Directory UMM :Data Elmu:jurnal:A:Applied Soil Ecology:Vol12.Issue2.Apr1999:
Applied Soil Ecology 12 (1999) 179±189
Equilibrated body metal concentrations in laboratory
exposed earthworms: can they be used to screen
candidate metal-adapted populations?
F. MarinÄoa, A.J. Morganb,*
a
Departamento de Ecologia y Biologia Animal, Facultad de Ciencias, Universidad de Santiago, 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
The accumulated metal (Ca, Cd, Cu, Pb, Zn) concentrations in seven different populations of the earthworm, Lumbricus
rubellus, maintained in the laboratory for 90 days on their `own' native polluted soils were compared with those accumulated
by two reference populations (sampled from uncontaminated calcareous and acidic sites, respectively), maintained for the
same period on the same series of seven polluted soils. Worms native to the more heavily polluted soils accumulated higher Cd
and Zn concentrations than their reference counterparts, the striking exception being worms maintained on the exceptionally
acidic, Ca-poor, Cwmystwyth Stream soil; tissue Cu concentrations were consistently low and similar in `native' and reference
worms; tissue Pb concentrations were signi®cantly higher in only one population of native worms, a site (Wemyss) that did not
contain the highest soil Pb concentration; Ca concentrations were generally similar in native and reference worms, but
reference worms normally inhabiting an acidic soil, Caerf®li (CF) tended to accumulate Ca more ef®ciently than reference
worms derived from a more calcareous soil, Dinas Powys (DP). These observations indicated that differentiation between
natural populations as expressed by metal accumulation patterns is probably a commoner earthworm response to Cd- or Znexposures than it is to Pb- or perhaps Cu-exposures. Measuring and comparing accumulated metal concentrations is a crude
method of early-stage screening for metal-tolerant ecotypes; having identi®ed candidate tolerant populations more de®nitive
genetic tests must be undertaken. # 1999 Elsevier Science B.V. All rights reserved.
Keywords: Earthworms; Lumbricus rubellus; Cd; Cu; Pb; Zn
1. Introduction
Abandoned metalliferous mine soils are hostile
habitats, not only because they are often severely
contaminated by one or more different toxic metals,
*Corresponding author. Tel.: +44-1222874190; fax: +441222874305; e-mail: fry@cardiff.ac.uk
but also because many are shallow with low nutrient
and moisture-retaining status. Colonizers of such soils
must be capable of resisting metal toxicity whilst
overcoming the concomitant ecophysiological challenges. Metal resistance can be achieved through
avoidance reactions (Tranvik and Eijsackers, 1989),
reduced assimilation (i.e., down-regulated transepithelial uptake and/or increased excretion) (Post-
0929-1393/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved.
PII: S 0 9 2 9 - 1 3 9 3 ( 9 9 ) 0 0 0 0 4 - 9
180
F. MarinÄo, A.J. Morgan / Applied Soil Ecology 12 (1999) 179±189
huma et al., 1996), detoxication by accumulative
sequestration within constitutive granules or inducible
proteins (Morgan, 1984; Morgan and Winters, 1987;
Donker and Bogert, 1991), increased excretion ef®ciency (Posthuma et al., 1996), and possibly by enzymic modi®cations (Bengtsson et al., 1992).
Whether the resistance expressed by populations
inhabiting metal-stressed sites is a consequence of
intraspeci®c inherited adaptations or, alternatively, of
acclimatory adjustments during the exposure history
of individual organisms is a dif®cult, but fundamentally important distinction to make (Greville and
Morgan, 1993; Klerks and Levinton, 1993; Posthuma
and Van Straalen, 1993). Brandon (1991) presented a
number of criteria that need to be satis®ed before a
metal-resistant population can be described as
adapted. These include: (i) survival in the presence
of toxic concentrations; (ii) modi®ed life-history parameters, typically a foreshortened life-cycle and
enhanced reproductive effort; (iii) changes in metal
metabolism, preferably recorded in second or later
generation offspring. In the case of the third criterion,
adapted populations within certain species limit their
tissue metal accumulation, but adaptation in other
species is characterized by increased accumulation
(Klerks and Weiss, 1987; Klerks, 1990). The freshwater oligochaete, Limnodrilus hoffmeisteri, inhabiting Cd-polluted sediments has been conclusively
shown to be genetically different from worms inhabiting adjacent but less Cd-polluted sediments (Klerks
and Levinton, 1989, 1993). Furthermore, the resistant
L. hoffmeisteri ecotype accumulates more, not less, Cd
than the Cd-sensitive conspeci®es (Klerks and Bartholomew, 1991).
Bengtsson et al. (1992) were unable to obtain ®rm
evidence that populations of the earthworm, Dendrobaena octaedra, inhabiting a soil contaminated with
Cu and Zn had evolved adaptive resistance. However,
this does not mean that earthworms cannot become
metal adapted. The work of Al-Hiyaly et al. (1993)
on zinc tolerance in the grass, Agrostis capillaris,
growing under a series of electricity pylons may be
instructive. They found that the products of evolutionary processes, in what are effectively replicated
islands-of-toxicity, were not uniform: populations
under some pylons were Zn-tolerant, others not, probably because of genetic differences in the founding
populations.
Posthuma and Van Straalen (1993) drew attention to
the fact that estimates of the correlation between metal
tolerance and the degree of site pollution should be
performed for at least three, not the usual two, populations. In the present preliminary investigation, we
compared the accumulation of metals by seven different populations of the earthworm, Lumbricus rubellus, maintained for a lengthy period on their `own'
native polluted soils, with the accumulation by two
reference populations maintained on the same series
of polluted soils. Whilst differential accumulation of
metals by different populations does not constitute
evidence of local adaptation, it is helpful for screening
candidate populations so that they can be bred in the
laboratory to yield offspring upon which the de®nitive
tolerance tests can be applied.
2. Materials and methods
2.1. Earthworm and soil samples
Soils were collected during April 1993 from seven
metalliferous sites in the UK: Cwmystwyth Stream
(CS; O.S. Grid Ref. SN 804747), Rhandirmwyn (RM;
SN 790455), Wemyss (WM; SN 716741), Llantrisant
(LL; ST 058822), Cwmystwyth Cottage (CC; SN
806748), Draethen Quarry (DT; ST 195617), Roman
Gravels (RG; SJ 338003). Each soil sample (approximately 0±5 cm) was thoroughly mixed by hand to
minimize heterogeneity, and they were used to ®ll
three 4 l-capacity plastic boxes.
Mature L. rubellus were collected by digging and
hand sorting from the abandoned mine sites (approx.
10 worms per site), and from two uncontaminated
reference sites (Dinas Powys, relatively calcareous
soil, ST 149723, [Ca] 2010 mg/g, pH 5.8; Caerf®li
acidic soil, ST 160855, [Ca] 120 mg/g pH 4.1).
Worms from the polluted sites were placed in a box
of their `own' soil (these are referred to as `native', or
`N' worms). The other boxes were occupied by either
Dinas Powys (DP) or CF worms (`introduced', or I-DP
and I-CF, respectively. The boxes were placed in a
constant temperature (178C), light-controlled, room
for 90 days, and checked regularly for moribund
worms. Soil moisture was maintained at approximately 50% (of dry weight). Worms were not fed
during the experiment.
F. MarinÄo, A.J. Morgan / Applied Soil Ecology 12 (1999) 179±189
2.2. Analytical methods
About 100 g of soil was removed from each box
before worms were introduced. These soil samples
were dried at room temperature, gently crushed and
passed through a 2 mm stainless steel sieve. Soil pH
was measured in triplicate in deionized water slurries
(3 g soil: 30 ml H2O) after stirring and equilibration
over 3 h. Soil organic matter content was estimated (in
triplicate) by igniting weighed dried-soil samples in
silica crucibles at 6008C for 2 h.
Metal (Cd, Pb, Cu, Zn, Ca) concentrations were
measured in conc. HNO3 ± digests of soils and earthworms by atomic absorption spectrophotometry in an
Instruments Laboratory AA/AE 457 with 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 ASS protocols does
not exceed 7% for any of the reported metals. These
protocols have been described elsewhere (Corp and
Morgan, 1991).
2.3. Statistical analysis
Data were expressed as mean SE. Differences
between the means of native and introduced worms,
and between the two introduced populations, were
determined by the Mann±Whitney U-test.
3. Results
Metal concentrations, pH and % loss on ignition
values for the seven polluted soils and two reference
soils are presented in Table 1. The soils were compositionally very different: the LL, CC, Draethen (DT)
and Roman Gravels (RG) soils were very calcareous,
especially DT and RG, with a pH of about 6.0 and a
relatively high organic content. These four soils were
also fairly heavily contaminated with Cd, Pb and Zn,
with DT containing a very high Pb concentration. The
RM and WM soils had relatively low calcium contents
and pH's of
Equilibrated body metal concentrations in laboratory
exposed earthworms: can they be used to screen
candidate metal-adapted populations?
F. MarinÄoa, A.J. Morganb,*
a
Departamento de Ecologia y Biologia Animal, Facultad de Ciencias, Universidad de Santiago, 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
The accumulated metal (Ca, Cd, Cu, Pb, Zn) concentrations in seven different populations of the earthworm, Lumbricus
rubellus, maintained in the laboratory for 90 days on their `own' native polluted soils were compared with those accumulated
by two reference populations (sampled from uncontaminated calcareous and acidic sites, respectively), maintained for the
same period on the same series of seven polluted soils. Worms native to the more heavily polluted soils accumulated higher Cd
and Zn concentrations than their reference counterparts, the striking exception being worms maintained on the exceptionally
acidic, Ca-poor, Cwmystwyth Stream soil; tissue Cu concentrations were consistently low and similar in `native' and reference
worms; tissue Pb concentrations were signi®cantly higher in only one population of native worms, a site (Wemyss) that did not
contain the highest soil Pb concentration; Ca concentrations were generally similar in native and reference worms, but
reference worms normally inhabiting an acidic soil, Caerf®li (CF) tended to accumulate Ca more ef®ciently than reference
worms derived from a more calcareous soil, Dinas Powys (DP). These observations indicated that differentiation between
natural populations as expressed by metal accumulation patterns is probably a commoner earthworm response to Cd- or Znexposures than it is to Pb- or perhaps Cu-exposures. Measuring and comparing accumulated metal concentrations is a crude
method of early-stage screening for metal-tolerant ecotypes; having identi®ed candidate tolerant populations more de®nitive
genetic tests must be undertaken. # 1999 Elsevier Science B.V. All rights reserved.
Keywords: Earthworms; Lumbricus rubellus; Cd; Cu; Pb; Zn
1. Introduction
Abandoned metalliferous mine soils are hostile
habitats, not only because they are often severely
contaminated by one or more different toxic metals,
*Corresponding author. Tel.: +44-1222874190; fax: +441222874305; e-mail: fry@cardiff.ac.uk
but also because many are shallow with low nutrient
and moisture-retaining status. Colonizers of such soils
must be capable of resisting metal toxicity whilst
overcoming the concomitant ecophysiological challenges. Metal resistance can be achieved through
avoidance reactions (Tranvik and Eijsackers, 1989),
reduced assimilation (i.e., down-regulated transepithelial uptake and/or increased excretion) (Post-
0929-1393/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved.
PII: S 0 9 2 9 - 1 3 9 3 ( 9 9 ) 0 0 0 0 4 - 9
180
F. MarinÄo, A.J. Morgan / Applied Soil Ecology 12 (1999) 179±189
huma et al., 1996), detoxication by accumulative
sequestration within constitutive granules or inducible
proteins (Morgan, 1984; Morgan and Winters, 1987;
Donker and Bogert, 1991), increased excretion ef®ciency (Posthuma et al., 1996), and possibly by enzymic modi®cations (Bengtsson et al., 1992).
Whether the resistance expressed by populations
inhabiting metal-stressed sites is a consequence of
intraspeci®c inherited adaptations or, alternatively, of
acclimatory adjustments during the exposure history
of individual organisms is a dif®cult, but fundamentally important distinction to make (Greville and
Morgan, 1993; Klerks and Levinton, 1993; Posthuma
and Van Straalen, 1993). Brandon (1991) presented a
number of criteria that need to be satis®ed before a
metal-resistant population can be described as
adapted. These include: (i) survival in the presence
of toxic concentrations; (ii) modi®ed life-history parameters, typically a foreshortened life-cycle and
enhanced reproductive effort; (iii) changes in metal
metabolism, preferably recorded in second or later
generation offspring. In the case of the third criterion,
adapted populations within certain species limit their
tissue metal accumulation, but adaptation in other
species is characterized by increased accumulation
(Klerks and Weiss, 1987; Klerks, 1990). The freshwater oligochaete, Limnodrilus hoffmeisteri, inhabiting Cd-polluted sediments has been conclusively
shown to be genetically different from worms inhabiting adjacent but less Cd-polluted sediments (Klerks
and Levinton, 1989, 1993). Furthermore, the resistant
L. hoffmeisteri ecotype accumulates more, not less, Cd
than the Cd-sensitive conspeci®es (Klerks and Bartholomew, 1991).
Bengtsson et al. (1992) were unable to obtain ®rm
evidence that populations of the earthworm, Dendrobaena octaedra, inhabiting a soil contaminated with
Cu and Zn had evolved adaptive resistance. However,
this does not mean that earthworms cannot become
metal adapted. The work of Al-Hiyaly et al. (1993)
on zinc tolerance in the grass, Agrostis capillaris,
growing under a series of electricity pylons may be
instructive. They found that the products of evolutionary processes, in what are effectively replicated
islands-of-toxicity, were not uniform: populations
under some pylons were Zn-tolerant, others not, probably because of genetic differences in the founding
populations.
Posthuma and Van Straalen (1993) drew attention to
the fact that estimates of the correlation between metal
tolerance and the degree of site pollution should be
performed for at least three, not the usual two, populations. In the present preliminary investigation, we
compared the accumulation of metals by seven different populations of the earthworm, Lumbricus rubellus, maintained for a lengthy period on their `own'
native polluted soils, with the accumulation by two
reference populations maintained on the same series
of polluted soils. Whilst differential accumulation of
metals by different populations does not constitute
evidence of local adaptation, it is helpful for screening
candidate populations so that they can be bred in the
laboratory to yield offspring upon which the de®nitive
tolerance tests can be applied.
2. Materials and methods
2.1. Earthworm and soil samples
Soils were collected during April 1993 from seven
metalliferous sites in the UK: Cwmystwyth Stream
(CS; O.S. Grid Ref. SN 804747), Rhandirmwyn (RM;
SN 790455), Wemyss (WM; SN 716741), Llantrisant
(LL; ST 058822), Cwmystwyth Cottage (CC; SN
806748), Draethen Quarry (DT; ST 195617), Roman
Gravels (RG; SJ 338003). Each soil sample (approximately 0±5 cm) was thoroughly mixed by hand to
minimize heterogeneity, and they were used to ®ll
three 4 l-capacity plastic boxes.
Mature L. rubellus were collected by digging and
hand sorting from the abandoned mine sites (approx.
10 worms per site), and from two uncontaminated
reference sites (Dinas Powys, relatively calcareous
soil, ST 149723, [Ca] 2010 mg/g, pH 5.8; Caerf®li
acidic soil, ST 160855, [Ca] 120 mg/g pH 4.1).
Worms from the polluted sites were placed in a box
of their `own' soil (these are referred to as `native', or
`N' worms). The other boxes were occupied by either
Dinas Powys (DP) or CF worms (`introduced', or I-DP
and I-CF, respectively. The boxes were placed in a
constant temperature (178C), light-controlled, room
for 90 days, and checked regularly for moribund
worms. Soil moisture was maintained at approximately 50% (of dry weight). Worms were not fed
during the experiment.
F. MarinÄo, A.J. Morgan / Applied Soil Ecology 12 (1999) 179±189
2.2. Analytical methods
About 100 g of soil was removed from each box
before worms were introduced. These soil samples
were dried at room temperature, gently crushed and
passed through a 2 mm stainless steel sieve. Soil pH
was measured in triplicate in deionized water slurries
(3 g soil: 30 ml H2O) after stirring and equilibration
over 3 h. Soil organic matter content was estimated (in
triplicate) by igniting weighed dried-soil samples in
silica crucibles at 6008C for 2 h.
Metal (Cd, Pb, Cu, Zn, Ca) concentrations were
measured in conc. HNO3 ± digests of soils and earthworms by atomic absorption spectrophotometry in an
Instruments Laboratory AA/AE 457 with 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 ASS protocols does
not exceed 7% for any of the reported metals. These
protocols have been described elsewhere (Corp and
Morgan, 1991).
2.3. Statistical analysis
Data were expressed as mean SE. Differences
between the means of native and introduced worms,
and between the two introduced populations, were
determined by the Mann±Whitney U-test.
3. Results
Metal concentrations, pH and % loss on ignition
values for the seven polluted soils and two reference
soils are presented in Table 1. The soils were compositionally very different: the LL, CC, Draethen (DT)
and Roman Gravels (RG) soils were very calcareous,
especially DT and RG, with a pH of about 6.0 and a
relatively high organic content. These four soils were
also fairly heavily contaminated with Cd, Pb and Zn,
with DT containing a very high Pb concentration. The
RM and WM soils had relatively low calcium contents
and pH's of