Directory UMM :Data Elmu:jurnal:A:Applied Soil Ecology:Vol12.Issue2.Apr1999:

Applied Soil Ecology 12 (1999) 169±177

The time-course of metal (Ca, Cd, Cu, Pb, Zn) accumulation
from a contaminated soil by three populations
of the earthworm, Lumbricus rubellus
F. MarinÄoa, A.J. Morganb,*
a

Departamento de EcoloxõÂa e Bioloxia Animal, Facultade de Ciencias, Universidade de Vigo, Vigo, Pontevedra, Spain
b
Cardiff 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 belonging to the families Mesostigmata and Gamasine (HaÊgvar and Abrahamsen, 1990), the springtails Orchesella
cincta (Posthuma et al., 1993), and the dipteran Drosophila melanogaster (Lauverjat et al., 1989). Genetic
*Corresponding author. Tel.: +44-12222874190; fax: +441222874305; e-mail: fry@cardiff.ac.uk

tolerance has been con®rmed for the marine polychaete, Nereis diversicolor (Grant et al., 1989) and the
freshwater oligochaete Limnodrilus hoffmeisteri
(Klerks and Levinton, 1989, 1993), but the unequivocal evidence for `heavy' metal adaptations in earthworm 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 assimilation ef®ciency, the binding/immobilization capacity, and/or the excretory rate. Very few studies on
terrestrial macroinvertebrates have distinguished

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F. MarinÄo, A.J. Morgan / Applied Soil Ecology 12 (1999) 169±177

between `adaptation' and `acclimation' (Klerks and
Weis, 1987) mainly because of the dif®culty of producing 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 toxicity data (Baird et al., 1990; Duan et al., 1997).
Corp and Morgan (1991) compared the metal concentrations in nine populations of the earthworm
L. rubellus maintained in the laboratory for 31 days
on their `own' metalliferous soils (native worms), with
those in batches of worms from a clean site (introduced worms) maintained on the same series of contaminated soils. Their data did not furnish evidence of
inter-population differentiation because it was not
possible to determine whether or not the metal concentrations in the introduced earthworms had equilibrated 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 population 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 equilibrate 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 `introduced' (presumptive intolerant) earthworms as
expressed by their accumulated metal burdens? (iii)
do the patterns of metal accumulation in laboratoryexposure experiments reveal physiological differentiation between `control' earthworm populations inhabiting contaminated calcareous and acidic soils,
respectively?

2. Materials and methods

from two uncontaminated sites in south Wales: Dinas
Powys (O.S. Grid Ref. ˆ ST149723; a relatively calcareous soil: pH ˆ 5.8; [Ca] ˆ 2000 mg/g dry weight;
[Zn] ˆ 130 mg/g; [Cu] ˆ 13 mg/g; [Pb] ˆ 45 mg/g;
[Cd] ˆ 0.8 mg/g) and Caerf®li (O.S. Grid Ref. ˆ

ST160555; an acidic soil: pH ˆ 4.1; [Ca] ˆ 120 mg/g
dry weight; [Zn] ˆ 60 mg/g; [Cu] ˆ 10 mg/g; [Pb] ˆ
25 mg/g; [Cd] ˆ 0.2 mg/g).
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
boxes  10 worms per population). The boxes were
placed in a constant temperature (178C), light-controlled room for the duration of the exposure experiment. 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-onignition 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 spectrophotometry in an Instruments Laboratory AA/AE
457 with automatic background adjustment. All solutions, including standards, used for Ca assay contained
1% lanthanum. Regular analysis of certi®ed sediments, plant materials and soft animal tissues indicated 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.

2.1. Earthworms and soil samples
2.3. Statistical analysis
Earthworms (L. rubellus) and soil were collected
during April 1993 from one contaminated site in
mid-Wales (Cwmystwyth Cottage, O.S. Grid
Ref. ˆ SN806748). Earthworms were also collected

Concentration data were expressed as mean  SE.
Differences between means were statistically evaluated by the Mann±Whitney non-parametric U-test.

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F. MarinÄo, A.J. Morgan / Applied Soil Ecology 12 (1999) 169±177

3. Results
Soils associated with disused mine spoils are notoriously heterogeneous in composition. This was con®rmed by the analyses of soils (all from Cwmystwyth
Cottage) taken from each plastic box (Table 1; concentrations of Cd, Pb, Cu, Zn and Ca ranged from 31±
120 mg/g dry weight, 1594±8688 mg/g, 9±144 mg/g,
6625±54 450 mg/g and 14 825±56 350 mg/g, respectively; 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-samples, the heterogeneity was unlikely to introduce a
systematic error.
3.1. Cadmium
Qualitative assessment indicates that Cd concentrations in the native worms (N) did not increase signi®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 earthworms 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 conclusive 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 concentration in the range from 0.5 to