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Resistance to arsenic-toxicity in a population of the earthworm Lumbricus rubellus
Soil Biology and Biochemistry 31 (1999) 1963±1967
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Resistance to arsenic-toxicity in a population of the earthworm Lumbricus rubellus Caroline J. Langdon a, Trevor G. Piearce a,*, Stuart Black b, Kirk T. Semple c a
Department of Biological Sciences, Institute of Environmental and Natural Sciences, Lancaster University, Lancaster LA1 4YQ, UK b Postgraduate Research Institute for Sedimentology, University of Reading, Whiteknights, P.O. Box 227, Reading RG6 6AB, UK c Department of Environmental Sciences, Institute of Environmental and Natural Sciences, Lancaster University, Lancaster LA1 4YQ, UK Accepted 24 June 1999
Abstract Specimens of the earthworms Lumbricus terrestris L. and L. rubellus Homeister from an uncontaminated site rapidly deteriorated in condition when kept in spoil rich in metal contaminants and arsenic. The site from which the spoil was collected supports several earthworm species, L. rubellus being dominant. Native L. rubellus survived for 12 weeks in spoil in the laboratory. L. rubellus collected from the spoil site and an uncontaminated site were kept for 28 d in uncontaminated soil and in soil containing 2000 mg sodium arsenate heptahydrate kgÿ1, the state of the specimens being recorded using a semi-quantitative assessment of earthworm condition (condition index, CI). The CI remained high for all specimens except those from the uncontaminated site kept in As-rich soil, for which mortality was 100% after 28 d. Tissue As concentrations in L rubellus from uncontaminated and contaminated sites were <1 mg kgÿ1 and 230 mg kgÿ1, respectively. In L. rubellus collected from the uncontaminated site and exposed to contaminated soil containing 2000 mg sodium arsenate heptahydrate kgÿ1, mean tissue As concentration was 92 mg kgÿ1. # 1999 Elsevier Science Ltd. All rights reserved. Keywords: Arsenic-sensitivity; Earthworm; Soil contamination; Metal contaminants; Toxicity testing
1. Introduction Arsenic is widely distributed in soils, with a mean concentration of around 6 mg kgÿ1. However, much higher concentrations can occur in mine spoils and areas contaminated by industrial wastes and biocides (Yan-Chu, 1994). Under aerobic conditions inorganic As is present predominantly as arsenate; however, arsenite, a more toxic form of As, predominates in anoxic soils (Cullen and Reimer, 1989). Earthworms have a particularly intimate contact with the soil, consuming large quantities and having little external barrier to the soil solution. For this reason, and because of their importance in terrestrial food webs, they are widely used as indicators of soil contamination (Greig* Corresponding author. Tel.: +44-1524-65201; fax: +44-1524843854. E-mail address: [email protected] (T.G. Piearce)
Smith et al., 1992). This, together with their major role in the incorporation and decomposition of dead organic matter in the soil, and in the development and maintenance of soil structure (Edwards and Bohlen, 1996), also makes them valuable indicators of soil health. Their in¯uence on soil drainage and aeration, through burrowing and casting, has well-known implications for soil fertility (Edwards and Bohlen, 1996). It may also have consequences for the speciation, solubility and toxicity of soil contaminants, seen, to some extent, in sediment bioturbation by polychaete worms (Doyle and Otte, 1997). Earthworms have been reported to inhabit As-rich metalliferous soils in SW England (Morgan and Janes, unpubl, in Morgan et al., 1994). Spoil at an abandoned tungsten mine at Carrock Fell, Cumbria, NW England, (NY 324 330), contains As, largely as arsenopyrite, in concentrations up to 53,000 mg kgÿ1 (Black et al., in press), three orders of magnitude above back-
0038-0717/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 8 - 0 7 1 7 ( 9 9 ) 0 0 1 1 8 - 2
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C.J. Langdon et al. / Soil Biology and Biochemistry 31 (1999) 1963±1967
ground contents for the Carrock Fell area. Since soil treated with sodium arsenate at substantially lower concentrations than this has been shown to be highly toxic to the earthworms Eisenia fetida (Savigny) (Fischer and Koszorus, 1992) and L. terrestris L. (Meharg et al., 1998), it is of interest that several earthworm species occur in the spoil, including L. rubellus, which is the dominant species. Arsenic-tolerance has been reported in plants (Porter and Peterson, 1975, 1977; Meharg and Macnair, 1991; Meharg and Macnair, 1994) and may also occur in the fauna of contaminated soils. Specimens of L. terrestris and L. rubellus Homeister from an uncontaminated site were kept in Carrock Fell mine spoil, as described below, for 15 d. The condition of many of the earthworms deteriorated substantially over the course of the experiment, although specimens of L. rubellus native to the mine site survived in good condition in spoil in the laboratory for several months. Since the mine spoil is rich in other metal contaminants (Black et al., in press) which might have contributed to the deterioration in the earthworms' condition, a second experiment, also described here, was conducted to evaluate the earthworms' tolerance of As per se. A semi-quantitative assessment of earthworm condition was developed, which provides ®ner discrimination of the state of the animals than simply recording mortalities. Tissue As concentration of L. rubellus from contaminated and uncontaminated soils were recorded, together with total tissue As concentrations for L. rubellus exposed to the As treated soil. 2. Materials and methods 2.1. Responses of L. terrestris and L. rubellus on exposure to As-contaminated mine spoil Sixty adult and large immature L. terrestris were collected by hand at night from the surface of mown grassland and 18 adult and large immature L. rubellus by hand-sorting in mixed deciduous woodland. After removal of stones, 500 g soil samples were weighed into 20 25 cm polythene bags and 200 mg of dried, ground mine spoil vegetation, dominated by grass and rush, was added as food. One earthworm was placed in each bag (experience having shown that, where several specimens are kept together, there is a serious risk of infection spreading from one moribund individual to another). The bags were sealed, stored in the dark at 98C and periodically opened for ventilation. Twenty L. terrestris and six L. rubellus were removed after 5, 10 and 15 d and their condition noted. The tissue As concentration in L. rubellus was determined (see below).Ten L. rubellus from Carrock Fell spoil were
kept under similar conditions in the laboratory for 84 d. 2.2. Soil and earthworm tissue analysis Quantities of major elements in soils from Carrock Fell and Lancaster University campus were determined using a Phillips PW 1400 XRF (Tertran and Claisse, 1982). One bulk sample of soil was used for analysis. The L. rubellus were weighed and depurated for 24 h in individual polythene bags containing moist tissue, then killed by dipping in hot water, placed in conical ¯asks and dried at 408C overnight and re-weighed. Mean dry body weights 2 S.E. were: university campus L. rubellus 221 215 mg, L. terrestris 693 2 25 mg, Carrock Fell L. rubellus 47 2 19 mg. Samples were acid-digested with 20 ml (Aristar grade) nitric acid, ®ltered using Whatman No. 540 ashless ®lter papers and stored at 98C. Mean total tissue contents of As were measured using a Perkin Elmer 2280 graphite furnace (AAS) with calibration standards of 0, 50, 100, 150 and 200 mg gÿ1. Dilutions ranging between 1/1000±1/ 50 were required for L. rubellus from contaminated soil and As-treated soils; no dilutions were required for L. rubellus from uncontaminated soil (see Beaty and Kerber, 1993 for machine keyboard entries). 2.3. Response of L. rubellus to As-treated soil Specimens of L. rubellus were collected from an uncontaminated grassland site on the University of Lancaster campus, by formalin expulsion, followed by washing, and from Carrock Fell mine spoil by a combination of formalin extraction, followed by washing, and by hand-sorting of soil. All were kept for several weeks in their parent soil before use. Soil from a mixed deciduous woodland site at Lancaster, (SD 488 574), which supported an abundant and diverse earthworm community, was partially air-dried, sieved through a 2.8 mm mesh and rewetted to a moisture content of 53% (dry weight equivalent) using distilled water, or with a solution of sodium arsenate heptahydrate to give a concentration of 2000 mg kgÿ1 dry weight of soil of the hydrated salt. Mean soil pH (aqueous suspension)2 S.E. was 4.97 2 0.05. Moistened soil (69 g) was weighed into each of a series of 20 25 cm polythene bags. One L. rubellus was weighed and introduced into each bag, the earthworms being assigned to treatments at random. Mean body weights 2 S.E. were: university campus 1087 2 120 mg, Carrock Fell 490 282 mg. Ten earthworms were used per treatment, except for the campus earthworms in uncontaminated soil, where N = 9. The experiment was repeated, using hand-sorting to collect the earthworms, to eliminate any possible detrimental eects on earthworm condition from for-
C.J. Langdon et al. / Soil Biology and Biochemistry 31 (1999) 1963±1967
1965
Table 1 Concentrations of major contaminants in soil. A single bulk sample was analysed Element
As W Zn Cu
Concentration (mg kgÿ1) uncontaminated soil
contaminated soil
< 0.5 0.8 48 49
10,277 4097 1092 725
malin, and to achieve a greater consistency in body size of L. rubellus from Carrock mine spoil and campus, with 10 earthworms per treatment. The numbers and sizes of specimens used re¯ect availability and ease of identi®cation. At the campus site several Lumbricus species were present and distinguishing juveniles was dicult; only adults, subadults and large juveniles could be assigned to L. rubellus with con®dence. The bags were sealed and stored in the dark at 98C. After 7 d, 200 mg dried, powdered nettle (Urtica dioica L.) leaves were added as a food supplement. Specimens were examined weekly and assigned one of the following condition index (CI) scores: 2 Ð good muscle tone, earthworm responding rapidly to stimulation, 1 Ð poor muscle tone, responding fairly rapidly to stimulation, or good muscle tone, but slow in responding to stimulation and 0 Ð poor muscle tone, no response to stimulation. Assessment was carried out `blind', with an assistant presenting the specimens for assessment to the recorder. Ten L. rubellus collected from the University of Lancaster campus site and 10 L. rubellus collected from Carrock Fell mine spoil site, were kept in soil from a mixed deciduous woodland site on the campus for 24 h to evacuate the gut contents. Mean body weights 2S.E. were: university campus 797 2 33 mg, Carrock Fell 582 2 31 mg. Total tissue As of these earthworms was determined, using AAS, as was that of 10 L. rubellus from campus soil kept in the Asenriched soil for 28 d and the Carrock Fell L rubellus that were removed for analysis at 7 d intervals, if dead, or at 28 d.
Fig. 1. Condition of Lumbricus rubellus from a Lancaster site (uncontaminated with As) when kept for 28 d in uncontaminated (*) and arsenate-enriched (Q) soil. Condition of Lumbricus rubellus from a Carrock Fell site (heavily contaminated with As) when kept for 28 d in uncontaminated (W) and arsenate-enriched (T) soil. S.E. bars omitted for clarity.
and after 15 d three L. terrestris and one L. rubellus were dead, with six L. terrestris and two L. rubellus alive but in poor condition. Pathological indications included yellow discolouration, poor muscle tone and in some cases lesions and swellings along the body. The Carrock Fell earthworms kept under similar conditions remained active and in good condition over 84 d. L. rubellus collected from campus soil and kept in As-enriched soil rapidly lost condition (Figs.1 and 2). After only 7 d, the CI was signi®cantly lower for these earthworms than in both sets of controls (low As treat-
3. Results Arsenic was one of the most abundant elements found at Carrock Fell. Tungsten was also abundant, as was zinc and copper (Table 1). In the ®rst experiment, no earthworms were moribund after 5 d, one L. terrestris was dead after 10 d
Fig. 2. Condition of Lumbricus rubellus from a Lancaster site (uncontaminated with As) when kept for 28 d in uncontaminated (*) and arsenate-enriched (Q) soil. Condition of Lumbricus rubellus from a Carrock Fell site (heavily contaminated with As) when kept for 28 d in uncontaminated (W) and arsenate-enriched (T) soil. S.E. bars omitted for clarity.
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C.J. Langdon et al. / Soil Biology and Biochemistry 31 (1999) 1963±1967
Table 2 As concentrations in L. rubellus. N = 10; means2S.E Experiment
Exposure to contaminated soil As-enriched soil (trial 2)
Material analysed
L. rubellus campus L. rubellus Carrock Fell L. rubellus campus L. rubellus Carrock Fell
ments) and L. rubellus collected from Carrock Fell. (ANOVA and Tukey test; P < 0.01). After 14 d, the CI was signi®cantly lower than in any other treatment (P < 0.01). All campus earthworms, kept in As-rich soil, were comatose or dead after 28 d. In contrast, all specimens in the other treatments were in good or moderate condition after 28 d (Fig. 1). Bayesian statistical analysis provided a lethal time (LT50) value of 20.7 d, 95% CI 18.4±22.3. When the experiment was repeated using only handsorted earthworms, L. rubellus collected from campus soil and kept in As-enriched soil rapidly lost condition (Fig. 2). After only 7 d, the CI was signi®cantly lower for these earthworms than in both sets of controls (low As treatments) (ANOVA and t-test; P < 0.01). After 14 d, 7 of the campus earthworms kept in Asrich soil were comatose or dead; all were in this state after 28 d (Fig. 2). In contrast, all specimens in the other treatments were in good condition (CI=2) after 28 d (Fig. 2). (LT50 13.4 d, 95% CI 9.89±16.8). Pathological symptoms observed in both trials were similar to those noted above. L. rubellus from uncontaminated soil contained signi®cantly less total As than in all other treatments (ANOVA and Tukey test; P < 0.001) (Table 2). The concentration of As in campus soil (<1 mg kgÿ1), was signi®cantly less than that in Carrock Fell mine spoil (t-test; P < 0.01). The As concentration in Carrock L. rubellus was not signi®cantly dierent from that in the moribund/dead campus L. rubellus exposed to Asenriched soil (230 mg kgÿ1 and 92 mg kgÿ1 respectively) (Table 2), (t-test; P > 0.05). 4. Discussion Morgan and Morgan (1992) have reviewed the factors that might in¯uence heavy metal uptake and accumulation in earthworms. The degree of exposure of earthworms to As in soil will depend on the form and concentration of the As and on the ecological characteristics of the earthworm species. The dominant species recorded at Carrock Fell, L. rubellus, is epigeic
Time
Element
0d 0d 0d 28 d 0d 7±28 d
Concentration (mg kgÿ1) uncontaminated soil
contaminated soil
As As As
<1 nd nd
As
nd
nd 2302 14 <1 922 20 2002 54 1302 27
(BoucheÂ, 1977), i.e. an inhabitant of the super®cial layers of the soil that consumes a proportionately greater amount of organic matter than deeper-burrowing species. Its surface-dwelling mode of life might lead to a lower exposure risk than for deeper burrowers, both from cutaneous uptake and in the diet. However, the plants growing in the spoil at Carrock Fell have been shown to take up large amounts of As (reaching tissue concentrations of 253±500 mg kgÿ1, Black and Retberg, unpubl). Meharg and Macnair (1991) have recorded high contents of As in plants at other mine spoil sites, so dietary exposure from litterfeeding is likely to be substantial. Yeates et al. (1994) found no earthworms in soils contaminated by As, derived from timber preservatives, at concentrations of 400 and 800 mg kgÿ1 and few earthworms at 100 mg kgÿ1. However, the soils that these workers examined contained other contaminants in addition to As. In the ®rst experiment of the present study, the loss of condition of L. terrestris and L. rubellus in mine spoil may not have been due to As alone, but to other metal contaminants, or a synergistic action of metals including As. The loss of condition in the second pair of experiments is likely to be a direct response to As. Meharg et al. (1998) have demonstrated an LC50 of 100 mg arsenate kgÿ1 for an 8 d-exposure of L. terrestris and 400 mg kgÿ1 for 2 dexposure. Fischer and Koszorus (1992) recorded an LC50 of 100 mg kgÿ1 for exposure of E. fetida to arsenate over 56 d. Factors in¯uencing arsenic speciation and mobility include soil pH, Eh and organic matter content (Masscheleyn et al., 1991; Bhumbla and Keefer, 1994; Meharg et al., 1998). The response of earthworms to toxicity can be expected to vary with temperature, which in our experiments was comparatively low, at 98C. Tissue concentrations of As in individual earthworms varied considerably, possibly partly due to the nonhomogeneous distribution of As in the soil. Mean tissue concentrations of As found in L. rubellus native to Carrock Fell mine spoil were much higher than those for L. rubellus found in uncontaminated soil. L. rubellus from uncontaminated soil exposed to 2000 mg
C.J. Langdon et al. / Soil Biology and Biochemistry 31 (1999) 1963±1967
sodium arsenate kgÿ1 achieved mean tissue concentrations similar to those in L. rubellus native to Carrock Fell spoil, but suered 100% mortality after 28 d. Our results suggest that L. rubellus that are native to the mine spoil site may have developed resistance to As-toxicity. This resistance might be behavioural, the earthworms reducing exposure by curling up, moving away, coming to the surface, or ceasing to feed. In our experiments, all specimens burrowed and (judging from body colour) ingested substantial amounts of soil. The only individuals to be found curled up were campus earthworms kept in As-rich soil. Resistance might be due to acclimatisation, i.e. physiological adaptation. Meharg et al. (1998) suggest that bioconcentration of As in earthworms is due to sequestering of As in tissues in forms that are not readily eliminated. Morgan et al. (1994) reported that As may be bound to sulphur-rich enzymes in the tissues of earthworms inhabiting highly contaminated soils. Some enzymes are known to be involved in the biotransformation of As, reducing its toxicity (Li et al., 1994). Resistance might have a microbiological basis: Morgan and Morgan (1992) proposed that metal-tolerant strains of soil micro¯ora might accumulate metals in the earthworm gut. Conditions in the gut favour the rapid proliferation of some groups of microorganisms (Edwards and Bohlen, 1996). It is also possible that, as in metaltolerant strains of plants, the earthworms' insensitivity to As-toxicity is adaptive i.e. genetically based. Further work is needed to determine the basis of this resistance to As-toxicity and to determine whether the resistance extends to other species and to metal contaminants. Earthworms resistant to high concentrations of As and metal contaminants could play a valuable role in restoration of contaminated soils, together with As-resistant plants. Acknowledgements We thank Dr. A. J. Morgan and Dr. A. A. Meharg for valuable comments on the manuscript, Miss C. Kenny for assistance in the blind evaluation of earthworm condition, Miss V. Burnett for her technical assistance in AAS and Dr. M. Green for his statistical advice. For part of the work C. J. Langdon was supported by a NERC CASE studentship and S. Black by the Leverhulme Trust. References Beaty, R.D., Kerber, J.D., 1993. Concepts, Instrumentation and
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Techniques in Atomic Absorption Spectrophometry. Perkin Elmer Corporation. Bhumbla, D.K., Keefer, R.F., 1994. Arsenic mobilization and bioavailability in soils. In: Nriagu, J.O. (Ed.), Arsenic in the Environment, Part I: Cycling and Characterization. John Wiley, New York, pp. 51±82. Black, S., Lees, K., Bullock, S., Semple, K.T. Arsenic contamination from abandoned tungsten mine tailings, Carrock Fell, The Lake District, Applied Geochemistry, in press. BoucheÂ, M.B., 1977. StrateÂgies lombriciennes. Ecological Bulletin (Stockholm) 25, 122±132. Cullen, W.R., Reimer, K.J., 1989. Arsenic speciation in the environment. Chemical Reviews 89 (4), 713±764. Doyle, M.O., Otte, M.L., 1997. Organism-induced accumulation of iron, zinc and arsenic in wetland soils. Environmental Pollution 96, 1±11. Edwards, C.A., Bohlen, P.J., 1996. Biology and Ecology of Earthworms, 3rd ed. Chapman and Hall, London. Fischer, E., Koszorus, L., 1992. Sublethal eects, accumulation capacities and elimination rates of As, Hg and Se in the manure worm, Eisenia fetida Oligochaeta, Lumbricidae). Pedobiologia 36, 172±178. Greig-Smith, P.W., Becker, H., Edwards, P.J., Heimbach, F., 1992. Ecotoxicology of Earthworms. Intercept, Andover. Li, W., Chien, P.K., Furst, A., 1994. Evaluation of three antidotes on arsenic toxicity in the common earthworm (Lumbricus terrestris ). Journal of Applied Toxicology 14, 181±183. Masscheleyn, P.H., Delaune, R.D., Patrick, W.H., 1991. Eect of redox potential and pH on arsenic speciation and solubility in a contaminated soil. Environmental Science and Technology 25, 1414±1419. Meharg, A.A., Macnair, M.R., 1991. Uptake, accumulation and translocation of arsenate in arsenate tolerant and nontolerant Holcus lanatus. New Phytologist 117, 225±231. Meharg, A.A., Macnair, M.R., 1994. Phosphorus nutrition of arsenate-tolerant and nontolerant phenotypes of Velvetgrass. Journal of Environmental Quality 23, 234±238. Meharg, A.A., Shore, R.F., Broadgate, K., 1998. Edaphic factors aecting the toxicity and accumulation of arsenate in the earthworm Lumbricus terrestris. Environmental Toxicology and Chemistry 17, 1124±1131. Morgan, J.E., Morgan, A.J., 1992. Seasonal changes in the tissue metal (Cd, Zn and Pb) concentrations in two ecophysiologically similar earthworm species: pollution monitoring implications. Environmental Pollution 82, 1±7. Morgan, A.J., Winters, C., Yarwood, A., 1994. Speed-mapping of arsenic distribution in the tissues of earthworms inhabiting arsenious soil. Cell Biology International 18, 911±914. Porter, E.K., Peterson, P.J., 1975. Arsenic accumulation by plants on mine waste (United Kingdom). Science of the Total Environment 4, 365±371. Porter, E.K., Peterson, P.J., 1977. Arsenic tolerance in grasses growing in mine waste. Environmental Pollution 14, 255±267. Tertran, R., Claisse, F., 1982. Principles of Quantitative X-ray Florescence Analysis. Heyden, London. Yan-Chu, H., 1994. Arsenic distribution in soils. In: Nriagu, J.O. (Ed.), Arsenic in the Environment, Part I: Cycling and Characterization. John Wiley, New York, pp. 51±82. Yeates, C.W., Orchard, V.A., Speir, T.W., Hunt, J.L., Hermans, M.C.C., 1994. Impact of pasture contamination by copper, chromium, arsenic timber preservative on soil biological activity. Biology and Fertility of Soils 18, 200±208.
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Resistance to arsenic-toxicity in a population of the earthworm Lumbricus rubellus Caroline J. Langdon a, Trevor G. Piearce a,*, Stuart Black b, Kirk T. Semple c a
Department of Biological Sciences, Institute of Environmental and Natural Sciences, Lancaster University, Lancaster LA1 4YQ, UK b Postgraduate Research Institute for Sedimentology, University of Reading, Whiteknights, P.O. Box 227, Reading RG6 6AB, UK c Department of Environmental Sciences, Institute of Environmental and Natural Sciences, Lancaster University, Lancaster LA1 4YQ, UK Accepted 24 June 1999
Abstract Specimens of the earthworms Lumbricus terrestris L. and L. rubellus Homeister from an uncontaminated site rapidly deteriorated in condition when kept in spoil rich in metal contaminants and arsenic. The site from which the spoil was collected supports several earthworm species, L. rubellus being dominant. Native L. rubellus survived for 12 weeks in spoil in the laboratory. L. rubellus collected from the spoil site and an uncontaminated site were kept for 28 d in uncontaminated soil and in soil containing 2000 mg sodium arsenate heptahydrate kgÿ1, the state of the specimens being recorded using a semi-quantitative assessment of earthworm condition (condition index, CI). The CI remained high for all specimens except those from the uncontaminated site kept in As-rich soil, for which mortality was 100% after 28 d. Tissue As concentrations in L rubellus from uncontaminated and contaminated sites were <1 mg kgÿ1 and 230 mg kgÿ1, respectively. In L. rubellus collected from the uncontaminated site and exposed to contaminated soil containing 2000 mg sodium arsenate heptahydrate kgÿ1, mean tissue As concentration was 92 mg kgÿ1. # 1999 Elsevier Science Ltd. All rights reserved. Keywords: Arsenic-sensitivity; Earthworm; Soil contamination; Metal contaminants; Toxicity testing
1. Introduction Arsenic is widely distributed in soils, with a mean concentration of around 6 mg kgÿ1. However, much higher concentrations can occur in mine spoils and areas contaminated by industrial wastes and biocides (Yan-Chu, 1994). Under aerobic conditions inorganic As is present predominantly as arsenate; however, arsenite, a more toxic form of As, predominates in anoxic soils (Cullen and Reimer, 1989). Earthworms have a particularly intimate contact with the soil, consuming large quantities and having little external barrier to the soil solution. For this reason, and because of their importance in terrestrial food webs, they are widely used as indicators of soil contamination (Greig* Corresponding author. Tel.: +44-1524-65201; fax: +44-1524843854. E-mail address: [email protected] (T.G. Piearce)
Smith et al., 1992). This, together with their major role in the incorporation and decomposition of dead organic matter in the soil, and in the development and maintenance of soil structure (Edwards and Bohlen, 1996), also makes them valuable indicators of soil health. Their in¯uence on soil drainage and aeration, through burrowing and casting, has well-known implications for soil fertility (Edwards and Bohlen, 1996). It may also have consequences for the speciation, solubility and toxicity of soil contaminants, seen, to some extent, in sediment bioturbation by polychaete worms (Doyle and Otte, 1997). Earthworms have been reported to inhabit As-rich metalliferous soils in SW England (Morgan and Janes, unpubl, in Morgan et al., 1994). Spoil at an abandoned tungsten mine at Carrock Fell, Cumbria, NW England, (NY 324 330), contains As, largely as arsenopyrite, in concentrations up to 53,000 mg kgÿ1 (Black et al., in press), three orders of magnitude above back-
0038-0717/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 8 - 0 7 1 7 ( 9 9 ) 0 0 1 1 8 - 2
1964
C.J. Langdon et al. / Soil Biology and Biochemistry 31 (1999) 1963±1967
ground contents for the Carrock Fell area. Since soil treated with sodium arsenate at substantially lower concentrations than this has been shown to be highly toxic to the earthworms Eisenia fetida (Savigny) (Fischer and Koszorus, 1992) and L. terrestris L. (Meharg et al., 1998), it is of interest that several earthworm species occur in the spoil, including L. rubellus, which is the dominant species. Arsenic-tolerance has been reported in plants (Porter and Peterson, 1975, 1977; Meharg and Macnair, 1991; Meharg and Macnair, 1994) and may also occur in the fauna of contaminated soils. Specimens of L. terrestris and L. rubellus Homeister from an uncontaminated site were kept in Carrock Fell mine spoil, as described below, for 15 d. The condition of many of the earthworms deteriorated substantially over the course of the experiment, although specimens of L. rubellus native to the mine site survived in good condition in spoil in the laboratory for several months. Since the mine spoil is rich in other metal contaminants (Black et al., in press) which might have contributed to the deterioration in the earthworms' condition, a second experiment, also described here, was conducted to evaluate the earthworms' tolerance of As per se. A semi-quantitative assessment of earthworm condition was developed, which provides ®ner discrimination of the state of the animals than simply recording mortalities. Tissue As concentration of L. rubellus from contaminated and uncontaminated soils were recorded, together with total tissue As concentrations for L. rubellus exposed to the As treated soil. 2. Materials and methods 2.1. Responses of L. terrestris and L. rubellus on exposure to As-contaminated mine spoil Sixty adult and large immature L. terrestris were collected by hand at night from the surface of mown grassland and 18 adult and large immature L. rubellus by hand-sorting in mixed deciduous woodland. After removal of stones, 500 g soil samples were weighed into 20 25 cm polythene bags and 200 mg of dried, ground mine spoil vegetation, dominated by grass and rush, was added as food. One earthworm was placed in each bag (experience having shown that, where several specimens are kept together, there is a serious risk of infection spreading from one moribund individual to another). The bags were sealed, stored in the dark at 98C and periodically opened for ventilation. Twenty L. terrestris and six L. rubellus were removed after 5, 10 and 15 d and their condition noted. The tissue As concentration in L. rubellus was determined (see below).Ten L. rubellus from Carrock Fell spoil were
kept under similar conditions in the laboratory for 84 d. 2.2. Soil and earthworm tissue analysis Quantities of major elements in soils from Carrock Fell and Lancaster University campus were determined using a Phillips PW 1400 XRF (Tertran and Claisse, 1982). One bulk sample of soil was used for analysis. The L. rubellus were weighed and depurated for 24 h in individual polythene bags containing moist tissue, then killed by dipping in hot water, placed in conical ¯asks and dried at 408C overnight and re-weighed. Mean dry body weights 2 S.E. were: university campus L. rubellus 221 215 mg, L. terrestris 693 2 25 mg, Carrock Fell L. rubellus 47 2 19 mg. Samples were acid-digested with 20 ml (Aristar grade) nitric acid, ®ltered using Whatman No. 540 ashless ®lter papers and stored at 98C. Mean total tissue contents of As were measured using a Perkin Elmer 2280 graphite furnace (AAS) with calibration standards of 0, 50, 100, 150 and 200 mg gÿ1. Dilutions ranging between 1/1000±1/ 50 were required for L. rubellus from contaminated soil and As-treated soils; no dilutions were required for L. rubellus from uncontaminated soil (see Beaty and Kerber, 1993 for machine keyboard entries). 2.3. Response of L. rubellus to As-treated soil Specimens of L. rubellus were collected from an uncontaminated grassland site on the University of Lancaster campus, by formalin expulsion, followed by washing, and from Carrock Fell mine spoil by a combination of formalin extraction, followed by washing, and by hand-sorting of soil. All were kept for several weeks in their parent soil before use. Soil from a mixed deciduous woodland site at Lancaster, (SD 488 574), which supported an abundant and diverse earthworm community, was partially air-dried, sieved through a 2.8 mm mesh and rewetted to a moisture content of 53% (dry weight equivalent) using distilled water, or with a solution of sodium arsenate heptahydrate to give a concentration of 2000 mg kgÿ1 dry weight of soil of the hydrated salt. Mean soil pH (aqueous suspension)2 S.E. was 4.97 2 0.05. Moistened soil (69 g) was weighed into each of a series of 20 25 cm polythene bags. One L. rubellus was weighed and introduced into each bag, the earthworms being assigned to treatments at random. Mean body weights 2 S.E. were: university campus 1087 2 120 mg, Carrock Fell 490 282 mg. Ten earthworms were used per treatment, except for the campus earthworms in uncontaminated soil, where N = 9. The experiment was repeated, using hand-sorting to collect the earthworms, to eliminate any possible detrimental eects on earthworm condition from for-
C.J. Langdon et al. / Soil Biology and Biochemistry 31 (1999) 1963±1967
1965
Table 1 Concentrations of major contaminants in soil. A single bulk sample was analysed Element
As W Zn Cu
Concentration (mg kgÿ1) uncontaminated soil
contaminated soil
< 0.5 0.8 48 49
10,277 4097 1092 725
malin, and to achieve a greater consistency in body size of L. rubellus from Carrock mine spoil and campus, with 10 earthworms per treatment. The numbers and sizes of specimens used re¯ect availability and ease of identi®cation. At the campus site several Lumbricus species were present and distinguishing juveniles was dicult; only adults, subadults and large juveniles could be assigned to L. rubellus with con®dence. The bags were sealed and stored in the dark at 98C. After 7 d, 200 mg dried, powdered nettle (Urtica dioica L.) leaves were added as a food supplement. Specimens were examined weekly and assigned one of the following condition index (CI) scores: 2 Ð good muscle tone, earthworm responding rapidly to stimulation, 1 Ð poor muscle tone, responding fairly rapidly to stimulation, or good muscle tone, but slow in responding to stimulation and 0 Ð poor muscle tone, no response to stimulation. Assessment was carried out `blind', with an assistant presenting the specimens for assessment to the recorder. Ten L. rubellus collected from the University of Lancaster campus site and 10 L. rubellus collected from Carrock Fell mine spoil site, were kept in soil from a mixed deciduous woodland site on the campus for 24 h to evacuate the gut contents. Mean body weights 2S.E. were: university campus 797 2 33 mg, Carrock Fell 582 2 31 mg. Total tissue As of these earthworms was determined, using AAS, as was that of 10 L. rubellus from campus soil kept in the Asenriched soil for 28 d and the Carrock Fell L rubellus that were removed for analysis at 7 d intervals, if dead, or at 28 d.
Fig. 1. Condition of Lumbricus rubellus from a Lancaster site (uncontaminated with As) when kept for 28 d in uncontaminated (*) and arsenate-enriched (Q) soil. Condition of Lumbricus rubellus from a Carrock Fell site (heavily contaminated with As) when kept for 28 d in uncontaminated (W) and arsenate-enriched (T) soil. S.E. bars omitted for clarity.
and after 15 d three L. terrestris and one L. rubellus were dead, with six L. terrestris and two L. rubellus alive but in poor condition. Pathological indications included yellow discolouration, poor muscle tone and in some cases lesions and swellings along the body. The Carrock Fell earthworms kept under similar conditions remained active and in good condition over 84 d. L. rubellus collected from campus soil and kept in As-enriched soil rapidly lost condition (Figs.1 and 2). After only 7 d, the CI was signi®cantly lower for these earthworms than in both sets of controls (low As treat-
3. Results Arsenic was one of the most abundant elements found at Carrock Fell. Tungsten was also abundant, as was zinc and copper (Table 1). In the ®rst experiment, no earthworms were moribund after 5 d, one L. terrestris was dead after 10 d
Fig. 2. Condition of Lumbricus rubellus from a Lancaster site (uncontaminated with As) when kept for 28 d in uncontaminated (*) and arsenate-enriched (Q) soil. Condition of Lumbricus rubellus from a Carrock Fell site (heavily contaminated with As) when kept for 28 d in uncontaminated (W) and arsenate-enriched (T) soil. S.E. bars omitted for clarity.
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C.J. Langdon et al. / Soil Biology and Biochemistry 31 (1999) 1963±1967
Table 2 As concentrations in L. rubellus. N = 10; means2S.E Experiment
Exposure to contaminated soil As-enriched soil (trial 2)
Material analysed
L. rubellus campus L. rubellus Carrock Fell L. rubellus campus L. rubellus Carrock Fell
ments) and L. rubellus collected from Carrock Fell. (ANOVA and Tukey test; P < 0.01). After 14 d, the CI was signi®cantly lower than in any other treatment (P < 0.01). All campus earthworms, kept in As-rich soil, were comatose or dead after 28 d. In contrast, all specimens in the other treatments were in good or moderate condition after 28 d (Fig. 1). Bayesian statistical analysis provided a lethal time (LT50) value of 20.7 d, 95% CI 18.4±22.3. When the experiment was repeated using only handsorted earthworms, L. rubellus collected from campus soil and kept in As-enriched soil rapidly lost condition (Fig. 2). After only 7 d, the CI was signi®cantly lower for these earthworms than in both sets of controls (low As treatments) (ANOVA and t-test; P < 0.01). After 14 d, 7 of the campus earthworms kept in Asrich soil were comatose or dead; all were in this state after 28 d (Fig. 2). In contrast, all specimens in the other treatments were in good condition (CI=2) after 28 d (Fig. 2). (LT50 13.4 d, 95% CI 9.89±16.8). Pathological symptoms observed in both trials were similar to those noted above. L. rubellus from uncontaminated soil contained signi®cantly less total As than in all other treatments (ANOVA and Tukey test; P < 0.001) (Table 2). The concentration of As in campus soil (<1 mg kgÿ1), was signi®cantly less than that in Carrock Fell mine spoil (t-test; P < 0.01). The As concentration in Carrock L. rubellus was not signi®cantly dierent from that in the moribund/dead campus L. rubellus exposed to Asenriched soil (230 mg kgÿ1 and 92 mg kgÿ1 respectively) (Table 2), (t-test; P > 0.05). 4. Discussion Morgan and Morgan (1992) have reviewed the factors that might in¯uence heavy metal uptake and accumulation in earthworms. The degree of exposure of earthworms to As in soil will depend on the form and concentration of the As and on the ecological characteristics of the earthworm species. The dominant species recorded at Carrock Fell, L. rubellus, is epigeic
Time
Element
0d 0d 0d 28 d 0d 7±28 d
Concentration (mg kgÿ1) uncontaminated soil
contaminated soil
As As As
<1 nd nd
As
nd
nd 2302 14 <1 922 20 2002 54 1302 27
(BoucheÂ, 1977), i.e. an inhabitant of the super®cial layers of the soil that consumes a proportionately greater amount of organic matter than deeper-burrowing species. Its surface-dwelling mode of life might lead to a lower exposure risk than for deeper burrowers, both from cutaneous uptake and in the diet. However, the plants growing in the spoil at Carrock Fell have been shown to take up large amounts of As (reaching tissue concentrations of 253±500 mg kgÿ1, Black and Retberg, unpubl). Meharg and Macnair (1991) have recorded high contents of As in plants at other mine spoil sites, so dietary exposure from litterfeeding is likely to be substantial. Yeates et al. (1994) found no earthworms in soils contaminated by As, derived from timber preservatives, at concentrations of 400 and 800 mg kgÿ1 and few earthworms at 100 mg kgÿ1. However, the soils that these workers examined contained other contaminants in addition to As. In the ®rst experiment of the present study, the loss of condition of L. terrestris and L. rubellus in mine spoil may not have been due to As alone, but to other metal contaminants, or a synergistic action of metals including As. The loss of condition in the second pair of experiments is likely to be a direct response to As. Meharg et al. (1998) have demonstrated an LC50 of 100 mg arsenate kgÿ1 for an 8 d-exposure of L. terrestris and 400 mg kgÿ1 for 2 dexposure. Fischer and Koszorus (1992) recorded an LC50 of 100 mg kgÿ1 for exposure of E. fetida to arsenate over 56 d. Factors in¯uencing arsenic speciation and mobility include soil pH, Eh and organic matter content (Masscheleyn et al., 1991; Bhumbla and Keefer, 1994; Meharg et al., 1998). The response of earthworms to toxicity can be expected to vary with temperature, which in our experiments was comparatively low, at 98C. Tissue concentrations of As in individual earthworms varied considerably, possibly partly due to the nonhomogeneous distribution of As in the soil. Mean tissue concentrations of As found in L. rubellus native to Carrock Fell mine spoil were much higher than those for L. rubellus found in uncontaminated soil. L. rubellus from uncontaminated soil exposed to 2000 mg
C.J. Langdon et al. / Soil Biology and Biochemistry 31 (1999) 1963±1967
sodium arsenate kgÿ1 achieved mean tissue concentrations similar to those in L. rubellus native to Carrock Fell spoil, but suered 100% mortality after 28 d. Our results suggest that L. rubellus that are native to the mine spoil site may have developed resistance to As-toxicity. This resistance might be behavioural, the earthworms reducing exposure by curling up, moving away, coming to the surface, or ceasing to feed. In our experiments, all specimens burrowed and (judging from body colour) ingested substantial amounts of soil. The only individuals to be found curled up were campus earthworms kept in As-rich soil. Resistance might be due to acclimatisation, i.e. physiological adaptation. Meharg et al. (1998) suggest that bioconcentration of As in earthworms is due to sequestering of As in tissues in forms that are not readily eliminated. Morgan et al. (1994) reported that As may be bound to sulphur-rich enzymes in the tissues of earthworms inhabiting highly contaminated soils. Some enzymes are known to be involved in the biotransformation of As, reducing its toxicity (Li et al., 1994). Resistance might have a microbiological basis: Morgan and Morgan (1992) proposed that metal-tolerant strains of soil micro¯ora might accumulate metals in the earthworm gut. Conditions in the gut favour the rapid proliferation of some groups of microorganisms (Edwards and Bohlen, 1996). It is also possible that, as in metaltolerant strains of plants, the earthworms' insensitivity to As-toxicity is adaptive i.e. genetically based. Further work is needed to determine the basis of this resistance to As-toxicity and to determine whether the resistance extends to other species and to metal contaminants. Earthworms resistant to high concentrations of As and metal contaminants could play a valuable role in restoration of contaminated soils, together with As-resistant plants. Acknowledgements We thank Dr. A. J. Morgan and Dr. A. A. Meharg for valuable comments on the manuscript, Miss C. Kenny for assistance in the blind evaluation of earthworm condition, Miss V. Burnett for her technical assistance in AAS and Dr. M. Green for his statistical advice. For part of the work C. J. Langdon was supported by a NERC CASE studentship and S. Black by the Leverhulme Trust. References Beaty, R.D., Kerber, J.D., 1993. Concepts, Instrumentation and
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Techniques in Atomic Absorption Spectrophometry. Perkin Elmer Corporation. Bhumbla, D.K., Keefer, R.F., 1994. Arsenic mobilization and bioavailability in soils. In: Nriagu, J.O. (Ed.), Arsenic in the Environment, Part I: Cycling and Characterization. John Wiley, New York, pp. 51±82. Black, S., Lees, K., Bullock, S., Semple, K.T. Arsenic contamination from abandoned tungsten mine tailings, Carrock Fell, The Lake District, Applied Geochemistry, in press. BoucheÂ, M.B., 1977. StrateÂgies lombriciennes. Ecological Bulletin (Stockholm) 25, 122±132. Cullen, W.R., Reimer, K.J., 1989. Arsenic speciation in the environment. Chemical Reviews 89 (4), 713±764. Doyle, M.O., Otte, M.L., 1997. Organism-induced accumulation of iron, zinc and arsenic in wetland soils. Environmental Pollution 96, 1±11. Edwards, C.A., Bohlen, P.J., 1996. Biology and Ecology of Earthworms, 3rd ed. Chapman and Hall, London. Fischer, E., Koszorus, L., 1992. Sublethal eects, accumulation capacities and elimination rates of As, Hg and Se in the manure worm, Eisenia fetida Oligochaeta, Lumbricidae). Pedobiologia 36, 172±178. Greig-Smith, P.W., Becker, H., Edwards, P.J., Heimbach, F., 1992. Ecotoxicology of Earthworms. Intercept, Andover. Li, W., Chien, P.K., Furst, A., 1994. Evaluation of three antidotes on arsenic toxicity in the common earthworm (Lumbricus terrestris ). Journal of Applied Toxicology 14, 181±183. Masscheleyn, P.H., Delaune, R.D., Patrick, W.H., 1991. Eect of redox potential and pH on arsenic speciation and solubility in a contaminated soil. Environmental Science and Technology 25, 1414±1419. Meharg, A.A., Macnair, M.R., 1991. Uptake, accumulation and translocation of arsenate in arsenate tolerant and nontolerant Holcus lanatus. New Phytologist 117, 225±231. Meharg, A.A., Macnair, M.R., 1994. Phosphorus nutrition of arsenate-tolerant and nontolerant phenotypes of Velvetgrass. Journal of Environmental Quality 23, 234±238. Meharg, A.A., Shore, R.F., Broadgate, K., 1998. Edaphic factors aecting the toxicity and accumulation of arsenate in the earthworm Lumbricus terrestris. Environmental Toxicology and Chemistry 17, 1124±1131. Morgan, J.E., Morgan, A.J., 1992. Seasonal changes in the tissue metal (Cd, Zn and Pb) concentrations in two ecophysiologically similar earthworm species: pollution monitoring implications. Environmental Pollution 82, 1±7. Morgan, A.J., Winters, C., Yarwood, A., 1994. Speed-mapping of arsenic distribution in the tissues of earthworms inhabiting arsenious soil. Cell Biology International 18, 911±914. Porter, E.K., Peterson, P.J., 1975. Arsenic accumulation by plants on mine waste (United Kingdom). Science of the Total Environment 4, 365±371. Porter, E.K., Peterson, P.J., 1977. Arsenic tolerance in grasses growing in mine waste. Environmental Pollution 14, 255±267. Tertran, R., Claisse, F., 1982. Principles of Quantitative X-ray Florescence Analysis. Heyden, London. Yan-Chu, H., 1994. Arsenic distribution in soils. In: Nriagu, J.O. (Ed.), Arsenic in the Environment, Part I: Cycling and Characterization. John Wiley, New York, pp. 51±82. Yeates, C.W., Orchard, V.A., Speir, T.W., Hunt, J.L., Hermans, M.C.C., 1994. Impact of pasture contamination by copper, chromium, arsenic timber preservative on soil biological activity. Biology and Fertility of Soils 18, 200±208.