Directory UMM :Data Elmu:jurnal:A:Applied Soil Ecology:Vol13.Issue1.Sep1999:
Applied Soil Ecology 13 (1999) 9±20
The accumulation of metals (Cd, Cu, Pb, Zn and Ca) by two
ecologically contrasting earthworm species (Lumbricus rubellus and
Aporrectodea caliginosa): implications for ecotoxicological testing
J.E. Morgana, A.J. Morganb,*
b
a
Department of Applied Science, Writtle College, Chelmsford Essex, CM1 3RR, UK
School of Pure and Applied Biology, University of Wales College of Cardiff, P.O. Box 915, Cardiff CF1 3TL, UK
Received 16 July 1998; received in revised form 12 February 1999; accepted 16 February 1999
Abstract
The metal (Cd, Cu, Pb, Zn and Ca) concentrations in the tissues, ingesta (crop contents) and egesta (faeces) were investigated
in two physiologically contrasting earthworm species (Lumbricus rubellus and Aporrectodea caliginosa) inhabiting soils
exhibiting various levels of heavy metal contamination. In addition, a complementary soil layering experiment, conducted
under laboratory conditions, was undertaken to investigate whether the distribution of Pb within a soil vertical pro®le
in¯uenced the relative metal accumulation patterns of these species. Generally, the Cd, Cu and Pb concentrations of ®eld
populations of A. caliginosa were signi®cantly greater than in L. rubellus, a pattern reversed for Ca. Concentrations of Zn were
signi®cantly greater in A. caliginosa in soils containing the lowest Zn concentrations, but no species differences were apparent
at high soil concentrations of this metal. Comparisons of metal concentrations between ingesta and soils indicate that both
species selectively ingest material from the soil matrix, although no signi®cant correlations were found between tissue metal
and ingesta metal concentrations. Differences in concentrations of Cd, Pb and Zn between the ingesta of the species were,
however, concomitant with observed differences in tissue concentrations of the respective metals, which cannot be explained
by excretion via the egesta. The soil strati®cation experiment indicated that Pb distribution within a soil pro®le affected the
pattern of species differences in tissue metal concentrations observed in ®eld populations. The evidence therefore suggests that
the difference in dietary intakes of these metals is an important factor in contributing to observed differences between these
species, although other factors are also contributory. The observations are discussed in the context of soil hazard assessment
monitoring, and in particular, the role of concentration factors in such applied surveys. # 1999 Elsevier Science B.V. All
rights reserved.
Keywords: Earthworms; Heavy metals; Species differences; Concentration factors; Ecotoxicology
1. Introduction
Different earthworm species inhabiting the same
polluted microhabitat clearly exhibit different dispo*Corresponding author. Tel.: +44-1245-24200; fax: +44-1245420456; e-mail: jem@writtle.ac.uk
sitions to accumulate essential and non-essential
metals (Ireland, 1979; Ireland and Richards, 1977;
Ash and Lee, 1980; Wright and Stringer, 1980; Andersen and Laursen, 1982; Morris and Morgan, 1986;
Morgan and Morgan, 1988, 1991, 1993; Morgan and
Morris, 1982; Morgan et al., 1986; Terhivuo et al.,
1994). It has been suggested that these observations
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 1 2 - 8
10
J.E. Morgan, A.J. Morgan / Applied Soil Ecology 13 (1999) 9±20
are a result of inter-speci®c differences in dietary
metal intakes and physiological utilisation (Andersen
and Laursen, 1982; Ireland, 1983; Beyer et al., 1985;
Morris and Morgan, 1986; Hopkin, 1989; Morgan and
Morgan, 1992, 1993), although little direct evidence
has been collected to substantiate these claims.
In addition, although there appears to be a degree of
inter-species consistency in the accumulation of particular metals from metalliferous mine soils (e.g.
Morgan and Morgan, 1991), such patterns become
obscured when soils polluted primarily by aerial
deposition are considered (e.g. Ash and Lee, 1980).
Such disparities appear to have received little attention, but clearly have implications for biomonitoring
and hazard assessment programmes.
The aims of this study were three-fold. First, to
con®rm our provisional observations, restricted to one
metalliferous site (Morgan and Morgan, 1993), that
differences exist in tissue Cd, Cu, Pb, Zn and Ca
concentrations between two ecophysiologically contrasting earthworm species (Lumbricus rubellus and
Aporrectodea caliginosa). L. rubellus is a litter dwelling (epigeic) species, which under normal conditions
lives outside of the mineral substrata, whereas A.
caliginosa lives permanently in essentially horizontal
burrows within the organo-mineral soil matrix, and is
thus categorised as an endogeic species (see Sims and
Gerard, 1985).
Second, to establish whether tissue metal concentrations in L. rubellus and A. caliginosa re¯ect the
respective metal concentrations of their ingested
materials, since it appears that absorption of metals
through the alimentary canal probably accounts for
most of the metal burdens of earthworms (e.g. Piearce,
1972). Although a number of studies (e.g. Ma, 1982;
Ma et al., 1983; Morgan and Morgan, 1988) have
demonstrated statistically that, for a number of heavy
metals, the concentration in the soil explains a large
proportion of the variance in accumulated tissue metal
concentrations of earthworms, there appears to be
little published information on the metal concentrations of material ingested by earthworms. This is
surprising since earthworms are known to ingest
material selectively (Piearce, 1978; Edwards and
Bohlen, 1996).
Third, to investigate, by means of a simple soil
layering experiment, whether the differences in Pb
accumulation by L. rubellus and A. caliginosa is
affected by the preferred vertical distribution of the
species. The source of contamination may in¯uence
metal distribution within the soil vertical pro®le
(Martin and Coughtrey, 1982), which could be a
factor determining the exposure and accumulation
by biota, and the consequent metal transfer through
food chains.
Recent laboratory exposure studies have suggested
that species differences exist in their sensitivities to
metals (Spurgeon and Hopkin, 1995) and organic
residues (Edwards and Coulson, 1992). Under ®eld
conditions the effects of metal contaminants are more
complicated: the toxicological effects of a given metal
on a given species should be coupled with the probability of encountering the metal during the earthworm's normal activities within the soil vertical
pro®le (i.e. exposure). Clearly this aspect of bioavailability of toxicants to de®ned target species has
important implications for toxicity testing programmes under ®eld conditions.
2. Materials and methods
2.1. Earthworms and soils
Mature (clitellate) specimens of L. rubellus and
A. caliginosa were collected from an abandoned,
non-acidic Pb±Zn mine at Llantrisant, S. Wales
(O.S. grid reference ST 048822) by formalin extraction (0.55%, 20 l). Samples were taken from ®ve 5 m2
`stations' across the site and represented areas within
the mine workings and adjacent pastures, which were
used for cattle grazing. Both species were collected in
a similar manner from a pasture adjacent to an abandoned Pb±Zn mine at Halkyn, N. Wales (O.S. grid
reference SJ 201704), and from an uncontaminated
`control' site at Dinas Powis, S. Wales (O.S. grid
reference ST 146723). All sampling occurred early
in November.
Extracted clitellate earthworms were immediately
rinsed in de-ionised water and transferred without
delay to the laboratory. They were rinsed clean of
adhering soil particles and maintained on moist Whatman No. 1 ®lter paper disks in plastic Petri dishes
(2 animals per dish) in the dark at 128C for 3 days. To
prevent coprophagy, the ®lter paper was changed at
least daily. The animals were then depurated for a
J.E. Morgan, A.J. Morgan / Applied Soil Ecology 13 (1999) 9±20
further day without ®lter paper but with a few drops of
deionised water to allow complete egestion of gut
contents. Dissection of several specimens con®rmed
that the guts were clean of consumed material.
The animals were quenched in liquid nitrogen,
placed in individual pre-weighed glass beakers, and
oven-dried (24 h at 708C) to constant weight. The
samples were allowed to cool in desiccators and reweighed in a moisture-free environment on a Mettler
HK60 semi-microbalance. The samples were wet
oxidised in 2 ml boiling concentrated `AnalaR' grade
nitric acid (BDH Chemicals, Poole, Dorset, UK), and
were made up to 10 ml with double de-ionised water.
Blank digests were also prepared.
At least 15 soil samples from two depths (0±5 and
10±15 cm) were taken randomly from within the
designated areas and pooled for each sampling area.
Each pooled soil was dried at 258C in a fan-oven,
gently crushed in an acid-washed porcelain pestle and
mortar, and passed through a 2 mm2 aperture stainless
steel sieve. Subsequent analysis was undertaken on
®ve sub-samples of each soil.
`Total' soil metal concentrations were determined
for both depths by extraction with boiling concentrated `AnalaR' grade nitric acid (Morgan and Morgan, 1988). Extracted metal concentrations (0±5 cm)
were determined after equilibration of 3 g of soil with
35 ml 0.5% acetic acid for 3 h (Morgan, 1987). Soil
pH (de-ionised water) was measured in soil slurries
(w/v, 1/2) following equilibration for 1 h (Peech,
1965). Soil organic carbon was determined by the
Walkley±Black method (Allison, 1965).
2.2. Ingesta (crop contents) and egesta (faeces)
Fresh specimens of A. caliginosa collected from
four Llantrisant stations were dissected and the contents of the crop and gizzard of each animal removed
and placed on individual pre-weighed Millipore
(Millipore S.A., Moshein, France) ®lter paper pieces.
The contents of the crop/gizzard of two animals were
combined for each sample. For comparative purposes,
the crop/gizzard contents were removed from L. rubellus at Station 5 of Llantrisant. Limited sample numbers precluded the crop contents being collected from
L. rubellus at all sites.
Egesta were collected from A. caliginosa taken
from Llantrisant stations during the ®rst day of starva-
11
tion. For comparative purposes egesta were collected
from L. rubellus from Station 5. Each sample was
derived by pooling faeces from two individuals for
each species, and prepared for analysis as described
for the ingesta.
Samples were oven-dried at 708C for 24 h, cooled in
a desiccator, and weighed on a Mettler HK60 semimicrobalance. Each sample was individually wet-oxidised in concentrated boiling `AnalaR' grade nitric
acid. Excess acid was evaporated to leave approximately 0.5 ml to which was added 1 ml double deionised water. Each sample was centrifuged at
2500 rpm for 2 min, and the supernatant removed.
A further 1 ml aliquot of de-ionised water was added
to the solid residue, the suspension again centrifuged,
and the supernatant added to that collected previously.
This procedure was repeated a third time. The ®nal
volume of the pooled supernatant solution was made
up to 5 ml with double de-ionised water. Blank digests
were also prepared.
2.3. Soil stratification experiment
This laboratory experiment was designed to determine whether the vertical distribution of Pb within
strati®ed combinations of naturally contaminated and
uncontaminated soils affects the relative concentrations of Pb accumulated by L. rubellus and A. caliginosa. Groups of between 12 and 16 similarly sized,
clitellate animals of each species, collected from the
uncontaminated Dinas Powis site, were exposed to
different soil treatments for 60 days in the dark at 48C.
Prior to their exposure to the appropriate soil, each
worm group was starved for 2 days to remove most of
their gut contents.
Surface soils (0±5 cm) used for the experiment
were collected from Dinas Powis (uncontaminated
site) and Llantrisant (Pb-contaminated site). The
individual soils were thoroughly mixed before being
placed in plastic containers to a depth of 8 cm, as
follows:
Expt. Group 1
Expt. Group 2
Expt. Group 3
Expt. Group 4
8 cm Dinas Powis soil
8 cm Llantrisant soil
5 cm Llantrisant soil covered by
3 cm Dinas Powis soil
5 cm Dinas Powis soil covered
by 3 cm Llantrisant Powis soil
12
J.E. Morgan, A.J. Morgan / Applied Soil Ecology 13 (1999) 9±20
At the end of the exposure period, the animals were
removed from the soils and processed as previously
described. Soil samples were taken for analysis from
0±3 and 3±8 cm horizons in each experimental container. The soils were processed for `total' soil Pb as
described above.
was prevalent in any of the soils investigated. The
acetic acid-extractable fraction of the heavy metals
(Table 1) was greatest for Cd (9±26%), followed by
Zn (2±10%), Cu (0.7±1.4%) and Pb (0.4±1.0%).
Extractability for Ca ranged from 11 to 41%. Organic
carbon contents of the soils (0±5 cm) ranged from 3 to
9% (Table 1).
2.4. Atomic absorption spectrophotometry (AAS)
3.2. Whole-worm field observations
Digests were quanti®ed for Cd, Cu, Pb, Zn and Ca
by ¯ame (air-acetylene) AAS in a Varian AA6 atomic
absorption spectrophotometer. Samples from the soil
layering experiment were analysed for Pb only. Background correction for Cd, Cu, Pb and Zn analysis was
made automatically with a hydrogen continuum lamp.
Samples and standards for determination of Ca were
made up containing 1% lanthanum.
2.5. Statistics
Statistical comparisons between the species for dry
weight and metal concentrations of tissue, ingesta and
egesta were made by the Mann±Whitney U-test. Signi®cance between metal concentrations in ingesta and
the whole worm was determined by Kendall's rank
correlation. For all statistical tests, differences were
regarded as signi®cant if p < 0.05.
3. Results
3.1. Soils
Soil pH, percent organic carbon content and `total'
(nitric acid-extractable) Cd, Cu, Pb, Zn and Ca concentrations (0±5 cm depth) are presented in Table 1.
Total concentrations of Cd, Pb and Zn were elevated in
the vicinity of the abandoned mine sites compared to
the uncontaminated control site (Dinas Powis), but the
concentrations of Cu were broadly similar at all sites
(Table 1). `Total' concentrations of Ca ranged from
1050 to 8300 mg/kg dw, and re¯ected the range of soil
pH (5±6.8). `Total' concentrations of metals in the 10±
15 cm depth (data not shown) ranged from 0.8 to
16 mg/kg dw for Cd, 21±60 mg/kg dw for Cu, 158±
10 020 mg/kg dw for Pb, 185±1870 mg/kg dw for Zn
and 980±8120 mg/kg dw for Ca. The data indicated
that no surface (0±5 cm) enrichment of heavy metals
Concentrations of Cd, Cu, Pb, Zn and Ca in L.
rubellus and A. caliginosa from the different sites are
presented in Table 2. The concentrations of Cd, Pb,
and Zn were greater in both species inhabiting the
mine soils than in the uncontaminated soil.
Mean concentrations of Cd in A. caliginosa were
between 2 and 5 times greater than those of L. rubellus
in six of the seven soils examined, with all the
differences statistically signi®cant. The trend for Pb
was similar to that for Cd; greater concentrations were
found in A. caliginosa, although the differences were
not always statistically signi®cant. No consistent pattern for Zn accumulation was apparent for these
species, although A. caliginosa accumulated signi®cantly greater concentrations of Zn than L. rubellus
from the soils containing the lowest (total and extractable) concentrations of Zn. Although concentrations
of Cu were broadly similar for both species (range 11±
20 mg/kg dw) in all soils, the concentrations in
A. caliginosa were signi®cantly greater in four of
the soils studied. Concentrations of Ca were signi®cantly greater in L. rubellus in all soils.
3.3. Metal concentrations in ingesta (crop contents)
and egesta (faeces)
The Cd, Cu and Ca concentrations in the ingesta of
both species were in general about 2±15 times greater
than total concentrations in bulk soil (compare
Tables 1 and 3). The pattern for Pb and Zn was less
consistent, although marked differences between
ingesta and soil were apparent for A. caliginosa.
The results suggest that both species ingest material
selectively.
The Cd, Cu and Ca concentrations of the ingesta
of both species were signi®cantly greater than those
in the faeces (Table 3). In contrast, the concentrations
of Pb and Zn were generally signi®cantly greater in
13
J.E. Morgan, A.J. Morgan / Applied Soil Ecology 13 (1999) 9±20
Table 1
Total (16N nitric acid-extractable) and extractable (0.5% acetic acid-extractable) concentrations of Cd, Cu, Pb, Zn and Ca (mg/kg dry wt.),
percentage organic matter and pH of soils (0±5 cm depth) from an uncontaminated site (Dinas Powis) and metalliferous mine sites
Total metal concentration (mg/kg dry wt.)
Sitea
Cd
Cu
Pb
Zn
Ca
DP
Halk
Ll1
Ll2
Ll3
Ll4
Ll5
0.9 0.1
3.6 0.1
14.7 0.3
6.5 0.1
10.5 0.1
17 0.5
2.7 0.1
26 1
40 3
62 1
27 1
30 1
31 1
23 1
166 7
1350 17
10110 280
2370 63
3820 38
6730 71
570 6
193 4
675 9
1550 27
770 21
830 15
1960 33
460 6
1790 48
6080 71
7560 212
5320 110
8300 166
5120 86
1050 21
Extractable metal concentration (mg/kg dry wt.)
DP
0.08 0.01
Halk
0.32 0.01
Ll1
3.0 0.1
Ll2
1.2 0.1
Ll3
1.6 0.1
Ll4
2.4 0.1
Ll5
0.7 0.1
DP
Halk
Ll1
Ll2
Ll3
Ll4
Ll5
Organic carbon (%)
4.7 0.3
9.0 0.5
5.6 0.2
4.1 0.1
5.0 0.2
6.5 0.3
2.9 0.1
0.4 0.1
0.4 0.1
0.3 0.1
0.4 0.1
0.3 0.1
0.2 0.1
0.2 0.1
1 0.2
8 0.1
100 4
18 1
21 1
29 1
31
4.5 0.1
38 12
155 4
31
35 1
97 2
24 1
680 17
2290 43
1730 20
1610 15
1920 25
2115 25
375 2
pH (de-ionised water)
5.19 0.01
6.52 0.03
6.54 0.01
6.49 0.01
6.78 0.01
6.72 0.01
4.98 0.01
Values are mean SE; n 5.
DP Dinas Powis; Halk Halkyn, Ll±Ll5 Llantrisant Stations 1±5.
a
the egesta. No signi®cant correlations (tested by
Kendall's rank correlation) were found between the
concentrations of metals in crop contents and earthworm tissue.
The concentrations of Cd, Pb and Zn in the crop
contents of A. caliginosa from Llantrisant 5 were
signi®cantly greater than those of L. rubellus
(Fig. 1, Table 3), and re¯ected the signi®cant species
differences in the whole-worm concentrations of
the respective metals. No clear pattern was evident
for the Cu concentrations in the ingesta and wholeworm, and no signi®cant species differences were
found. Concentrations of Ca were greater in the crop
contents of L. rubellus, although the difference was
not statistically signi®cant; signi®cant differences
were apparent between the species in the whole-worm
Ca concentration.
No signi®cant differences between the species were
apparent in the concentrations of Cd, Cu and Pb of
the egesta (Fig. 1). The concentration of Zn in the
egesta of A. caliginosa was signi®cantly greater than
in L. rubellus, with the pattern being reversed for Ca.
3.4. Soil stratification experiment
The concentrations of Pb in A. caliginosa were
signi®cantly greater than in L. rubellus for Treatments
1 to 3 (Table 4) following the pattern found in the ®eld
studies. However, in Treatment 4, where the surface
soil was greatly contaminated with Pb, the concentrations in both species were similar. The results indicate
that the pattern of Pb accumulation by the two earthworm species is affected by the distribution of soil Pb
within the vertical soil pro®le.
14
J.E. Morgan, A.J. Morgan / Applied Soil Ecology 13 (1999) 9±20
Table 2
Dry weights (mg) and metal concentrations (mg/kg dw) in A. caliginosa (Ac) and L. rubellus (Lr) from an uncontaminated soil (Dinas Powis)
and 6 soils from abandoned metalliferous mines (see Table 1 for key) (mean SE; n number of samples analysed). Statistical differences
were determined by the Mann±Whitney U-test
Site
Species
Dry wt. (mg)
Cd
Cu
Pb
Zn
Ca
DP
Ac (n 21)
Lr (n 11)
Halk
Ac (n 12)
Lr (n 11)
Ll1
Ac (n 9)
Lr (n 8)
Ll2
Ac (n 17)
Lr (n 5)
Ll3
Ac (n 8)
Lr (n 8)
Ll4
Ac (n 16)
Lr (n 13)
Ll5
Ac (n 17)
Lr (n 27)
94.6 3.6
186.9 12.8
****
86.4 4.3
105.2 8.5
NS
85.8 8.5
128.8 16.7
NS
94.3 6.3
120.4 18.2
NS
71.2 4.5
129.3 12.5
****
88.0 6.1
117.1 11.7
NS
70.6 1.6
100.5 4.3
****
24 2
12 1
****
90 8
42 5
****
233 49
123 12
**
135 11
144 31
NS
229 45
57 6
****
344 30
147 16
****
214 11
46 3
****
17 0.6
14 1.1
NS
17 0.6
14 0.4
***
20 1.2
14 1.2
**
14 1
10 1
*
12 1.1
13 1.2
NS
13 0.8
11 0.5
*
12 0.4
16 1.5
NS
81
51
NS
2010 306
238 31
****
1940 320
460 37
****
1270 208
330 65
**
310 109
300 44
NS
1460 267
900 115
NS
662 49
71 6
****
660 32
330 25
****
1835 169
1495 235
NS
1160 174
1375 87
NS
1200 129
1120 78
NS
770 127
1350 214
NS
1505 131
1745 198
NS
1315 53
770 59
****
1610 32
3225 225
****
3420 302
5853 452
****
2270 168
4815 227
****
2250 83
4740 374
****
3000 50
8300 661
****
2000 199
4810 309
****
3260 73
5280 1690
****
NS Not significant.
* p < 0.02.
** p < 0.01.
*** p < 0.002.
**** p < 0.001.
4. Discussion
Earthworms are known to be selective consumers
(Edwards and Bohlen, 1996). Although relatively few
studies have measured the concentrations of contaminants within material ingested by earthworms, published ®ndings con®rm selective consumption because
there are marked differences between residue concentrations in bulk soil and ingesta, both in the case of
trace organics (Diercxsens et al., 1985) and heavy
metals (Ireland, 1976; Morgan and Morgan, 1992).
The present study also demonstrated that there are
signi®cant differences in concentrations of certain
essential and non-essential metals between the ingesta
and bulk soil.
Such observations pose questions in relation to the
interpretation of concentration factors (the ratio of
metal concentration of earthworm tissue and bulk
soil), which have been cited as being representative
indicators of metal bioavailability in soils (Neuhauser
et al., 1995). Although there exist clearly demonstrable relationships between the concentrations in
earthworms and bulk soil for several metals (Morgan
and Morgan, 1988; Neuhauser et al., 1995), the partition coef®cients of a metal within bulk soil have been
shown to be an important parameter in determining
metal bioavailability (Janssen et al., 1997). An advantage of the concentration factor approach is that it
integrates the bioavailable metal fraction and the
kinetics of the transport system (Janssen et al.,
1997) and thus may have practical application for
the evaluation of the potential toxicological impact
of metals in soils. However, the data from the present
study would suggest that without careful consideration, the use of such indices could lead to spurious
conclusions in relation to toxicological testing of
xenobiotics on different earthworm species. Furthermore, it is probable that earthworm exposure to resi-
15
J.E. Morgan, A.J. Morgan / Applied Soil Ecology 13 (1999) 9±20
Table 3
Dry weight (mg) and concentrations of metals (mg/kg dw) in ingesta and egesta of A. caliginosa (Ac) and L. rubellus (Lr) from Llantrisant
soils (mean SE; n nos of samples). Statistical differences in metal concentrations were determined by the Mann±Whitney U-test
Species
Site
Component
Dry wt. (mg)
Cd
Cu
Pb
Zn
Ca
Ac
Ll1
Ingesta (n 10)
Egesta (n 6)
4.5 0.6
43.7 3.1
Ac
Ll2
Ingesta (n 10)
Egesta (n 10)
4.9 0.4
26.2 3.1
Ac
Ll3
Ingesta (n 9)
Egesta (n 9)
5.7 0.3
48.9 5.3
Ac
Ll5
Ingesta (n 10)
Egesta (n 9)
5.6 0.8
24.3 3.2
Lr
Ll5
Ingesta (n 10)
Egesta (n 15)
6.4 0.7
40.0 6.0
33 4
11 0.7
**
35 4
9 0.8
***
47 9
13 1.1
***
31 5
6 0.4
**
91
61
**
69 6
39 2
**
41 4
31 1
*
35 2
27 2
*
52 7
24 1
***
37 3
27 4
*
4520 306
3720 301
NS
2100 117
3425 255
**
1970 77
3400 238
***
740 51
940 74
**
570 41
810 35
***
1005 47
1100 95
NS
645 42
990 65
***
560 39
920 86
**
550 15
685 21
***
415 22
605 19
***
10 400 1190
3430 720
**
7340 311
2500 294
***
8250 750
6940 610
NS
13 560 1932
1760 132
***
11 950 1883
3320 409
***
NS not significant.
* p < 0.05.
** p
The accumulation of metals (Cd, Cu, Pb, Zn and Ca) by two
ecologically contrasting earthworm species (Lumbricus rubellus and
Aporrectodea caliginosa): implications for ecotoxicological testing
J.E. Morgana, A.J. Morganb,*
b
a
Department of Applied Science, Writtle College, Chelmsford Essex, CM1 3RR, UK
School of Pure and Applied Biology, University of Wales College of Cardiff, P.O. Box 915, Cardiff CF1 3TL, UK
Received 16 July 1998; received in revised form 12 February 1999; accepted 16 February 1999
Abstract
The metal (Cd, Cu, Pb, Zn and Ca) concentrations in the tissues, ingesta (crop contents) and egesta (faeces) were investigated
in two physiologically contrasting earthworm species (Lumbricus rubellus and Aporrectodea caliginosa) inhabiting soils
exhibiting various levels of heavy metal contamination. In addition, a complementary soil layering experiment, conducted
under laboratory conditions, was undertaken to investigate whether the distribution of Pb within a soil vertical pro®le
in¯uenced the relative metal accumulation patterns of these species. Generally, the Cd, Cu and Pb concentrations of ®eld
populations of A. caliginosa were signi®cantly greater than in L. rubellus, a pattern reversed for Ca. Concentrations of Zn were
signi®cantly greater in A. caliginosa in soils containing the lowest Zn concentrations, but no species differences were apparent
at high soil concentrations of this metal. Comparisons of metal concentrations between ingesta and soils indicate that both
species selectively ingest material from the soil matrix, although no signi®cant correlations were found between tissue metal
and ingesta metal concentrations. Differences in concentrations of Cd, Pb and Zn between the ingesta of the species were,
however, concomitant with observed differences in tissue concentrations of the respective metals, which cannot be explained
by excretion via the egesta. The soil strati®cation experiment indicated that Pb distribution within a soil pro®le affected the
pattern of species differences in tissue metal concentrations observed in ®eld populations. The evidence therefore suggests that
the difference in dietary intakes of these metals is an important factor in contributing to observed differences between these
species, although other factors are also contributory. The observations are discussed in the context of soil hazard assessment
monitoring, and in particular, the role of concentration factors in such applied surveys. # 1999 Elsevier Science B.V. All
rights reserved.
Keywords: Earthworms; Heavy metals; Species differences; Concentration factors; Ecotoxicology
1. Introduction
Different earthworm species inhabiting the same
polluted microhabitat clearly exhibit different dispo*Corresponding author. Tel.: +44-1245-24200; fax: +44-1245420456; e-mail: jem@writtle.ac.uk
sitions to accumulate essential and non-essential
metals (Ireland, 1979; Ireland and Richards, 1977;
Ash and Lee, 1980; Wright and Stringer, 1980; Andersen and Laursen, 1982; Morris and Morgan, 1986;
Morgan and Morgan, 1988, 1991, 1993; Morgan and
Morris, 1982; Morgan et al., 1986; Terhivuo et al.,
1994). It has been suggested that these observations
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 1 2 - 8
10
J.E. Morgan, A.J. Morgan / Applied Soil Ecology 13 (1999) 9±20
are a result of inter-speci®c differences in dietary
metal intakes and physiological utilisation (Andersen
and Laursen, 1982; Ireland, 1983; Beyer et al., 1985;
Morris and Morgan, 1986; Hopkin, 1989; Morgan and
Morgan, 1992, 1993), although little direct evidence
has been collected to substantiate these claims.
In addition, although there appears to be a degree of
inter-species consistency in the accumulation of particular metals from metalliferous mine soils (e.g.
Morgan and Morgan, 1991), such patterns become
obscured when soils polluted primarily by aerial
deposition are considered (e.g. Ash and Lee, 1980).
Such disparities appear to have received little attention, but clearly have implications for biomonitoring
and hazard assessment programmes.
The aims of this study were three-fold. First, to
con®rm our provisional observations, restricted to one
metalliferous site (Morgan and Morgan, 1993), that
differences exist in tissue Cd, Cu, Pb, Zn and Ca
concentrations between two ecophysiologically contrasting earthworm species (Lumbricus rubellus and
Aporrectodea caliginosa). L. rubellus is a litter dwelling (epigeic) species, which under normal conditions
lives outside of the mineral substrata, whereas A.
caliginosa lives permanently in essentially horizontal
burrows within the organo-mineral soil matrix, and is
thus categorised as an endogeic species (see Sims and
Gerard, 1985).
Second, to establish whether tissue metal concentrations in L. rubellus and A. caliginosa re¯ect the
respective metal concentrations of their ingested
materials, since it appears that absorption of metals
through the alimentary canal probably accounts for
most of the metal burdens of earthworms (e.g. Piearce,
1972). Although a number of studies (e.g. Ma, 1982;
Ma et al., 1983; Morgan and Morgan, 1988) have
demonstrated statistically that, for a number of heavy
metals, the concentration in the soil explains a large
proportion of the variance in accumulated tissue metal
concentrations of earthworms, there appears to be
little published information on the metal concentrations of material ingested by earthworms. This is
surprising since earthworms are known to ingest
material selectively (Piearce, 1978; Edwards and
Bohlen, 1996).
Third, to investigate, by means of a simple soil
layering experiment, whether the differences in Pb
accumulation by L. rubellus and A. caliginosa is
affected by the preferred vertical distribution of the
species. The source of contamination may in¯uence
metal distribution within the soil vertical pro®le
(Martin and Coughtrey, 1982), which could be a
factor determining the exposure and accumulation
by biota, and the consequent metal transfer through
food chains.
Recent laboratory exposure studies have suggested
that species differences exist in their sensitivities to
metals (Spurgeon and Hopkin, 1995) and organic
residues (Edwards and Coulson, 1992). Under ®eld
conditions the effects of metal contaminants are more
complicated: the toxicological effects of a given metal
on a given species should be coupled with the probability of encountering the metal during the earthworm's normal activities within the soil vertical
pro®le (i.e. exposure). Clearly this aspect of bioavailability of toxicants to de®ned target species has
important implications for toxicity testing programmes under ®eld conditions.
2. Materials and methods
2.1. Earthworms and soils
Mature (clitellate) specimens of L. rubellus and
A. caliginosa were collected from an abandoned,
non-acidic Pb±Zn mine at Llantrisant, S. Wales
(O.S. grid reference ST 048822) by formalin extraction (0.55%, 20 l). Samples were taken from ®ve 5 m2
`stations' across the site and represented areas within
the mine workings and adjacent pastures, which were
used for cattle grazing. Both species were collected in
a similar manner from a pasture adjacent to an abandoned Pb±Zn mine at Halkyn, N. Wales (O.S. grid
reference SJ 201704), and from an uncontaminated
`control' site at Dinas Powis, S. Wales (O.S. grid
reference ST 146723). All sampling occurred early
in November.
Extracted clitellate earthworms were immediately
rinsed in de-ionised water and transferred without
delay to the laboratory. They were rinsed clean of
adhering soil particles and maintained on moist Whatman No. 1 ®lter paper disks in plastic Petri dishes
(2 animals per dish) in the dark at 128C for 3 days. To
prevent coprophagy, the ®lter paper was changed at
least daily. The animals were then depurated for a
J.E. Morgan, A.J. Morgan / Applied Soil Ecology 13 (1999) 9±20
further day without ®lter paper but with a few drops of
deionised water to allow complete egestion of gut
contents. Dissection of several specimens con®rmed
that the guts were clean of consumed material.
The animals were quenched in liquid nitrogen,
placed in individual pre-weighed glass beakers, and
oven-dried (24 h at 708C) to constant weight. The
samples were allowed to cool in desiccators and reweighed in a moisture-free environment on a Mettler
HK60 semi-microbalance. The samples were wet
oxidised in 2 ml boiling concentrated `AnalaR' grade
nitric acid (BDH Chemicals, Poole, Dorset, UK), and
were made up to 10 ml with double de-ionised water.
Blank digests were also prepared.
At least 15 soil samples from two depths (0±5 and
10±15 cm) were taken randomly from within the
designated areas and pooled for each sampling area.
Each pooled soil was dried at 258C in a fan-oven,
gently crushed in an acid-washed porcelain pestle and
mortar, and passed through a 2 mm2 aperture stainless
steel sieve. Subsequent analysis was undertaken on
®ve sub-samples of each soil.
`Total' soil metal concentrations were determined
for both depths by extraction with boiling concentrated `AnalaR' grade nitric acid (Morgan and Morgan, 1988). Extracted metal concentrations (0±5 cm)
were determined after equilibration of 3 g of soil with
35 ml 0.5% acetic acid for 3 h (Morgan, 1987). Soil
pH (de-ionised water) was measured in soil slurries
(w/v, 1/2) following equilibration for 1 h (Peech,
1965). Soil organic carbon was determined by the
Walkley±Black method (Allison, 1965).
2.2. Ingesta (crop contents) and egesta (faeces)
Fresh specimens of A. caliginosa collected from
four Llantrisant stations were dissected and the contents of the crop and gizzard of each animal removed
and placed on individual pre-weighed Millipore
(Millipore S.A., Moshein, France) ®lter paper pieces.
The contents of the crop/gizzard of two animals were
combined for each sample. For comparative purposes,
the crop/gizzard contents were removed from L. rubellus at Station 5 of Llantrisant. Limited sample numbers precluded the crop contents being collected from
L. rubellus at all sites.
Egesta were collected from A. caliginosa taken
from Llantrisant stations during the ®rst day of starva-
11
tion. For comparative purposes egesta were collected
from L. rubellus from Station 5. Each sample was
derived by pooling faeces from two individuals for
each species, and prepared for analysis as described
for the ingesta.
Samples were oven-dried at 708C for 24 h, cooled in
a desiccator, and weighed on a Mettler HK60 semimicrobalance. Each sample was individually wet-oxidised in concentrated boiling `AnalaR' grade nitric
acid. Excess acid was evaporated to leave approximately 0.5 ml to which was added 1 ml double deionised water. Each sample was centrifuged at
2500 rpm for 2 min, and the supernatant removed.
A further 1 ml aliquot of de-ionised water was added
to the solid residue, the suspension again centrifuged,
and the supernatant added to that collected previously.
This procedure was repeated a third time. The ®nal
volume of the pooled supernatant solution was made
up to 5 ml with double de-ionised water. Blank digests
were also prepared.
2.3. Soil stratification experiment
This laboratory experiment was designed to determine whether the vertical distribution of Pb within
strati®ed combinations of naturally contaminated and
uncontaminated soils affects the relative concentrations of Pb accumulated by L. rubellus and A. caliginosa. Groups of between 12 and 16 similarly sized,
clitellate animals of each species, collected from the
uncontaminated Dinas Powis site, were exposed to
different soil treatments for 60 days in the dark at 48C.
Prior to their exposure to the appropriate soil, each
worm group was starved for 2 days to remove most of
their gut contents.
Surface soils (0±5 cm) used for the experiment
were collected from Dinas Powis (uncontaminated
site) and Llantrisant (Pb-contaminated site). The
individual soils were thoroughly mixed before being
placed in plastic containers to a depth of 8 cm, as
follows:
Expt. Group 1
Expt. Group 2
Expt. Group 3
Expt. Group 4
8 cm Dinas Powis soil
8 cm Llantrisant soil
5 cm Llantrisant soil covered by
3 cm Dinas Powis soil
5 cm Dinas Powis soil covered
by 3 cm Llantrisant Powis soil
12
J.E. Morgan, A.J. Morgan / Applied Soil Ecology 13 (1999) 9±20
At the end of the exposure period, the animals were
removed from the soils and processed as previously
described. Soil samples were taken for analysis from
0±3 and 3±8 cm horizons in each experimental container. The soils were processed for `total' soil Pb as
described above.
was prevalent in any of the soils investigated. The
acetic acid-extractable fraction of the heavy metals
(Table 1) was greatest for Cd (9±26%), followed by
Zn (2±10%), Cu (0.7±1.4%) and Pb (0.4±1.0%).
Extractability for Ca ranged from 11 to 41%. Organic
carbon contents of the soils (0±5 cm) ranged from 3 to
9% (Table 1).
2.4. Atomic absorption spectrophotometry (AAS)
3.2. Whole-worm field observations
Digests were quanti®ed for Cd, Cu, Pb, Zn and Ca
by ¯ame (air-acetylene) AAS in a Varian AA6 atomic
absorption spectrophotometer. Samples from the soil
layering experiment were analysed for Pb only. Background correction for Cd, Cu, Pb and Zn analysis was
made automatically with a hydrogen continuum lamp.
Samples and standards for determination of Ca were
made up containing 1% lanthanum.
2.5. Statistics
Statistical comparisons between the species for dry
weight and metal concentrations of tissue, ingesta and
egesta were made by the Mann±Whitney U-test. Signi®cance between metal concentrations in ingesta and
the whole worm was determined by Kendall's rank
correlation. For all statistical tests, differences were
regarded as signi®cant if p < 0.05.
3. Results
3.1. Soils
Soil pH, percent organic carbon content and `total'
(nitric acid-extractable) Cd, Cu, Pb, Zn and Ca concentrations (0±5 cm depth) are presented in Table 1.
Total concentrations of Cd, Pb and Zn were elevated in
the vicinity of the abandoned mine sites compared to
the uncontaminated control site (Dinas Powis), but the
concentrations of Cu were broadly similar at all sites
(Table 1). `Total' concentrations of Ca ranged from
1050 to 8300 mg/kg dw, and re¯ected the range of soil
pH (5±6.8). `Total' concentrations of metals in the 10±
15 cm depth (data not shown) ranged from 0.8 to
16 mg/kg dw for Cd, 21±60 mg/kg dw for Cu, 158±
10 020 mg/kg dw for Pb, 185±1870 mg/kg dw for Zn
and 980±8120 mg/kg dw for Ca. The data indicated
that no surface (0±5 cm) enrichment of heavy metals
Concentrations of Cd, Cu, Pb, Zn and Ca in L.
rubellus and A. caliginosa from the different sites are
presented in Table 2. The concentrations of Cd, Pb,
and Zn were greater in both species inhabiting the
mine soils than in the uncontaminated soil.
Mean concentrations of Cd in A. caliginosa were
between 2 and 5 times greater than those of L. rubellus
in six of the seven soils examined, with all the
differences statistically signi®cant. The trend for Pb
was similar to that for Cd; greater concentrations were
found in A. caliginosa, although the differences were
not always statistically signi®cant. No consistent pattern for Zn accumulation was apparent for these
species, although A. caliginosa accumulated signi®cantly greater concentrations of Zn than L. rubellus
from the soils containing the lowest (total and extractable) concentrations of Zn. Although concentrations
of Cu were broadly similar for both species (range 11±
20 mg/kg dw) in all soils, the concentrations in
A. caliginosa were signi®cantly greater in four of
the soils studied. Concentrations of Ca were signi®cantly greater in L. rubellus in all soils.
3.3. Metal concentrations in ingesta (crop contents)
and egesta (faeces)
The Cd, Cu and Ca concentrations in the ingesta of
both species were in general about 2±15 times greater
than total concentrations in bulk soil (compare
Tables 1 and 3). The pattern for Pb and Zn was less
consistent, although marked differences between
ingesta and soil were apparent for A. caliginosa.
The results suggest that both species ingest material
selectively.
The Cd, Cu and Ca concentrations of the ingesta
of both species were signi®cantly greater than those
in the faeces (Table 3). In contrast, the concentrations
of Pb and Zn were generally signi®cantly greater in
13
J.E. Morgan, A.J. Morgan / Applied Soil Ecology 13 (1999) 9±20
Table 1
Total (16N nitric acid-extractable) and extractable (0.5% acetic acid-extractable) concentrations of Cd, Cu, Pb, Zn and Ca (mg/kg dry wt.),
percentage organic matter and pH of soils (0±5 cm depth) from an uncontaminated site (Dinas Powis) and metalliferous mine sites
Total metal concentration (mg/kg dry wt.)
Sitea
Cd
Cu
Pb
Zn
Ca
DP
Halk
Ll1
Ll2
Ll3
Ll4
Ll5
0.9 0.1
3.6 0.1
14.7 0.3
6.5 0.1
10.5 0.1
17 0.5
2.7 0.1
26 1
40 3
62 1
27 1
30 1
31 1
23 1
166 7
1350 17
10110 280
2370 63
3820 38
6730 71
570 6
193 4
675 9
1550 27
770 21
830 15
1960 33
460 6
1790 48
6080 71
7560 212
5320 110
8300 166
5120 86
1050 21
Extractable metal concentration (mg/kg dry wt.)
DP
0.08 0.01
Halk
0.32 0.01
Ll1
3.0 0.1
Ll2
1.2 0.1
Ll3
1.6 0.1
Ll4
2.4 0.1
Ll5
0.7 0.1
DP
Halk
Ll1
Ll2
Ll3
Ll4
Ll5
Organic carbon (%)
4.7 0.3
9.0 0.5
5.6 0.2
4.1 0.1
5.0 0.2
6.5 0.3
2.9 0.1
0.4 0.1
0.4 0.1
0.3 0.1
0.4 0.1
0.3 0.1
0.2 0.1
0.2 0.1
1 0.2
8 0.1
100 4
18 1
21 1
29 1
31
4.5 0.1
38 12
155 4
31
35 1
97 2
24 1
680 17
2290 43
1730 20
1610 15
1920 25
2115 25
375 2
pH (de-ionised water)
5.19 0.01
6.52 0.03
6.54 0.01
6.49 0.01
6.78 0.01
6.72 0.01
4.98 0.01
Values are mean SE; n 5.
DP Dinas Powis; Halk Halkyn, Ll±Ll5 Llantrisant Stations 1±5.
a
the egesta. No signi®cant correlations (tested by
Kendall's rank correlation) were found between the
concentrations of metals in crop contents and earthworm tissue.
The concentrations of Cd, Pb and Zn in the crop
contents of A. caliginosa from Llantrisant 5 were
signi®cantly greater than those of L. rubellus
(Fig. 1, Table 3), and re¯ected the signi®cant species
differences in the whole-worm concentrations of
the respective metals. No clear pattern was evident
for the Cu concentrations in the ingesta and wholeworm, and no signi®cant species differences were
found. Concentrations of Ca were greater in the crop
contents of L. rubellus, although the difference was
not statistically signi®cant; signi®cant differences
were apparent between the species in the whole-worm
Ca concentration.
No signi®cant differences between the species were
apparent in the concentrations of Cd, Cu and Pb of
the egesta (Fig. 1). The concentration of Zn in the
egesta of A. caliginosa was signi®cantly greater than
in L. rubellus, with the pattern being reversed for Ca.
3.4. Soil stratification experiment
The concentrations of Pb in A. caliginosa were
signi®cantly greater than in L. rubellus for Treatments
1 to 3 (Table 4) following the pattern found in the ®eld
studies. However, in Treatment 4, where the surface
soil was greatly contaminated with Pb, the concentrations in both species were similar. The results indicate
that the pattern of Pb accumulation by the two earthworm species is affected by the distribution of soil Pb
within the vertical soil pro®le.
14
J.E. Morgan, A.J. Morgan / Applied Soil Ecology 13 (1999) 9±20
Table 2
Dry weights (mg) and metal concentrations (mg/kg dw) in A. caliginosa (Ac) and L. rubellus (Lr) from an uncontaminated soil (Dinas Powis)
and 6 soils from abandoned metalliferous mines (see Table 1 for key) (mean SE; n number of samples analysed). Statistical differences
were determined by the Mann±Whitney U-test
Site
Species
Dry wt. (mg)
Cd
Cu
Pb
Zn
Ca
DP
Ac (n 21)
Lr (n 11)
Halk
Ac (n 12)
Lr (n 11)
Ll1
Ac (n 9)
Lr (n 8)
Ll2
Ac (n 17)
Lr (n 5)
Ll3
Ac (n 8)
Lr (n 8)
Ll4
Ac (n 16)
Lr (n 13)
Ll5
Ac (n 17)
Lr (n 27)
94.6 3.6
186.9 12.8
****
86.4 4.3
105.2 8.5
NS
85.8 8.5
128.8 16.7
NS
94.3 6.3
120.4 18.2
NS
71.2 4.5
129.3 12.5
****
88.0 6.1
117.1 11.7
NS
70.6 1.6
100.5 4.3
****
24 2
12 1
****
90 8
42 5
****
233 49
123 12
**
135 11
144 31
NS
229 45
57 6
****
344 30
147 16
****
214 11
46 3
****
17 0.6
14 1.1
NS
17 0.6
14 0.4
***
20 1.2
14 1.2
**
14 1
10 1
*
12 1.1
13 1.2
NS
13 0.8
11 0.5
*
12 0.4
16 1.5
NS
81
51
NS
2010 306
238 31
****
1940 320
460 37
****
1270 208
330 65
**
310 109
300 44
NS
1460 267
900 115
NS
662 49
71 6
****
660 32
330 25
****
1835 169
1495 235
NS
1160 174
1375 87
NS
1200 129
1120 78
NS
770 127
1350 214
NS
1505 131
1745 198
NS
1315 53
770 59
****
1610 32
3225 225
****
3420 302
5853 452
****
2270 168
4815 227
****
2250 83
4740 374
****
3000 50
8300 661
****
2000 199
4810 309
****
3260 73
5280 1690
****
NS Not significant.
* p < 0.02.
** p < 0.01.
*** p < 0.002.
**** p < 0.001.
4. Discussion
Earthworms are known to be selective consumers
(Edwards and Bohlen, 1996). Although relatively few
studies have measured the concentrations of contaminants within material ingested by earthworms, published ®ndings con®rm selective consumption because
there are marked differences between residue concentrations in bulk soil and ingesta, both in the case of
trace organics (Diercxsens et al., 1985) and heavy
metals (Ireland, 1976; Morgan and Morgan, 1992).
The present study also demonstrated that there are
signi®cant differences in concentrations of certain
essential and non-essential metals between the ingesta
and bulk soil.
Such observations pose questions in relation to the
interpretation of concentration factors (the ratio of
metal concentration of earthworm tissue and bulk
soil), which have been cited as being representative
indicators of metal bioavailability in soils (Neuhauser
et al., 1995). Although there exist clearly demonstrable relationships between the concentrations in
earthworms and bulk soil for several metals (Morgan
and Morgan, 1988; Neuhauser et al., 1995), the partition coef®cients of a metal within bulk soil have been
shown to be an important parameter in determining
metal bioavailability (Janssen et al., 1997). An advantage of the concentration factor approach is that it
integrates the bioavailable metal fraction and the
kinetics of the transport system (Janssen et al.,
1997) and thus may have practical application for
the evaluation of the potential toxicological impact
of metals in soils. However, the data from the present
study would suggest that without careful consideration, the use of such indices could lead to spurious
conclusions in relation to toxicological testing of
xenobiotics on different earthworm species. Furthermore, it is probable that earthworm exposure to resi-
15
J.E. Morgan, A.J. Morgan / Applied Soil Ecology 13 (1999) 9±20
Table 3
Dry weight (mg) and concentrations of metals (mg/kg dw) in ingesta and egesta of A. caliginosa (Ac) and L. rubellus (Lr) from Llantrisant
soils (mean SE; n nos of samples). Statistical differences in metal concentrations were determined by the Mann±Whitney U-test
Species
Site
Component
Dry wt. (mg)
Cd
Cu
Pb
Zn
Ca
Ac
Ll1
Ingesta (n 10)
Egesta (n 6)
4.5 0.6
43.7 3.1
Ac
Ll2
Ingesta (n 10)
Egesta (n 10)
4.9 0.4
26.2 3.1
Ac
Ll3
Ingesta (n 9)
Egesta (n 9)
5.7 0.3
48.9 5.3
Ac
Ll5
Ingesta (n 10)
Egesta (n 9)
5.6 0.8
24.3 3.2
Lr
Ll5
Ingesta (n 10)
Egesta (n 15)
6.4 0.7
40.0 6.0
33 4
11 0.7
**
35 4
9 0.8
***
47 9
13 1.1
***
31 5
6 0.4
**
91
61
**
69 6
39 2
**
41 4
31 1
*
35 2
27 2
*
52 7
24 1
***
37 3
27 4
*
4520 306
3720 301
NS
2100 117
3425 255
**
1970 77
3400 238
***
740 51
940 74
**
570 41
810 35
***
1005 47
1100 95
NS
645 42
990 65
***
560 39
920 86
**
550 15
685 21
***
415 22
605 19
***
10 400 1190
3430 720
**
7340 311
2500 294
***
8250 750
6940 610
NS
13 560 1932
1760 132
***
11 950 1883
3320 409
***
NS not significant.
* p < 0.05.
** p