Equilibrated body metal concentrations in laboratory
exposed earthworms: can they be used to screen
candidate metal-adapted populations?
F. MarinÄo
a, A.J. Morgan
b,*aDepartamento de Ecologia y Biologia Animal, Facultad de Ciencias, Universidad de Santiago, 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
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 Zn-exposures than it is to Pb- or perhaps Cu-Zn-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,
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 chal-lenges. Metal resistance can be achieved through avoidance reactions (Tranvik and Eijsackers, 1989), reduced assimilation (i.e., down-regulated trans-epithelial uptake and/or increased excretion)
(Post-*Corresponding author. Tel.: 1222874190; fax: +44-1222874305; e-mail: fry@cardiff.ac.uk
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 enzy-mic 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 fundamen-tally 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 para-meters, 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 fresh-water oligochaete,Limnodrilus hoffmeisteri, inhabit-ing Cd-polluted sediments has been conclusively shown to be genetically different from worms inhabit-ing adjacent but less Cd-polluted sediments (Klerks and Levinton, 1989, 1993). Furthermore, the resistant
L. hoffmeisteriecotype accumulates more, not less, Cd than the Cd-sensitive conspeci®es (Klerks and Bartho-lomew, 1991).
Bengtsson et al. (1992) were unable to obtain ®rm
evidence that populations of the earthworm,
Dendro-baena 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 evolu-tionary processes, in what are effectively replicated islands-of-toxicity, were not uniform: populations under some pylons were Zn-tolerant, others not, prob-ably 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, popula-tions. In the present preliminary investigation, we compared the accumulation of metals by seven dif-ferent populations of the earthworm,Lumbricus rubel-lus, 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 (approxi-mately 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]2010mg/g, pH 5.8; Caerf®li
acidic soil, ST 160855, [Ca]120mg/g pH 4.1).
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
earth-worms 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 meanSE. Differences
between the means of native and introduced worms, and between the two introduced populations, were
determined by the Mann±WhitneyU-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 compo-sitionally 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 <5.5, and were fairly heavily contami-nated with Pb, but only moderately contamicontami-nated with Zn, Cd and Cu. The CS soil contained low concentra-tions of Cd, Pb, Cu and Zn, but was distinguished by
possessing low calcium concentrations, low pH (<4.0) and organic matter content (<20%). The major differ-ences in the two reference soils were that CF contained a much lower Ca concentration and pH than DP.
Table 2 summarizes the metal composition of earth-worms at the end of the 90 days exposure period. DP worms did not survive for the full duration of the experiment on the three (CS, RM, WM) relatively acidic soils or on the heavily Pb-contaminated DT soil. Reference worms from the acidic CF soil survived in reasonable numbers on all the contaminated soils.
Some inter-population differences in accumulated metal concentrations were observed (Fig. 1, Table 3), but these were not consistent for all the metals under consideration. Population differences were particu-larly striking in the case of Cd (Fig. 1(A)), where worms native to the three most heavily Cd-contami-nated soils (LL, DT, RG; Table 1) had signi®cantly higher tissue Cd concentrations than both (i.e., when both survived the exposure) reference populations maintained on the same soils. Worms native to the RM and WM soils also had signi®cantly higher Cd concentrations than their CF counterparts, but the small number of surviving worms in these experi-mental groups (Table 2) diminished observational con®dence.
Pb concentrations were higher in native worms than in their introduced (CF) counterparts only at WM
(Fig. 1(B), Table 3). Cu (Fig. 1(C)) and Ca
(Fig. 1(E)) concentrations were generally similar in native and introduced populations after 90 days, but it may be noteworthy that the reference worms from the acidic soils (CF) tended to accumulate higher Ca concentrations than reference worms from the rela-tively Ca-rich site (DP) when both were maintained on calcareous LL, CC and RG soils (Fig. 1(E)). This may be consistent with the ®nding that the two reference populations maintained on their `own' soils for 90 days contained similar tissue Ca concentrations (Table 2) despite the over 10-fold difference in the Ca concentrations of their native soils.
Table 1
Metal concentrations (mg/g dry weight), pH and % organic matter content (o.m.) of the soil samples to which native (N) and introduced (I-CF;
I-DP) earthworms were exposed for up to 90 days
pH OM Cd Pb Cu Zn Ca
Cwmystwyth Stream (CS)*
Median 3.9 18.8 0.5 184 10.7 69 175
Range 3.7±4.0 17.9±20.5 0.3±0.6 68±506 8.6±16.7 60±93 110±217
Q1 3.8 18.4 0.4 147 9.6 65 155
Q3 3.9 19.2 0.6 242 11.3 73 182
MeanSE 3.80.03 1890.3 0.50.0 21235 11.30.8 723 16910
Rhandirmwyn (RM)*
Median 4.9 26.9 3.5 3750 42.3 570 660
Range 4.5±5.3 20.5±31.0 0.3±5.7 2344±8437 32.1±63.3 100±847 325±862
Q1 4.6 23.6 2.1 2734 33.3 371 392
Q3 5.0 29.9 4.4 4015 49.9 680 792
MeanSE 4.80.08 26.71.3 3.20.5 3900479 44.23.2 52565 62168
WEMYSS (WM)*
Median 5.5 29.4 0.5 4312 31.5 297 552
Range 5.3±5.6 26.0±36.3 0.1±1.6 3562±12219 22.8±40.1 235±915 490±980
Q1 5.4 27.9 0.3 3969 27.4 285 542
Q3 5.5 32.3 0.6 5234 35.1 361 895
MeanSE 5.40.03 30.31.1 0.50.1 5517782 31.31.4 37554 69163
Llantrisant (LL)
Median 6.0 29.3 89 1531 23.5 15775 20175 Range 5.9±6.0 25.6±33.7 79±102 575±3625 14.6±28.1 12500±18425 12550±39150
Q1 5.9 27.8 88 1008 18.8 14700 17500
Q3 6.0 32.1 95 1997 25.8 16987 21975
MeanSE 5.90.02 29.80.8 912 1639245 22.21.3 15741516 209142425
Cwmystwyth Cottage (CC)
Median 6.1 38.2 82.0 4219 26.9 25425 34975 Range 5.9±6.3 32.9±41.1 69.2±93.0 3156±6125 22.8±89.9 20375±54450 31050±40175
Q1 6.0 34.6 76.0 3359 25.2 21462 33287
Q3 6.2 39.3 89.0 4859 36.1 30425 38912
MeanSE 6.10.04 37.30.9 82.22.21 4253304 34.84.8 292633123 358001247
Draethen Quarry (DT)*
Median 6.1 32.0 110.5 79375 45.1 8050 126000 Range 5.9±6.3 28.7±37.7 50.2±123.1 40000±109688 24.3±52.3 4075±9300 53000±179000 Q1 6.0 30.2 103.1 49999 42.3 6412 112000 Q3 6.2 37.2 114.6 84062 47.3 8437 132000 MeanSE 6.10.04 33.31.2 105.25.5 720746418 43.22.1 7314487 12094410456
Roman Gravels (RG)
Median 6.4 35.6 117.5 1875 137.2 17375 127600 Range 6.3±6.5 23.6±46.2 89.2±219.2 1281±4906 103.5±230.7 12525±59950 71200±243900 Q1 6.3 25.30 106.1 1609 112.2 14875 113800 Q3 6.4 39.2 163.6 2703 162.1 20637 193400 MeanSE 6.40.02 34.42.6 136.711.9 2258315 146.811.6 214023658 14851119527
Caerffili (CF)
Median 4.1 19.92 0.2 17 8.3 57 132
Range 4.1±4.2 19.9±20.5 0.2±0.3 13±49 7.4±14.7 49±68 87±135
Q1 4.1 19.9 0.2 14 7.7 55 110
Q3 4.1 20.2 0.2 27 10.2 60 133
Table 1 (Continued)
pH OM Cd Pb Cu Zn Ca
Dinas Powys (DP) Median
Median 5.8 17.8 0.8 42 13.3 128 2100
Range 5.8±5.9 17.6±18.4 0.6±0.8 35±65 11.1±15.4 112±133 1735±2187
Q1 5.8 17.7 0.7 36 12.5 123 1917
Q3 5.8 18.1 0.8 52 14.2 130 2144
MeanSE 5.80.03 17.90.2 0.70.04 467 13.30.9 1254 2007138 Q1: first quartile; Q3: third quartile
Table 2
Metal concentrations (mg/g dry weight) in the tissues of native (N) and introduced (I-CFCaerffili; I-DPDinas Powys) earthworms at the
end of the 90 day exposure to seven contaminated and two reference soils
Cd Pb Cu Zn Ca nand % mortality
Cwmystwyth Stream (CS)a
(N-CS)
Median 11 5193 20.9 809 3629
Range 4±13 2200±8152 13.79±26.4 477±1706 2977±4909 6 Q1/Q3 7/12 3783/7325 16.5/25.1 574/945 3260/4603
MeanSE 91 5348972 20.612.2 881183 3868334 40% (I-CF)
Median 3 4105 18.9 1193 4179
Range 1±7 3237±8312 10.6±37.9 983±2065 2784±5742 6 Q1/Q3 3/5 3340/6274 12.6/20.9 1050/1384 3151/5350
MeanSE 40.8 4973867 19.74.1 1314165 4239526 40%
Rhandirmwyn (RM)a
(N-RM)
Median 209 3473 23.8 5403 5001
Range 134±261 736±13944 16.9±29.7 2546±5705 2583±5318 5 Q1/Q3 146/212 2311/13753 20.0/26.1 3786/5573 4842/5113
MeanSE 19223 68442893 23.32.3 4603620 4571503 50% (I-CF)
Median 31 4085 14.9 1081 4625
Range 30±48 2269±7466 8.4±16.2 99±1544 1191±5470 3 Q1/Q3 31/39 3177/5775 11.6/15.6 590/1313 2908/5047
MeanSE 366 46061523 13.22.4 908426 37621308 70%
Wemyss (WM)a
(N-WM)
Median 27 15735 16.8 1545 3500
Range 19±39 10516±32131 15.1±16.8 1112±1628 3272±4907 3 Q1/Q3 23/33 13215/23933 16.0/18.0 1328/1586 3385/4203
MeanSE 296 194616512 17.11.9 1428160 3893511 70% (I-CF)
Median 10 3745 9.4 769 4316
Range 9±15 2741±11569 7.3±20.8 353±973 3596±5210 5 Q1/Q3 10/13 3130/9592 8.4/10.7 526/882 3955/5136
MeanSE 121 61561840 11.32.4 701115 4442319 50%
Llantrisant (LL)
(N-LL)
Median 789 631 13.2 4797 3607
Range 623±1470 323±1481 9.7±23.5 2788±11224 2376±5425 5 Q1/Q3 636/1225 453/811 12.3/17.6 3702/5530 3321/5164
Table 2 (Continued)
Cd Pb Cu Zn Ca nand % mortality
(I-CF)
Median 199 544 14.3 2180 6209
Range 154±431 111±1319 7.7±30.3 1638±4176 3213±7742 6 Q1/Q3 180/325 421/781 12.1/16.4 1725/2453 4764/7322
MeanSE 25447 624171 15.83.1 2382387 5891739 40% (I-DP)
Median 238 538 14.5 4645 4038
Range 117±309 197±610 10.1±22.0 2604±6362 30365625 7 Q1/Q3 220/270 454/544 14.1/18.1 3194/5481 3760/4385
MeanSE 24416 47751 15.81.5 4423561 4145300 30%
Cwmystwyth Cottage (CC)
(N-CC)
Median 121 3736 20.6 10916 4872
Range 80±197 3521±14411 18.9±36.9 4809±21236 2758±7834 5 Q1/Q3 97/135 3629/6383 19.2/29.1 6366/17771 4859/6625
MeanSE 12620 63362088 24.93.5 122203186 5390865 50% (I-CF)
Median 79 3213 17.5 8723 9520
Range 63±95 1859±4568 17.1±17.8 7487±9959 9058±9982 2 Q1/Q3 71/87 2536/3890 17.3/17.6 8104/9341 9289/9751
MeanSE 7916 32131354 17.50.3 87231236 9520462 80% (I-DP)
Median 53 1669 16.8 5677 5439
Range 42±89 1183±4858 7.8±34.7 4141±15729 3113±8139 4 Q1/Q3 47/66 1506/2508 12.3/23.6 4226/9258 4095/6876
MeanSE 6010 2345846 19.05.8 78062728 55321107 60%
Draethen Quarry (DT)a
(N-DT)
Median 736 11037 11.4 7560 6905
Range 580±872 2804±17777 5.7±16.6 6107±7927 6235±15567 4 Q1/Q3 604/863 6004/15697 7.4/15.4 7004/7844 6588/9220
MeanSE 73178 106643466 11.32.6 7289417 89032228 60% (I-CF)
Median 231 9184 14.8 2813 4551
Range 215±320 2653±9688 9.9±27.8 2358±3396 4387±5568 4 Q1/Q3 217/264 7453/9408 12.8/18.8 2361/3297 4501/4814
MeanSE 25024 76771680 16.83.8 2845281 4764270 60%
Roman Gravels (RG)
(N-RG)
Median 539 888 26.1 6530 6577
Range 383±952 349±1416 12.1±38.1 3064±10470 4454±10916 6 Q1/Q3 501/676 655/1188 24.0/29.0 4852/8614 5097/8253
MeanSE 60482 901163 25.93.5 67001138 69901007 40% (I-CF)
Median 208 546 21.6 1979 5032
Range 133±350 210±971 9.9±51.3 1287±4187 2920±10534 8 Q1/Q3 155/273 385/561 16.2/28.0 1689/2134 4737/6964
MeanSE 22330 51282 24.14.6 2109319 6061909 20% (I-DP)
Median 361 604 20.8 2933 4327
Range 263±438 496±1301 16.1±31.9 2303±3823 3705±6064 8 Q1/Q3 327/381 557/659 18.2/24.4 2554/3661 3873/5402
4. Discussion
Metal adaptation is extant in a number of species belonging to several terrestrial invertebrate groups (Posthuma and Van Straalen, 1993). Genetically dif-ferentiated metal-resistant earthworm populations have not yet been identi®ed, but it is possible to construct ana prioricase for their existence. Firstly, estuarine polychaetes (Grant et al., 1989) and fresh-water oligochaetes (Klerks and Levinton, 1989, 1993) can respond to metal-induced stress in their natural environments by evolving resistant ecotypes. Sec-ondly, Klerks and Levinton (1993) proposed that
one reason why an oligochaete (L. hoffmeisteri)
became adapted to Cd-toxicity, whilst a chironomid (Tanypus neopunctipennis) inhabiting the same heav-ily contaminated sediment did not, is because the former is a much more ef®cient metal accumulator (sensuDallinger, 1993), and is subject to direct selec-tion pressures. Earthworms are also metal accumula-tors (Morgan et al., 1993). Thirdly, certain earthworm species are prominent components of the macro-invertebrate fauna of metalliferous soils (Morgan and Morgan, 1988; Corp and Morgan, 1991), their distribution restricted perhaps more frequently by soil nutrient and moisture de®ciencies rather than exces-sive metal concentrations.
The present study was not designed to furnish direct evidence for metal adaptations in earthworms. The objective was to determine whether or not populations of an earthworm species resident on their `own'
metal-contaminated soils accumulate different tissue metal concentrations than reference populations exposed to the same polluted soils. Before embarking on such a comparative exercise it was essential to establish that the accumulated metal concentrations in the `native' and `introduced' earthworms reached their respective equilibrium levels within the exposure span. This requirement was explored in a previous study (MarinÄo and Morgan, 1999), which indicated that equilibration levels for the metals of interest are approached under the stated exposure conditions in about 60 days, i.e., after this period the degree of further metal accumula-tion is minor. On this basis the exposure period for the present study was 90 days.
Three aspects of our observations on metal accu-mulation are worthy of comment.
1. Interpopulation differences between `native' and `introduced' worms were more frequently encoun-tered for Cd than for the other metals studied. This was a revealing ®nding when taken with those reported by Bengtsson et al. (1992) showing that the Zn and Cu resistance ofD. octaedrainhabiting a soil heavily contaminated with both metals was weak, but Cd-resistance in the same worms was high even though Cd was a minor constituent of the soil. Cadmium in earthworms is bound by cysteine-rich, Cd-induced metallothionein pro-teins (Morgan et al., 1989; Bengtsson et al., 1992). Copper certainly (Bengtsson et al., 1992; MarinÄo et al., 1998), and Zn possibly, are poor
Table 2 (Continued)
Cd Pb Cu Zn Ca nand % mortality
Caerffili (CF)
(N-CF)
Median 13 1027 12.9 1012 3907
Range 12±14 895±1842 11.3±19.9 713±1402 2759±5813 5 Q1/Q3 12/13 942/1333 12.1/15.6 929/1022 3092/4506
MeanSE 130.4 1208176 14.41.7 1016111 4015544 50%
Dinas (DP)
(N-DP)
Median 23 13 16.4 1244 3705 6
Range 18±31 7±20 2.4±25.1 540±3369 2368±6296 Q1/Q3 22/25 11/15 13.7/17.4 787/1856 3406/5224
MeanSE 242 132 15.13.0 1515429 4185609 40%
aThe earthworms from the Dinas Powys reference site (I-DP) did not survive for 90 days in these soils.
Fig. 1. Median metal concentrations (mg/g dry tissue weight) in native and introduced (reference sites) worms maintained on seven different
inducers of metallothionein gene expression in earthworms. Thus, it follows that Cd-resistance in earthworms, even in those not subjected to high exposure levels in their native soils, is latent and inducible. But this leaves unanswered the crucial question of the existence of genetically modi®ed local populations with enhanced Cd-resistance. It is reasonable to suppose that earthworms, like certain populations of freshwater oligochaetes (Klerks and Levinton, 1993), can evolve Cd tolerance through metallothionein gene ampli®ca-tion. This hypothesis is currently being investi-gated. Alternatively, it is possible that some of the observed differences between the Cd burdens of native and introduced earthworms may re¯ect the very slow rate of Cd-elimination from earthworm tissues revealed by recent depuration experiments (Sheppard et al., 1997; Spurgeon, 1997).
2. Metal adaptation may impose a cost, where `cost' has been defined (Klerks and Levinton, 1993) as `the reduction of fitness of a metal-adapted geno-type'. The costs can be of several different types (Klerks and Levinton, 1993; Posthuma and Van
Straalen, 1993). These include, in the specific case of Cd-resistance, the metabolic cost of synthe-sising metallothionein and transporting the metal to the tissue and subcellular sequestration sites, and the possible `loss' of certain essential metals incidentally sequestered. Our observations indi-cated that Zn could be a case in point. If Cd-adapted earthworm populations with high levels of metallothionein expression do exist, it does not follow that these populations are co-adapted to Zn via the same physiological mechanism unless Zn itself is capable of activating the metal-lothionein gene(s). In other words, if offspring of Cd-adapted parents were exposed to soils of high Zn but background Cd concentrations, they would not necessarily display higher Zn resistance and accumulation capacity than reference popula-tions. An intriguing possibility raised by a report on Cd and Cu sequestration by metallothionein in the land snail, Helix pomatia, (Dallinger et al., 1997) is that different isoforms of the protein may be involved in the tissue handling of specific metals.
Table 3
Summary of the statistical analysis (Mann±WhitneyU-test) of the differences between the concentrationsaof metals in native and introduced
earthworms, and (where applicable) between the two introduced populations, after maintenance on seven polluted soils for 90 days Paired comparisons Cd Pb Cu Zn Ca Soil
N-CS vs. I-CF * ns ns * ns Cwmystwyth Stream (CS) N-RM vs. I-CF * ns * * ns Rhandirmwyn (RM)
N-WM vs. I-CF * * ns * ns Wemyss (WM)
N-LL vs. I-CF ** ** ns * ns
N-LL vs. I-DP ** ns ns * ns
I-CF vs. I-DP ns ns ns ns ns Llantrisant (LL)
N-CC vs. I-CF ns ns ns ns ns
N-CC vs. I-DP * ns ns ns ns Cwmystwyth Cottage (CC)
I-CF vs. I-DP ns ns ns ns ns
N-DT vs. I-CF * ns ns * * Draethen Quarry (DT)
N-RG vs. I-CF ** * ns * *
N-RG vs. I-DP ** ns ns ** * Roman Gravels (RG)
I-CF vs. I-DP * ns ns * ns
aFor concentration values refer to Table 2.
*P< 0.05; **P< 0.01; ***P< 0.001; n.s.±non significant.
3. In one instance only (Wemyss) was a difference recorded in the capacity of native and reference populations, respectively, to accumulate Pb. High proportions of the Pb (and Zn) burdens of earth-worms are bound by constitutive (i.e., non-induced) chloragosome granules in the chloragog (Morgan et al., 1993). In this sense earthworms may be considered pre-disposed to withstand Pb toxicity. However, the accumulation of Pb within chloragosomes could compromise the normal phy-siological functions of these organelles. There is no evidence that worm populations that have colo-nized Pb-enriched soils possess a genetically-deter-mined capacity to synthesise more chloragosomes.
5. Conclusions
In conclusion, this study was undertaken to screen for candidate metal tolerant earthworm populations on the basis of differential tissue accumulation. This simple and perhaps simplistic approach clearly over-looked adaptations that are not physiologically expressed as altered accumulation. This study con-®rmed that Cd resistance is possibly ubiquitous (Bengtsson et al., 1992), but further studies on key populations need to be completed before the mole-cular genetics of Cd-resistance by earthworms is better understood.
Acknowledgements
We thank Mrs. Nicola Bassett for typing the manu-script.
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