelomic fluid: polymorphic system of the natural hemolysin of Eisenia fetida andrei. Developmental and Comparative Immunology 3, 599–608. Scott-Fordsmand, J. J., Weeks, J. M. (2000) Biomarkers in earthworms. Environmental Contamination Toxicology 165, 17–59. Spurgeon, D. J., Hopkin, S. P. (1999) Tolerance to Zinc in populations of the earthworm Lumbricus rubellus from uncontaminated and metal-contaminated ecosystems. Archives of Environmental Contamination and Toxicology 37, 332–337. Stankiewicz, A., Plytycz, B. (1998a) Effects of in vitro conditions and in vitro thermal adaptation on viability of the earthworm (Eisenia fetida) coelomocytes. Folia Biologica (Cracow) 46, 3–4. Stankiewicz, A., Pl/ ytycz, B. (1998b) Optimalization of in vitro assays for activity of earthworm (Eisenia fetida) coelomocytes. Scientific Papers of Agricultural Academy in Cracow 334, 183–190 (in Polish). Weltje, L. (1997) Mixture toxicity and tissue interactions of Cd, Cu, Pb and Zn in earthworms (Oligochaeta) in laboratory and field soils: a critical evaluation of data. Chemosphere 12, 2643–2660.
Pedobiologia (2003) 47, 640–645
The 7th international symposium on earthworm ecology · Cardiff · Wales · 2002
Effect of heavy metals on coelomocytes of the earthworm Allolobophora chlorotica Joanna Homa1*, Maria Niklinska2 and Barbara Plytycz1 1 2
Department of Evolutionary Immunology, Institute of Zoology, Jagiellonian University, Ingardena 6, 30-060 Cracow, Poland Department of Ecotoxicology, Institute of Environmental Sciences, Jagiellonian University, Gronostajowa 3, 30-387 Cracow, Poland
Submitted September 6, 2002 · Accepted September 4, 2003
Summary Earthworms are sensitive bioindicators of soil pollution. The aim of present investigations was to study the effects of heavy metals on earthworms and on their coelomocytes involved in the defence reactions. Adult individuals of Allolobophora chlorotica collected in Krakow (K) soil were kept in the laboratory either in the K soil, or were transferred to unpolluted soil from the rural area Sierbowice (S) or to the heavily polluted (Zn>Pb>Cd>Cu) soil from the industrial area, Bukowno (B). They were kept there at 22 °C for up to 8 weeks. Cocoons and juveniles appeared in S and K soil samples. The number and activity of the coelomocytes of worms maintained in S and K soils were unaffected despite some accumulation of heavy metals in the earthworm tissues. In contrast, in the B soil samples, bioaccumulation of metals was strongest, high mortality of adults was recorded, body mass was reduced, and reproduction completely inhibited. Coelomocytes retrieved from the B soil survivors exhibited significant impairment of pinocytosis and plastic adherence. Perhaps impairment of immune functions contributed to the poor survival under conditions of heavily polluted B soil samples. Key words: Coelomocytes, copper, lead, zinc, cadmium, in vivo metal accumulation, in vitro
Introduction Earthworms play a major role in physical, chemical, and biological processes in the soil. They are keystone species within ecosystems and are used extensively as bioindicators of environmental contamination (Bunn et al. 1996; Fitzpatrick et al. 1996; Scott-Fordsmand & Weeks 2000). Soil organisms inhabiting contaminated habitats are usually exposed to various chemicals simultaneously (Weltje 1997). Earthworms may affect the chemical forms of pollutants (organic and inor-
*E-mail corresponding author: [email protected]
0031-4056/03/47/05–06–640 $15.00/0
ganic residues) during food consumption, metabolism, and excretion (Mariño et al. 1992; Morgan et al. 1992). Furthermore, metal contaminants usually interact with each other, and with an array of edaphic factors, such that the bioviabilities of the metals, and their potential toxicities, are modulated in ways that are difficult to predict from individual concentration values (Marinussen et al. 1996; Weltje 1997). Immunoactive coelomocytes and their viability is one of our most promis-
Effect of heavy metals on coelomocytes
ing surrogate assays to assess immunotoxic risks (Bunn et al. 1996; Burch et al. 1999; Fugère et al. 1996). The aim of present experiments was to check the effects of unpolluted and contaminated soil samples on the earthworm species Allolobophora chlorotica. We investigate the viability, reproduction, and accumulation of heavy metals (Zn, Pb, Cd, Cu) in the body and its effects on viability and activity of immunoactive coelomocytes.
Materials and Methods Animals
Sexually mature earthworms (with well-developed clitella) of Allolobophora chlorotica were field-collected from the soil in the garden of the Institute of Zoology, in Krakow (K). They were transferred to the laboratory and maintained for 2, 4 or 8 weeks (under a constant temperature of 22 °C, and a 12 h light/12 h dark lighting regime) in groups of 6 animals in plastic boxes with 0.4 l of the soil samples from 3 different localities. Soil samples
Soil samples were collected from the top 15 cm mineralised layers at three geographical locations: the urban area Krakow (K), the industrial area Bukowno (B), and the uncontaminated rural area Sierbowice (S). Prior to analysis, the soils were air-dried. Soil pH was measured by a glass-calomel electrode in suspension of 16 g soil with 40 ml distilled H2O, or 40 ml 1 M KCl equilibrated for 24 h. Organic carbon (OC) and organic matter (OM) were determined by Tiurin method (Oleksynowa et al. 1991). (a-b)f 0.0006 × 100 % O.C = ——————————— q (a-b)f 0.0006 × 1.724 × 100 % O.M = ——————————————— q a – amount of 0.2 n FeSO4 (ml) used to titration blank test b – amount of 0.2 n FeSO4 (ml) used to titration sample f – normality of 0.2 n FeSO4 solution (f = 0.98) q – amount of soil in grams
641
Heavy metal accumulation in the soil and in earthworm tissues
Earthworms were placed on wet filter paper in Petri dishes for 48 h to allow the depuration of their gut contents (Helling et al. 1999). After this period the worms were washed in distilled water and dried in 70 °C. Heavy metal (Zn, Pb, Cd, and Cu) content in earthworm tissues and in soil samples was analysed according to the methods described by Hopkin (1989) using an Instrumentation Laboratory Model Analyst 800 spectrophotometer (Perkin Elmer). Three replicates were run for all samples. Metal concentration factors (CF) were estimated according to the formula: whole earthworm metal content / soil metal content. Earthworm viability, body weight and reproduction
Viability, body weight, and the presence of cocoons and juveniles in particular soil samples were checked every 2 weeks. Coelomocyte retrieval and viability
Extrusion fluid: Cells were extruded to 1 % NaCl solution supplemented with the mucolytic agent guiacol glyceryl ether (10mg.ml–1, Sigma Chemical Co.) and 2.5mg.ml–1 EDTA (Sigma Chemical Co.) to prevent cell aggregation as described previously (Cossarizza et al. 1996; Roch 1979; Stankiewicz & Plytycz 1998a). Incubation fluid: Cells were incubated in Hank’s balanced salt solution – HBSS (10 g.l–1 NaCl, 0,5 g.l–1 KCl, 1,25 g.l–1 glucose, 75 mg.l–1 KH2PO4, 475 mg.l–1 Na2HPO4 × 2H2O) with osmolarity adjusted with NaCl to 320mOsm (osmometer Trident) and pH 7.4 (1N NaOH, 1M HCl; CP-315 Elmetron) (Cossarizza et al. 1996; Roch 1979; Stankiewicz & Plytycz 1998b). Cell retrieval: The earthworms were gently massaged to remove an intestine contents, surface cleaned by dipping them in water and blotting them dry. Then they were placed into Petri dishes (60 mm diameter) containing 3 ml of extrusion fluid. Coelomocytes were extruded through dorsal pores after stimulation with 5V electric current for 1 min (Roch 1979). The fluid with the extruded cells was transferred into centrifuge tubes previously treated with Sigmacote (Sigma Chemical Co.) to avoid cell adhesion to glass. Cells were washed in HBSS (10 min, 2000 rpm), counted and adjusted to 106 cell.ml–1. The viability of cells was estimated by the trypan blue exclusion test. Plastic adherence: After 1 hour of incubation in 96-well flat-bottomed plates, the nonadherent cells were removed. Cells, which adhered to the bottom of plates, were fixed with 2 % paraformaldehyde and Pedobiologia (2003) 47, 640–645
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Table 1. Physico-chemical characteristics (mean±SD) of soil samples from Krakow (K), Sierbowice (S) and Bukowno (B) K
S
Table 2. Metal concentration (mean±SD) in tissues of Allolobophora chlorotica kept in Krakow soil samples (K), or transferred for 4 weeks to soil samples from Bukowno (B).
B
pH H2O 7.5 5.9 7.6 pH KCl 7.2 5.7 7.4 Metals (µg/g) Zn 331.2 ± 2.20 172.6 ± 14.50 9820.5 ± 345.8 Pb 94.1 ± 2.7 3.2 ± 2.9 1174.8 ± 126.8 Cd 1.4 ± 0.1 3.4 ± 0.2 72.2 ± 1.7 Cu 40.4 ± 0.7 7.6 ± 1.4 18.6 ± 2.6 O.C (%) 2.9 1.4 4.4 O.M (%) 5.0 2.5 7.7
Metals
Locality
Tissue [µg/g]
Zn
K B K B K B K B
478.8 ± 139.3 3250.0 ± 1178.1 7.2 ± 1.0 325.2 ± 109.2 3.2 ± 16.0 27.7 ± 3.1 9.4 ± 3.7 22.4 ± 2.3
Pb Cd Cu
O.C = organic carbon, O.M = organic matter
a)
S
K
B
earthworms viability
7,5 a
6
a
a
a
4,5
a
a ***
3 b
1,5 0
b) 0,5 body weight [g]
tested by crystal violet (CV; Sigma Chemical Co.) staining/extraction (Plytycz et al. 1992; Plytycz & Jozkowicz 1994; Stankiewicz & Plytycz 1998b). Pinocytosis ability: Cells were incubated for 1 hour in 96-well flat-bottomed plates in HBSS supplemented with 500 µg.ml–1 of neutral red (NR; Sigma Chemical Co.). Supernatant containing nonadherent cells and free NR was removed and wells with adherent cells were washed with HBSS. Then NR was extracted from adherent cell with acid alcohol (3 % HCl in 95 % ethanol) (Plytycz et al. 1992; Plytycz & Jozkowicz 1994; Stankiewicz & Plytycz 1998b). Absorbance measurement: The results were read on Uniskan II spectrophotometer (Labsystem, Helsinki) at 570nm (CV) and 540nm (NR). The relative values were compared using a Student’s t-test; the level of significance was established at P<0.05.
Results
b
0,4 0,3
a
0,2
* a * b
b a
*a
a
a
*
c
0,1 0
Soil characteristics
Heavy metal accumulation in Allolobophora chlorotica tissues
Heavy metal accumulation at 4 weeks after transfer of the animals from the K to B soil in tissues of A. chlorotica are given in Table 2. Zn, Pb, Cd and Cu concentration in the earthworms were highest in B soil but Pedobiologia (2003) 47, 640–645
cocoons number
c)
Standard soil properties (pH, organic carbon, organic matter), and soil heavy metal contamination are included in Table 1. Soil pH was similar in K and B soil, and lower in S soil. The content of Zn, Pb, and Cd was negligible in K and S but high in B soil (Zn>Pb>Cd), while Cu contamination was generally low and increased in the sequence S
30
c
c
24 ***
18
b
**
c
d
12 6
a
*b
***
***
***
0 0
2
4
6
8
time [weeks]
Fig. 1. Effects of soil pollution on Allolobophora chlorotica kept in the K, S and B soil samples for 0-8 weeks on (a) viability; (b) body weight; and (c) cocoons production. Different letters in given groups indicate values significantly different according to Sudent’s t-test *P<0,05, ** P<0,01, *** P<0,005. (n=8-24)
Effect of heavy metals on coelomocytes
S
K
c)
pinocytosis OD 540 nm
b)
cell number/body weight x 10 4 cell number/earthworm x 10 4
a)
50 0 010
B
175 140
a
105
c a
a ab
70
a
0
c * b * a
750 600 450 a 300
a
a
150
* b
*
a
a
a
a **
ab
bc
0
0,03 0,024
a
0,018 0,012
b
a a ** c
Coelomocyte number and ex vivo activity
The number of coelomocytes was constant from 0 to 8 weeks in animals from S soil. Total number was temporarily (at 2–4 weeks) reduced in K soil, and especially in animals from the B soil. When recalculated per body mass, the number of coelomocytes was constant in the S samples and increased between 4 and 8 weeks in K and B samples (Fig. 2a–b). At week eight, cell activity measured by plastic adherence and pinocytosis of neutral red was lower in B than in S and K samples. Activities in K and S samples were similar (Fig. 2c–d).
Discussion
0,006 0
d) plastic adherence OD 570 nm
Animal viability, body weight and reproduction
Animals were fully vital and reproduced (as evidenced by the presence of cocoons) in S and K soil, while high mortality and no reproduction were recorded in heavily polluted B soil (Fig. 1a–c). Earthworm body mass was reduced after 2 weeks in B soil, remained constant in the K soil, and was increased during 2–4 weeks in S soil (Fig. 1).
b
b
concentration factors for B were lower for Zn and Cd. Concentration factors for K soil were: 1.44, 0.08, 2.23, 0.23 for Zn, Pb, Cd and Cu respectively (Table 2). Respective factors for B soil were: 0.33, 0.28, 0.38 and 1.2.
a
*b
*
35
643
2 1,6
a
b * a
1,2 0,8
a
a
* b
a *** c
0,4 0 0
2
4
6
8
time[weeks] Fig. 2. Effects of soil pollution on coelomocytes of Allolobophora chlorotica retrieved after 0–8 weeks animal maintenance in K, S, or B soil samples; (a) number of coelomocytes; (b) number of coelomocytes per body weight; (c) in vitro pinocytosis of neutral red; (d) in vitro plastic adherence. OD – optical density. Different letters in given groups indicate values significantly different according to Sudent’s t-test. *P<0,05, **P<0,01, ***P<0,005. (n=8-24)
Organisms inhibiting contaminated habitats are almost always exposed to various chemicals simultaneously (Lock & Janssen 2002). Therefore, in some ecotoxicological tests the animals are collected in the field from various areas or animals reared in the knows soil samples are transferred to the more or less polluted natural soil samples (Mariño et al. 1998; Mariño & Morgan 1999; Marinussen et. al 1997). Mariño et al. (1998) showed significant interactions of Cu and Cd ions on Lumbricus rubellus. Another attempt is to use an artificial soil (OECD) deliberately contaminated with the known solution of one or several contaminants, in the latter case applied simultaneously or in sequence. The earthworms can accumulate the heavy metal ions (Maboeta et al. 1999; Maenpaa et al. 2002). For example, Maboeta et al. (1999) showed negative effects of lead nitrate on the growth of Asian earthworm Perionyx excavatus with no effects on maturation and cocoon production, while Posthuma et al. (1997) investigated the effects of copper and zinc applied singly or in mixtures in the artificial soil on reproduction of Enchytraeus crypticus. Pedobiologia (2003) 47, 640–645
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Mortality and cocoon production are the main endpoints in acute toxicity tests (Reinecke et al. 2001; Spurgeon & Hopkin 1999) and were used also in our present experiments. As expected, animals were fully vital and successfully reproduced in a relatively unpolluted K and S soil samples while their reproduction was inhibited and viability was significantly decreased in the B soil samples from the industrial area (Fig. 1a–c). The impairment of earthworm viability and reproduction in the B soil samples corresponded with a high bioaccumulation of Zn, Pb, Cd and Cu in the earthworm bodies leading to high metal concentration in their tissues (Table 2). Similar body concentrations of the investigated heavy metals were obtained for Dendrobaena veneta from a commercial supplier transferred to the K or B soil samples (Olchawa-Wieczorek et al. in press). In our experiments individuals of A. chlorotica were much more vital in the polluted B soil samples than D. veneta. Perhaps the field-collected individuals of A. chlorotica from the city area were genetically more resistant to some of contaminating heavy metals. Earthworms living in the field can develop a tolerance to metal ions (Fugère et al. 1996; Spurgeon & Hopkin 1999). The toxic effects of heavy metals are partly determined by soil pH and the content of organic matter, high values being protective (Peredney & Williams 2000). Perhaps this was in a case of B soil samples with pH around 7.5 and 7.7 % of organic matter (Table 1). These factors can explain the relatively good survival of animals despite high soil contamination with Zn and Pb ions. Perhaps also others factors, not investigated here, had a protective effects on earthworm viability as, for example, bioavailibity of Zn, Pb and Cd is limited in the presence of phosphorus compounds (Maenpaa et al. 2002). Several studies concerned the effects of environmental pollution, including heavy metals, on earthworm immune functions mediated by coelomocytes (Kurek et al. 2002; Scott-Fordsmand & Weeks 2000). For example, Fugère et al. (1996) recorded inhibition of phagocytic activity of coelomocytes exposed in vitro on heavy metal (Cd, Zn) solutions. Also, Burch et al. (1999) showed toxic effects of Cu ions (at 11×106 µg.l–1 Cu) on coelomocyte viability and phagocytosis. Therefore, it was not surprising that in our experiments coelomocyte viability and activity was significantly affected in A. chlorotica maintained in the B soil samples, being transiently decreased in number. However, in animals surviving in such a condition for a longer period the number of coelomocytes increased but their pinocytic activity and plastic adherence were significantly impaired. The parallel experiments performed by (Olchawa–Wieczorek et al. in press) on D. veneta revealed a better bacterial survival in the Pedobiologia (2003) 47, 640–645
coelomic cavity of earthworms kept in the polluted B soil samples. In conclusion, perhaps in both D. veneta and A. chlorotica, metal contamination of the soil caused their accumulation in the earthworm body including their coelomocytes which, in turn, affects their health and viability. Acknowledgements. This work was supported by KBN grant 6PO4G01318 and IZ/DS/ZIE. We would like to thank Professor Stefan Skiba and his co-workers the valuable help in soil characteristics.
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