The time-course of metal (Ca, Cd, Cu, Pb, Zn) accumulation
from a contaminated soil by three populations
of the earthworm,
Lumbricus rubellus
F. MarinÄo
a, A.J. Morgan
b,*aDepartamento de EcoloxõÂa e Bioloxia Animal, Facultade de Ciencias, Universidade de Vigo, Vigo, Pontevedra, Spain bCardiff School of Biosciences, University of Cardiff, P.O. Box 915, Cardiff CF1 3TL, Wales, UK
Received 4 September 1997; accepted 7 October 1998
Abstract
Adult earthworms (Lumbricus rubellus), sampled from a metal-contaminated site (Cwmystwyth Cottage) and two reference sites (acidic, Caerf®li; calcareous, Dinas Powys), were maintained on metalliferous Cwmystwyth Cottage soil in the laboratory for up to 90 days. All three populations accumulated Cd, Pb and Zn for at least the ®rst 60 days of laboratory exposure, while Cu and Ca concentrations were maintained fairly constant throughout. The experiment did not prove conclusively that the native worms maintained on their own contaminated soil had the capacity to accumulate higher burdens of non-essential toxic metals (Cd and Pb) than the introduced reference worms. Finally, the reference population naturally inhabiting the Ca-poor acidic soil (Caerf®li) accumulated or maintained tissue Ca concentrations more ef®ciently than the second reference population (Dinas Powys) and `native' population (Cwmystwyth Cottage), both naturally inhabiting relatively Ca-rich soils.#1999 Elsevier Science B.V. All rights reserved.
Keywords: Lumbricus rubellus; Cd; Pb; Zn; Cu; Ca
1. Introduction
Metal tolerance has been fairly conclusively demonstrated in terrestrial isopods (Donker and Bogert, 1991), the centipede Lithobius variegatus (Hopkin and Martin, 1984), some mite species belong-ing to the families Mesostigmata and Gamasine (HaÊg-var and Abrahamsen, 1990), the springtailsOrchesella cincta(Posthuma et al., 1993), and the dipteran Dro-sophila melanogaster(Lauverjat et al., 1989). Genetic
tolerance has been con®rmed for the marine poly-chaete,Nereis diversicolor(Grant et al., 1989) and the freshwater oligochaete Limnodrilus hoffmeisteri (Klerks and Levinton, 1989, 1993), but the unequi-vocal evidence for `heavy' metal adaptations in earth-worm populations is non-existent (Posthuma and Van Straalen, 1993).
Organisms can resist the toxic effects of metals by any one of several means (Klerks, 1990; Posthuma and Van Straalen, 1993), including adjusting metal assim-ilation ef®ciency, the binding/immobilization capa-city, and/or the excretory rate. Very few studies on terrestrial macroinvertebrates have distinguished *Corresponding author. Tel.: 12222874190; fax:
+44-1222874305; e-mail: fry@cardiff.ac.uk
between `adaptation' and `acclimation' (Klerks and Weis, 1987) mainly because of the dif®culty of produ-cing suf®cient F1 and F2 offspring. There are many compelling ecotoxicological reasons for making this distinction (Posthuma and Van Straalen, 1993). For example, the indiscriminate testing of adapted and non-adapted populations could yield misleading toxi-city data (Baird et al., 1990; Duan et al., 1997).
Corp and Morgan (1991) compared the metal con-centrations in nine populations of the earthworm L. rubellusmaintained in the laboratory for 31 days on their `own' metalliferous soils (native worms), with those in batches of worms from a clean site (intro-duced worms) maintained on the same series of con-taminated soils. Their data did not furnish evidence of inter-population differentiation because it was not possible to determine whether or not the metal con-centrations in the introduced earthworms had equili-brated within the exposure period.
The present paper describes the time-course of metal accumulation by three earthworm populations maintained for up to 90 days on a soil from a single metalliferous source. While one earthworm popula-tion was native to the polluted soil, the others were collected from a relatively calcareous soil and an acidic soil, respectively. Three questions were addressed: (i) do tissue metal concentrations equili-brate in native and introduced worms within 90 days, i.e. is uptake eventually balanced by elimination? (ii) is there evidence of population differentiation between `native' (presumptive tolerant) earthworms and `intro-duced' (presumptive intolerant) earthworms as expressed by their accumulated metal burdens? (iii) do the patterns of metal accumulation in laboratory-exposure experiments reveal physiological differen-tiation between `control' earthworm populations inha-biting contaminated calcareous and acidic soils, respectively?
2. Materials and methods
2.1. Earthworms and soil samples
Earthworms (L. rubellus) and soil were collected during April 1993 from one contaminated site in mid-Wales (Cwmystwyth Cottage, O.S. Grid Ref.SN806748). Earthworms were also collected
from two uncontaminated sites in south Wales: Dinas Powys (O.S. Grid Ref.ST149723; a relatively cal-careous soil: pH5.8; [Ca]2000mg/g dry weight;
Soil from Cwmystwyth was thoroughly mixed to minimize heterogeneity problems and distributed between 27 plastic boxes (4 l capacity). Ten adult worms from Cwmystwyth (native worms, `N'), Dinas Powys (introduced, `I-DP'), or Caerf®li (introduced, `I-CF') were placed in assigned boxes (i.e. 9 boxes10 worms per population). The boxes were placed in a constant temperature (178C), light-con-trolled room for the duration of the exposure experi-ment. Every 10th day up to 90 days, ten worms were removed from one container per site and prepared for metal analysis. No food was added to the boxes throughout the period of exposure. Soil moisture was maintained at approximately 50% (of dry weight).
2.2. Analytical methods
Soil pH was measured in triplicate in deionised water slurries (3 g soil : 30 ml H2O) after stirring and
equilibration over 3 h. Soil organic matter content was estimated (in triplicate) from the percentage loss-on-ignition during 18 h in a muf¯e furnace at 6008C. Soil and earthworm `total' metal (Cd, Ca, Cu, Pb, Zn) concentrations were determined by digestion in 5 ml and 2 ml conc. `Analar' HNO3, respectively. Metal
analyses were performed by atomic absorption spec-trophotometry in an Instruments Laboratory AA/AE 457 with automatic background adjustment. All solu-tions, including standards, used for Ca assay contained 1% lanthanum. Regular analysis of certi®ed sedi-ments, plant materials and soft animal tissues indi-cated that the overall analytical error using our standard acid-digestion and A.S.S. protocols does not exceed 7% for any of the reported metals.
2.3. Statistical analysis
3. Results
Soils associated with disused mine spoils are notor-iously heterogeneous in composition. This was con-®rmed by the analyses of soils (all from Cwmystwyth Cottage) taken from each plastic box (Table 1; con-centrations of Cd, Pb, Cu, Zn and Ca ranged from 31± 120mg/g dry weight, 1594±8688mg/g, 9±144mg/g, 6625±54 450mg/g and 14 825±56 350mg/g, respec-tively; pH was 5.9±6.3; % loss on ignition was 31.85%±51.19%). However, since the earthworm groups were randomly assigned to the soil sub-sam-ples, the heterogeneity was unlikely to introduce a systematic error.
3.1. Cadmium
Qualitative assessment indicates that Cd concentra-tions in the native worms (N) did not increase sig-ni®cantly over the 90 day period and equilibrated in the two introduced populations (I-CF, I-DP) after 60 days (Fig. 1).
Up to about 70 days, the differences in the tissue Cd concentrations between native and introduced earth-worms were statistically signi®cant, while between 70 and 90 days the differences were usually non-signi®-cant (Table 3). In general, the earthworms sampled from calcareous soil (DP) did not accumulate Cd at a rate different from that of the earthworms sampled from acidic soil (CF). The data did not yield con-clusive evidence about population divergence.
3.2. Lead
Despite some sample-to-sample ¯uctuations, there was no overall qualitative increase in the Pb
concen-trations or Pb contents in native worms during the 90 day period (Table 2, Fig. 1). After an initial lag period of about 30 days, the Pb concentrations and contents in the introduced worms (DP and CF) increased steadily up to 90 days (Table 2, Fig. 1), with few notable differences in the Pb concentrations between the two populations (Table 3).
Towards the end of the laboratory exposure period of 90 days, the Pb concentrations in the introduced worms appeared to converge on those of the native worms (Table 2, Fig. 1). This observation, together with the generally insigni®cant differences in the accumulated Pb concentrations in I-DP (i.e. worms of calcareous soil origin) and I-CF (worms of acidic soil origin) populations, indicates that there were no pronounced population-speci®c differences in Pb metabolism.
3.3. Copper
Cu tissue concentrations and contents like Ca, but unlike Cd, Pb and Zn, did not differ initially between the native and introduced populations (Tables 2 and 3, Fig. 1). Indeed the soil in which the worms were maintained (i.e. Cwmystwyth Cottage soil) had Cu concentrations at near background levels (Table 1).
Tissue Cu concentrations were low in each worm population (bioaccumulation factor, whole-worm Cu concentration/conc. HNO3-extractable soil Cu
con-centration in the range from 0.5 to <1.0). They remained fairly constant throughout the 90 day main-tenance period. Thus, the data indicated that earth-worms are not ef®cient Cu accumulators, at least from soils of relatively low `total' Cu concentrations, and that there is no evidence of physiological differentia-tion between the three populadifferentia-tions.
Table 1
Metal concentrations (mg/g dry weight), pH and % organic matter content (o.m.) of the metalliferous (Cwmystwyth) soil to which earthworms
were exposed in the laboratory
Parameters Cadmium Lead Copper Zinc Calcium pH o.m.
Median 74 3937 26 22 938 30 725 6.1 40
Q1±Q3 62.81/84.30 2992/5242 19.03/40.62 15 944/33 750 24 900/36 150 6/6 37.55/42.72 Range 31±120 1594±8688 9±114 6625±54 450 14 825±56 350 5.9±6.3 31.85±51.19 MeanS.E. 741.4 4242156 342 25 6311120 31 061936 6.10.01 400.46
n 108 108 108 108 108 81 81
n: Number of observations.Q1: first quartile;Q3: third quartile;Q1±Q3: interquartile range.
3.4. Zinc
The Zn temporal relationships for the three popula-tions resembled those for Cd and Pb (Table 3, Fig. 1). The native worms accumulated Zn very gradually over the 90-day period. The two introduced populations resembled each other and accumulated Zn steadily, so that after 80 and 90 days there were no signi®cant differences between the tissue Zn concentrations in native and introduced worms (Table 3).
3.5. Calcium
The Ca concentrations in each earthworm popula-tion were very similar at the start of the laboratory
exposure period and were regulated at fairly constant levels during the 90-day exposure (Table 2, Fig. 1). However, it is worth noting that worms sampled from the acidic clean soil (CF) tended to have higher tissue Ca concentrations between Days 20 and 90 (signi®-cant, only on Days 50 and 60) as compared with worms sampled from a calcareous metalliferous soil (CC) and a calcareous uncontaminated soil (DP) (Table 3).
4. Discussion
The Cwmystwyth Cottage earthworms maintained on their `own' contaminated soil increased their tissue Fig. 1. Metal concentrations (mg/g dry weight) in threeL. rubelluspopulations (native: Cwmystwyth Cottage, CC (open squares); introduced
metal contents (Cd, Pb, Zn) over a period of 60 days, albeit at a rate slower than those exhibited by `intro-duced' earthworms sampled from clean sites. There are several possible reasons for this. First, the ambient physicochemical conditions within the soil under normalized laboratory conditions may exert important
effects on metal bioavailabilities. For example, an increase in temperature is accompanied by increased Pb uptake by the earthworm Lumbricus terrestris (Bengtsson and Rundgren, 1992), while it has been reported that a rise in temperature increases Cd uptake, but does not affect Cd elimination by the Table 2
Metal concentrations (mg/g dry weight) in the tissues of native (N: Cwmystwyth) and introduced (I-CF: Caerffili; I-DP: Dinas Powys)
earthworms at ten day intervals after exposure to Cwmystwyth soil
Cadmium Lead Copper Zinc Calcium n
10 days
N 9215 4050470 171 4320530 5390330 10
I-CF 92 710230 161 1150360 6290870 9
I-DP 74 380100 162 1250410 61501160 9
20 days
N 4616 2690640 141 3670620 6320870 10
I-CF 112 49040 161 1580150 5700300 9
I-DP 165 33060 141 1480260 5190560 8
30 days
N 9015 69701190 222 83701740 5380390 10
I-CF 143 45090 245 2270440 77101550 9
I-DP 232 36030 192 1640130 3980380 8
40 days
N 7710 3740760 212 6660910 4970410 10
I-CF 294 93090 486 3510490 6110620 10
I-DP 415 66033 334 2530300 4010320 9
50 days
N 12515 63601050 292 5940460 4480330 10
I-CF 263 1350170 303 2100570 6000380 5
I-DP 413 1160110 272 2570300 4050340 10
60 days
N 14337 71101590 272 79901290 54901060 6
I-CF 446 1690310 352 4450390 7140560 6
I-DP 555 140070 282 4290410 4230250 7
70 days
N 13525 6730840 252 92901040 5610320 9
I-CF 463 2070420 3311 52101310 78101250 5
I-DP 474 1590270 272 4190790 53301150 5
80 days
N 16420 75901010 294 10 9901000 6090440 9
I-CF 7011 2890750 193 83603960 91902910 4
I-DP 7713 3260660 509 13 3502520 87901370 4
90 days
N 12620 63402090 254 12 220 3190 5390870 5
I-CF 7916 32101350 170 87201240 9520460 2
I-DP 6011 2350850 196 78102730 55301110 4
Data expressed as meanSE.
oribatid mite,Platynothrus peltifer(Janssen and Ber-gema, 1991). Soil moisture content may also be a modulating factor (Van Gestel, 1997). Secondly, there are some circumstantial grounds for considering that biological factors may contribute to the uptake of metals into the tissues of earthworms maintained on their own soils in small chambers. Hopkin (1990) observed that isopods, when fed metal-contaminated litter from their `own' sites under laboratory condi-tions, had a higher net accumulation of some metals than their ®eld counterparts. Net metal accumulation
can increase in the laboratory-maintained isopods because they display hyperphagy, and because they also selectively consume fungal hyphae that infest and amplify the metal concentrations in laboratory sub-strates (Hopkin, 1990). Hyperphagy and the consump-tion of soil micro¯ora were unlikely to be determinants of the steady increase in the metal con-centrations in the Cwmystwyth Cottage earthworms, mainly because the fairly restricted soil volumes in which they were maintained were not supplemented with food at any stage. This maintenance protocol was Table 3
Statistical comparison of the metal concentrations in the three earthworm populations (N, I-CF, I-DP) at ten day intervals after exposure to a metalliferous soil
Comparisons Cd Pb Cu Zn Ca
10 days N vs. I-CF a a ns a ns
N vs. I-DP a a ns a ns
I-CF vs. I-DP ns ns ns ns ns
20 days N vs. I-CF ab abc ab abc ns
N vs. I-DP ns a ns abc ns
I-CF vs. I-DP ns ns ns ns ns
30 days N vs. I-CF a a ns a ns
N vs. I-DP a a ns a ab
I-CF vs. I-DP ab ns ns ns ns
40 days N vs. I-CF a a a abc ns
N vs. I-DP ab a ab abc ns
I-CF vs. I-DP ns ab ns ns abc
50 days N vs. I-CF a abc ns abc ab
N vs. I-DP a a ns a ns
I-CF vs. I-DP ab ns ns ns abc
60 days N vs. I-CF ab abc ab ab ns
N vs. I-DP ab abc ns ab ns
I-CF vs. I-DP ns ns ns ns abc
70 days N vs. I-CF ab ab ns ab ns
N vs. I-DP ab abc ns abc ns
I-CF vs. I-DP ns ns ns ns ns
80 days N vs. I-CF ab abc ns ns ns
N vs. I-DP ab abc ns ns ns
I-CF vs. I-DP ns ns ab ns ns
90 days N vs. I-CF ns ns ns ns ns
N vs. I-DP ab ns ns ns ns
I-CF vs. I-DP ns ns ns ns ns
aP< 0.001. abP< 0.05. abcP< 0.01. ns: Non significant.
not unlike that adopted by Bengtsson et al. (1992) for even longer-term metal exposure experiments, and by Janssen et al. (1997). Probably, in the case of our laboratory exposure experiments with a natural metal-liferous soil, earthworms lost rather than gained body mass during the 90 day maintenance period, although this was not monitored explicitly. The metal contents (Cd, Pb and Zn) in `resident' and `introduced' earth-worms did not, however, increase appreciably during the 90 day period (data not presented). Spurgeon (1997a) found that inEisenia fetida sampled from a clean site and exposed to contaminated soils in the laboratory, the essential metals Zn and Cu were accu-mulated and eliminated during depuration much faster than non-essential Cd and Pb. These observations indicate thatL. rubellusin our experiment probably did not experience inadvertent depuration because the tissue Zn concentrations and contents did not decline systematically during the exposure period.
The metal accumulation experiments performed in the present study failed to furnish any ®rm evidence about population differentiation in terms of metal relationships. Bengtsson et al. (1992) examined the effect of long-term metal exposure on growth, repro-duction and metalloprotein composition in Dendro-baena octaedra, but were unable to demonstrate conclusively that the stress of metal toxicity had engendered local adaptive changes. Our observations, together with those of Bengtsson et al. (1992), should not be interpreted as proof that earthworm populations cannot evolve metal-resistance ecotypes. Even from the restricted point of view of metal sequestration, there may be several factors that are pertinent in this regard. First, the identity of the primary metal stres-sor(s) in a local habitat may be crucial. Lead and zinc are sequestered predominantly by non-inducible ligands within the matrix of earthworm chloragosome granules (Morgan et al., 1993). There is no evidence available presently indicating that this important metal accumulating compartment can be `expanded' (i.e. more chloragocytes and/or more chloragosomes per cell) or that its kinetics is altered by metal exposure in a genetically-determined fashion. These two metals are the major contaminants of Cwmystwyth Cottage soil. Cd, in contrast to Pb and Zn, binds to inducible metallothionein-like proteins in the posterior alimen-tary fraction of earthworms (Suzuki et al., 1980; Morgan et al., 1989, 1993; Bengtsson et al., 1992)
and freshwater oligochaetes (Bauer-Hilty et al., 1989; Klerks and Bartholomew, 1991; Klerks and Levinton, 1993). Heavy selection pressure imposed by high environmental Cd concentrations could, therefore, lead to genetic modi®cations such as the metallothio-nein gene duplications suspected to underpin Cd-tolerance in freshwater oligochaetes (Klerks and Levinton, 1993). However, it should be noted that the sediment Cd concentration thatL. hoffmeisteriwas exposed to at Foundry Cove (Klerks and Levinton, 1993) exceeded by at least an order of magnitude, the highest recorded `natural' soil Cd concentration in soils inhabited by earthworms (Morgan and Morris, 1992).
A second factor that may make it dif®cult to identify locally-adapted earthworm populations is that metal-resistance responses may not be uniform. A good example of evolutionary patchiness is the pre-sence of Zn-tolerant populations of the grassAgrostis capillaris (tenuis) under some galvanized electricity pylons but not under others in a `linked' series (Al-Hiyaly et al., 1988).
(Eijsackers, 1997; Posthuma, 1997). Recent observa-tions on cadmium tolerance in F1 juveniles of a freshwater snail do, however, raise cautionary advice concerning the effects environmental factors exert on the expression of metal tolerance (Lam, 1996).
Acknowledgements
We thank Mrs. Nicola Bassett for typing the manu-script.
References
Al-Hiyaly, S.A., McNeilly, T., Bradshaw, A.D., 1988. The effects of zinc contamination from electricity pylons ± evolution in a replicated situation. New Phytol. 110, 571±580.
Baird, D.J., Barber, I., Calow, P., 1990. Clonal variation in general responses ofDaphnia magnaStraus to toxic stress I. Chronic life-history effects. Functional Ecology 4, 399±407.
Bauer-Hilty, A., Dallinger, R., Berger, R., 1989. Isolation and partial characterization of a cadmium-binding protein from
Lumbriculus variegatus(Oligochaeta Annelida). Comp. Bio-chem. Physiol. 94C, 373±379.
Bengtsson, G., Rundgren, S., 1992. Seasonal variation of lead uptake in the earthwormLumbricus terrestrisand the influence of soil liming and acidification. Arch. Environ. Contam. Toxicol. 23, 198±205.
Bengtsson, G., Ek, H., Rundgren, S., 1992. Evolutionary response of earthworms to long-term metal exposure. Oikos 63, 289± 297.
Corp, N., Morgan, A.J., 1991. Accumulation of heavy metals from polluted soils by the earthworm, Lumbricus rubellus: can laboratory exposure of `control' worms reduce biomonitoring problems? Environ. Pollut. 74, 39±52.
Donker, M.H., Bogert, C.G., 1991. Adaptation to cadmium in three populations of the isopodPorcellio scaber. Comp. Biochem. Physiol. 110C, 143±146.
Duan, Y., Guttman, S.I., Oris, J.T., 1997. Genetic differentiation among laboratory populations ofHyalella azteca: implications for toxicology. Environ. Toxicol. Chem. 16, 691±695. Eijsackers, H., 1997. Soil ecotoxicology: still new ways to explore
or just paving the road? In: van Straalen, N.M., Lùkke, H. (Eds.), Ecological Risk Assessment of Contaminants in Soil, Chapman & Hall, London, pp. 323±330.
Grant, A., Hateley, J.G., Jones, N.V., 1989. Mapping the ecological impact of heavy metals on the estuarine polychaete Nereis diversicolorusing inherited metal tolerance. Mar. Pollut. Bull. 20, 235±238.
HaÊgvar, S., Abrahamsen, G., 1990. Microarthropoda and Enchy-traeidae (Oligochaeta) in naturally lead-contaminated soils: a gradient study. Environ. Entomol. 19, 1263±1277.
Hopkin, S.P., 1990. Species-specific differences in the net assimilation of zinc, cadmium, lead, copper and iron by the
terrestrial isopods Oniscus asellus and Porcellio scaber. J. Appl. Ecol. 27, 460±474.
Hopkin, S.P., Martin, M.H., 1984. The assimilation of zinc, cadmium, lead and copper by the centipede Lithobius variegatus. J. Appl. Ecol. 21, 535±546.
Janssen, M.P.M., Bergema, W.F., 1991. The effect of temperature on cadmium kinetics and oxygen consumption in soil arthropods. Environ. Contam. Toxicol. Chem. 10, 1493±1501. Janssen, R.P.T., Posthuma, L., Baerselman, R., Den Hollander,
H.A., Van Veen, R.P.M., Peijnenburg, W.J.G.M., 1977. Equilibrium partitioning of heavy metals in Dutch field soils II. Prediction of metal accumulation in earthworms. Environ. Contam. Toxicol. Chem. 16, 2479±2488.
Klerks, P.L., 1990. Adaptation to metals in animals. In: Shaw, A.J. (Ed.), Heavy Metal Tolerance in Plants: Evolutionary Aspects, CRC Press, Boca Raton, Florida, USA, pp. 313±321. Klerks, P.L., Bartholomew, P.R., 1991. Cadmium accumulation and
detoxification in a Cd-resistant population of the oligochaete
Limnodrilus hoffmeisteri. Aquat. Toxicol. 19, 97±112. Klerks, P.L., Levinton, J.S., 1989. Rapid evolution of metal
resistance in a benthic oligochaete inhabiting a metal polluted site. Biol. Bull. (Woods Hole) 176, 135±141.
Klerks, P.L., Levinton, J.S., 1993. Evolution of resistance and changes in community composition in metal-polluted environ-ments: a case study in Foundry Cove. In: Dallinger, R., Rainbow, P.S. (Eds.), Ecotoxicology of Metals in Invertebrates, Lewis Publishers, Boca Raton, Florida, USA, pp. 223±241. Klerks, P.L., Weis, J.S., 1987. Genetic adaptation to heavy metals
in aquatic organisms: a review. Environ. Pollut. 45, 173±205. Lam, P.K.S., 1996. Interpopulation differences in acute response of
Brotia hainanensis (Gastropoda, Prosobranchia) to cadmium: genetic or environmental variance? Environ. Pollut. 94, 1±7. Lauverjat, S., Ballan-Dufrancais, C., Wegnez, M., 1989.
Detoxi-fication of cadmium. Ultrastructural study and electron-microprobe analysis of the midgut in a cadmium-resistant strain ofDrosophila melanogaster. Biol. Metals 2, 97±107. Morgan, A.J., Morgan, J.E., Turner, M., Winters, C., Yarwood, A.,
1993. Heavy metal relationships of earthworms. In: Dallinger, R., Rainbow, P.S. (Eds.), Ecotoxicology of Metals in Inverte-brates, Lewis Publishers, Boca Raton, Florida, USA, pp. 333± 358.
Morgan, A.J., Morris, B., 1992. The accumulation and intracellular compartmentation of cadmium, lead, zinc and calcium in two earthworm species (Dendrobaena rubida and Lumbricus rubellus) living in highly contaminated soil. Histochemistry 75, 269±285.
Morgan, J.E., Norey, C.G., Morgan, A.J., Kay, J., 1989. A comparison of the cadmium-binding proteins isolated from the posterior alimentary canal of the earthwormDendrodrilus rubidus and Lumbricus rubellus. Comp. Biochem. Physiol. 92C, 15±21.
Posthuma, L., Van Straalen, N.M., 1993. Heavy-metal adaptation in terrestrial invertebrates: a review of occurrence, genetics, physiology and ecological consequences. Comp. Biochem. Physiol. 106C, 11±36.
Posthuma, L., Verweij, R.A., Widianarko, B., Zonneveld, C., 1993. Life-history patterns in metal adapted Collembola. Oikos 67, 235±249.
Spurgeon, D.J., 1997a. Comparison of cadmium, copper, lead, and zinc kinetics in the earthworm (Eisenia fetida). Abstracts of the 2nd Int. Workshop on Earthworm Ecotoxicology, Amsterdam, p. 40.
Spurgeon, D.J., 1997b. Extrapolation of laboratory toxicity results to the field: a case study using the OECD artificial soil earthworm toxicity test. In: van Straalen, N.M., Lùkke, H.
(Eds.), Ecological Risk Assessment of Contaminants in Soil, Chapman & Hall, London, pp. 253±273.
Suzuki, K.T., Yamamura, M., Mori, T., 1980. Cadmium-binding proteins induced in the earthworms. Arch. Environ. Contam. Toxicol. 9, 415±424.
Van Gestel, C.A.M., 1997. Scientific basis for extrapolating results from soil ecotoxicity tests to field conditions and the use of bioassays. In: van Straalen, N.M., Lùkke, H. (Eds.), Ecological Risk Assessment of Contaminants in Soil, Chapman & Hall, London, pp. 25±50.
