Camp. Biochem. Physiol. Vol. lOfX, No. I, pp. 11-38, 1993 0742~8413/93 $6.00 + 0.

00
Printed in Great Britain 0 1993 Pergamon Press Ltd

MINI REVIEW

HEAVY-METAL ADAPTATION IN TERRESTRIAL

INVERTEBRATES: A REVIEW OF OCCURRENCE,
GENETICS, PHYSIOLOGY AND ECOLOGICAL
CONSEQUENCES
LEO POSTHUMA* and NICO M. VAN STRAALEN~
*Laboratory for Ecotoxicology, National Institute of Public Health and Environmental Protection,
P.O. Box 1, 3720 BA Bilthoven, The Netherlands; and TDepartment of Ecology and Ecotoxicology,
Vrije Universiteit, De Boelelaan 1087, 1081 HV Amsterdam, The Netherlands

(Received 22 March 1993; accepted for publication 7 May 1993)

Ahstract-1. The occurrence of genetic adaptation to heavy metals in natural populations of terrestrial
invertebrates is evaluated from literature data. Five criteria for adaptation evidence are applied, with
concepts from ecotoxicology, ecology, life-history theory and quantitative genetics.
2. There is strong evidence for the occurrence of adaptation in natural populations of the isopod
Porcellio scaber (Isopoda), the springtails Isotoma notabilis, Onychiurus armatus and Orchesella cincta
(Collembola), the blowfly Lucilia cuprina and the fruit fly Drosophila melanogaster (Dip&a). Adaptation
to metal-containing pesticides has been demonstrated in ticks (Acarina). Population divergence indicates
acclimation or adaptation in many other species.
3. Metal adaptation has been achieved within a few generations under laboratory conditions in some
species; adapted populations occur at field sites that have been polluted for decades, or longer.
4. Genetic variation for tolerance and life-history characteristics, allowing for adaptation, was
quantified in a reference population of Orchesella cincta. Tolerance and life-history patterns in exposed
field populations matched predictions from genetic variation.
5. Adaptation involves modification and intensification of existing physiological mechanisms for metal
assimilation, excretion, immobilization or compartmentalized storage. There are indications of inter-popu-
lation divergence in metal-binding proteins in a snail. In the fruit fly Drosophila melanogaster metal
adaptation is achieved by duplication of the metallothionein gene.
6. An altered life-history is often part of the complex adaptation syndrome. Metal-adapted invert-
ebrates have a shorter life-cycle and a higher reproductive effort.
7. Possible consequences of adaptation, consisting of costs of tolerance determined by genetic
correlations, and probably of reduced genetic variation for tolerance and other features, are discussed.
Reduced genetic variation is suggested by results for the springtail Orchesella cincra.
8. The distinction between “costs of tolerance” on the one hand and linkage disequilibrium or direct
selection for altered life-history patterns on the other hand is discussed.
9. Species with high sensitivity (i.e. a low NOEC), that do not have populations maintaining sufficient
genetic variation to evolve tolerance or modified life-history characteristics, or that have costly tolerance
mechanisms, or both, are most at risk for extinction at sites with increasing metal pollution.
10. Metal adaptation in terrestrial invertebrates appears to be of degree rather than of kind: indications
for a specific metal-fauna, equivalent to metal-vegetation, are lacking.

1. HEAVY-METALADAPTATION present, and such animal populations may be. metal-

1.1. What about terrestrial invertebrates?
In this extensive review volume on the ecophysi-
ology of heavy metals in terrestrial invertebrates,
Hopkin (1989), in his summary of conclusions, stated
that “it is still not known whether any terrestrial
invertebrate (with the exception of Drosophila spec.)
has evolved genetic tolerance to metals in the diet,
despite the fact that this phenomenon has been
demonstrated clearly in plants”. This held true in
1989, but, nonetheless, subsequent research has
shown that populations of terrestrial animals inhabit
sites where specific heavy-metal tolerant vegetation is
adapted.
Aspects of heavy-metal adaptation have been
reviewed for fungi (kshida, 1965), bacteria (Trevors
et al., 1985), aquatic organisms (Klerks and Weis,
1987; Klerks, 1990) and terrestrial plants (Antonovics
et al., 1981; Baker, 1987; Macnair, 1987; 1990). Up
to now, attention has particularly been focused on
plant species, which have been recognized as indi-
caters for the presence of metals since ancient times
(Ernst, 1974). Furthermore, classical adaptation
studies aimed to analyze pesticide tolerance in insects
for economic reasons (Georghiou, 1972; Roush,
1987). Invertebrate heavy-metal adaptation contrasts

11
12 L. POSTHUMA and N. M. VANSTRAALEN

with the evolution of pesticide tolerance, since almost a correlation study of pollution and population
all pesticides are xenobiotics that usually affect a characteristics at various sites. The occurrence of
single target site within the organism, whereas vari- heavy-metal adaptation is substantiated by corre-
ous metals act as micro-nutrients and potentially lations between tolerance characteristics of popu-
have many target sites. Moreover, heavy-metal adap- lations and local site pollution (Endler, 1986). The
tation may reduce adverse effects on ecosystem func- presence of such correlations makes random environ-
tioning, e.g. by counteracting metal-induced mental factors (such as humidity) or population
inhibition of litter breakdown (Tyler et al., 1989). processes (such as genetic drift) less plausible as a
Several mechanisms can contribute to the major explanation of the observations. Comparisons
resistance of an individual to a pollutant, viz. avoid- of field populations allow conclusions on the results
ance behaviour, detoxification (or sequestration), of selection, without assigning causes, i.e. conclusions
compartmentation, and excretion. A metal-tolerant on selection of characteristics (sensu Sober, 1984).
individual is able to prevent, decrease, or repair Selection for increased metal tolerance (sensu Sober,
effects of metals that have entered the body (Levitt, 1984) pertains only to populations where natural
1980), which bears on the latter three phenomena selection directly operated on tolerance. Subsequent
only. Behavioural avoidance occurs in terrestrial studies usually consist of extensive work on genetics,
invertebrates (Joosse et al., 1981; Russell et al., 1981; biochemistry and physiology of tolerance
Joosse and Verhoef, 1983; Tranvik and Eijsackers, development.
1989). It has, however, not been incorporated in the The importance of metal-adaptation studies is de-
present review, because changes of avoidance in termined by theoretical and practical considerations.
response to selection have not been studied. It de- From the theoretical point of view, firstly, local
serves further study, because avoidance is important adaptation indicates the presence of toxic effects at a
for stress evasion in animals (Hoffmann and Parsons, site. Secondly, the studies demonstrate the presence
1991). or absence of genetic variability of tolerance and
Since the review of Hopkin (1989), several cases of other features within a population. Genetic variabil-
heavy-metal adaptation in terrestrial invertebrates ity determines the potential for responses to future
have been reported. In the present review we aimed environmental variation, both from pollution events
to present theory and practice of research on heavy- and from natural origin. Thirdly, the studies indicate
metal adaptation, to summarize avail- the critical physiological pathways involved in metal
able literature data on the occurrence, genetics, tolerance. Fourthly, the studies may identify conse-
physiology and consequences of heavy-metal quences of strong directional selection for fitness, the
adaptation in natural populations of terrestrial so-called “costs of tolerance”: heavy-metal adap-
invertebrates, and to indicate gaps in knowledge. tation is expected to cause a reduction of fitness,
Gaps have been identified for some topics in metal- expressed by non-tolerance characteristics, compared
adaptation literature, namely for “costs of toler- to reference animals.
ance”, for toxicity parameters related to tolerance From the practical point of view, firstly, studies on
and adaptation, and for genetic variation related to pollutant adaptation are relatively easy to interpret in
the success or failure to evolve tolerance when metal comparison to evolutionary studies on natural
concentrations around pollution sources increase. selective agents, while the basic processes are similar.
Literature evidence is evaluated in view of the Secondly, there are implications of adaptation for
strength of the applied methods, and suggestions are regulatory decisions regarding maximum acceptable
made for further research. levels for pollutants, if risks of pollution at the
ecosystem level are extrapolated from toxicity data
1.2. Theory and practice of heavy-metal adaptation for a sample of test species (e.g. Van Straalen and
research Denneman, 1989). The validity of the extrapolation is
Heavy-metal adaptation of terrestrial plants is diminished in two ways, namely by incorporation of
considered to be one of the best examples of natural adapted populations in the phase of data-collection,
selection in action (Endler, 1986; Bradshaw et al., or by the assessment of actual risks at polluted sites.
1990; Brandon, 1991). Selection for metal tolerance is Adaptation, thirdly, renders a species unsuitable for
considered to be directional, continuous and strong, monitoring aims, since test species must form a
since metals are non-degradable substances, that are genetically uniform group that responds in a
toxic at high concentrations, and that accumulate in predictable (or known) manner to contaminants.
the organic layer of the soil. Metal-adaptation often Monitoring bias may result from altered assimilation
occurs quickly (Bradshaw and McNeilly, 1981), and or accumulation in special organs. Fourthly,
is expected to be determined, to a large extent, by adaptation in one species may affect other species
single major genes in contrast to adaptation to indirectly, since it may change exposure of species at
natural stressors (Macnair, 1991; Hoffmann and other trophic levels, which need not be adapted. Both
Parsons, 1991; Schat and Ten Bookum, 1992). positive and negative effects are possible, for example
Studies on metal-adaptation in field populations for a predator hunting prey adapted through reduced
have often started with a comparative approach, by assimilation, or through increased accumulation in
 Metal adaptation in invertebrates
compartments, respectively. The analysis of patterns
of such indirect effects of adaptation is outside the
scope of this review.
1.3. The evidence required
The principal problem in adaptation research is the
identification of the cause of tolerance: metal
tolerance can arise from individual exposure (acclim-
ation), and from natural selection (adaptation), or
both. Physiological acclimation implies that an
individual has acquired a degree of tolerance after
pre-exposure to a sub-lethal concentration in some
period of its life; tolerance is not passed on to
offspring by genetic means. Acclimation, therefore, is
a type of phenotypic plasticity, and acclimatory
tolerance may be induced and lost within a
generation. Genetic adaptation, however, implies
that a population has evolved an increased mean
tolerance through natural selection operating on
genetically based inter-individual variation in toler-
ance; tolerance is inherited in the offspring. Selection
may also operate on tolerance characteristics which
are phenotypically plastic, so that increased tolerance
will be expressed only upon exposure.
Tolerance present in offspring does not necessarily
imply adaptation as a female may influence the
tolerance of her offspring by maternal effects.
Maternal effects are pre- and post-natal influences of
a female parent on her young (Falconer, 1981). Such
influences can be accomplished by transfer of metals
through yolk, or by transfer of substances produced
after exposure (e.g. Munkittrick and Dixons, 1988).
Therefore, population comparison studies with
second- or later generation offspring are preferred
(Klerks and Levinton, 1989a). The term “acclimatiz-
ation” should be used if an individual adjusts
physiologically to an array of environmental factors,
for example acclimatization to laboratory conditions
(Hoffmann and Parsons, 1991).
The recognition of adaptation in natural
populations requires detailed knowledge of several
parameters. An “ideally complete adaptation
explanation” should consist of the following com-
ponents, which read [modified for metal tolerance
from Brandon (1991)]:
(1) evidence that selection for tolerance has
occurred in the past, i.e. that some types are better
adapted to the polluted environment than others, and
that this has resulted in different reproductive success
of the types: this criterion bears on the presence of
potential toxic effects;
(2) an ecological explanation of the fact that some
types are better adapted than others, i.e. why selec-
tion has occurred and on which characteristic it has
been operating: this criterion bears on relationships
with life-history characteristics, it does not necessarily
incorporate the question how tolerance is improved
physiologically;
13
(3) evidence that the characteristics involved in
divergence of tolerance are heritable: strong evidence
for a potential to adapt is present if genetic variation
prior to selection can be demonstrated or reasonably
extrapolated;
(4) information about the distribution of the
species in the polluted habitat and the surrounding
areas: patterns of adaptation result from both gene-
flow and selection; and
(5) phylogenetic information to enable distinction
between the original and the derived (adapted) state
of the characteristic, i.e. the direction of adaptation.
These criteria serve as a main line of reasoning in this
review. The criteria indicate that evidence should
consist of data from ecotoxicology (1) ecology
[life-history (2), and temporal and spatial distribution
pattern (4)], quantitative genetics (3) geology or
industrial history (45) and phylogenetic systematics
(5). The criteria are partly interconnected, for
example (2) presupposes (1). Several criteria require
extrapolation back in time [especially (1) and (5)] or
through comparisons with contemporary reference
sites [especially (3)], which in turn requires that the
selection factor presently observed is similar to past
or contemporary reference sites. Brandon (1991)
argues that the criteria have largely been taken into
account in evolutionary studies of metal-adaptation
in plants, so that present day plant research tends to
focus on physiology of tolerance and on the number
of genes controlling tolerance. Metal-tolerance in
terrestrial invertebrates, in contrast, has been studied
only in a few species, and few of Brandon’s criteria
have been explicitly mentioned; research interests
have focused particularly on the criteria (l), (2) and
(3) so that less attention is paid below to data on
(4) and (5). Ecophysiological data are, however,
already available (Hopkin, 1989; Dallinger, 1993)
and these may serve to identify the key physiological
mechanisms in the evolution of tolerance.
For the review, we have chosen to incorporate only
reports in which the presence or absence of adap-
tation within a species is explicitly discussed, or which
indicate an increased tolerance or altered metal-
physiology in an exposed population. We focused
particularly on the criteria which can be empirically
addressed, viz. the presence of toxic metal-concen-
trations (l), of life-history differences (2) and of
genetic variation for tolerance or other features (3).
The present and past distribution of the terrestrial
invertebrates at or near polluted sites (4) is often
badly or not documented, and exact data on
phylogeny (5) are lacking. Circumstantial evidence
for this criterion is treated with data on “costs of
tolerance” (see section 5.2).
2. THE SPECIES THAT SHOW ADAPTATION
Table 1 presents a summary of the literature data,
and our evaluation of evidence for physiological or
genetic changes, or both, of exposed populations. The
 Table 1. Occttrrence of heavy-“ieta adaptation in lenestial invertebrates, obtained fro”t reports ~o”st~ting divergence of physiology or tolerance between reference and exposed populations or strarns

Comparison type
-
Capture site history; Remarks
number of sites* Physiology TOlera”Ce Inheritance structureg Genetic Physiology on previous
Speciff Metal (criterion I) (criterion 2) (cnterion 2) (criterion 3) (criteria” 4) component component colum”s (7) Author

ProtouM
Paramecium prbrrawelia several (?): I4 (3 Survival Olkptlltg 0) Yes? Possible Special inheritance Nyberg and Bishop (1983)
Oligochaeta
cogrwia sphagfrerorum Pb Ore; I; Gradient (I&tad, N) (?) Medrum? Field Gradient Possible Possible Author’s catqory Hggvar and Abrahamsen (t990)
Eirenia f&da Cd (?); I (control site) Mt-BP/MTt~ 0 (?) (7) m Yes lnducable Suzukr et a/. ( 1980)
Lwnbricw n&l/w Pb Mine; 2 (U.K.) Accumulationt Cytotoxiciry Field Distant Possible Possible Compartmentalion Morgan and Morgan (1988)
Pb, Ca Mine; 4 (U.K.) Accum./excret. of Pbt Field Distant Possible Possible Compartmentalto” Morean and Morean (19901
&WA Mine; IO (U.K.) Accumulationt ;; Field Distant Possible Possible Compartmentalion CoGand Morgan(lYbl) ’
cd Mine: (Draethen.
I U.K.1 Ml-BP/MTt: (?) Field Distant (7) Yes Inducible Morgan er a/. (I 989)
Pb, Co Mine; (Cwmystwyth,
I U.K.) Accum./Energy reserve+ (?) Field Distant Possible Possible Glycogen affected Ireland and Richards (1977)
Lkndrodrilw rubi& Cd Mine; (Draethen,
I U.K.) Mt-BPt: (?) Field Distant (?) Yes Inducible Morgan er a/. (1989)
Lhmdrobaeno rubida Z”, cu Brass mill; 2 (Gusum, S) Ml-BP: Life-history Field Gradient Not likely+ Possible Low divergence Bengtsson er al. (1992)
Pb, Cu Mine: (Cwmystwyth,
I U.K.) Acc”m./Energy reservest (?) Field Distant Possible Possible Glycogen affected Ireland and Richards (1977)

Co Spraying; 2 lab. Strainst (3 Survival Yes (?) Adaptation? Possible A&i&I selection Sullivan et a/. (1984)li
Pb Car-par)t; I(London) Accum.iexcretion (7) Field Distant Likely? Possible No acclimation Beeby and Richmond (1987)
Pb. Ca Car-park; I(London) Accum./excretion of Pb (?) Field Distant Likely? Possible Ca metabolism Beeby and Richmond (1988)
Z”, Co, Cd Smelter; I (Avonmouth) Accumulation (?) Field Gradient Not No Tolerant gut flora Simkiss and Watkins (1991)
Z”, Mn Mine; I (Cwnystwyth, U.K.) Excretiont (7) Field Distant Possible Possible Zn/Mn in mww Ireland 979)
(I
Cd Mine: I 1Braubach. GI Ml-BP/MTt: (?) Field Distant Likely Ye5 Inducible Dallinger et al. (lY89)
Pb, Z&d Mine: I (Llantris& U.K.) Accumulation/storage (?) Field Distant Possible Possible Greville and Morgan (1991)
Pb, Zn/Cd Mine; I (Llantrisant, U.K.) ACCl”“ldatio”/StOr”ge (?) Field Distant Possible Possible Greville and Morgan (1991)

CU Mine; I (Cornwall, U.K.) ~umo~tio” (?f Lab-off3pr.t Distant Possible (1) Not sure Wiener (1961)
Zn Smelter; NL)
1(Budel, Accumulationt (7) Field Distant Possible Possible Z” “cccl Joosse er al. (1981. 1983)
Z”, Cd Smeller: (Bud& NL)
I Accum., Mt.BPt$ Life-history Field Distant Possible Possible Inducible; costs Van Capelleveen (1985, 1987)
Cd, co Mine; I (Braubach. G) Accumulation (7) (?) Distant Possible Possible Dallinger and Prosy (1988)
Several Smelter/Ore; 2 (Lludel, Plom) Energy reserves Survival Field Distant Possible Possible Donker (1992)
Cd Smeller/Ore; 2 (Budel, Plom) Accumulationt Growth Fl Distant Adaptation Possible Compartmentatio” Donker and Bogert (1991)
Cu/Zn/Cd Smelter/Ore; 2 (Budel, Plom) Ml-BP+: (7) Field Distant NO No Inducible Danker er II/, ( 1990)
Z” Smeller: I (BodeI. NLl Accum./excretion 0) Field, FI Distant Adaptation? Yes Compartm./excret. Donker er al. (1992)
Several Smeller; iBwJel, NL)
I 0) Phenology Field Distant Possible Possible Danker er a/. (I993a)
Several Smelter/Ore; 2 (Bodel, Piom) (7) Life-history FL FZ Distant Yes Possible Danker er d (i993b)
Several Ore/Smelter; 5 resp. (U.K.) 1 A~“m”lariont (?I Field/offspr. Distant Possible YeS Hopkin and Martin (1982, 1984a)
Several Mine; (U.K.)
I Energy reserves (?) Field Distant Possible Possible Morgan et o/. f 199+)

Several Smeller; I(Avonmoulh, U.K.) Survival Fteld Nearby Possible Possible Hopkin and Martin (1984b)
Arachnida
Boodibu deco/orarw AS Smwina:(?) _.. Mechamsm S”r%%,el Strain (?) Adaptation Possible WhItehead (1961)
S.outof 8tick species AS Spraying:(?) Mechanism Survival Strain (?) Adaptation Possible Matthewson and Baker (1975)
4 species Pb Ore: I: Gradient (Kastad. Ni (?I Lowf Field Gradient Not likely Possible Authors’ category HBzvar and Abrahamsen (19901
5 swcies Pb Oref Ii Gradient ?Kastad, Nj (?) Medrumt Field Gradient Possible Pomible Authors’ cate8ory H&r and Abrahamsen il99Oj
s&al Pb Ore: I; Gradient (Kastad. N) 17) High+ Field Gradient Possible Possible Authors’ category Hdgvar and Abrahamsen (1990)
Tectocepheur t&xur Several Smelter; (Bud& NL)
1 Allotymes (7) Yes Distant Possible (?) H. Siepel (pas. common.)
Othoptera
Chorthippus spec. Cd. Zn. Pb Industrial; 2 (P) Accumulatron Feedingt Field Distant Possible Yes Avoidance Migula and Bmkowska (1993)
 Table I (continued)

Field Gradient Not likely Not likely Author’s category HBgvar and Abrahamsen (1990)
Pb Ore; I: Gradient (Kastad, N) (3 Louq Field Distant Possible Possible Nottrot er a/. (1987)
Fe. Mn Blast-furnace; I (NL) Accum./Excretion Moultine Field Distant Likely Not likely Van Straalen et a/. (1987)
Pb. Cd Ore/Smelter: 4-5 (NL,B,G)** Excretion (?) - Fl Distant Adaptation Possible Posthuma (1990)
Cd 0re;Smelter 6 (NL,B.G)*’ (?I Growth Fl Distant Adaptation Yes? Zn burden Posthuma e, al. (1992a)
Cd, Zn, Pb Ore/Smelter 6 (NL.9.G)” Excretion Growth Heritabibty Distant Yes§t Possible Decrease Heritability Posthuma el al. (1993a)
Cd SII& (stdb&, Gj ’ Excretion (?) FI Distant Adaptationt Possible Survival costs Posthuma et al. (1993b)
Cd Ore/Smelter 6 (NL.9.G)” (7) Life-history Heritabilities Distant Posthuma and Janssen (1993)
2 rcferenoe sites U) LifehIstory Field, F3 Dist./nearby Yes (?, Correlation Frati el al. (1992)
SeV%Il Ore/Smelter; 6 (NL.B,G,I)*’ Allozymest Growth? Field Gradient Not (?) Due to migration Tranvik er i/. (1992)
Orchesellabifascbfa cu. Zn Brass mill; I (Gusurn, S) Alloqmes/proteins (?) Field Gradient Possiblet Possible Not for Pb Bengtsson and Rundgren (1988)
Onychiurus armatu.v Cu. Pb Brass mill; I (Gusum, S) Accumulation (?) Field Gradient Possible Possible Author’s category H&par and Abrahamsen (1990)
Pb Ore; I; Gradient (Kastad. N) C) High? FI Gradient Adaptation Possible Tranvik er d. (1993)
cu. Zn Brass mill; I (Gusum, S) 0) Life-history FI Gradient Adaptation Possible Tranvik P, al. (1993)
1soromo Nombilir cu. Zn Brass mill; I (Gusurn, S) (3 Life-history Field Gradient Notlikely Possible Author’s category HIgvar and Abrahamsen (1990)
Pb ore; I; Gradient (Kastad. N) Lowt Field Gradlent Not likely Possible Author’s category Hagvar and Abrahamsen (1990)
5 species Pb Ore; I; Gradient (Kastad. N) 0) Lwt Field Gradient Possible Possible Author’s category H&par and Abrahamsen (1990)
2 swcies Pb On; I; Gradient (Kastad. N) (3 High?
Hemipt& f?) (2 Possiblet Possible Co-evolution Ernst er al. (1990)
Brochvcmuhu lvcbnidiv Several Mine; I (Blankenrode, G) Accumulation
Lepid&ra . 0) (?) Possible Powble Ernst er al. (1990). pen. commu”.
Plualla maculipennis Several. Zn Mine; I (Blankenrode, G) 0) Presence
Diptera Strain (2 Adaptation Possible Blackman and Baker (1975)
Lucilia cuprina As Spwing; (7) 0) Survival Yes Gradient Yes (?) Lower (1975)
Drosophilrr mdaNogvrrer Several Smelter: I (Bixbv. U.S.A.) Allozymes (?) Yes YCS Possible Nassar (1976)
Pb Labor&~; 4 &ins 0) Ufe-history Yes Yest Possible Genotype tests Chapco el al. (1978)
Zn, Se Laboratory; 4 strains (7) Reproduction Yes (9 Yesf Possible Artificial selection Wallace (1982)
C” Laboratory strains (?) Survival Yes 0 Yes Possible Christie et al. (1983, 1985)
SWed strains; 3 Accumulation Survival Yes 0 Yest Possible Artificial selection Magnusson and Ramel(l986)
Hg Strains; several (?) Survival OffSOti”e. Distant Responset Possible Survival costs BaJraktari e, a/. (1987% b)
SWeId Industrial: I (Y) 0 Life-history Yes’ - Distant Y& Possible Maroni et al. (1987)
cu. Gi Global; ma.; Mt-BP/MT?: Survival Yes C) Response Possible Glycogen Lauvejat et al. (1989)
cd Laboratory; 2 strains Gram&i/energy rerrvet (?) Yes (?) Yes Possible 2 MTs Gill a al. (1989)
cd Laboratory; 2 strains Ml-BP/MTtf Survival
Coleoptera Field Gradient Possible Possible Read e, al. (1987)
Nebrio braricollis !kVEd Smelter; I (Avonmouth, U.K.) (?) Diapause Field Distant Not likely Not likely Haefelfinger (1991)
SubcocciMI1I124-pwcmln cd, Zn Mine plant; I (Pedrozzio,
I) Uptake Life-history Field - (?) (7) Haefeltinger (1991)
_._
cassidll (IZ”IP(I cd. Zn Reference; I Uptake Llte-t”story

Column I: the species or genus names.

Column 2: the metals used in the studies; these are not necessarily similar to the combination of metals in the field.
Column 3: the capture sites of exposed populations, the number of investigated sites (excluding the reference site) and the types of field study (gradient. or independent nearby or distant sites), these data are used to evaluate criterion I.
Columns 4 and 5: the type of population comparisons made: both factors causal to tolerance (“physmlogy”) and performance resulting from some (unknown) mechanism (“tolerance”) have been studied. “Physiology” comprised metal-binding proteins (Mt-BP),
aaumulation and excretion patterns. energy reserves, wmpartmentation in body- or cell. and probably allozymes. “tolerance” comprised three classes of tolerance at a small long-term polluted site (H&a and Abraham%% 1990). and of performance
of cell damage, survival, growth, and reproduction during exposure (criterion 2, particularly life-history data).
Column 6: informalton on inheritance: laboratory strains of unknown origin, field captured animals or their offspring (Fl) have been compared (critenon 3).
Column 7: information on the field situation, distinguishing separate populations (distant or nearby) with probably low inter-dispersal, and gradient studies (criterion 4).
Column 8: our evaluation on the presena of a genetic component in population diKerentiation: “adaptation” indicates sufficient evidence, “yes” indicates genetic elTects without additional evidence for criteria I, 2,4, and 5; “likely” indicates that acclimation
is not likely to be the cause of divergence.
Column 9: our evaluation on the presence of a physiological component in population ditTerentiation.
Column IO: tlndicates that a further specification of a remark in a previous column IS given here.
Column I I literature reference.
:
Evidence was judged with Brandon’s (1991) criteria, which were modified to mcorparate circumstantial evidence. The evidence is summarized for each study: “genetic component” to indicate whether differences were. at least partly, inherited, and “physiology
component” to indicate whether etTects of individual exposure could influence differences. “Adaptation” was used for cases where both inheritance and a reasonable ecological explanation are gjven. (9 = Belgi’um; G = Germany; I = Italy; N = Noway;
NL = The Netherlands; P = Poland; S = Sweden; U K. = United Knngdom; U.S.A. = United States of America; Y = Yugoslavia). tMetal binding protein or metallothionem; *The histones of the numbers of polluted site are given (sometimes there are
several reference sites); &XI maternal etTect; in Klerks and Weis (1987); lldistance to nearest reference site; **includes Budel (NL), Stolberg (G). Plombieres (9).
l/not

.- -...“.I.._.., _.._.-^._... . ._._ _.“._... . _““.“._ __..”

..,._ .~il._lx_.-..i~~~.~-,~l-~.~.~~-.-”~”-.-‘-”
. I.“I
16 L. POSTHUMA and
reasoning for our evaluation of the evidence is
elaborated in section 3.
Aspects of heavy-metal adaptation have been
studied in Protozoa, and in representatives of
Oligochaeta, Mollusca, Crustacea, Myriapoda,
Arachnida, Apterygota, Orthoptera, Hemiptera,
Lepidoptera, Diptera and Coleoptera (Table 1,
column 1); most of the studies are recent (Table 1,
column 11).
Genetic divergence for metal tolerance, particularly
copper, was found in the protozoan Paramecium
primaurelia between 26 stocks from 14 locations
(Nyberg and Bishop, 1983). Tolerance divergence
among sublines of the same genotype was also
observed (Nyberg and Bogar, 1986). Such sub-geno-
typic variation does, however, not occur in the other
species studied, and specific details are further
omitted here.
In earthworms, conclusive evidence to demonstrate
a response to selection is absent. Transplant
experiments with field captured Lumbricus rubellus
demonstrated differences in accumulation patterns
for cadmium and zinc, and sensitivity divergence for
lead (Morgan and Morgan, 1988). Morgan et al.
(1989) demonstrated that L. rubeilus and Dendro-
drilus rubidus from a polluted site were able to
synthesize cadmium-binding proteins. Suzuki et al.
(1980) and Yamamura et al. (1981) demonstrated
that there is phenotypic plasticity for metallothionein
production in Eisenia fitida, i.e. they are produced
upon exposure. Metal-binding proteins were also
found by Bengtsson et al. (1992) for Dendrobaena
rubida; there was neither divergence of these proteins,
nor of sensitivity differences between exposed and
reference worms near the Gusum (S) brass mill. The
lack of evidence for adaptation in terrestrial annelids
contrasts with the occurrence of adaptation in the
aquatic oligochaete Limnodrilus hoffmeisteri (Klerks
and Levinton, 1989b), and in two other aquatic worm
species, Nereis diversicolor and Nephtys hombergi (see
Klerks and Weis, 1987). Indirect adaptation through
calcium regulation, affecting the regulation of toxic
metals is suggested for Lumbricus rubellus (Morgan
and Morgan, 1990).
In the terrestrial mollusc Helix aspersa, inter-
population differences have been observed for
accumulation patterns of lead in animals captured in
a lead polluted car-park, compared to reference
animals (Beeby and Richmond, 1987; 1988). The
inheritance of the phenomenon and its relationship
to improved ecological function remain to be
established. Dallinger et al. (1988) compared metal-
binding proteins in field captured Arion lusitanicus.
Reference animals produced metal inducible proteins
of 2O,O~D, whereas the proteins in field-exposed
animals were 10,000 D; it is not clear whether a
similar pattern would be observed in long-term ex-
posed reference animals. In Helix aspersa, the pres-
ence of metal-tolerant gut-flora at a polluted site
determined population differences in zinc uptake, and
N. M. VAN STRAALEN
0.01 0.10 1.00
Cd @no& food)
Fig. 1. Example of an approach to find evolutionary effects
of heavy metals in natural populations: comparisons of
laboratory offspring from populations of different sites,
reared on clean or polluted substrate. Dose-effect relation-
ships for body growth of Pore&o scuber, exposed for 3
weeks to cadmium, in first generation laboratory animals
from a reference site (open circles), a zinc smelter site (black
triangles) and a former mining site (black squares), the latter
two contaminated with cadmium. Dose-effect relationships
differ significantly among populations: populations are sen-
sitive, Iess sensitive, or indifferent to cadmium exposure,
respectively. Bars indicate standard errors. (Modified from
Donker and Bogert, 1991; reproduced with permission of
M. H. Donker.)
adaptation was considered to be absent (Simkiss and
Watkins, 1991). Greville and Morgan (1991) demon-
strated differences in metal uptake patterns in trans-
plant experiments with Arion subfucus and Deroceras
reticulatum, but these may be related to adaptation,
acclimation, or other soil factors. For aquatic mol-
lusts, increased tolerance has been demonstrated by
comparisons of toxicological parameters in several
species; Klerks and Weis (1987) reported for Serobic-
u/aria plana convincing evidence for adaptation,
while Sullivan et al. (1984) demonstrated a doubling
of copper tolerance upon artificial selection in the
aquatic snail Biomphalaria glabrata (Pulmonata).
Inherited tolerance has been demonstrated for
terrestrial crustaceans, particularly the terrestrial
isopod For~e~lio scaber (Fig. 1). Apparently, genetic
variation for tolerance is present in reference popu-
lations of this species. Inter-population differences
consist of modified regulation, storage and excretion
of metals, and life-history changes, which may have
influenced energy reserves of exposed animals
(Donker, 1992; Donker and Bogert, 1991; Donker
et al,, 1992; 1993a; 1993b). Similar results were found
previously for field animals (Van Capelleveen, 1987),
for which, moreover, differences in bioconcentration
factors were found (Joosse et al., 1981; 1983;
Dallinger and Prosi, 1988). Hopkin and Martin
(1982) demonstrated that the size of the hepatopan-
cress of the isopod Oniscw belly from two contami-
nated sites was high compared to that of reference
animals, indicating an enlarged sequestration
capacity. However, Hopkin and Martin (1984a)
showed that accumulation patterns in a controlled
experiment with iaboratory reared offspring were
 Metal adaptation in invertebrates
similar, indicating that possible divergence in this
species cannot be attributed to sequestration capacity
per unit of time. A change of energy reserves was also
found in field captured Oniscus asellus (Morgan et al.,
1990). Inducible metal-binding proteins in hepato-
pancreas and haemolymph of the isopod Porcellio
scaber were similar in animals from polluted sites
and a reference site (Donker et al., 1990). Adap-
tation studies in aquatic arthropods are restricted to
crustaceans and to the midge Chironomus tentans,
an insect species with an aquatic larval stage (Klerks
and Weis, 1987). Genetic components of change of
metal tolerance were likely for several crustaceans,
including the aquatic isopod Asellus meridianus.
For the following taxa, only representatives of the
terrestrial environment have been studied. Large
differences in sensitivity were observed between popu-
lations of the centipede Lithobius variegatus after
individual exposure (Hopkin and Martin, 1984b).
The presence of genetic differences in this species
for metal-sequestration capacity remains to be
established.
Several species of arachnids (ticks, mites and
spiders) have been studied. Strong evidence for the
occurrence of adaptation is present for adaptation of
the tick BoophiIus decoloratus to arsenic-containing
pesticides (Thompson and Johnston, 1958; Haring-
ton, 1959; Whitehead, 1961); inherited tolerance was
also reported for five out of eight other tick species
(Matthewson and Baker, 1975). In mites, sensitivity
differences between species have been found, and the
presence of some mite species in the centre of a site
naturally polluted by surface lead ore effluent
[Kastad, Norway: Hagvar and Abrahamsen (1990)]
may be an indication for the possibility for adap-
tation in some of them (particularly some Meso-
stigmata and Gamasina). Siepel (pers. commun.) has
demonstrated low genetic variation for the usually
variable esterase locus in an exposed field population
of the parthenogenetic mite, Tectocepheus velatus.
Apparently, some resistant clones increased in
frequency at the polluted site.
Among Orthoptera, metal accumulation differed
between field populations, but inheritance has not
been studied (Migula and Binkowska, 1993).
Adaptation has conclusively been demonstrated in
Apterygota, particularly in Collembola; Protura are
considered to be a lead sensitive group (Hagvar and
Abrahamsen, 1990). Adaptation seemed to be species
rather than family dependent, since some species of
a single family are absent from the naturally polluted
ore effluent site, whereas others have colonized it
successfully (Hagvar and Abrahamsen, 1990). Popu-
lation comparisons in the springtail Orchesella cincta
revealed evolutionary changes for cadmium excretion
(Posthuma et al., 1992) cadmium tolerance
(Posthuma, 1990), the equilibrium concentration of
zinc in the body (Posthuma et al., 1992) and life
history (Posthuma et a!., 1993b); there was no
evidence for increased zinc tolerance, when judged by
17
body growth during exposure. The difference between
adaptation to cadmium and zinc may be related to
the fact that zinc is a micronutrient, which is
regulated at slightly different levels in reference and
adapted animals. Life-history divergence was also
demonstrated in the springtails Onychiurus armatus
and Isotoma notabiks (Tranvik et al., 1993; Fig. 2).
The latter two species are parthenogenetic, and the
increased mean tolerance probably resulted from the
increase of the frequency of tolerant clones.
The potential of a continuously variable character-
istic to respond to selection is determined by its
genetic variation, expressed as the heritability, h* (see
for further explanation: Fig. 3 and Section 3.3). This
parameter has been quantified for various tolerance
and life-history characteristics in 0. cincta. The
presence of genetic variation has been established for
Cd excretion efficiency (Posthuma et al., 1993a;
Fig. 3) growth rate (h* k SE = 0.75 k 0.56) moult-
ing frequency (0.46 + 0.31) and age at maturity
(0.36 f 0.24) (all P < 0.05) (Posthuma and Janssen,
in press). The amount of genetic variation, and the
presence of adapted populations at industrially
contaminated sites, demonstrate that adaptation in
0. cincta may occur within years or decades. Non-
genetic maternal effects did not contribute to
adaptation through cadmium excretion (Posthuma
et al., 1993a); negative maternal effects have, how-
ever, been found for age at maturity (Janssen et al.,
1988), but this apparently did not affect the response
of field populations. In 0. cincta, the presence of
genetic variation allows for an evolutionary response.
Therefore, the apparent absence of adaptation at low
and recently polluted sites (Breinigerberg, Germany,
and Mook, The Netherlands) can be caused by low
100 I
BG
0.16M.76 l&7.6 16+76
Exposure concentration Cu+Zn
(pmoI/g soil)
Fig. 2. Example of life-history adaptation associated with
metal exposure, and of evolutionary effects in a partheno-
genetic species. The springtail Zsotoma notabilisshows an
increased reproductive output in a population from a site
polluted by a brass mill since 1968 (black circles) compared
to reference animals (white circles), both in control and
exposed conditions (bars indicate standard deviations).
Characteristics were measured in first generation laboratory
animals. BG indicates background concentration treatment.
(Data from Tranvik er al., 1993; reproduced with permission
from L. Tranvik.)
 L. POSTHUMA and N. M. VAN STRAALEN

oxaloacetate transaminase (Got) and cadmium toler-

ance in the springtail Orchesella cincta from various
polluted sites (Frati et al., 1992; Fig. 4). In
0. bifasciata, however, such correlations were absent
for the frequencies of alleles of two enzymes (Pgi and
Pgm) in relation to metal concentrations in a gradient
around the Gusum brass mill (Tranvik et al., 1992).
The role of specific Got-alleles in metal tolerance in
0. cincta, however, remains to be established.
Among Hemiptera there is evidence for the
lead smelter occurrence of co-evolution of mine-plants and the
phloem-sucking aphid Brachycaudus lychnidis feeding
on them (Ernst et al., 1990), but additional ecological
and genetic parameters are necessary to demonstrate
adaptation. This also holds for the lepidopteran
Plutella maculipennis, which feeds on extremely metal
30
burdened leaves and stems of Cardaminopsis halleri
in a mining area (W. H. 0. Ernst, pers. commun.).

10 30 50 70 0.0

Excretion efficiency male parent (%)

0.6
Fig. 3. Example of an approach to study genetic variation
for tolerance characteristics. Offspring-father regression
analyses for cadmium excretion efficiency in the springtail
Orchesellu cincta: genetic variation (the heritability h2) is OA
calculated as twice the slope of the regression line of
offspring on father (Falconer, 1981); axes indicate gener-
ation means in the experiment groups. If the slope is larger
than zero, selection for tolerance in the parent generation 0.2

results in increased tolerance in the offspring; if the slope

reduces to zero, then the speed of evolutionary change is
also reduced, until a heritability of zero. In that case, even
strong selection in the parent generation will not cause a
g 0.6
response of the population mean in the next generation (see
further: 3.3). In the reference population (top) genetic
variation is present (the heritability is 0.35, P < 0.001); in a f 04
highly exposed population from Stolberg (bottom), genetic
variation could not be demonstrated (the estimate is 0.03,
P>>O.O5). Regression on male parents is not biased by
0.2
maternal effects (from: Posthuma et al., 1993a.)

0.06
exposure, by the presence of some threshold value
or
below which a response to selection cannot be distin-
0.04
guished from phenotypic plasticity.
Bengtsson and Rundgren (1988) found that
0.02
Onychiurus armatus captured near the new brass mill
in Gusum (S) contained a relatively low body concen-
tration of copper, but not of lead, compared to 0.00

reference animals. The inheritance of the phenom- -15 -1.0 -05 0.0
enon has not been established.
Index of Growth Reduction
Hagvar and Abrahamsen (1990) distinguished
collembolan sensitivity classes at the lead-polluted Fig. 4. Example of an approach to analyse effects on
Kastad site: “sensitives” were Mesaphorura yosii, allozyme variation. Both tolerance (Index of Growth
Reduction, see Fig. 6) and allozyme frequencies were
Folsomia sensibilis, Isotoma notabilis, Isotomiella
measured in natural populations of the springtail Orchesellu
minor, Anurida pygmaea and Willemia anophthalma. cincra with different histories of metal exposure. Frequencies
“Tolerants” were Folsomia quadrioculata, Onychiurus of alleles of glutamate-oxaloacetate transaminase (Got) are
armatus and Friesea mirabilis. It is interesting to note correlated with tolerance. Alleles were designated A, B, and
that 0. armatus has also been marked as a “life- C; allele A was the most frequent morph; it was positively
correlated with population mean tolerance (r = 0.91,
history adapter” (see above). P < 0.01); B and C were less frequent, and frequencies were
Analyses of 22 allozymes revealed a correlation negatively correlated with tolerance (r = -0.90, and
between the frequency of alleles for glutamate- r = -0.94, resp., both P < 0.01) (from: Frati et al., 1992).
 Metal adaptation in invertebrates
Metal adaptation has been demonstrated for
natural populations of the dipteran Drosophila
melanogaster, and could also be induced in labora-
tory strains. Adaptation involved, at least partly, the
duplication of the metallothionein gene Mtn (Maroni
et al., 1987). Genetic divergence among sites or
strains was demonstrated with various other tech-
niques by several independent authors, e.g. through
selection experiments (Nassar, 1976; Magnusson and
Ramel, 1986; Otto et al., 1986; Fig. S), through the
change of allozyme patterns along a gradient (Lower,
1975) and through the comparisons of genotypes in
relation to exposure (Chapco et al., 1987; Lauverjat
et al., 1989). Some data are not readily applicable to
field populations, since origin or composition of the
base strains was not documented. Life-history
divergence was demonstrated in laboratory reared
offspring from a polluted site (Bajraktari et al.,
1987b), and artificial selection for 25 generations with
tetraethyllead induced an increased performance of
fecundity, proportion hatching and viability (Nassar,
1979). The latter finding contrasts with the findings
from Bajraktari et al. (1987a), indicating an increased
genetic load, expressed by increased mortality, in
offspring from exposed field populations. Adaptation
in fruit flies seemed to be metal-specific, probably due
to metal-specific metallothionein induction. The
dipteran Lucilia cuprina, moreover, appeared to
adapt to pesticides containing arsenic (Blackman and
Baker, 1975).
For Coleopteran species, clear evidence for adap-
tation was lacking. Life-history changes were found
in the species Nebria brevicollis along a gradient
around a smelter, but adaptation could not be distin-
guished from acclimation or toxic effects (Read et al.,
0 2 4 6 8 10
Methylmercury in substrate (m&l)
Fig. 5. Example of artificial selection causing an increase
in metal tolerance. Change of dose-effect relationships
for tolerance to methylmercury in a composite strain orig-
inally composed of four wild-type strains of Drosophila
melanogaster. Doseeffect relationships for hatching success
are shown after three (circles), seven (triangles) and twelve
(squares) generations rearing on methylmercury in the sub-
strate. The toxicity parameter ECSO (the concentration
ing 50% reduction of performance) increased from c. 1.8 to
c. 6ma/l (from Mannusson and Ramel. 1986: renroduced
II .
with permission of J. Magnusson). 1 however, body concentrations
19
1987). Haefelfinger (1991) did not find divergence of
feeding rates between field populations of Subcoc-
cinefla 24-punctata fed with metal contaminated
leaves.
3. METHODS AND EVIDENCE EVALUATED
3.1. Circumstantial evidence .for past selection by
metals
The first criterion of Brandon (1991) requires
ecotoxicological evidence for past selection as the
cause of tolerance. Past selection has not been
demonstrated in natural animal populations. Circum-
stantial evidence for past selection can be obtained
from indications for present-day selection (3.1. I),
from the presence of adapted plant populations
(3.1.2) and through the correlation between site and
population characteristics in the comparative
approach (3.3.3). Within this approach, various
methods have been applied, revealing evidence for
adaptation of different strength.
3.1.1. Present-day selection. The presence of an
actual selection force can be used to assess the
strength of selection in the past. A present-day selec-
tion force at a field site can be assessed from
dose-effect studies. A concentration exceeding the No
Effect Concentration (NEC) for some relevant
characteristic, by definition, causes adverse effects.
Due to inter-individual differences in sensitivity,
exposure of a population beyond the NEC implies
selection, promoting the performance of the most
tolerant individuals. The hypothesis that adaptation
may occur in populations where a NEC is exceeded
is supported by the occurrence of adaptation in
various populations for which at least the NOEC (No
Observed Effect Concentration) for one metal was
exceeded (Table 2), and the absence of divergence in
opposite cases. In some cases, it is possible to identify
which metal, or which combination of metals, may
have contributed to the selection response. The
evolution of co-tolerance for a metal of minor
importance may occur. Exceptional is Dendrobaena
rubida, for which the NOEC for copper was exceeded,
and which was not adapted, but which was absent
from the most polluted site. A second exception is
Onychiurus armatus, which showed life-history adap-
tation at a site with no known exceeded NOEC. Zinc
pollution may, however, exceed the species’ NOEC
for this metal; this illustrates that the observations are
indicative rather than conclusive.
Data on the bodv concentrations of exnosed field
animals, compared to body concentrations in artifi-
cially exposed animals of similar origin (offspring
generation), may indicate the relevance of laboratory
toxicity data for the field situation. If exposure to
similar concentrations in the field and laboratorv ,
causes similar body concentrations, then toxicity in
caus-
the field can reasonably be extrapolated from labora-
tory dose-effect relationships. In Orchesella cincta,
of field animals were
20 L. POSTHUMA and N. M. VAN STRAALEN

‘Table 2. Overview of relative sensitivities of species to metals in the food, expressed as the No Observed Effect Concentration
(NOECs) for
reproduction characteristics (from: Van Straalen, 1993a), and site pollution data (total metal concentrations in substrate), collected from
literature of Table I, at various sampling sites; additional literature source for Helix aspwsa: Beeby and Eaves (1983)
Metal concentrations (fimo1.g“) Genetic
Cd Zn Pb CU component
~-..lll.. - “.._. Table 1
Species Site NOEC Soil NOEC Soil NOEC Soil NOEC Soil (column 8)
Dendrobaena rubida Gusum (S)* 0.89 0.03 n.d. 29.8 2.70 1.4 1.9 23.5 Not likely?
Helix aspma London(UK) 0.09 0.05 n.d. 10.0 n.d. 0.4 n.d. n.d. Likely
Arion Iusiranicus Braubach (G) n.d. 0.40 1.5 10.6 n.d. n.d. n.d. n.d. Likely
Porcellio scaber Budel (NL) 0.09 0.04 6.1 20.5 n.d. 1.7 n.d. 0.8 Likelyf
Plombi&es (B) 0.09 0.53 6.1 90.1 n.d. 67.5 n.d. 3.5 Yes
OrcheseNa cincra$ Breinigerberg (G) 0.04 0.03 n.d. 2.6 n.d. 3.9 n.d. 1.0 Not likely
Bud.4 (NL) 0.04 0.05 n.d. 14.5 n.d. 1.5 n.d. 0.7 Likely%
Ptombi&res (B) 0.04 0.24 n.d. 75.2 n.d. 54.9 n.d. 2.7 Yes
Stolberg (G) 0.04 0.58 n.d. 23.9 n.d. 41.1 n.d. 20.2 Yes
Onychiurus amtatw Gusum (S)* n.d. 0.03 n.d. 29.8 5.29 I.4 41.1 23.5 Yes
Kastad (N) n.d. n.d. n.d. n.d. 5.29 >loo 41.1 n.d. Likely
Bold values indicate that the NOEC for the metal has been exceeded at the site. The conclusions of Table 1are summarized in the last column,
to enable comparisons of the ocurrence of adaptation to the concentration data.
*Two brass mills; sampling site for observation in the most polluted old brass mill soil.
Qndicates absence of the species at the site nearest to the brass mill, interpreted as not-adapted.
fAuthors indicated that adaptation at this site is less than in higher polluted sites.
$NOEC for body growth is given.

much lower than expected from site pollution
(Posthuma, 1990). The assessment of selection from
NGEC data and site pollution is, therefore, indica-
tive, since substrate characteristics and combination
toxicity may influence actual exposure and toxicity by
an order of magnitude.
3.1.2. Association of animals with metal ffora.
Adapted plant populations near adapted animals
have been reported for a zinc smelter site in The
Netherlands, Budel [Porc&o sea&r and probably
Orchesella cincta (Table t) near Agrostis ten& and
Moeiiniu caerulea (Dueck et al., 1984)], for an
abandoned lead/zinc mine in Belgium, Plombieres
[P. scuber and 0. cincta near a Viola caIaminariae
association (Simon, 197.5; 1977)], and for a lead
smelter site in Germany, Stolberg [O. c&eta near a
Viola ~aIami~ariae association (Posthuma et al.,
1993a)]. Adapted plant populations at a former
mining area in Germany, Breinigerberg, are spatially
separated from the investigated non-adapted spring-
tail population, which is exposed to low metal
concentrations. For other sites no data are available
on the association between animal adaptation and
metal flora. These observations indicate that metal-
adaptation in plants is often accompanied by metal-
adaptation in animals, and that, apparently,
avoidance behaviour has not precluded the occur-
rence of adaptation in animals. The suggestion of
Ernst et al. (1990) that coevolution between metal
adapted plants and their herbivores may occur is of
particular interest, and needs further investigation.
3.1.3. The correlation between site and population
churacter~st~cs. The ~puIation comparisons ap-
proach has been applied with only two populations in
most cases (Table I, cotumn 3). At least three popu-
lations are needed, however, to estimate correlation
between tolerance characteristics and site pollution.
Real replication, i.e. replicated sampling of sites with
similar pollution, should be applied to allow for
identitication of environmental factors causing devi-
ations from the correlation (Eberhardt and Thomas,
1991).
Within the comparisons-approach, reference
populations under investigation were studied either at
distant sites or far away in a gradient around a point
source (see Table 1). In the gradient approach the
contribution of environmental factors disturbing the
correlation is minimum, so that metals can be ident-
ified as the main selective agents compared to the
“distant-site” approach. The strongest evidence for
metals as selective agents, in the gradient approach,
is obtained from real replication of gradient
sampling, at various polluted sites.
An additional dichotomy of methods is associated
with the experimental exposure, namely reciprocal
transplantation or artificial exposure. In the latter
case, high metal concentrations are usually applied,
to induce toxic effects within the experiment time-
span (e.g. Posthuma, 1990). The applied concen-
tration need not be relevant for field conditions, in
contrast to the transplant approach. The transplant-
approach may reveal field-relevant population
divergence (e.g. Tranvik et af., 1992), but phenotypic
variability is often large compared to tolerance diver-
gence, especially when time, space or equipment are
limiting. To avoid wrong interpretations, the species’
sensitivity in relation to site pollution can be used as
a guide to choose the experiment approach:
“sensitive” species allow a transplant approach, and
inherently “tolerant” species require artificially high
exposure (see Table 2).
In conclusion, the intensity of present selection at
a field site has not been quantified for any terrestrial
animal species. Crucial to the assessment of past or
present selection is a proper experimental approach,
taking into account site pollution, some measure of
actual exposure of field animals, disturbing factors,
and species sensitivity characteristics. We believe that
the study of actual exposure or “bioavaiIability” in
the field on the one hand, and of dose-effect
 Metal adaptation in invertebrates
relationships, including estimation of toxicological
parameters, such as EC&,.NOEC, LC,, or NOLC, on
the other hand, is crucial to assess the ecotoxicity-side
of adaptation processes at polluted sites.
3.2. Life-history changes accompanying tolerance
The second criterion of Brandon addresses the
question why some types are better adapted than
others. This requires measurement of ecologically
relevant parameters, such as survival, reproduction
and growth. Selection for metal tolerance is expected
to improve fitness in exposed conditions, to be
expressed by improved performance of these
characteristics when measured in exposed conditions,
compared to exposed sensitive conspecifics. The
evolutionary response, i.e. tolerance, is the direct
cause of the improvements, and the fitness character-
istics are indirectly affected. In the springtail
Orchesella cincta, for example, tolerance is accom-
plished by improved immobilization and excretion of
cadmium, and maintenance of growth during
exposure is the indirect effect (Fig. 6). Other mechan-
isms underlying tolerance are metal-binding proteins,
altered accumulation patterns, conservation of
energy and other resources, increased excretion or
immobilization in granula, and possibly alleles coding
for proteins with decreased sensitivity for metal ions
in the cell [see Table 1; Klerks (1990)].
There are, however, three alternative explanations
for altered performance of fitness or life-history
characteristics. Their distinction is important, be-
cause one alternative has mainly positive implications
(3.2.1), while the other two are expected to have
negative implications (3.2.2, 3.2.3). These alternatives
will be illustrated mainly by springtail data (3.2.4),
since both genetic data and population characteristics
are available in this case; other species are presented
thereafter.
0 exhibited a decreased metabolic rate and slow
lcadadtcr~ ,
3 251
-2.0 -15 -1.0 -05 0.0
Relative Index of Growth Reduction
Fig. 6.
effects in metal tolerance. In a comparison of four popu-
lations of the springtail Orchesellacincta, the reduction of
body growth during cadmium exposure is associated with
the capacity to excrete cadmium: a high excretion efficiency
is considered to be the main determinant of low growth
reduction (a Relative Index of Growth Reduction of “zero”
indicates absence of toxic effects, negative values indicate
toxic effects (from: Posthuma et al., 1993a)).
21
3.2.1. Selection for life-history characteristics.
Improved performance is expected as a response to
direct selection for characteristics associated with
fitness (Posthuma et al., 1993b). Life-history theory
is concerned with evolutionary responses to
environmental factors that affect life-history
characteristics, such as development time and clutch
size. It usually deals with the effects of selection forces
from natural origin, rather than of pollutants.
Responses of life-history characteristics are, however,
to be expected from any factor that affects
age-specific survival or reproduction (Michod, 1979;
Charlesworth, 1980; Stearns, 1992) including pollu-
tants. Reduced adult survival and reproductive
allocation is expected to induce earlier maturation
and increased reproductive allocation; the opposite is
true for increased adult survival and reproductive
allocation. In soil, heavy-metal exposure is chronic,
and toxic effects consist of impairment of growth and
reproduction, and mortality (see, e.g. Joosse and
Verhoef, 1983; Bengtsson et al., 1983; 1985a).
Toxic effects will increase with age in exposed field
animals, because metals often accumulate at a rate
comparable to development rate (Janssen et al.,
1991). For metal-exposed field populations, theory
thus predicts that the evolutionary response to
chronic exposure to toxic metals will consist of earlier
maturation and an increased reproductive allocation.
The prediction from life-history theory only holds if
the modification can be achieved by the population in
the conditions of the polluted environment. The
distribution of energy over tolerance and other
functions is of central importance (Sibly and Calow,
1989), and may constrain the array of possible
responses. Crucial for any evolutionary response is
also the presence of genetic variation for the charac-
teristics involved. Various life-history changes have
been observed in pollution-exposed populations,
probably due to such constraints. In fact, it has been
demonstrated that some metal-tolerant populations
growth, to conserve energy or other resources
(Antonovics et al., 1981), and this may apply to
tolerant isopods (Donker, 1992). Expectations, more-
over, are different from the above if a pollutant
specifically affects juvenile life-stages. Predictions
should therefore be based upon knowledge of
ecology, toxicology and genetics, and generalizations
should be made with care.
0.5 3.2.2. Correlated responses. Correlated responses
of selection for tolerance may arise through
pleiotropic effects of genes. This implies that selection
for tolerance may affect performance of other, genet-
ically correlated, continuously variable character-
istics, usually in a negative sense. A genetic
correlation expresses the extent to which such charac-
teristics reflect what is genetically the same character-
istic (Falconer, 1981). Such effects are known as
“costs of tolerance” (M. R. Macnair, pers. com-
mun.). For a further discussion see section 5.2.
22 L. POSTHUMA
and N. M. VANSTRAALEN
3.2.3. Linkage disequilibrium. Environmental
factors other than metals may contribute to the
performance of fitness characteristics, since they also
impose selection forces; such factors include changes
of predatory species responding to pollution. Metal-
polluted sites are often stressful in many aspects, and
species inhabiting the sites may also be tolerant to the
usually adverse nutrient and humidity conditions.
Such adaptations may consist of reduced growth rate
or com~titive ability. Linkage disequilibrium will
result if the environmental factors covary with the
degree of pollution. Correlations between tolerance
and fitness characteristics caused by linkage disequi-
librium are transient, and may be lost after few
generations.
3.2.4. Alternatiue causes of population di~eren-
tiution in Orchesella, and other species. In Or~hesella
cincta, all four explanations may be important to
explain the various components of adaptation of
exposed field populations. Metal excretion improved
tolerance in exposed conditions (Fig. 6, explanation
l), but growth and reproduction characteristics also
differed among pop~ations in clean cultured
offspring: tolerant populations exhibit fast growth,
and early maturation (Posthuma et al., 1993b). The
presence of these responses matches with the presence
of genetic variation for these characteristics in a
reference population (Posthuma and Janssen, in
press). Direct selection for these characteristics may
have caused these effects, in accordance with expec-
tations from life-history theory (Posthuma et al.,
1993b, alternative explanation 2): the change is
considered to be adaptive, since the phenology of
exposed tolerant populations shows closer
resemblance to the normal situation than without
life-history adaptation. Life-history adaptation may
balance the adverse effects of exposure, independent
of- or in addition to adaptation of metal regulation.
With respect to correlated responses, Posthuma et al.
(1993a) demonstrated a positive genetic correlation in
0. cincta between growth and excretion efficiency, so
that the responses observed in the field may also have
been caused by correlated responses to selection for
increased excretion efficiency (explanation 3). Finally,
two populations from reference sites appeared to
differ in clean culture, indicating that other factors
than metals are involved (explanation 4, Posthuma
er al., 1992). One possible confo~ding factor is the
calcium content of the field soils, since the population
from the soil with the lowest calcium concentration
exhibited the lowest excretion efficiency, calcium
being involved in granula formation (Humbert, 1978;
see also: Adaptation in snails: Beeby and Richmond,
1988).
Life-history adaptation has been demonstrated in
metal-exposed populations of several other species,
namely Isotoma notabilis and Onychiurus armatus
(Tranvik et al., 1993), Porcellio scaber (Donker et al.,
1993a,b), Drosophila melanogaster (Bajraktari et al.,
1987b), the fish Fund&s heteroclytus (Weis and Weis,
1987) and the aquatic isopod Asellus meridianus
(Maltby, 1991). In Drosophila, Porcellio and
Orchesella, components of inherited divergence
consisted of both life-history adaptation and of
adaptation of characteristics causal to physiological
tolerance (altered metal regulation); in Drosophila,
artificial selection for lead-tolerance was demon-
strated to result in altered life-history patterns
(Nassar, 1976). The involvement of multiple charac-
teristics in adaptation to metals has been called
“complex adaptation” by Posthuma et ai. (1993b), a
phenomenon also described for plants (Ernst, 1983;
Macnair, 1990). The improvement of pollutant
tolerance through modification of life-history
characteristics has been called “indirect adaptation”
by Mahby (1991), which causes semantic problems in
view of the difficulty to distinguish cause and effects.
We propose to use “life-history adaptation” instead,
for populations where selection of life-history charac-
teristics in exposed conditions is demonstrated.
“Life-history adaptation” is probably more wide-
spread than recognized at present for populations
inhabiting sites polluted with metais or other toxi-
cants. If adaptation depends only on life-history
modification, then the risk for extinction is highest
for populations that do not maintain sufficient
genetic variation for life-history characteristics.
Risks, moreover, are also high if the involved charac-
teristics have a high sensitivity to the metal.
3.2.5. Ailozymes studies and their ecological
relevance. Methods allowing the study of genetic
variation in relation to tolerance do not necessarily
shed light on its ecological relevance or the physio-
logical mechanisms. Aliozyme studies may indicate
correlations between allele frequencies and site
pollution (Lower, 1976) or even tolerance (Frati
et al., 1992). In the latter case, the altered frequencies
of Got-alleles in exposed populations of Orchesella
cincta may be associated with an altered energy use,
as was suggested for other Krebs-cycle enzymes in the
mosquitofish Gambusia ho~brook~ (Kramer et al.,
1992). In turn, this change may have contributed to
fast growth and early reproduction of adapted ani-
mals. In such research, however, complex exper-
iments are needed to establish the role of specific
allozymes. Ecologically relevant habitat differences
are not necessarily accompanied by changes of al-
lozyme patterns, for example in the mite P~atynothrus
pelttjer (Ziegler et al., 1990), the moss Funaria hygro-
metrica (Shaw, 1991) and the plant Silene cucubaius
(Verkleij et al., 1985). This may result from allozymes
being constitutive enzymes not directly involved in
tolerance to the stress factor under investigation. An
experimental approach, addressing the role of the
specific alleles or marker genes in tolerance, is needed
(Koehn, 1978; Nevo et al., 1983), but this approach
has only been applied with some success with aquatic
animals through analysis of genotype characteristics
in relation to tolerance (Chagnon and Guttman,
1989a,b; Gillespie and Guttman, 1989; Benton and
 Metal adaptation in invertebrates
Guttman, 1990; Kopp et al., 1992; Heagler et al.,
1993).
In conclusion, looking at the state of adaptation of
populations, we think that analysis of life-history
characteristics, if possible through determination of
dose-effect relationships (Fig. 2, Posthuma et al.,
1993b), is crucial to assess the fitness of populations
at polluted sites.
3.3. The quantitative genetics of adaptation
Quantitative genetic studies are central to studies
on the evolution of continuously variable character-
istics, such as tolerance and life-history characteristics
(Macnair, 1990; Willis et al., 1991). Genetic par-
ameters may help to distinguish the four causes of
differences between populations from polluted and
reference sites (see 3.2.). Estimates of genetic
variation serve, furthermore, for prediction of the
potential speed of evolution, which is particularly
important for adaptation to industrial pollution, if
the speed of metal emission exceeds the potentials for
evolution of tolerance. Due to their predictive value,
genetic parameters may be both prognostic and diag-
nostic parameters to estimate pollutant-mediated
damage in ecosystems, as recently suggested also for
insecticide tolerance (Firko and Hayes, 1990):
prognostic in the sense of prediction of future
performance, diagnostic in the sense of indicating a
loss of genetic variation during prolonged periods of
directional selection.
Indications for the presence of genetic variation
can be obtained from comparisons of field animals
from several populations or from offspring cultured
in a common environment. Quantification of genetic
variation is, however, possible. A measure of genetic
variability of continuously variable characteristics,
such as tolerance and life-history characteristics, is
heritability (see Fig. 3). The narrow-sense heritability
(h*) is defined as the ratio of additive genetic variance
to phenotypic variance (Falconer, 1981). The herita-
bility is a measure to predict the rate at which a
population may adapt to environmental parameters,
since the evolutionary response to selection (R)
depends on the selection intensity (S) through the
heritability: R = h*S.
Several quantitative genetic methods have been
applied to study genetic variation of metric character-
istics, namely comparisons of relatives by offspring-
parent regression or half-sib analysis and artificial
selection. The strength of evidence for the presence of
genetic variation is considered to increase in the
following order: phenotypic comparisons of field-or
laboratory reared animals (no quantification of vari-
ation); quantitative genetics, estimates from relatives;
laboratory selection experiments; selection exper-
iment in the field (see Stearns, 1976; Bell and Koufo-
panou, 1986; Hoffmann and Parsons, 1991; Willis
et al., 1991).
3.3.1. Tolerance characteristics. Genetic variation
for metal-tolerance in a reference population of
23
animals has only been presented for the benthic
oligochaete Limnodrilus hoflmeisteri (Klerks and
Levinton, 1989) and for the springtail Orchesella
cincta (Fig. 3). For the oligochaete, tolerance was
expressed as survival time during exposure, and the
heritability of this characteristic was high (h* > 0.9).
For the springtail, tolerance was expressed as
cadmium excretion efficiency, and its heritability was
estimated as 0.33 and 0.48 in two experiments;
maternal effects were absent (Posthuma et al., 1993a).
Heritability estimates for metal tolerance character-
istics in both species were consistent with evolution-
ary responses demonstrated in field populations.
3.3.2. Life-history characteristics. In some species,
adaptation involved a change of life-history, and this
offers the opportunity to incorporate estimates of
genetic variation for life-history characteristics in the
inventory of heritability data. Life-history character-
istics, in general, are considered to have low heritabil-
ities compared to morphological, physiological and
behavioral characteristics, since selection for fitness
will lead to fixation of alleles of characteristics closely
associated with fitness (Falconer, 1981, Ch. 10; Price
and Schluter, 1991). This suggests that the potential
to show evolutionary responses of life-history charac-
teristics may be limited. Nonetheless, genetic
variation may be maintained by environmental
sources (spatial and temporal heterogeneity) or
genetic structure (antagonistic pleiotropy) (Hoffmann
and Parsons, 1991).
Genetic variation for life-history characteristics has
been reported for various terrestrial invertebrates,
but data for the species that show life-history diver-
gence in response to metal exposure (Table 1) are
restricted to Orchesella cincta (see above) and
Drosophila. Genetic variation for various character-
istics has been shown in natural and laboratory
populations of Drosophila melanogaster by various
authors, namely life-history characteristics (Rose and
Charlesworth, 1980; Rose, 1983; Hillesheim and
Stearns, 1991; 1992; Graves et al., 1992; Partridge and
Fowler, 1992; Rose et al., 1992) and energy allocation
(Clark, 1990). These data match with the divergence
of life histories of metal-exposed field populations of
D. rnelanogaster (Bajraktari et al., 1987b), despite the
phenomenon that the proportion of heritable vari-
ation in natural populations may be lower than in
laboratory conditions due to increased environmental
variance (Hoffmann, 1991).
Apart from Drosophila and Orchesella cincta, none
of the species listed in Table 1 has been subjected to
genetic analysis of metal tolerance. Genetic variation
not related to metal tolerance has, however, been
quantified for life-history characteristics in various
other terrestrial invertebrates, for example in the
carabids Callosobruchus macuiatus (Msller et al.,
1989) and Pogonus chalceus (Desender, 1989), the
milkweed bugs Oncopeltus fasciatus and Lygaeus
kalmii (Dingle et al., 1988; Dingle, 1991), and the
snails Arianta arbustorum (Cook, 1965) and Cepaea
24 L. POSTHUMA
and N. M. VANSTRAALEN
nemoralis (Cook, 1967). In the cotton stainer bug
Dysdercus bimaculatus, genetic variation was absent
for characteristics related to timing of reproduction,
but present for some other characteristics (Derr,
1980). Circumstantial evidence for genetic variation,
derived from geographical comparisons, is available
for the isopod Armadillidium vulgare (Souty-Grosset
et al., 1988) and the isopod Philoscia muscarum (e.g.
Grundy and Sutton, 1989). These examples illustrate
that genetic variation for life history may be
maintained in populations of various terrestrial
invertebrates, despite the expectation that variation
should be low, but that genetic variability is not
ubiquitous. If variability is absent, phenotypic
plasticity may contribute to survival at contaminated
sites. High plasticity has been reported for isopods
(Grundy and Sutton, 1989), snails (Baur and
Raboud, 1988) and slugs (Rollo and Shibata, 1991).
An unknown proportion of species may not be able
to show life-history adaptation, or they may show
alternative responses determined by genetic variation
and correlation patterns.
In conclusion, a generalized pattern on the
presence or absence of genetic variation within
(groups of) species cannot be established for either
tolerance- or life-history characteristics due to a lack
of data. In most cases, circumstantial evidence
indicates the presence of genetic variation in the past,
since population comparisons of laboratory reared
offspring showed divergence. This scarcity of data
hampers both the distinction between acclimation
and adaptation, and the identification of “costs of
tolerance”.
3.4. Study sites of adapted popuiat~on~
The fourth criterion introduced in section 1.3 refers
to the interactions of selection and gene-flow, as
determined by two spatial components, namely
pollution and population heterogeneity. Pollution
and habitat heterogeneity are often documented
relatively well (3.4.1), but little is known about the
population structure of animal populations and
mobility in relation to spatial heterogeneity (3.4.2).
3.4.1. The abiotic component. Investigated popu-
lations originated from two types of sites, namely
sites contaminated by aerial deposition originating
from human activities (smelting, leakage, transport,
spraying), or by surface ores (Table 1, column 3).
This may cause differences in the compositions of
adapted species between industrially and geologically
contaminated sites. Surface ores need to be colonized
prior to or during adaptation, whereas aerially
polluted sites may have originally been inhabited by
a population which has experienced a gradually
increasing selection force. Nonetheless, adapted
populations occur at both types of sites; investigated
sites are mainly located in Europe.
For both types of sites, the duration of pollution
may be quantified in retrospect from the history of
human activities. The duration of selection is, how-
ever, difficult to estimate, since both the date of
colonization of the species and the historical rate of
concentration increase are difficult to quantify. In
general, the duration of selection for animals can be
given an upper limit only, namely the duration of the
pollution period. Due to the lack of exact data,
indications for selection histories are often restricted
to the qualifications “reference”, “industrial” or
“mine” site (Table 1, column 3). For sites with
natural, undisturbed surface ores, such as Kastad
(HBgvar and Abrahamsen, 1989) one can reasonably
state that inhabitants common in the direct surround-
ings are either very insensitive, or adapted: only at
such sites, with nearby uncontaminated refugia, the
time to colonize the site is long compared to the time
necessary to adapt, and not limiting. For all other
types of sites, a low dispersal capacity may be the
factor causing the absence of some species at
contaminated sites, rather than the species’ potential
to adapt.
3.4.2. The biotic component. At some polluted sites
different species have been studied. Results for two
species o~ginating from a single site are present for
the springtail Orchesefla cincta and the isopod
Porcellio scaber (both in Plombi&res and Budel), and
for two springtail species and the worm Dendrobaena
rubida (in Gusum). In Plombi&es and Budel, both
species were adapted, whereas in Gusum, both
springtail species were adapted, but the worm was
not. The observations in Gusum indicate that different
species inhabiting a single site may show different
evolutionary responses, either due to species charac-
teristics or to differences in exposure. Therefore, adap-
tation appears to be a species specific phenomenon.
Frequent dispersal of sensitive animals from
uncontaminated refugia to a site inhabited by an
adapted population may reduce inter-population
differences. Estimates for actual gene flow in a
polluted area are, however, restricted to the values of
Tranvik et al. (1993) for Orchesella bifasciata around
the Gusum brass mill: estimates of c. 2-7% of actual
immigrants were reported between the popuIations
along the pollution gradient, on the basis of allozyme
patterns (maximum distance between populations: c.
9 km). This may have caused the observed similarity
of allozyme patterns along the pollution gradient. On
the other hand, the congeneric species 0. cincta,
which occasionally climbs in trees, showed consider-
able tolerance divergence along a smaller distance
(Posthuma, 1990). This indicates that the effect of
gene-flow, estimated from allozyme patterns, may be
small compared to the effect of selection for
tolerance.
Populations structure is an important source of
info~ation, both for the recognition of adaptation
and for assessments on costs of tolerance (see 5.2.).
Now that adaptation has been established for some
species, the gradient approach may be used to assess
the influence of gene-flow and actual dispersal of
individuals on adaptation processes.
 Metal adaptation in invertebrates 25

3.5. The direction of adaptation heritable variation between individuals in the efficacy
or the efficiency of the physiological machinery, in
The last criterion discussed in section 1.3 requires
other words, variation in “reaction norms” (Stearns,
historical evidence for the original state of the
1992) to metal exposure. The folIowing paragraphs
characteristic. It will be clear that observations on
do not attempt to review all aspects of metal
past sensitivity of adapted natural populations of
physiology, but only discuss those cases where
invertebrates are lacking. Moreover, the methods
physiological mechanisms underlying differences
usually applied in phylogenetic systematics to derive
between adapted and non-adapted populations have
the original state of a characteristic cannot be applied
been suggested.
on the small data-base present. For example, out-
group comparisons on the state of the characteristic
in closely related taxa is a method which could be 4.1. Assimilation from the diet
applied in the future on a large data-base (e.g. Sluys, Unlike aquatic invertebrates, terrestrial invert-
1988). Brandon (1991) argues, however, that in ebrates will take up a significant part of their body
plants, the original state is considered to be “non- burden from ingested food. However, for animals in
tolerance”, and the derived state, “tolerance”, since close contact with the soil (earthworms, nematodes
tolerance evolution has been observed “in action” at and small spring~ils), uptake from the soil pore
polluted sites, and since there is evidence that there water may be more important, especially when the
are “costs” associated with tolerance. There is no a integument is permeable to water. For uptake from
priori reason to believe that animals would have ingested food, metal complexes have to pass the
opposite characteristics (see also section 5.2.), and intestinal cells; this could provide the first possibility
the possibility to induce inherited tolerance in for a physiological mechanism contributing to
selection experiments with animals matches with this adaptation.
generalization. The efficiency of metal assimilation, relative to the
If non-tolerance is assumed to be the original state, amount ingested, varies greatly between species
then it is possible to assess the speed of adaptation (Janssen et al., 1991). Values between almost 0 and c.
towards tolerance. This speed can be estimated for 90% have been found for the assimilation efficiency
some species from pollution histories. In the fruit fly, for cadmium, being the element most frequently
adaptation to methylmercury increased considerably studied (Table 3). These inter-specific differences
within 12 generations of artificial selection (Magnus- relate to different feeding physiologies and trace
son and Ramel, 1986; Fig. 5). Adapted animal element requirements.
populations have been found in the field at sites In none of the species discussed in section 2 and
polluted on an industrial time scale (max. c. 150 listed in Table 1 are there indications that adaptation
year), and in mines abandoned for decades, The could be due to alterations in the assimilation
estimates for genetic variation in the springtail efficiency. This may appear peculiar at first sight,
Orcheseffa cirtcta, moreover, indicate that adaptation since it would seem to be one of the most simple
may occur within a few (tens of) generations, and at solutions possible. However, adjustment of the assim-
least within the time-span of the operation of the ilation process may be difficult to achieve, since it
metal emitting source. Observations of fast adap would require constitutive changes in the intestinal
tation are also known from the benthic oligochaete cell membrane, which could easily interfere with the
Limnodrifus hoffmeisteri (Klerks and Levinton, uptake of digestion products and essential trace
1989b) and from vascular plants (e.g. Coulaud and elements.
McNeilly, 1992). In conclusion, there are indications
that adaptation may occur quickly, but the actual
speed in exposed field populations is unknown. Table 3. Assimilation efficiencies (%) and excretion constants
(day-‘) for cadmium in various terrestrial invertebrates. Values
indicate differences in uptake and excretion, and thus equilibrium
concentrations, between species (data from Janssen ef al., 1991)
4. PHYSIOLOGY OF HEAVY-METAL ADAPTATION
Assimilation Excretion
Soecies efficiencv (%f constant Idav- ‘t
Physiological mechanisms involved in metal toler-
Mollusca
ance of animals, reviewed by Hopkin (1989) and Arianta arbustorum 55-92
Dallinger (1993), have revealed important aspects Helix porn&t 0.000
Cepaea nemoralis 0.007
of uptake, distribution over tissues, binding to Crustaoea
organelles and macromolecules, interactions with Porcellio scaber IO-60
vital processes and elimination pathways. In this way, Myriapoda
Lithobiw uartegatus 0.0-7.2
a better understanding has been obtained of how an Glomeris marghara 8.2-40.6
individual animal may avoid negative effects of a high Arachnida
exposure to metals, by adjusting its physiological Platynothrus pelt@ 17.2 0.013
Apterygota
machinery. As explained in section 1.3, this type of Orchesella cincta 8.3-9.4 0.0614.087
adjustment is named “acclimation” in the present Coleoptera
Notiophilus biguttatus 35.5 0.375
review. For adaptation to occur, there must be
26 L. POSTHUMA and N. M. VAN
The study of assimilation as a mechanism for
adaptation is complicated by the fact that in many
invertebrates, the intestinal cells are involved not only
with uptake, but also with excretion of metals into the
gut lumen (Hopkin, 1989). Therefore, if the net
transfer of metals over the gut epithelium is decreased,
this could be due to both decreased assimilation and
to increased excretion (Posthuma et al., 1992).
4.2. Distribution over tissues
During circulation in the haemolymph (insects,
crustaceans and molluscs) or in the blood (annelids),
metals are most probably bound to protein carriers.
In Porceflio scaber Donker et al. (1990) demonstrated
that both Cd and Zn in the haemolymph were bound
to haemocyanin, a protein with an apparent molecu-
lar weight of 70 kD which represented 80% of the
haemolymph protein content and contained a high
amount of copper. Further studies (Donker, 1992)
suggested that the transport of zinc from the
haemolymph to excretory organs is enhanced in
metal-adapted isopods.
The distribution of metals over the various
invertebrate organs is usually far from uniform.
Many species have special organs that accumulate the
metals more than other organs, such as the chloroga-
gene tissue in earthworms, the midgut gland in
isopods, centipedes and snails, and the midgut
epithelium in apterygotes and insects (Hopkin, 1989).
Metal-accumulating organs may receive metals not
only from circulation, but also directly from assimila-
tion. This is the case, for example, with the midgut
gland in isopods and snails, where digestion products
enter the lumen of the gland, and with the unicellular
gut epithelium of apterygotes and insects, which may
take up metals both from the basal and the proximal
sides of the cells.
There are some indications that the internal
distribution of metals is different in metal-adapted
populations compared to reference animals. Hopkin
and Martin (1982) provided data to suggest that the
storage capacity, and the total weight of the hepato-
pancreas in isopods living in highly contaminated
areas was greater than in isopods from a less polluted
site. Donker (1992) showed that the distribution of
zinc over hepatopancreas, haemolymph, head and
carcass of isopods differed between populations: in a
smelter population with a large body burden of zinc,
the distribution of this element was shifted towards
the hepatopancreas.
The accumulation of one element in a certain organ
may induce secondary changes in the tissue
distribution of another element. For example, the
accumulation of lead in the chloragogene tissue of
earthworms may disturb the calcium homeostasis
(Morgan and Morgan, 1988), the accumulation of
zinc in the isopod hepatopancreas decreased its cop-
per content (Donker, 1992), and the accumulation of
cadmium in the oribatid mite Hatynothrus peltifer,
decreased the zinc content (Van Straalen et al., 1989).
STRAALEN
The evidence for changed tissue distribution as a
mechanism for adaptation is not (yet) convincing.
Although some suggestions have been made, most
data may also be explained as a non-linear effect from
increasing accumulation, which appears in metal-
adapted field animals only because they are highly
exposed.
4.3. Intracellular regulation and sequestration
Once inside the cell, the distribution of metals over
the various intracellular binding sites is determined
by the metal affinity of the ligands, the number of
binding sites and the presence of competing metals.
One of the most frequently studied ligands is the
protein metallothionein (MT). The rate of synthesis
of this protein is considered as a key-factor in metal
regulation, because metal binding to MT will dimin-
ish the binding to other structures, including the
targets for metal toxicity.
A well known property of MT is its inducibility
(Roesijadi, 1992; Fig. 7). Free metal ions in the cell
will be taken up by a metal regulating factor, which
A. MT-gene
q TV’
MREs coding region
*
IMC?+-
B.
J
Fig 7. Pathways of subcellular metal distribution in metai-
uninduced (A) and induced (B) states. In the uninduced
state, metallothionein (MT) synthesis is low and metals
partition over MT and nonthionein ligands (L). Induction
by metals involves binding of metal ions to a metal regulat-
ory factor (MRF), that in turn binds to the MT regulatory
element (MRE) of the MT gene. Transcription is initiated
resulting in increased synthesis of MT. In adapted fruit flies,
the MT gene is amplified (see Maroni et al., 1987) which
results in a higher MT production, as indicated in situation
B. Binding to L continues following MT induction, but is
moderated by binding of metals to MT. Protection against
metal toxicitv is considered to be the outcome of this effect.
Heavy lines-and large boxes indicate increased fluxes or
pools. (Modified from Roesijadi, 1992; reproduced with
permission of G. Roesijadi.)
 Metal adaptation
will then be able to bind to the metal regulating
element of the MT-gene, triggering transcription of
the coding region and the synthesis of MT (Hamer,
1986; Maroni, 1990; Roesijadi, 1992). In the induced
state, the cell will be more tolerant to high metal
exposure (Fig. 7).
Metal binding proteins similar to mammalian MT
have now been identified for various terrestrial
invertebrates, including the slug Arion lusitanicus
(Dallinger et al., 1989), the midge Chironomus yoshi-
matsui, the fleshfly Sarcophaga peregrina, and the
cockroach Blatella germanica (see Maroni, 1990), and
earthworms (Suzuki et al., 1980; Morgan et al., 1989).
The inducibility of metal binding proteins may differ
between species, and this correlates well with their re-
sistance or susceptibility to metal exposure (Aoki et al.,
1989). Moreover, non-metallothionein glycoproteins
with metal-binding properties have been demon-
strated in the stonefly Peronarcys californica and the
orthopteran Locusta migratoria (see Maroni, 1990).
The involvement of MT in the evolution of metal
tolerance has been well documented in the fruitfly
Drosophila melanogaster (Maroni et al., 1986; 1987).
Natural populations of this species differed in the
amplification of the MT gene. Four types and two
variants of the duplication were observed in speci-
mens from various sites all over the world. The
duplications were attributed to a selection pressure
from environmental contamination. Strains with
amplified MT genes had a higher resistance to copper
and cadmium (Maroni et al., 1987). This study is one
of the best examples of a biochemical explanation for
mechanisms involved in metal tolerance and
adaptation.
The role of MT (or of compounds with similar
functions; see Stone and Overnell, 1985) in metal
adaptation is more difficult to demonstrate on the
protein level, compared to the DNA level. Donker et
al. (1991) using FPLC gel filtration of hepatopan-
cress homogenates, were unable to detect differences
between metal-adapted isopods and reference
isopods. Dallinger et al. (1989) however, found that
slugs sampled from a mine area produced different
metal-binding proteins upon exposure to cadmium,
compared to reference animals.
Another intracellular component capable of metal
sequestration is formed by membrane enclosed
granules. Electron-microscopical studies using
electron-induced X-ray emission have revealed that
granules may be of four different types, depending on
their morphology and the dominating metal (Brown,
1982; Hopkin, 1989). They are present in all metal
accumulating tissues of invertebrates, including the
hepatopancreas of isopods (Hopkin et al., 1989) the
chloragogene tissue of earthworms (Morgan and
Morris, 1982) and the midgut epithelium of
apterygotes and insects (Humbert, 1978; Lauverjat et
al., 1989).
The role of granules in metal adaptation has yet to
be demonstrated. Between-species differences in
in invertebrates 27
metal accumulation potential have been described for
Porcellio scaber and Oniscus asellus, and these could
relate to a difference in sequestration capacity of two
types of granules (Hopkin, 1991). Within-species
variation in granule number, composition or seques-
tration, however, has not been demonstrated. The
role of granules probably cannot be separated from
the function of MT-like proteins, as these serve as the
major intracellular carrier. One of the granule types,
abundant in the “S-cells” of the isopod hepatopan-
cress, represents a residual body from the lysosomal
system, in which sulphur-rich metal binding proteins,
or katabolic products of them, accumulate (Dallinger
and Prosi, 1988; Hopkin, 1989).
4.4 Excretion
Similar to the existence of assimilation differences
between species, there are also excretion differences,
as demonstrated by the values for net excretion,
estimated from the change of body burden of metal-
loaded animals on clean substrate (Table 3).
Excretion pathways of metals in invertebrates are,
however, badly described. This may be due to the fact
that the tissues involved in excretion (gut epithelium,
midgut gland) often perform other functions as well
(production of digestive enzymes, phagocytosis). The
nephridia present in the head of isopods and spring-
tails have never been investigated with respect to their
possible role in metal kinetics, although their function
in maintaining the water balance is well known.
For the springtail Orchesellu cincta, there is clear
evidence that excretion of metals, through exfoliation
of the midgut epithelium at every moult, is an
important component of cadmium tolerance and
adaptation (Van Straalen et al., 1987; Posthuma et
al., 1992; Fig. 6). This species has a peculiar habit of
depositing the degenerated midgut epithelium as an
easily recognizable pellet on the substrate. The pellet
has a high mineral content, and is fully loaded with
excretory granules (Fig. 8), which most probably
contain the metals. Metal analyses of the pellet, using
micromethods for graphite-furnace AAS, allow ex-
cretion to be quantified directly, a unique possibility
offered by this species.
Prior to holocrine excretion, metals will have to be
bound in the cells, either to MT-like proteins, or in
granules. Therefore, the increased excretion efficiency
of metal-adapted springtails reflects a greater ability
of the midgut epithelium to effectively sequester
metals. How this is achieved remains as yet unknown,
but it cannot be excluded that similar mechanisms as
in the fruit fly are involved.
5. CONSEQUENCES OF HEAVY-METAL ADAPTATION
5.1. The possible consequences
Adaptation to heavy metal exposure improves
fitness in contaminated habitats, but two potentially
deleterious consequences are identified, compared to
fitness of sensitive populations. These consequences
28 L. POSTHUMA and N.M. VANSTRAALEN

Fig. 8. Ei~tron-micro~aph showing a quarter of a cross-section of the midgut of ~~eheseliu cincta as

it appears three days after shedding the previous epithelium. The thickness of the cell layer of the new
epithelium is c. lo-40 pm, depending on the day after moulting. The animal was fed with clean algae.
OE = old midgut epithelium cells; NE = new midgut epithelium cells; lmf = longitudinal muscle fibers;
n = nucleus; g = granulum. The old midgut is loaded with granula (Lupetti et al., 1992). The picture is
a product of original research in the Dipartimento di Biologia Evolutiva, Universita di Siena, Italia. R.
Dallai, P. Lupetti and G. Roest are acknowledged for the use of this photograph in this review.

of selection determine future performance, and there-
fore population persistence at contaminated sites.
Despite the fact that these consequences can be
explained with firm theoretical considerations, the use
of terminology is far from uniform. Strictly speaking,
strong evidence for consequences of metal-adaptation
will only be present if adaptation has been demon-
strated with application of Brandon’s (1991) criteria.
Strong evidence for both the deleterious
consequences mentioned below is scarce, since metal-
adaptation has been demonstrated for only few
species. The following discussion (sections 5.1-5.3),
therefore, serves the dual purpose of both definition
and inventory of data; it has a descriptive nature
rather than that it recognizes patterns in data.
Firstly, maintenance of a tolerance mechanism
may be physiologically or energetically costly, so that
allocation of nutrients or energy to other functions is
reduced. Such “costs of tolerance” are frequently
suggested as an inevitable consequence of being
tolerant. For example, tolerance for the herbicide
triazine increased heat sensitivity in five plant species
(Ducruet and Lemoine, 1985), and tolerance for the
insecticide malathion reduced reproductive perform-
ance of moths (Halliday, 1990).
Two types of costs can be distinguished (cf. Sibly
and Calow, 1989; Hoffmann and Parsons 1991).
Expenditures may increase as a consequence of
individual exposure: this is a plastic response and it
is often expressed in terms of physiology. On the
other hand, there may be expenditures associated
with the maintenance of a tolerance mechanism
evolved by genetic adaptation: this is a constitutive,
inherited characteristic of a tolerant individual,
caused by negative genetic correlations between
tolerance and other features. The latter costs are the
 Metal adaptation in invertebrates
real “costs of tolerance”, whereas the former are in
fact “costs of individual exposure”. The fitness of an
individual at a contaminated site is affected by both
types of costs: “costs of tolerance” are superimposed
upon “costs of exposure”. The constitutive fitness
deficit expected for tolerant individuals in control
conditions, in comparison to sensitive conspecifics, is
determined by the permanent type only. Thus, genetic
evidence for “costs” is confirmed by rearing tolerants
and sensitives in a clean environment, if the tolerants
have a lower fitness than the sensitives.
The second possible consequence is a change of
genetic variation due to strong directional selection.
Up until now, the approach to study this consequence
explicitly consisted of analyses of allozyme frequen-
cies. One possible alternative is the study of altered
genetic variation for ecologically relevant character-
istics. The restriction to the latter characteristics
implies that the results can be directly interpreted as
an alteration of the possibilities to respond to future
environmental variation, or to adapt to neighbouring
habitats for extension of the home-range. For such
characteristics, the possible changes of genetic vari-
ation are expressed through an altered heritability.
5.2. “Costs of tolerance” and individual jtness
The main problem in demonstrating “costs of
tolerance” is its distinction from other causes that
may alter characteristics of populations in metal-
polluted habitats (see 3.2). This problem is analogous
to the distinction between acclimation and adap-
tation. Brandon’s (1991) criteria apply again to ident-
ify costs, since evidence for costs is based upon
evidence for adaptation. For most animals, however,
observations on metal tolerance are restricted to
comparisons of field populations, and “costs” have
hardly been mentioned explicitly. The inventory of
“costs of tolerance”, therefore, mainly resulted in
circumstantial evidence (5.2. l), and in observations
conflicting to the cost hypothesis in some cases
(52.2).
52.1. Circumstantial evidence for costs. A general
argument to expect costs is obtained from the absence
of metal tolerant plant populations nearby tolerant
ones, despite evidence for considerable gene flow in
some species (Wilson, 1988; and references therein).
This phenomenon is attributed to deleterious effects
of tolerance genes on fitness characteristics not being
tolerance itself. The absence of adapted populations
of animal species in clean habitats nearby polluted
sites can, in some cases, even be explained by an
evolved dependency of high metal concentrations.
For example, a population of the isopod Porcellio
scaber captured near the zinc smelter in Budel per-
formed badly on reference diet, but performance
increased when it was reared on cadmium and zinc
enriched food [van Capelleveen (1985); (1987, ch. 2);
see also Fig. 11;a shortage of zinc for metal-mediated
enzyme function due to the high sequestration
capacity may have caused this effect. Culturing
29
adapted animals on reference substrate may be
difficult, if they depend on increased metal exposure,
particularly for micro-nutrients such as zinc and
copper. This may be an explanation for the some-
times reduced survival of animals from exposed
populations in control treatments in experimental
conditions. Usually these observations have not been
mentioned explicitly in reports (e.g. Posthuma, 1990)
but in a systematic mortality assay, 0. cincta
appeared to show an increased mortality rate of
adapted animals in control conditions (Posthuma et
al., 1993b). Drought tolerance was, furthermore,
suggested to be a major criterion influencing the
distribution of springtail species around the Gusum
brass mill: a population of the inherently tolerant
species Folsomia jimetarioides, dominant in polluted
soil, was drought sensitive compared to the less
tolerant Isotomiella minor, dominant in reference soil
(Tranvik and Eijsackers, 1989). These data match
with the cost-expectancy, but evidence from field
animals should be considered indicative rather than
strong, since correlations might be caused by other
factors. Stronger evidence can be obtained from
studies incorporating the genetics of tolerance, but
such studies revealed conflicting evidence (see 5.2.2).
5.2.2. Genetics as a central criterion. A strong
method to test cost hypotheses is artificial selection
for tolerance: correlated responses for fitness charac-
teristics indicate the presence of costs through
(negative) genetic correlations (e.g. Drosophila;
Nassar, 1979; Rose et al., 1992). Such methods have
not been applied yet to natural populations of
animals or plants (Macnair and Watkins, 1983;
Nicholls and McNeilly, 1985, M. R. Macnair, pers.
commun.), so that costs can best be inferred from
genetic parameters estimated from comparisons of
relatives.
The presence of heritable variation for tolerance in
a reference springtail population (Posthuma et al.,
1993a) indicates that the costs of maintenance of a
mechanism effective in excreting cadmium are low in
reference conditions. Similar reasoning may apply to
many invertebrates, since various tolerance mechan-
isms appear to be modifications of existing “cheap”
metal-regulation mechanisms (see section 4). The
presence of genetic variation of some magnitude for
metal tolerance has only recently been established for
the grass Deschampsia cespitosa on clean soil
(Coulaud and McNeilly, 1992) but the authors
suggest that genetic variation for metal tolerance may
be more widespread than expected previously from
the low incidence of tolerant individuals in reference
populations.
Deviations from “costs” expectations are ex-
pressed through the “vigorous” life-histories of
isopods and springtails, which were attributed to
direct selection for life-history characteristics, and for
springtails, attributed to positive genetic correlations
with tolerance. There may, however, still be costs,
that remained undetected, since the relevant charac-
30 L. POSTHUMA and N.
teristics were not compared among populations.
Ernsting et al. (1993) have shown that there is a
trade-off between growth rate and longevity in 0.
&era: ageing appears to be fast in springtails with a
high growth rate. This trade-off has also been
observed in other species (Hoffmann and Parsons,
1992). The importance of this possible trade-off can,
however, not be evaluated for polluted sites with the
Orchesella data, since only a very small fraction of the
hatchlings reaches sexual maturity in field conditions
(Van Straalen, 1985). The increased mortality of
adapted springtails may be caused by metal-depen-
dency, as indicated above, but it may also be an
expression of the trade-off of fast growth for ageing.
In the moss Funaria hygrometrica, linkage disequi-
librium and pleiotropy have been effectively disentan-
gled by comparisons of populations (clones) from
several reference and mining sites, in combination
with assessments of genetic correlations. Similar to 0.
cincta, positive genetic correlations were found be-
tween tolerance and growth rate, and these were
substantiated by high growth rates in natural
populations from contaminated sites (Shaw, 1988).
Indications for costs can also be obtained from
population comparisons, but such data do not necess-
arily indicate negative genetic correlations. Weis and
Weis (1989) demonstrated that increased embryonic
resistance in the fish Fund&s heteroclytus was associ-
ated with decreased adult growth rate and longevity.
Wilson (1988) found reduced growth rate in metal
tolerant clones of Agrostis capillaris. In these species,
observations matched expectations.
The examples mentioned above indicate that both
positive (0. cincta, F. hygrometrica) and negative
(F. heteroc~ytus and A. cap~llar~s) effects on life-
history parameters occur. This observation may
result from several factors diminishing the success
rate of studies on costs of tolerance. Firstly,
potentially important characteristics may have been
omitted in the reports, suggesting only positive effects
on life-history characteristics. The array of possible
cha~cte~stics showing costs should be carefully
chosen, to avoid missing the effect. Secondly, lack of
evidence for costs may result from adaptation to site
characteristics other than metals. Thirdly, costs may
be low in comparison to phenotypic variation of the
characteristic under investigation. Fourthly, selection
will operate to minimize costs, so that costs will be
most clearly expressed directly after the pollution
event, in early stages of adaptation (see Ernst, 1983;
see also section 5.3.3). Finally, phenomena may be
falsely identified as costs, while they actually are a
response to direct selection for the characteristic (see
“life-history changes”). Despite the apparent general-
ity of the principle of costs, very few examples have
been documented. Population comparisons may
reveal the characteristics possibly paying the costs in
a relatively simple experimental effort, but further
genetic research is needed to demonstrate costs with
certainty.
M. VAN STRAALEN
5.2.3 Physiology of costs. At the level of physi-
ology, costs may be derived from the mechanism
involved in tolerance (see section 4). There are,
however, no data quantifying the nutrient or energy
drain for the maintenance of a tolerance mechanism,
to estimate the importance of the drain relative to
normal performance of life-history characteristics.
The sum of use and maintenance of a tolerance
mechanism seems to be energetically costly in various
species, when judged by energy content of (exposed)
field animals. Only in Porcellio scaber, however. are
there indications that metabolic rates differ inherently
among populations, and in Drosophila attribution of
energy to different functions is genetically variable
(Clark, 1990). In all other species, the loss of energetic
reserves may have been caused by individual
exposure supe~mposed on possibly evolved tolerance
characteristics. It seems to be extremely difficult to
identify the generation of costs through physiology
without a proper analysis, taking into account adap-
tation, linkage disequilibrium, and other causes of
altered performance of metal-exposed populations
(see 3.2.). Costs may be not measurable through
comparisons, but they may be ecologically very
important. These factors may explain the absence of
physiological explanations for costs for metal-
adapted micro-organisms (P. Van Beelen, pers.
commun.) and plants (M. R. Macnair, pers. com-
mun.).
5.3. Altered genetic variation and future performance
In theory, several changes of genetic variation can
be expected. The pattern of change is determined by
the starting condition, the mode of inheritance, by the
strength and duration of selection, population size
and the influence of gene-flow. Assuming an isolated
large population, both increased and decreased
genetic variation can be expected, despite the fact that
directional selection is expected to reduce variation
eventually (Bulmer, 1971). An increased genetic
variation for tolerance is to be expected if very rare
tolerant variants increase in frequency in few gener-
ations upon starting selection (e.g. IIolloway ef at.,
1990). This may particularly apply to heavy-metal
tolerance, due to the possible influence of major genes
for tolerance with initially low frequencies (Macnair,
1991). A reduced variation is to be expected if
selection ultimately drives the tolerance alleies to
fixation. If populations are small, or not isolated,
then large influences from random processes will
occur: random genetic drift may alter allele frequen-
cies in a random way, and immigration from neigh-
bouring populations may maintain or restore initial
variation.
Up to now, changes of genetic variation have been
studied through comparisons of allozyme patterns
(5.3.1) or heritabilities (5.3.2).
5.3.1. Altered allozyme variability. Comparisons of
allozyme patterns for populations of terrestrial
invertebrates have demonstrated a change of allele
 Metal adaptation in invertebrates
frequencies in relation to metal pollution or tolerance
at a single locus (Frati et al., 1992), or such relations
were absent (Tranvik et al., 1993). Indications for a
reduction of overall genetic variation have not been
obtained in these cases. Similar findings were re-
ported for the plant Silene cucubalus (Verkleij et al.,
1985). The phenomenon can probably be explained
by a lack of loci involved in tolerance in the sample
of enzymes studied. If major genes are the major
determinants of metal adaptation (as expected), then
effects on allozyme patterns may be restricted to only
few loci, which need not be involved in tolerance, but
which are linked to the tolerance gene on the chromo-
some, and which can act as “marker” of tolerance.
Only in one case was a reduced allozyme variation for
the highly variable esterase locus demonstrated,
namely in a metal exposed population of the
parthenogenetic mite Tectocepheus uelatus (H. Siepel,
pers. commun.). This may indicate the selection of a
single clone, rather than an indication for a function
of specific esterase allozymes in metal tolerance.
Genotype-dependent differences of survival associ-
ated with metal exposure were reported for aquatic
species by various authors (see 3.2). In general, the
interpretation of such allozyme studies based on allele
frequencies only is hampered by a lack of evidence for
the ecological role in tolerance of the enzymes under
investigation (criterion 2).
53.2. Altered heritabilities. In animals, two
estimates for heritability of metal tolerance in refer-
ence populations have been reported for reference
populations (Klerks and Levinton, 1989b; Posthuma
et al., 1993a). In contrast to exposed populations,
these heritability estimates are not biased by gene-
flow from nearby populations with different tolerance
characteristics. In both animal species, and contrast-
ing to observations reported for many plant species,
a considerable amount of genetic variation for toler-
ance is apparently maintained in reference conditions.
Genetic variation in animals was assumed to be of
quantitative kind in both cases. Metal tolerance in
animals may, however, be governed by major genes:
tolerance in Drosophila is partly governed by dupli-
cation of a metallothionein-gene Mtn (Maroni et al.,
1987). It cannot be excluded that tolerance in other
species basically depends on a similar mechanism, for
example in the midgut of springtails. Nonetheless,
from the presence of variation in reference popu-
lations, we expect that long-term directional selection
will ultimately lead to a reduction of genetic variation
for characteristics selected for, namely tolerance- and
life-history characteristics, either due to direct selec-
tion, due to correlated responses, or due to both.
Reports on changed patterns of genetic variation
for metric characteristics are restricted to mosses and
springtails. In the moss, Funaria hygrometrica, the
heritability was reduced for 10 out of 14 character-
istics in populations from mining sites, compared to
populations from reference sites (Shaw, 1988). In the
springtail Orchesellu cincta, Posthuma et af. (1993a)
31
found a tendency for reduced genetic variation for
tolerance in a springtail population from a smelter
site exposed for c. 150 years, which is equivalent to
c. 300 generations. The findings of both groups
corroborate the expectation that, eventually, strong
directional selection will force the alleles conferring
tolerance to fixation, causing heritability to decrease.
Hoffmann and Parsons (1991) evaluated nine
alternative hypotheses to explain the limits of range
expansion of populations at the margins of the
distribution of the species. This evaluation bears on
the present topic, since populations under pollution
stress may be locally at the margin of their distri-
bution. The authors concluded that, from the genetic
point of view, the main cause reducing colonization
success of marginal populations to neighbouring
habitats is low genetic variation. This implies that the
colonization ability of species may be seriously
affected, if genetic variation would be increasingly
lost due to local pollution. An even worse case is
diffuse pollution, which occurs with metals worldwide
(see Boutron et al., 1991) and which may cause the
loss of populations of sensitive species over large
areas.
The duration of selection, and the resulting change
of genetic variation, influences the chance to show the
opposite evolutionary responses towards non-toler-
ance. If alleles are not driven to fixation, then
reduction of selection intensity may be followed by
recovery of original variation, as suggested by Baker
et al. (1986). In the case of insecticide resistance,
recovery after relaxation of the treatment seems to
occur, but tolerance genes appear to be maintained in
heterozygous conditions for long periods (Keiding,
1967). If alleles are eventually driven to fixation, then
one expects that the future response to environmental
variation will be hampered, reducing the persistence
of adapted populations compared to sensitive conspe-
cifics (Hoffmann and Parsons, 1991; e.g. Kopp et al.,
1992).
53.3. Fluctuating asymmetry. A method which has
not been applied to populations of metal-exposed
terrestrial invertebrates is the analysis of fluctuating
asymmetry (FA). FA is expressed as asymmetry of
usually bilateral symmetrical characteristics, such as
bristle number on the abdomen of insects. It has been
observed in stressed populations, and in populations
which have passed a bottleneck of population size, of
normally bilateral-symmetrical animals. Examples
are the occurrence of FA after insecticide application
in the blowfly Luciliu cuprina (Clarke and McKenzie,
1987), a species that also adapts to arsenic containing
pesticides (Table 1), and in the small populations of
the cheetah after a bottleneck (O’Brien et al., 1985;
Wayne et al., 1986). For further examples see Leary
and Allendorf (1989). FA is ascribed to perturbation
of physiological processes in developing individuals,
and the asymmetry reduces if the population reaches
equilibrium in the new environment. As expressed by
Clarke and McKenzie (1987), FA has at least a
32 L. POSTHUMA and N. M. VAN STRAALEN
potential to give insight into the state of adaptation
of a population, FA being indicative for a continued
selection pressure. From all abovementioned
methods, indications for the state of adaptation can
only be derived from the results of selection
experiments, but that requires highly complex and
time-consuming observations compared to the
possibilities of single-generation FA observations.
In conclusion, various factors influence the
possible change of genetic variation, but none of
them has been studied in enough detail to enable full
explanations, or even predictions, of the observed
patterns of genetic variation associated with metal
tolerance. The divergence of responses in artificial
selection experiments between selection lines originat-
ing from reference sites and polluted sites seems to us
a valuable approach to study changes of ecologically
relevant adaptation potential, with the comparison of
reference and long-term exposed populations as a
prime goal.
6. DISCUSSION
6.1. Occurrence
The occurrence of heavy-metal adaptation in
natural populations of terrestrial invertebrates has
been demonstrated conclusively, on the basis of Bran-
don’s criteria, in several taxa, namely for the fruit fly
Drosophila melanogaster, and for four soil inhabi-
tants, viz. the isopod Porcellio scaber, and the spring-
tails Orchesella cincta, Isotoma notabilis and
Onychiurus armatus. Evolution of metal-tolerance
has also been demonstrated for ticks and a fly species,
in response to the application of metal-based pesti-
cides. There was some evidence for increased heavy-
metal tolerance or altered metal-regulation in
populations of many other species exposed to high
levels of metals in their habitat (Table 1), but
acclimation and adaptation could not be distin-
guished in these cases.
Within-species divergence of tolerance was absent
in some species, but this does not necessarily imply
that such species are not able to adapt. Similarity of
populations may simply indicate that exposure was
low in comparison with the species’ ability to regulate
or tolerate heavy metals. A reduction of the number
of animal species, however, has been observed at
several contaminated sites (Bengtsson and Rundgren,
1988; Tyler et al., 1989; Hggvar and Abrahamsen,
1990), which indicates that many species fail to adapt
to local concentrations. This matches the findings
reported for terrestrial plants (Antonovics et al.,
1981; Macnair, 1990) and aquatic species (Klerks and
Weis, 1987). Reports on unsuccessful adaptation are
scarce, probably since non-adapting species are
absent at polluted sites, and do not attract research
attention. Other biases against reports on unsuccess-
ful adaptation may also occur (Klerks and Weis,
1987). Therefore, proposals to increase the trigger
concentrations for remedial action based on the idea
that many species will adapt, are not supported by the
reviewed adaptation data.
6.2. Species at risk for extinction
A guide towards the species that are locally most
at risk for extinction, sensu being not able to adapt,
consists of various data. Associated with the potential
to show adaptation, data are needed on sensitivity for
metal exposure and on genetic variation, in relation
to the rate of increase of metal contamination and the
generation time; the influence of generation time is
not straightforward, but it depends on interactions
between this characteristic and a variety of genetic
and ecological factors (Rosenheim and Tabashnik,
1991). Successful adaptation depends on the NOEC
and the heritability of characteristics relevant for
metal tolerance; species with a low NOEC (a high
sensitivity), a low heritability and a high generation
time, or all three are more at risk of extinction than
species with the opposite. Such species may be said to
have a high “risk profile”. Moreover, if metal-
concentrations increase fast, adaptation may be
restricted to species with a high evolutionary poten-
tial, as determined by a major gene for tolerance. The
costs of maintaining or using a tolerance mechanism
may, furthermore, exceed the profits of increased
tolerance if resources are limiting. It is obvious that
species with physiologically costly tolerance mechan-
isms, or strong negative genetic correlations between
tolerance and fitness characteristics, or possibly both,
are more at risk than species with the opposite. For
sites with a constant metal concentration in the soil
(surface ores), the species with the “low-risk profile”
will also be the better colonizers. This profile is often
expressed through a high reproduction potential, a
short generation time, a wide ecological amplitude for
various characteristics, and omnipresence in different
habitats. Such euryoekoes or “opportunistic” species
may ultimately dominate at polluted sites. It is not
known whether the opportunists are species with a
particularly high incidence of major genes for
tolerance.
The absence of many species at contaminated sites
(Tyler et al., 1989) can be explained if a large
proportion of the populations “at risk” did not
maintain sufficient genetic variability for sensitive or
costly characteristics to respond at an adequate speed
to selection by heavy metals. This hypothesis can be
evaluated with a survey of the species composition at
reference and contaminated sites or in a pollution
gradient, in combination with data on the occurrence
of adaptation and the sensitivity of those species. At
present, the NOEC component is the only one which
has been investigated in some detail. The presence of
various adapted species at contaminated sites
indicates that genetic variation in those species
allowed for a fast adaptation, apparently without
costs being limiting (e.g. 0. cincta, D. melanogaster).
 Metal adaptation in invertebrates
6.3. The importance of other stress factors
An additional possibility to explain the loss of
species at contaminated sites is the exposure to other
stress factors, i.e. multiple stress. Various examples
indicate increased sensitivity of stressed populations
exposed to another stress. On the one hand, within a
single generation, sulphur-dioxide exposure makes
the grass Lo&m perenne more sensitive to low tem-
peratures (Davison and Bailey, 1982), and parasitism
makes the snail Lymnaea stagnal~ more sensitive to
zinc (Guth et al., 1977). On the other hand, in
subsequent generations, strong directional selection
may ultimately cause a reduction of genetic variation
of tolerance characteristics, which causes the speed of
potential responses to stress to decline over gener-
ations. If tolerance is genetically correlated with
fitness, then the genetic variation of fitness character-
istics will also reduce proportionally to the strength
of the correlation. This may reduce the potentials to
maintain a viable population during “natural”
environmental extremes or during exposure to other
pollutants. Whether this is the case should be investi-
gated with appropriate genetic techniques.
6.4. Ecological consequences
The recognition of the occurrence of heavy-metal
adaptation, and of the fundamental processes
associated with it, may have various consequences.
We identify, for example, the use of adaptation
knowledge for the recognition of metal flora to
prospect metal-ore deposits in ancient times; for the
development and application rate of metal-contain-
ing pesticides to reduce risk of adaptation; for recol-
onization of derelict areas, to start some biological
activity leading to recovery; for the recognition of a
need for increased micronutrients in cultures of
tolerant populations; for the use of a species in
biomonitoring; and for the assessment of effects of
trophic levels other than that of the adapted species.
The exact considerations for a species are numerable,
and fall outside the scope of this review. However,
important ecological consequences have been recog
nized in relation to the protection of species at
polluted sites.
Data on toxicity of metals to soil invertebrates may
be used for deriving maximum acceptable concen-
trations for clean soils and trigger values for remedial
action. In one of the approaches, data obtained from
a small representative group of species are taken as
input for a statistical extrapolation model (Van
Straalen, 1993a). These data are used to assess the
frequency distribution of species’ sensitivities in the
ecosystem as a whole.
Incorporation of sensitivity data from adapted
populations may shift the distribution towards toler-
ance, so that protection criteria may be biased. On
the other hand, the output of the extrapolation
method consists of the percentage of species in an
ecosystem which may be exposed beyond their
33
NOEC at a given concentration of a pollutant. Slight,
intermediate, or serious toxic effects may occur in
these species. The nature of the affected species
remains, however, unclear. The method does not give
a clue towards the ecological consequences associated
with intoxicating a few sensitive species. There may
be serious consequences for ecosystem function if the
affected species are a biased sample from the eco-
system, for example, when all the sensitive species
have a key role in nutrient cycling, or when they
represent Red Data Book species. Moreover, field
populations are usually exposed to a mixture of
pollutants, and to other environmental stresses. Sug
gestions to improve the present-day method of risk
analysis are given in Hopkin (1993) and Van Straalen
(1993b).
Part of the intoxicated species may be able to
adapt, since indications for adaptation processes were
found for a large number of species in the present
inventory. These species have the above mentioned
“low risk profile”, and the constituents of this profile
may give an explanation for the shift of species
composition at polluted sites. Such fast growing
species might initially prosper and disrupt the eco-
system. The distribution of species into “adapters”
and “non-adapters” may, however, prove to be
invaluable if selection alters the potential for future
responses to selection, so that “adapters” eventually
become extinct too. This is especially true if diffuse
pollution affects a large area, when recovery by
dispersal from refuge sites is impossible. On the other
hand, species composition may change due to
beneficial effects of slight increases of micronutrient
concentrations (e.g. Bengtsson and Rundgren, 1988).
The above examples show that some consider-
ations of low-level chronic exposure of a community
are clearly different from the quantitative percentual
end-points of the presently applied risk-analyses. Van
Straalen (1993b) therefore argued, that future risk-
analyses methods should incorporate the change of
populations or communities under toxicant stress,
and adaptation studies based on the application of
the criteria to demonstrate adaptation may provide
part of the theoretical background to develop such
methods.
6.5. Is there a heavy-metal fauna?
Strong evidence for metal-adaptation was obtained
for species which inhabit contaminated sites, and
which could be cultured in laboratory conditions to
obtain the evidence. Detailed studies are lacking, for
many species, partly due to culturing problems. The
collected evidence is, due to the paucity of data, not
representative for the soil community, and
conclusions on the proportions and kinds of species
that may show a response to selection by heavy
metals are not possible. One general remark is
justified. Typical heavy-metal vegetation types have
been distinguished for plant societies on heavy-metal
soil throughout the world {Ernst, 1974); evidence for
34 L. POSIWJMA and N.M. VANSTRAALEN

specific metal-fauna at metal-polluted sites, however, Bengtsson G., Gunnarsson T. and Rundgren S. (1983)
is absent. In 0. cincfa, we observed that animals from Growth changes caused by metal uptake in a population
mine and smelter populations were able to produce of Onychiurus armatus (Collembola) feeding on metal
polluted fungi. Oikos 40, 216-225.
fertile offspring after crossing to reference animals (L. Bengtsson G., Gunnarsson T. and Rundgren S. (1985a)
Posthuma, unpublished), so that the populations Influence of metals on reproduction, mortality and popu-
have to be considered ecotvues
*. of a single
- sDecies
. lation growth in Onychiurus armatus (Collembola).
rather than (sub)species. Increased tolerance in ani- .I. Appl.-Ecol. 22, 967-978.
Bengtsson G., Ohlsson L. and Rundgren S. (1985b). Influ-
mals has been achieved by alterations which are of
ence of fungi on growth and survival of Onychiurus
degree rather than of kind. This appears to be true for armatus (Collembola) in metal polluted soil. Oecologia
both aauatic (Klerks and Weis, 1987: Klerks, 1990), (Berl.) 68. 63-68.
and terrestrial species (this review). Bengtss& 6. and Rundgren S. (1988) The Gusum case: a
brass mill and the distribution of soil Collembola. Can.
J. Zool. 66, 1518-1526.
Acknowledgements-We thank M. H. Donker, S. P.
Benton M. J. and Guttman S. I. (1990) Relationship of
Hopkin, E. N. G. Joosse-van Damme, M. R. Macnair and
allozyme genotype to survivorship of mayflies (Sfenonema
the members of our Departments for stimulating discussions
femoratum) exposed to copper. J. N. Am. Benthol. Sot. 9,
and/or valuable comments on a previous draft of this
271-276.
manuscript. We gratefully acknowledge R. Dallai, M. H.
Blackman G. G. and Baker J. A. F. (1975) Resistance of the
Donker, P. Lupetti, J. Magnusson, G. Roesijadi and L.
sheep blowfly Lucilia cuprina to insecticides in the Repub-
Tranvik, who permitted us to use and reproduce their data
lic of South Africa. J. S. Afr. Vef. Ass. 46, 337-339.
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Boutron C. F., Gorlach U., Candelone J.-P., Bolshov M. A.
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