Aquatic Toxicology 60 (2002) 85–99 www.elsevier.com/locate/aquatox

Genetic variability in sublethal tolerance to mixtures ofcadmium and zinc in clones of Daphnia magna Straus

Carlos Barata a,c,*, Scott J. Markich b, Donald J. Baird c, Graeme Taylor c,Amadeu M.V.M. Soares a

a Departamento de Biologia, Uni�ersidade de A�eiro, Campus de Santiago, 3810-193 A�eiro, Portugalb En�ironment Di�ision, Australian Nuclear Science and Technology Organisation, Pri�ate Mail Bag 1, Menai,

New South Wales 2234, Australiac Institute of Aquaculture, Uni�ersity of Stirling, En�ironment Group, Stirling, Scotland FK9 4LA, UK

Received 11 July 2001; received in revised form 22 November 2001; accepted 23 November 2001

Abstract

To date, studies on genetic variability in the tolerance of aquatic biota to chemicals have focused on exposure tosingle chemicals. In the field, metals occur as elemental mixtures, and thus it is essential to study whether the geneticconsequences of exposure to such mixtures differs from response to single chemicals. This study determined thefeeding responses of three Daphnia magna Straus clones exposed to Cd and Zn, both individually and as mixtures.Tolerance to mixtures of Cd and Zn was expressed as the proportional feeding depression of D. magna to Cd atincreasing zinc concentrations. A quantitative genetic analysis revealed that genotype and genotype×environmentalfactors governed population responses to mixtures of both metals. More specifically, genetic variation in tolerance tosublethal levels of Cd decreased at those Zn concentrations where there were no effects on feeding, and increasedagain at Zn concentrations that affected feeding. The existence of genotype×environmental interactions indicatedthat the genetic consequences of exposing D. magna to mixtures of Cd and Zn cannot be predicted from the animals’response to single metals alone. Therefore, current ecological risk assessment methodologies for predicting the effectsof chemical mixtures may wish to incorporate the concept of genetic variability. Furthermore, exposure to low andmoderate concentrations of Zn increased the sublethal tolerance to Cd. This induction of tolerance to Cd by Zn wasalso observed for D. magna fed algae pre-loaded with both metals. Furthermore, in only one clone, physiologicalacclimatisation to zinc also induced tolerance to cadmium. These results suggest that the feeding responses of D.magna may be related to gut poisoning induced by the release of metals from algae under low pH conditions. Inparticular, both induction of metallothionein synthesis by Zn and competition between Zn and Cd ions for uptakeat target sites on the gut wall may be involved in determining sublethal responses to mixtures of both metals. © 2002Elsevier Science B.V. All rights reserved.

Keywords: Daphnia ; Genetic variation; Cadmium; Zinc; Mixture toxicity; Feeding

* Corresponding author. Tel.: +44-1786-467-874; fax: +44-1786-472-133.E-mail address: cb5@stir.ac.uk (C. Barata).

0166-445X/02/$ - see front matter © 2002 Elsevier Science B.V. All rights reserved.

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C. Barata et al. / Aquatic Toxicology 60 (2002) 85–9986

1. Introduction

It has long been recognised that to extrapolatecredibly from individuals in the laboratory tofield populations, it is essential to understandhow genetic factors in the field modify responsesmeasured in the laboratory (Forbes, 1998). Cur-rent knowledge about genetic variability in toler-ance, or adaptability, is typically based uponexposure of organisms to single chemicals(Forbes, 1998), however, field populations of or-ganisms are invariably exposed to chemical mix-tures. The occurrence of differences in toleranceto single chemicals amongst populations, individ-uals and clones within a species, is a well-knownphenomenon (Forbes, 1998). Reported differ-ences in the degree of genetic variation in re-sponse to different chemicals, within singlechemicals across toxic stress levels or across spe-cies (Baird et al., 1991; Barata et al., 1998,2000b; Forbes 1998), indicate that genotype-envi-ronmental interactions play an important role indetermining population responses to toxicants.Therefore, it is likely that genetic variability intolerance to single chemicals could vary with thepresence of other toxicants.

Two competing hypotheses have been pro-posed to explain how exposure to a chemicalmixtures will affect genetic variability in toler-ance within populations, and hence, the likeli-hood of tolerance evolution. Klerks (1998)postulated that an increase in number of chemi-cals (in a mixture) should bring about a decreasein genetic variation, as population sizes would befurther reduced and more phenotypes would beeliminated (the ones sensitive to any of the chem-icals or their interactions) relative to a singlechemical exposure. As a result, exposure to mix-ture of chemicals may reduce selection intensityfrom individual chemicals, and hence, retard theevolution of tolerance. Conversely, Hoffmannand Hercus (2000) postulate that increase instress from simultaneous exposure to multiplechemicals may influence evolutionary rates bygenerating genetic variability through the induc-tion of novel gene expression or increased levelsof mutation rates. Although neither of these hy-potheses is clearly supported by existing studies

on pesticide and antibiotic resistance, there ap-pears to be a widespread assumption that geneticvariability in tolerance to chemicals will reducein those cases in which combinations of antago-nistically acting chemicals are used (Klerks,1998).

Several studies have shown Cd and Zn to actantagonistically in freshwater biota, includingDaphnia magna Straus (Wicklund et al., 1988;Bennett and Brooks, 1989; Bodar et al., 1990;Stuhlbacher et al., 1992, 1993; Canizares-Vil-lanueva et al., 2000). The study of the sublethalresponses of different clones of D. magna ex-posed to Cd and Zn, both individually and as amixture, offers a working model to determinewhether genetic variation in the tolerance of D.magna to chemicals may decrease when exposedto mixtures of two antagonistically acting metals,relative to exposure of the single metals. Since D.magna can reproduce asexually by ameioticparthenogenesis (Hebert, 1987), single genotypescan be tested across a range of Cd and Zn con-centrations. Therefore, genotype, environmental,and genotype×environment components of vari-ance can be assessed (Barata et al., 1998,2000a,b). Cadmium and Zn co-occur in manyaquatic environments as a result of industrialand municipal effluents and urban runoff (Hare,1992). Cadmium is a non-essential metal that ishighly toxic to D. magna at low concentrationsin freshwater systems (Barata et al., 1998; Barataand Baird, 2000). In contrast, Zn is an essentialmetal that is moderately toxic to D. magna(Barata et al., 1998).

Response variation to single metals has beenrelated to the ability of individual organisms todevelop resistance through physiological acclima-tisation (Klerks and Weis, 1987). Previous stud-ies conducted on Daphnia have reported that zincinduces resistance to cadmium through synthesisof metallothionein-like proteins (Stuhlbacher etal., 1992, 1993; Canizares-Villanueva et al.,2000). Thus, it is predicted that the presence ofZn will ameliorate the toxicity of Cd in a mix-ture. Hence, variability in sublethal responses tomixtures of Cd and Zn could be related to ge-netic differences in the ability of Zn to induceresistance to Cd. Nevertheless, most studies on

C. Barata et al. / Aquatic Toxicology 60 (2002) 85–99 87

induction of Cd resistance by Zn have beenbased on survival data conducted at high metalconcentrations in the absence of food. Thus it ispossible to expect that other mechanisms mayalso be involved in determining sublethal re-sponses to mixtures of cadmium and zinc.

Recent studies have found that although theuptake or sublethal toxicity of metals like Cd ingrazers or predators, such as D. magna, are usu-ally correlated to Cd concentrations dissolved inwater, direct uptake or toxicity from Cd in wa-ter is probably not the most important pathway.Rather, the correlation is more likely a conse-quence of Cd uptake from water by organismsat a lower level in the food chain (e.g. algae fora grazer) (Hare, 1992; Munger and Hare, 1997;Taylor et al., 1998). Therefore, the sublethaltoxicity of mixtures of metals such as Cd andZn to grazers like D. magna may be governedby indirect metal uptake from water by algae,instead of direct uptake from water by D.magna.

In the present study, the feeding responses ofdifferent clones of D. magna exposed to Cd andZn were determined under three different expo-sure regimes:1. Exposure of D. magna to Cd and Zn, both

individually and as a mixture, in the presenceof food (algae);

2. Exposure of D. magna to single solutions ofCd, following pre-exposure to Zn, in the pres-ence of food (algae); and

3. Exposure of D. magna to algae (pre-loadedwith Cd and Zn) in water.

The hypothesis tested was: (1) genetic variationin tolerance of D. magna should decrease whenexposed to mixtures of Cd and Zn (two antago-nistically acting chemicals), relative to single metalexposures. We also investigate until which extent(2) induction of tolerance to cadmium by zincand/or (3) the metal fraction sorbed by a foodsource (algae) determined the observed feedingresponses across mixtures of cadmium and zinc.Feeding rate was selected to assess sublethal toxic-ity, since feeding impairment is one of the causalmechanisms of population responses of D. magnato chemicals (Barata and Baird, 2000).

2. Material and methods

2.1. Experimental animals

Three genetically distinct genotypes (S-28, G-19, G-62), or clones, of D. magna were selectedfrom 80 clones obtained from two field popula-tions originating from Spain and Germany(Barata et al., 2002). The three clones were se-lected as extreme genotypes on the basis of theirrelative (i.e. low to high) sublethal tolerance toCd. Therefore, differences in tolerance to Cdamongst these clones may indicate about howmuch genetic variation in tolerance to Cd existsamongst D. magna field populations. For eachclone, two 2 l bulk cultures of 15 animals eachwere maintained in ASTM hard synthetic water(APHA-AWWA-WEF, 1995) as described byBarata et al. (1998). Animals were fed daily withChlorella �ulgaris Beijerinck (5×105 cells ml−1,corresponding to 1.8 mg C l−1, Barata and Baird,1998). The culture medium was maintained at20 °C and changed every 48 h. Approximately,100–120 neonates (�24 h old) were transferredto 2 l tanks and reared under the same conditionsas their mothers until they reached the 4th instarstage (4–5 days). Once animals reached this stagethey were used in feeding tests. To minimise ma-ternal effects such as differences in toxicity due tochanging culture conditions (Stuhlbacher et al.,1992), only neonates that originated from third tosixth brood females from clonal lines previouslyacclimated to laboratory conditions for one yearwere used.

2.2. Feeding experiments

The effects of single solutions and mixtures ofCd and Zn on the feeding behaviour of three D.magna clones were investigated by exposinggroups of four 4th-instar juveniles. Feeding exper-iments were conducted using the protocol de-scribed by Barata et al. (2000b). A control (no Cdand Zn added, hereafter referred as 0 �g l−1) andfive concentrations of Cd (0.5, 1.2, 4, 10 �g l−1)and Zn (10, 100, 200, 400, 1000 �g l−1) were used,each with 5–6 replicates. A Zn concentration of10 �g l−1 was used to approximate dissolved Zn

C. Barata et al. / Aquatic Toxicology 60 (2002) 85–9988

measured in minimally polluted freshwater sys-tems (Hare and Tessier, 1998). Total metal con-centrations were prepared by adding appropriateamounts of Cd and Zn stock solutions to ASTMhard water (see Section 2.4). C. �ulgaris wasadded to the test solutions at a cell density of5×105 cells ml−1. Tests were conducted in 170ml borosilicate glass vessels with 150 ml of testsolution. Each group of replicates consisted offour or five vessels with animals and one blankwith no animals. Blanks were used to ensure thatthe initial algal cell density did not increase sig-nificantly over the exposure period.

At the start of each experiment, groups of fourjuveniles were randomly distributed among thereplicates of each feeding treatment and allowedto feed for a period of 24 h, after which they wereremoved from the vessels. Juveniles were trans-ferred immediately after their third molt to avoidmolting during the experiment. Feeding experi-ments were conducted in darkness to minimisealgal growth. Feeding experiments using the threeclones were either conducted simultaneously, orwithin two days. Individual feeding rates (cellsanimal−1 h−1) were determined as the change inalgal cell density over 24 h, according to themethod of Allen et al. (1995). Algal cell densitywas measured using a Coulter Multisizer particlecounter.

2.3. Exposure scenarios

To test the three objectives outlined in Section1, D. magna clones were exposed to Cd and Znunder three different scenarios:

2.3.1. Scenario (1)To test the hypothesis that genetic variation in

tolerance of D. magna will decrease when exposedto mixtures of Cd and Zn relative to single metalexposures, D. magna clones were exposed to Cdand Zn, both individually and as a mixture, in thepresence of algae. Test solutions consisted in allpossible combinations (30) of six (0, 0.5, 1, 2, 4,10 �g l−1) and five (0, 10, 100, 200, 400 �g l−1)concentrations of cadmium and zinc, respectively.Concentrations of Zn with no or moderate (�EC50) effects on feeding were selected.

2.3.2. Scenario (2)To test the second hypothesis that pre-exposure

of D. magna to Zn induces tolerance to Cd,feeding responses of D. magna to Cd followingpre-exposure to Zn (pre-acclimated animals), werecompared with the feeding responses obtained inscenario 1 under exposure to mixtures of cad-mium and zinc (no pre-acclimated animals). Pre-exposure to Zn was conducted by exposinggroups of 100–120 third instar juveniles to fiveconcentrations of Zn (0, 10, 100, 200, 400) for 24h under the same conditions as juveniles used toassess the tolerance to mixtures of Cd and Zn (inSection 2.3.1). After pre-exposure to Zn, animalswere transferred to clean ASTM hard water twiceto remove any residual Zn and then used to assessfeeding responses to Cd exposures (0, 0.5, 1, 2, 4,10). This procedure minimised any differences inexposure regimes, since juveniles of the same agewere reared under similar conditions.

2.3.3. Scenario (3)To test the third hypothesis, that the metal

fraction sorbed by a food source governs thefeeding responses of D. magna, feeding responsesof D. magna to algae pre-loaded with both metals(i.e. food only) in clean ASTM medium werecompared with individuals exposed to solutions ofsingle-element and mixtures of Cd and Zn in thepresence of algae (i.e. food+water). Within eachtreatment, animals were exposed to a single Cdconcentration (10 �g l−1) and five zinc concentra-tions (0, 10, 100, 200, 400 �g l−1). In the foodonly treatment, algae cells were pre-loaded byadding Cd and Zn to a 1 l sample of algae with acell density of 5×105 cells ml−1 and left for 24 hin darkness. The algae were then centrifuged(washed in ASTM water) and re-suspended inASTM hard water.

2.4. Preparation of stock solutions

Stock solutions of Cd and Zn were prepared byadding analytical reagent grade (BDH Poole, UK)salts (i.e. 3CdSO4·8H2O and ZnSO4·7H2O, respec-tively) to deionised water (Milli-Q; 18 M� cm−1

resistivity). Nominal test concentrations rangingfrom 0 to 10 �g l−1 for Cd and from 0 to 1000

C. Barata et al. / Aquatic Toxicology 60 (2002) 85–99 89

�g l−1 for Zn, were subsequently prepared byadding appropriate aliquot volumes of eachmetal stock solution to the synthetic ASTMhard water. Prior to preparing the nominal testconcentrations, stock solutions were filteredthrough a 0.2 �m filter (Millipore) to removeparticulate matter. Duplicate unfiltered andfiltered (0.2 �m filter) water samples (10 ml)were collected from experimental vessels at thebeginning (t=0 h) and end (t=24 h) of thetests to assess total and dissolved aqueous metalconcentrations. Water samples were then aci-dified (pH�1) with concentrated nitric acid(Merck, Ultrapur).

2.5. Metal sorption by algae

The sorption of Cd and Zn by C. �ulgariswas determined using the protocol described byTaylor et al. (1998). Algal cells were exposed toeight concentrations of Cd and Zn (0–100�g l−1 for Cd and 0–2000 �g l−1 for Zn). Eachtest solution was prepared by adding appropri-ate amounts of Cd or Zn stock solutions toASTM hard water. C. �ulgaris was added at acell density of 5×105 cells ml−1. Experimentaltreatments consisted of a control (no algal cellsadded) plus the test concentrations, each withtwo or three replicates. Analysis of controls al-lowed a determination of any Cd or Zn thatwas a product of sample carryover or contami-nation.

Treatments were prepared in 50 ml NalgeneTeflon fluorinated ethylene propylene centrifuga-tion tubes with 40 ml of solution. Tubes con-taining each treatment were shaken and left indarkness for 24 h before being centrifuged at3600 rpm for 20 min at 5 °C. The supernatantwas then carefully decanted, leaving the pelletedcells at the bottom of the tube. To each of thetubes, 1 ml of ASTM hard water was added,and the tube contents mixed for 30 s. The sus-pension was then layered onto 0.5 ml of 40%sucrose solution in 2.5 ml microcentrifugesilanised tubes. The samples were then cen-trifuged at 13 000 rpm for 5 min in an M.S.E.Micro Centaur bench ultra-centrifuge (Fisons,UK). The resulting supernatant was drawn off

and the remained pellet was then treated with 1ml of 50% double-distilled HNO3 and stored inthe refrigerator at 4 °C until analysis. Overall,the experimental conditions used in algal sorp-tion experiments reflected those used in feedingexperiments.

2.6. Element analyses

Concentrations of Na, K, Ca and Mg weremeasured using inductively coupled plasmaatomic-emission spectrometry (Perkin–ElmerOptima 3000 DV). Cadmium and Zn concentra-tions were measured using graphite furnaceatomic absorption spectrometry (ATI UnicamModel 939). Calibration standards and a reagentblank were analysed with every 10 samples tomonitor signal drift. In every instance, the signaltypically changed by 3–5% throughout an ana-lytical run. Although measured metal concentra-tions at the end of tests (t=24 h) were within10% of their initial measured values (t=0), thetwo measures were pooled for data analysis.

Duplicate water samples were also collectedfor the measurement of anions and generalphysico-chemical parameters. Chloride, sulphateand nitrate were determined by ion chromatog-raphy using an anion exchange column (DionexAS4A) with suppressed conductometric detec-tion, as described by Hansbury and Dyke(1992). Total hardness and alkalinity were deter-mined using standard methods 2320B and2340C, respectively (APHA-AWWA-WEF,1995). Bicarbonate concentrations were deter-mined nomographically from alkalinity measure-ments using standard method 4500-CO2

(APHA-AWWA-WEF, 1995). pH was measuredusing an epoxy-body combination electrode,coupled to a Chemtrix pH meter, and calibratedwith standard pH buffer solutions (BDH). Thedissolved oxygen (DO) concentration was mea-sured using a polarographic oxygen electrodecoupled to a YSI model 57 DO meter. Both pHand DO concentration were measured at the be-ginning and end of the tests. Dissolved organiccarbon (DOC) was measured using the photo-catalytic oxidation method described byMatthews et al. (1990).

C. Barata et al. / Aquatic Toxicology 60 (2002) 85–9990

2.7. Geochemical speciation modelling

The speciation of Cd and Zn in the test waterswas calculated using the HARPHRQ geochemi-cal speciation model (Brown et al., 1991). Theinput parameters for HARPHRQ were based onphysico-chemical data measured from the testwaters (Table 1). Stability constants for Zn andCd were taken from Markich and Brown (1999).

2.8. Data analysis

The tolerances of D. magna to sublethal con-centrations of Cd and Zn, in single and mixedmetal exposures were expressed as effective con-centrations that inhibit feeding by 50% (EC50),relative to a control, or as proportional feedingdepression at a given measured metal concentra-tion. EC50 values for Zn exposures were obtainedby fitting feeding responses to the allosteric de-cay regression model (Allen et al., 1995), whilstEC50 values for Cd exposures were determinedfrom the square-root-y regression model (Barataet al., 2000b). Since the Zn concentrations hadno or low sublethal effects (�EC50) on feeding,tolerance to mixtures of Cd and Zn were ex-pressed as tolerance to Cd across varying Zn

concentrations. Therefore, EC50s to mixtures ofboth metals were also determined by fitting feed-ing responses to the square-root-y regressionmodel. Prior to fitting data to the regressionmodels, metal concentrations were ln trans-formed, and feeding responses converted intoproportions relative to mean control values andsquare-root transformed.

By transforming proportional feeding rates,residuals were normalised and the square-root-ycurves were linearised. Model accuracy and sig-nificance of the regression models and coeffi-cients were determined by using the coefficient ofdetermination r2, F and t tests, respectively (Zar,1996). Confidence intervals associated with toler-ance to Cd (95% of EC50) were determined asinverse predictions of feeding responses (Zar,1996), whereas those associated with tolerance toZn were estimated directly from the allostericregression model by the least-squares method(Barata et al., 2000b). The three objectives pro-posed in this study were specifically tested asfollows:

2.8.1. Objecti�e 1Since differences in the tolerance of D. magna

clones to Cd varied across the Zn concentrationstested, it was necessary to quantify the effects ofgenotype, environment and their interaction(Soares et al., 1992). Thus, the variance compo-nents of Cd concentration–proportional feedingdepression curves were determined across the Znconcentrations by using a two-way analysis ofcovariance (ANCOVA), with Zn concentrationsand clones as fixed and random factors, respec-tively, and Cd concentrations entered as a co-variate (Lynch and Walsh, 1998). Clones withquantitatively different tolerances to Cd acrossthe Zn concentrations should show a significantgenotype×environmental interaction effect (Fal-coner, 1990). Once the interaction between geno-type and environment was characterised, thevariability due to genetic heterogeneity in toler-ance to Cd at each Zn concentration was as-sessed. For each Zn concentration, genetic andenvironmental components of variation of toler-ance were estimated using one-way ANCOVA,using clone as a factor and Cd concentrations as

Table 1Measured physico-chemical parameters in the test water

Test waterParameter

Temperature 20.0�0.58.3�0.1 (48)pH

Conductivity (�S cm−1) 547�12 (5)162�2 (60)Hardness (mg l−1 CaCO3)

Alkalinity (mg l−1 CaCO3) 110�4 (5)49.8�0.3 (60)Na (mg l−1)

K (mg l−1) 3.2�0.1 (60)Ca (mg l−1) 27.0�0.3 (60)Mg (mg l−1) 23.1�0.3 (60)SO4 (mg l−1) 156�5 (8)

133�5 (5)HCO3 (mg l−1)2.9�0.1 (8)Cl (mg l−1)

NO3 (�g l−1) 257�98 (8)PO4 (�g l−1) 14�5 (8)Dissolved organic carbon (mg l−1) 0.4�0.1 (12)

Values are reported as the mean�95% confidence limits, withthe number of samples (n) analysed in parentheses.

C. Barata et al. / Aquatic Toxicology 60 (2002) 85–99 91

a covariate. Genetic variability in tolerance to Cdacross Zn concentrations was then characterisedusing the standardised genetic coefficients of vari-ation (CVg) (Barata et al., 2000a,b) and broadsense heritabilities (H) (Falconer, 1989) as shownin Eqs. (1) and (2), respectively.

CVg=�Vg

X(1)

H=Vg

Vg+Ve

(2)

where Vg and Ve are the genetic and environmen-tal components of variance and X is the meanproportional feeding depression rate. By usingCVg, changes of variance due to scaling effects(changes of the mean) are avoided, while H ex-presses the proportion of variability explained bygenetic factors. Coefficients of variation were thencompared using Miller and Miller and Feltz tests,whereas Chi-square and Fisher exact tests wereused to compare heritability values (Zar, 1996).Prior to analyses, proportional feeding responsesand Cd concentrations were square-root and lntransformed, respectively, to linearise concentra-tion– feeding response curves.

2.8.2. Objecti�e 2For each D. magna clone, Cd concentration–

proportional feeding depression curves were com-pared between pre-acclimated and nopre-acclimated animals across Zn concentrationsusing a two-way ANCOVA with Zn concentra-tions and ‘acclimatisation scenario’ as fixed fac-tors, and Cd concentrations entered as a covariate(Lynch and Walsh, 1998). Proportional feedingdepression responses and Cd concentrations weresquare-root and ln transformed, respectively, tolinearise the curves.

2.8.3. Objecti�e 3For each D. magna clone, proportional feeding

depression responses (square-root transformed)between food only and food+water treatmentsacross zinc concentrations were compared by us-ing two-way ANOVA with exposure treatmentand Zn as fixed factors.

Table 2Nominal and measured concentrations (�g l−1) of Cd and Znin the test waters

Nominal Measured

Total Dissolveda AlgaeTotal Dissolvedb

Zinc0 1.91�0.06 0.91�0.06 0.96�0.38 0.80�0.02

8.58�0.31 6.43�0.3210 2.06�0.54 1.22�0.1948.1�2.6 14.6�1.2100 2.03�0.1464.3�2.5

122�7.4200 95.5�7.5 27.9�6.0 4.68�0.80266�17.6400 217�19.5 41.6�1.8 7.33�1.46

120�40.2571�45.61000 697�42.4

Cadmium0.02�0.010 0.02�0.01 �0.01 �0.01

0.47�0.080.53�0.050.5 0.05�0.021.16�0.11 1.01�0.141 0.13�0.03

1.88�0.112 0.24�0.022.28�0.094.54�0.21 3.93�0.254 0.50�0.04

10 9.86�0.86 0.28�0.0111.0�0.79 0.91�0.10

Values are reported as the mean�95% confidence limits;n=14 for total metal, n=4 for dissolved metal, n=2–3 formetal in algae. Metal in algae was obtained from Fig. 1.

a Dissolved aqueous metal concentration in those experi-ments where animals were exposed to Cd and Zn in food andwater.

b Dissolved aqueous metal concentrations in the food onlytreatment.

3. Results

3.1. Water chemistry

Measured physico-chemical data for ASTMhard water are given in Table 1. The water hard-ness, alkalinity and pH were within the rangeexpected for hard synthetic water (APHA-AWWA-WEF, 1995). Measured concentrations ofmajor cations and anions were within 10% of theirnominal concentrations. The initial pH values inthe test waters were not significantly (P�0.05;t-test) different to the final pH values (i.e. �0.1pH unit). Dissolved oxygen concentrations alwaysapproached near-saturation. Measured back-ground concentrations of DOC were very low (0.4mg l−1); the small amount present may have beenfrom exudates of the test animals and/or algae(Fish and Morel, 1983).

C. Barata et al. / Aquatic Toxicology 60 (2002) 85–9992

Measured concentrations of Cd and Zn in thetest were within 10 and 30% of their nominalconcentrations, respectively (Table 2). Meanmeasured background concentrations of Cd(0.025 �g l−1) and Zn (1.9 �g l−1) were very low,indicating that contamination was minimal(Table 2). In experiments where animals wereexposed to Cd and Zn in water, the dissolvedconcentrations of Cd and Zn were 87 and 79%of their total concentrations, respectively, indicat-ing that most of the metal was dissolved in thewater. Conversely, when animals were exposed towater with metal-exposed algae, the dissolvedconcentrations of Cd and Zn were between oneand two orders of magnitude lower than thoseobserved in exposures to water only. The lowdissolved Cd and Zn concentrations in the foodonly treatment suggests that metal losses fromalgae, or carry over from spiking samples, wereminimal. Thus, there was little dissolved Cd andZn available to affect feeding. As expected, onlya small fraction of total measured Cd and Znwas sorbed onto algal cells (10 and 20% of totalCd and Zn, respectively).

3.2. Metal sorption kinetics by algae

The sorption kinetics of Cd and Zn for C.�ulgaris cells, which include both the adsorbedand absorbed fraction, are shown in Fig. 1. Sam-ple carryover of Cd and Zn from laboratoryware or supernatant was 0.5 and 3%, respec-tively, from analysis of treatments where no algalcells were present. Data was fitted to theMichaelis–Menten equation. The saturation con-stant (Kmax) for Cd was 13.9×10−9 and 36.9×10−8 �g cell−1, for Zn. The corresponding halfsaturation values were determined to be 64.8�g Cd l−1 and 889.8 �g Zn l−1. Fig. 1 shows thatacross the concentrations used in the feeding tri-als (�11 �g Cd l−1 and �697 �g Zn l−1), therewas a rapid increase in the mass of Cd and Znsorbed onto the cells as dissolved metal concen-tration increased.

3.3. Geochemical speciation modelling

The free metal ion (M2+) is predicted to be

the major species of both Cd (64%) and Zn(45%) in the test waters, with minor proportionsof Cd and Zn carbonate and sulfate. The pro-portion of all metal species remained constantwithin the range of test concentrations across allexperiments. This means that differences in toxic-ity between exposure sources (i.e. exposure tometal mixtures in algae only or in both waterand algae) are unlikely to be related to differ-ences in speciation. It is unclear which metalsource (water or algae) regulates sublethal re-sponses to metals in Daphnia (Hare, 1992;Munger et al., 1999). Moreover, we did not mea-sure which metal fraction was taken up by algae.Therefore, all results will be referred as totalmetal concentration.

Fig. 1. Concentration of Cd (A) and Zn (B) per cell ofChlorella �ulgaris at increasing concentrations of total ionicmetal at cell density of 5×105 cells ml−1. Curves were fittedaccording to Michaelis–Menten kinetics.

C. Barata et al. / Aquatic Toxicology 60 (2002) 85–99 93

Fig. 2. Feeding rates of each of the three D. magna clones asa function of (A) Cd and (B) Zn concentration. Absolute feedingrates were converted to proportions by dividing by the mean ofthe control rate and fitting to a square-root-y regression model(A) or to an allosteric decay non-linear model (B). Effectiveconcentrations (EC50�5% CI) estimated for each clone aredepicted beside each graph. Error bars indicate 95% CI.

were the most tolerant to Cd and Zn, respectively,and clone S-28 was the most sensitive to bothmetals.

Exposure concentrations of Cd (�11 �g l−1)and Zn (�266 �g l−1) with high and low sub-lethal effects on feeding, respectively, were se-lected to explore whether genotype responses tometal mixtures varied from those occurring inresponse to single metal exposures. For brevity,the 24 concentration– feeding response curvesderived from exposure to mixtures of Cd and Zn,and of Cd after acclimatisation to Zn are notshown. Nevertheless, all feeding responses to Cdacross Zn concentrations or after acclimatisationto Zn follow the same pattern as those shown inFig. 2A. In all cases, the regression models weresignificant (P�0.05), and their residuals met theassumption of normality and variance homo-cedasticity (Shapiro–Wilk and Bartlett tests, re-spectively, P�0.05).

3.5. Genetic �ariation in sublethal tolerance tosingle metal and mixed metal exposures

Sublethal tolerance (EC50) of D. magna clonesto Cd across increasing Zn concentrations isshown in Fig. 3. Overall, tolerance to Cd acrossZn concentrations showed a bell shaped pattern.For clones G-19 and G-62, tolerance to Cd in-creased significantly (P�0.05) with Zn, until athreshold level of 122 �g l−1, above which toler-ance to Cd decreased with Zn. Clone S-28 showeda similar pattern but its threshold Zn concentra-tion was lower (64 �g l−1). The high 95% confi-dence interval associated with the EC valuesobtained for clones G-19 and G-62 at some Znconcentrations were a consequence of the ob-served low effects of Cd on feeding responses.

Only the genotype and genotype×environmentcomponents of variation of the feeding responsesof the clones across single and mixed metal expo-sures were significant (P�0.05) (Table 3). Bothgenetic and interaction components contributedequally to the observed variability (Table 3).These results indicate that clonal differences intolerance to Cd varied within and across Zn con-centrations (Fig. 3). A further characterisation ofthe feeding responses of the clones to Cd, within

3.4. Feeding responses to single and mixtures of Cdand Zn

Proportional feeding rates (square-root trans-formed) of the three clones showed a decreasinglinear and threshold response to increasing con-centrations of Cd and Zn, respectively (Fig. 2Aand B). For Zn, the allosteric decay model waschosen because it closely matched the pattern ofan initial period of no or low response (�122�g l−1), followed by an accelerated negative re-sponse as concentrations increase (Fig. 2B). TheEC50 derived from the concentration– feedingcurves (Fig. 2) showed substantial and marginaldifferences in tolerance to Cd and Zn, respec-tively, amongst the clones. Clones G-19 and G-62

C. Barata et al. / Aquatic Toxicology 60 (2002) 85–9994

Fig. 3. Tolerance to Cd (expressed as effective concentrationswith 50% of proportional depression of feeding, EC50) of threeD. magna clones across increasing Zn concentrations. Cad-mium and Zn concentrations are shown in ln scale. EC50

(�95% CI) are not significant different (P�0.05) if they bearthe same letter. Significance was determined by an ANCOVAtest followed by Tukey multiple comparison test among eleva-tions.

Table 4Genetic coefficients of variation (CVg) and heritabilities (H)for proportional feeding depression responses of the three D.magna clones to Cd at varying Zn concentrations

CVgnZn (�g l−1) H

1481.91 7.9AB 32.4B

22.1B6.4AB8.58 10810864.3 3.0A 6.8A

13.4BC 72.4C122 10875.3C26.0C266 108

n=sample size. Letters following the CVg and H that differ invalue indicate whether groups differed significantly at the 5%levels.

0.05). A comparison of standardised genetic com-ponents of variance and heritabilities across Znconcentrations revealed a significant (P�0.05)decrease of genetic variability in tolerance to Cdat intermediate concentrations of Zn, followed bya significant increase of tolerance with increasingZn concentrations (Table 4).

3.6. Effect of Zn acclimatisation on Cd tolerance

Tolerance to Cd (expressed as EC50 on feeding)of animals from the three D. magna clones ex-posed simultaneously to Cd and Zn mixtures (re-ferred as no pre-acclimated individuals) comparedwith that of animals pre-acclimated to Zn andexposed to single Cd solutions are depicted in Fig.4. Within each D. magna clone, ANCOVA resultsindicated that tolerance to cadmium after pre-ac-climatisation to zinc differed significantly (P�0.05) from that observed from direct exposure tomixtures of cadmium and zinc (F-ratios for expo-sure scenario and interaction effects, respectively,were F1, 279=16.4, F3, 279=27.1 for clone S-28;F1, 279=14.4, F3, 279=2.9 for clone G-19; F1, 279=26.9, F3, 279=5.7 for clone G-62). In particularonly in clone G-19 was there any evidence ofinduction of cadmium resistance through pre-ex-posure to zinc (Fig. 4). In the remaining twoclones (G-62, S-28), tolerance to Cd did not varysignificantly across increasing pre-exposure levelsof Zn.

each Zn concentration, denoted significant geno-type variability for most concentrations except at64 �g l−1 (based on one-way ANCOVAs, P�

Table 3Genotype (clonal), environment (Zn concentration), geno-type×environment and residual (error) variance componentsfor the ANCOVA carried out on proportional feeding re-sponses (square-root transformed) to mixtures of Cd and Znconcentrations

% VarianceSource F-ratiodf

257.1***Genotype 31.024 16.8 2.9Environment

38.6***25.7Genotype×environment 826.5565Residual

Log-transformed cadmium concentrations were entered as acovariate, and clone and Zn concentrations as random andfixed factors, respectively. ***Significance at P�0.001.

C. Barata et al. / Aquatic Toxicology 60 (2002) 85–99 95

3.7. Comparison of water and food as a metalsource in tolerance

Feeding rates of three D. magna clones exposedto mixtures of Cd and Zn concentrations, either inalgae only or in water+algae are shown in Fig. 5.According to Table 2, in the algae only treat-ments, animals were exposed to 0.91 �g l−1 of Cdand to 0.96–41.6 �g l−1 of Zn, whereas in thewater+algae treatment, metal concentrationswere much higher, 11 �g l−1 for Cd and 1.91–

Fig. 5. Feeding rates of three D. magna clones exposed to Cd(11 �g l−1) and increasing concentrations of Zn either frompre-loaded algae (algae only treatment) or from both waterand algae (water+algae treatment). For clarity only totalconcentrations of Zn, depicted in ln scale are shown. Mea-sured total metal concentrations in algae and dissolved inwater are shown in Table 2. Absolute feeding rates wereconverted to proportions by dividing by the mean of thecontrol rate and square-root transformed. For each Zn treat-ment, significant (P�0.05) differences between EC50 (�95%confidence intervals) from algae only and algae+water treat-ments are indicated by an asterisk. Significance was deter-mined by ANOVA followed by Tukey multiple comparisontest.

Fig. 4. Effective Cd concentrations of proportional depressionof feeding (EC50) in three D. magna clones under simultaneousexposure to Cd and Zn (referred as no pre-acclimated individ-uals, Fig. 3) compared with animals pre-acclimated to Zn andexposed to Cd. For each Zn treatment significant (P�0.05)differences between EC50 (�95% CI) from pre-acclimated andno pre-acclimated individuals are indicated by an asterisk.Significance was determined by ANCOVA followed by Tukeymultiple comparison test among elevations.

1266 �g l−1 for Zn. For each D. magna clone,feeding responses to mixtures of Cd and Zn dif-fered significantly (P�0.05) between exposuresources (algae vs. algae+water) within and acrossincreasing concentrations of Zn (F-ratios for ex-posure source and interaction effects, respectively,were F1, 40=227.7, F4, 40=38.7 for clone S-28;F1, 40=511.2, F4, 40=316.1 for clone G-19;

C. Barata et al. / Aquatic Toxicology 60 (2002) 85–9996

F1, 40=90.7, F4, 40=37.7 for clone G-62). Overall,feeding rates were less affected by Cd and Znwhen animals were exposed to metal-exposed al-gae than when exposed to algae in metal-spikedwater. Nevertheless, for clones G-19 and G-62feeding responses in three out of five mixturecombinations of Cd and Zn were similar whenexposed to metal-exposed algae or water+algae(Fig. 5).

4. Discussion

This study found that genetic variation in toler-ance to sublethal exposure levels of cadmium,expressed as standardised genetic components ofvariance and heritability levels, decreased at thoseexposure levels of zinc with no effects on feeding,and increased again across those zinc concentra-tions that affected feeding. These results partlysupport two competing hypotheses: In accordancewith the hypothesis postulated by Klerks (1998),this study provided evidence that an increase inchemical complexity (i.e. mixture of Cd and Zn)resulted in a decrease in genetic variation, at lowZn concentrations. Moreover, the results alsoagree in part with the hypothesis postulated byHoffmann and Hercus (2000), that genetic vari-ability in tolerance to chemical stress should in-crease with increasing stressful conditions (i.e. athigh Zn concentrations). Nevertheless, the datafrom this study, as well as those of Klerks (1998)and Hoffmann and Hercus (2000), agree with thegeneral hypothesis that the relative response ofgenotypes across an environmental stress gradient(reaction norms sensu Falconer 1990), are likelyto cross among optimal, low and extreme stresslevels (Hoffmann and Parsons, 1991).

Studies on insect resistance to pesticides haveshown that combinations of pesticides preventedthe development of resistance when there wascross-resistance (negative genetic correlations)among the chemicals used (Georghiou, 1990; Im-maraju et al., 1990; Huang et al., 1994). Althoughgenetic variability in tolerance to pesticides wasnot directly assessed in previous studies, the rateof evolution of resistance to a given chemical isdirectly related to the amount of genetic variabil-

ity in tolerance to that chemical (Falconer, 1989).This means that the slower the rate of evolutionof resistance, the less genetic variability in toler-ance. This study showed that the relative re-sponses of D. magna clones varied across singlesolutions of Cd or Zn (Fig. 2), thus indicating theexistence of negative genetic correlations. In par-ticular clones G-19 and G-62 were the most toler-ant to Cd and Zn, respectively (Fig. 2). As aresult, when both clones were exposed to mixturesof Cd and Zn, their tolerance curves crossed withincreasing contaminant complexity (EC50 to Cdacross Zn concentrations, Fig. 3). Hence, geneticvariability in tolerance to mixtures of both metalsdecreased at those metal combinations where re-action norms crossed, and increased again as mix-ture combinations moved away from the point ofintersection. Thus, the data reported for Cd andZn support the hypotheses proposed by bothKlerks (1998) and Hoffmann and Parsons (1991).

Studies on many organisms suggest that envi-ronmental stress may influence evolutionary ratesby increasing the expression of genetic variabilityin life-history traits (Hoffmann and Parsons,1991; Barata et al., 2000a,b; Stanton et al., 2000).Experimental studies on Drosophila populationshave reported that exposure to combinations ofenvironmental stresses with a common physiologi-cal mechanism, increased the expression of geneticvariation for traits such as fecundity or dryweight, relative to unexposed controls (Gebhardtand Stearns, 1992; Sgro and Hoffmann, 1998).These results support the Hoffmann and Hercus(2000) hypothesis that genetic variability in life-history traits, and hence, the response to selectionshould be greater under some combinations ofextreme stressful conditions. The present studyfound that at Cd and Zn concentrations thatdepressed feeding, the combined effects of bothmetals on feeding were additive. As a result, thetoxicity and genetic variability in tolerance at highconcentrations of Cd and Zn mixtures weregreater than that reported for single metals. Assuch, the results of this study also support theHoffmann and Hercus (2000) hypothesis.

Previous studies have reported that physiologi-cal acclimation to Zn increases lethal tolerance toCd through the induction of methallothionein-like

C. Barata et al. / Aquatic Toxicology 60 (2002) 85–99 97

protein synthesis (Klerks and Weis, 1987; Bodaret al., 1990; Stuhlbacher et al., 1992, 1993).Nevertheless, most of these studies were con-ducted under environmentally unrealistic expo-sure conditions, since lethal responses weremeasured in absence of food and chemical con-centrations were much higher than those affect-ing sublethal responses. Furthermore, as foodwas not present, water was the only metalsource for the test animals. The present studyfound that exposure to low and moderate con-centrations of Zn increased sublethal toleranceto Cd in all three D. magna clones (Fig. 3).However, this could be explained in one cloneonly (G-62) via physiological acclimatisation toZn (Fig. 4). For the other two D. magna clones,alternative mechanisms may explain the in-creased tolerance to Cd across moderate levelsof Zn.

In the present study Cd and Zn werebioavailable to D. magna from both water andfood (Hare 1992; Munger et al. 1999). Thismeans that the observed feeding behaviour of D.magna to mixtures of Cd and Zn could also beexplained by: (1) effects of Cd and Zn ions re-leased from algae on gut physiology and/or (2)interactive effects of Zn and Cd (i.e. competi-tion, internal distribution) for sorption sites onalgae cells. In all three D. magna clones, induc-tion of tolerance to Cd across varying Zn con-centrations was also observed when both metalswere sorbed by algal cells (Fig. 5). This meansthat feeding responses to mixtures of Cd and Zncould be regulated by the metal fraction sorbedby algal cells, probably by poisoning the gut. Inparticular, the observed induction of toleranceto Cd by Zn could be due to competition be-tween Cd and Zn ions released from algae foruptake or target sites on the gut. Two sourcesof evidence support this argument. First, a num-ber of studies indicate that gut poisoning by themetal fraction present in the algae might be oneof the underlying mechanisms governing sub-lethal responses of Daphnia to metals: Taylor etal. (1998) reported that algae pre-loaded with2.5 �g l−1 of bound Cd inhibited feeding inDaphnia, with animals not recovering after 24 hof exposure in clean medium with uncontami-

nated algae; Griffiths (1980) found that afterjust 2 h of exposure to Cd in both water andalgae at a concentration of 12 �g l−1 the gutdiverticula of D. magna were paralysed. Bodaret al. (1990), after exposing Daphnia individualsto Cd solutions of 10 �g l−1 and algae for 7days, observed a reduction of the algal contentin the digestive tract, with faeces only present inthe posterior end of the gut, combined with alower recycling rate of the epithelial lining. Sec-ond, studies conducted with mussels indicatethat Cd and Zn share similar uptake pathways(Wang and Dei, 1999; Wang and Fisher, 1999)and that both metals compete for uptake sites inthe digestive system (Vercauteren and Blust,1999).

Induction of tolerance to Cd across varyingZn concentrations may also be due to reduc-tions of bioavailable Cd in algal cells. It is wellknown that not all metal present in food isavailable to organisms. Results from Griffiths(1980) and Taylor et al. (1998) indicate thatfeeding depression in Daphnia by Cd was almostentirely due to Cd bound to the algal cell sur-face. Okamura and Aoyama (1994), after cultur-ing Chlorella cells with mixtures of Cr and Cdfor 6 days, found that the presence of Cr al-tered the proportion of Cd present in the cellwall. Therefore, competition of Cd and Zn ionsfor uptake sites on algal cells, or changes in theinternal distribution of Cd by Zn, may affectthe availability of Cd to Daphnia. The sorptionkinetics of Cd and Zn by algal cells (Fig. 1)showed that competition of both metals forsorption cell sites was unlikely to occur, since inall cases the experimental concentrations weremuch lower than the saturation constants. Fur-thermore, Kmax values reported by Taylor et al.(1998) for adsorption of Cd into algal cells werewithin 30% of those shown in Fig. 1. Therefore,in the present study, an important part of Cd,and probably Zn, were bound to the cell surfaceof algae, rather than transported into the cyto-plasm. Although the effects of Zn on Cd distri-bution were not assessed, changes of thebioavailable fraction of Cd by Zn are unlikelyto occur because the experimental conditions(clean water, darkness, short exposure time) didnot allow algal cells to grow.

C. Barata et al. / Aquatic Toxicology 60 (2002) 85–9998

5. Conclusions

Although limited to three distinct clones andtwo metals the results showed the existence ofgenetic and genotype×environmental interac-tions in determining population responses to mix-tures of chemicals (Forbes, 1998). This means thatin the field, temporal and spatial variability in thecomposition of mixtures of metals may preventnatural populations from developing resistance tosingle metals and hence to adapt to metal contam-inated habitats (Klerks and Weis, 1987; Posthumaand van Straalen, 1993). Furthermore, geneticfactors (differences among genotypes within andacross exposure toxic levels) were found to governpopulation responses to mixtures of chemicals.Therefore, current risk assessment procedures formultiple chemical mixtures should incorporate ge-netic variability (Poch, 1993).

Exposure to low and moderate Zn concentra-tions increased sublethal tolerance to Cd, andinduction of tolerance to Cd by Zn was alsoobserved when both metals were sorbed to algalcells. Furthermore, in only one clone, physiologi-cal acclimatisation to zinc induced tolerance tocadmium. These results suggest that in Daphnia,gut poisoning, by release of metals from algae,might also govern feeding responses. In particularboth induction of methallothionein synthesis byZn and competition of Zn and Cd ions for uptakeat target sites on the gut might be involved indetermining sublethal responses to mixtures ofboth metals.

Acknowledgements

Carlos Barata was supported by a PortugueseFundacao para a Ciencia e Tecnologia grantPRAXIS XXI/1629/98 while carrying out thisresearch.

References

Allen, Y., Calow, P., Baird, D.J., 1995. A mechanistic modelof contaminant-induced feeding inhibition in Daphniamagna. Environ. Toxicol. Chem. 14, 1625–1630.

APHA-AWWA-WEF, 1995. Standard Methods for the Exam-ination of Water and Wastewater. nineteenth ed. AmericanPublic Association/America Water Works Association/Wa-ter Environment Federation, Washington, DC.

Baird, D.J., Barber, I., Bradley, M., Soares, A.M.V.M.,Calow, P., 1991. A comparative study of genotype sensitiv-ity to acute toxic stress using clones of Daphnia magnaStraus. Ecotoxicol. Environ. Saf. 21, 257–265.

Barata, C., Baird, D.J., 1998. Phenotypic plasticity and con-stancy of life-history traits in laboratory clones of Daphniamagna Straus: effects of neonatal length. Funct. Ecol. 12,412–419.

Barata, C., Baird, D.J., 2000. Determining the ecotoxicologicalmode of action of toxicants from measurements on individ-uals: results from short duration chronic tests with Daphniamagna Straus. Aquat. Toxicol. 48, 195–209.

Barata, C., Baird, D.J., Markich, S.J., 1998. Influence ofgenetic and environmental factors on the tolerance ofDaphnia magna Straus to essential and non-essentialmetals. Aquat. Toxicol. 42, 115–137.

Barata, C., Baird, D.J., Amat, F., Soares, A.M.V.M., 2000a.Population responses to contaminants, between laboratoryand field: an approach using Daphnia magna ephippial eggbanks. Funct. Ecol. 14, 513–523.

Barata, C., Baird, D.J., Minarro, A., Soares, A.M.V.M.,2000b. Do genotype responses always converge from lethalto non-lethal toxicant exposure levels? A hypothesis testedusing laboratory Daphnia magna Straus clones. Environ.Toxicol. Chem. 19, 2314–2322.

Barata, C., Mitchell, S.E., Baird, D.J., Soares, A.M.V.M.,2002. Among and within population variability in toler-ance to toxic stress in natural populations of Daphniamagna. Implications for ecological risk assessment, Envi-ron. Toxicol. Chem., in press.

Bennett, W.N., Brooks, A.S., 1989. Measurement of zincamelioration of cadmium toxicity in Chlorella pyrenoidosausing turbidostat culture. Environ. Toxicol. Chem. 8, 877–882.

Bodar, G.W.N., Kluytmans, J.H., van Montford, J.C.P.,Voogt, P.A., Zandee, D.I., 1990. Cadmium resistance inDaphnia magna. Aquat. Toxicol. 16, 33–40.

Brown, P.L., Haworth, A., Sharland, S.M., Tweed, C.J., 1991.HARPHRQ: An extended version of the geochemical codePHREEQE. Nirex Safety Studies Report, 188. UnitedKingdom Atomic Energy Authority, Oxon.

Canizares-Villanueva, R.O., Martınez-Jeronimo, F., Espinosa-Chavez, F., 2000. Acute toxicity to Daphnia magna ofeffluents containing Cd, Zn and mixtures Cd–Zn, aftermetal removal by Chlorella �ulgaris. Environ. Toxicol. 15,160–164.

Falconer, D.S., 1989. Introduction to Quantitative Genetics,second ed. Longman, London.

Falconer, D.S., 1990. Selection in different environments: ef-fects on environmental sensitivity (reaction norm) and onmean performance. Genet. Res. 56, 57–70.

Fish, W., Morel, F.M.M., 1983. Characterisation of organiccopper-complex agents released by Daphnia magna. Can. J.Fish. Aquat. Sci. 40, 1270–1277.

C. Barata et al. / Aquatic Toxicology 60 (2002) 85–99 99

Forbes, V.E., 1998. Genetics and Ecotoxicology. Taylor andFrancis, Ann Arbor.

Gebhardt, M.D., Stearns, S.C., 1992. Phenotypic plasticity forlife-history traits in Drosophila melanogaster. III. Effect ofthe environment on genetic parameters. Genet. Res. 60,87–101.

Georghiou, G.P., 1990. Factors influencing the evolution ofresistance. In: National Research Council Committee onStrategies for the Management of Pesticide Resistant PestPopulations (Ed.), Pesticide Resistance: Strategies and Tac-tics for Management. National Academia Press, Washing-ton, DC, pp. 157–169.

Griffiths, P.R.E., 1980. Morphological and ultrastructural ef-fects of sublethal cadmium poisoning on Daphnia. Environ.Res. 22, 277–284.

Hansbury, E., Dyke, J.T., 1992. Anion chromatography: com-parison of eluant pH, concentration and flow rate. LA12378. Los Alamos National Laboratory, Los Alamos,New Mexico.

Hare, L., 1992. Aquatic insects and trace metals: bioavailabil-ity, bioaccumulation, and toxicity. Crit. Rev. Toxicol. 22,327–369.

Hare, L., Tessier, A., 1998. The aquatic insect Chaoborus as abiomonitor of trace metals in lakes. Limnol. Oceanogr. 43,1850–1859.

Hebert, P.D.N., 1987. Genotypic characteristics of the Clado-cera. Hydrobiologia 145, 183–193.

Hoffmann, A.A., Hercus, M.J., 2000. Environmental stress asan evolutionary force. Bioscience 50, 217–226.

Hoffmann, A.A., Parsons, P.A., 1991. Evolutionary Geneticsand Environmental Stress. Oxford University Press, NewYork.

Huang, H., Smilowitz, Z., Saunders, M.C., Weisz, R., 1994.Field evaluation of insecticide application strategies ondevelopment of insecticide resistance by Colorado potatobeetle (Coleoptera: Chrysomelidae). J. Econ. Entomol. 87,847–857.

Immaraju, J.A., Morse, J.G., Hobza, R.F., 1990. Field evalua-tion of insecticide rotation and mixtures as strategies forcitrus thrips (Thysanoptera: Thripidae) resistance manage-ment in California. J. Econ. Entomol. 83, 306–314.

Klerks, P.L., 1998. The influence of contamination complexityon adaptation to environmental contaminants. In: Forbes,V.E. (Ed.), Genetics and Ecotoxicology. Taylor and Fran-cis, Ann Arbor, pp. 103–121.

Klerks, P.L., Weis, J.S., 1987. Genetic adaptation to heavymetals in aquatic organisms: a review. Environ. Pollut. 45,173–205.

Lynch, M., Walsh, B., 1998. Genetics and Analysis of Quanti-tative Traits. Sinauer Associates Inc., Sunderland, MA.

Markich, S.J., Brown, P.L., 1999. Thermochemical Data forEnvironmentally-Relevant Elements. 1. Na, K, Ca, Mg,Mn, Fe, Al, U, Cu, Cd, Zn and Pb with Model Fulvic Acid(Aspartate, Citrate, Malonate, Salicylate and Tricarbally-ate). ANSTO/E735. Australian Nuclear Science and Tech-nology Organisation, Sydney.

Matthews, R.W., Abdullah, M., Low, G.K.C., 1990. Photo-

catalytic oxidation for total organic carbon analysis. Anal.Chim. Acta 233, 171–179.

Munger, C., Hare, L., 1997. Relative importance of water andfood as cadmium sources to an aquatic insect (Chaoboruspunctipennis): implications for predicting Cd bioaccumula-tion in nature. Environ. Sci. Technol. 31, 891–895.

Munger, C., Hare, L., Craig, A., Charest, P.M., 1999. Influ-ence of exposure time on the distribution of cadmiumwithin the cladoceran Ceriodaphnia dubia. Aquat. Toxicol.44, 195–200.

Okamura, H., Aoyama, I., 1994. Interactive toxic effect anddistribution of heavy metals in phytoplankton. Environ.Toxicol. Water Qual. 9, 7–15.

Poch, G., 1993. Combined effects of drugs and toxic agents.Modern evaluation in theory and practice. Springer-Ver-lag, New York.

Posthuma, L., van Straalen, N.M., 1993. Heavy-metal adapta-tion in terrestrial invertebrates: a review of occurrence,genetics, physiology and ecological consequences. Comp.Biochem. Physiol. 106C, 11–38.

Sgro, C.M., Hoffmann, A.A., 1998. Effects of stress combina-tions on the expression of additive genetic variation forfecundity in Drosophila melanogaster. Genet. Res. 72, 13–18.

Soares, A.M.V.M., Baird, D.J., Calow, P., 1992. Interclonalvariation in the performance of Daphnia magna Straus inchronic bioassays. Environ. Toxicol. Chem. 11, 1477–1483.

Stanton, M.L., Roy, B.A., Thiede, D.A., 2000. Evolution instressful environments. I. Phenotype variability, phenotypeselection, and response to selection in five distinct environ-mental stresses. Evolution 54, 93–111.

Stuhlbacher, A., Bradley, M.C., Naylor, C., Calow, P., 1992.Induction of cadmium tolerance in two clones of Daphniamagna Straus. Comp. Biochem. Physiol. 101C, 571–577.

Stuhlbacher, A., Bradley, M.C., Naylor, C., Calow, P., 1993.Variation in the development of Cadmium resistance inDaphnia magna Straus: effect of temperature, nutrition, ageand genotype. Environ. Pollut. 80, 153–158.

Taylor, G., Baird, D.J., Soares, A.M.V., 1998. Surface bindingof contaminants by algae: consequences for lethal toxicityand feeding to Daphnia magna Straus. Environ. Toxicol.Chem. 17, 412–419.

Vercauteren, K., Blust, R., 1999. Uptake of cadmium and zincby the mussel Mytilus edulis and inhibition by calciumchannel and metabolic blockers. Mar. Biol. 135, 615–626.

Wang, W.X., Dei, R.C.H., 1999. Factors affecting trace ele-ment uptake in the black mussel Septifer �irgatus. Mar.Ecol. Prog. Ser. 186, 161–172.

Wang, W.X., Fisher, N.S., 1999. Effects of calcium andmetabolic inhibitors on trace element uptake in two marinebivalves. J. Exp. Mar. Biol. Ecol. 236, 149–164.

Wicklund, A., Runn, P., Norrgren, L., 1988. Cadmium andzinc interactions in fish: effects of zinc on the uptake,organ distribution, and elimination of 109Cd in the ze-brafish, Brachydanio rerio. Arch. Environ. Contam. Toxi-col. 17, 345–354.

Zar, J.H., 1996. Biostatistical Analysis. Prentice-Hall, NewJersey.