RESEARCH ARTICLE

Toxic effect of metal cation binary mixtures to the seaweedGracilaria domingensis (Gracilariales, Rhodophyta)

Luiz Fernando Mendes & Cassius Vinicius Stevani &Leonardo Zambotti-Villela & Nair Sumie Yokoya &

Pio Colepicolo

Received: 4 November 2013 /Accepted: 10 March 2014 /Published online: 29 March 2014# Springer-Verlag Berlin Heidelberg 2014

Abstract The macroalga Gracilaria domingensis is an im-portant resource for the food, pharmaceutical, cosmetic, andbiotechnology industries. G. domingensis is at a part of thefood web foundation, providing nutrients and microelementsto upper levels. As seaweed storage metals in the vacuoles,they are considered the main vectors to magnify these toxicelements. This work describes the evaluation of the toxicity ofbinarymixtures of available metal cations based on the growthrates of G. domingensis over a 48-h exposure. The interactiveeffects of each binary mixture were determined using a toxicunit (TU) concept that was the sum of the relative contributionof each toxicant and calculated using the ratio between thetoxicant concentration and its endpoint. Mixtures of Cd(II)/Cu(II) and Zn(II)/Ca(II) demonstrated to be additive; Cu(II)/Zn(II), Cu(II)/Mg(II), Cu(II)/Ca(II), Zn(II)/Mg(II), and Ca(II)/Mg(II) mixtures were synergistic, and all interactions studiedwith Cd(II) were antagonistic. Hypotheses that explain thetoxicity of binary mixtures at the molecular level are also

suggested. These results represent the first effort to character-ize the combined effect of available metal cations, based onthe TU concept on seaweed in a total controlled medium. Theresults presented here are invaluable to the understanding ofseaweed metal cation toxicity in the marine environment, themechanism of toxicity action and how the tolerance of theorganism.

Keywords Interactive effect . Algal bioassay .Macroalgae .

Metal speciation . Toxic unit . Pollutants

Introduction

Algae play several important roles in the marine environment.As photosynthetic organisms, they absorb CO2 and release O2

to the surrounding seawater. They have nitrate and nitritereductases and therefore are able to reduce nitrate to nitriteand NH3 in order to synthesize amino acids and othernitrogen-containing compounds (Granbom et al. 2004). Theyare at the foundation of the marine food web and transferessential compounds such as lipids, aminoacids, minerals, andpigments to upper levels. In addition, algae are becomingimportant for the pharmaceutical, cosmetic, agricultural, food,and biotechnology industries. They produce large quantities ofunique substances with economic importance such as polysac-charides, polyunsaturated fatty acids, and carotenoids (Gressleret al. 2010). The red macroalga Gracilaria domingensis iseconomically important as a food source and for agar produc-tion. This alga is able to store large amounts of metals invacuoles and therefore is an appropriate target organism toevaluate the toxicity of metallic species in the marine environ-ment (Guaratini et al. 2012; Mendes et al. 2013a).

Most of the research conducted on aquatic organism metaltoxicity involves the use of bacteria (Fulladosa et al. 2005),crustaceans (Barata et al. 2006), duckweed (Horvat et al.

Responsible editor: Elena Maestri

Electronic supplementary material The online version of this article(doi:10.1007/s11356-014-2763-5) contains supplementary material,which is available to authorized users.

L. F. Mendes (*) : L. Zambotti-Villela : P. ColepicoloDepartamento de Bioquímica, Instituto de Química, Universidade deSão Paulo, 26077, 05599-970 São Paulo, SP, Brazile-mail: lfm@iq.usp.br

L. F. Mendese-mail: lfmendes1979@icloud.com

C. V. StevaniDepartamento de Química Fundamental, Instituto de Química,Universidade de São Paulo, São Paulo, SP, Brazil

N. S. YokoyaInstituto de Botânica, Núcleo de Pesquisa em Ficologia, São Paulo,SP, Brazil

Environ Sci Pollut Res (2014) 21:8216–8223DOI 10.1007/s11356-014-2763-5

2007), and macroalgae (Chaisuksant 2003; Pavasant et al.2006) by measuring the total uptake concentration of non-speciated single or binary metal cations by the organism(Pavasant et al. 2006; Collén et al. 2012). The formation offree metal cations in an aqueous solution depends not only onmetal abundance, but also on the pH, ionic strength, salinity,presence of organic matter, and other parameters of the seawater(Mendes and Stevani 2010; Mendes et al. 2010, 2013a, b).Hydrated cations are considered the most bioreactive metalspecies, their concentration in a sample represents a moremeaningful and consistent parameter for the evaluation oftoxic effects (Mendes et al. 2013a; Stevani et al. 2013).

Relatively little attention has been given to the studyof the toxic effects of mixtures of binary metals onmarine organisms (Chaisuksant 2003; Kumar et al.2008). The toxic unit (TU) metric has become widelyused to measure the toxicological effect of binary mix-tures of metal cations (Fulladosa et al. 2005; Barataet al. 2006). The TU is the sum of the relative contri-bution of each toxicant and is calculated using the ratioof the toxicant concentration vs. its endpoint. Initially,the effect is assumed to be additive; the toxic effect issimply the sum of the toxicological contributions ofeach toxicant. When the calculated value is the sameof that determined experimentally, the effect is additive(ADD). If the value is higher, the effect is synergistic(SYN), and if the value is lower, the effect is antago-nistic (ANT).

In this work, we evaluate the toxic effects of binary mix-tures of the speciated metal cations Cd(II), Cu(II), Zn(II),Mg(II), and Ca(II) on the red seaweed G. domingensis in asynthetic seawater medium. The artificial seawater mediumwas selected to assure a complete control of the culture andassay conditions which are crucial to the accurate determina-tion of the toxicological effects of the added cation mixtures(Mendes et al. 2012). The median inhibitory concentration(IC50) values were obtained by measuring the mass variation(i.e., daily growth rate observation) of the apical segmentsexposed to single and binary metal cation mixtures over 48 h(Mendes et al. 2013a). Several hypotheses that explain thetoxicity of binary mixtures at the molecular level are alsoproposed. To our knowledge, this is the first binary cationtoxicological study conducted with relevant seaweed undertotal controlled medium conditions.

Materials and methods

Chemicals and solutions

The standard reference materials for the inductively coupledplasma-atomic emission spectroscopy (ICP-AES) determina-tion of metals Cd(II), Cu(II), Zn(II), Mg(II), and Ca(II) were

purchased from PerkinElmer (Table S1). The exact concen-trations of several solutions were determined with ICP-AES(Spectro Genesis SOP) using a procedure described in thestandard methods by EPA method 6010C. In order to mini-mize spectral and transport interferences in ICP-AES deter-mination of metal cation concentrations, the standard solu-tions were matrix-matched and prepared using the same syn-thetic seawater of the toxicological experiments. The workingsolutions were prepared using ZnSO4.7H2O, CuSO4.5H2O,3(CdSO4.8H2O), CaCl2.2H2O, and MgCl2.6H2O purchasedfrom Sigma-Aldrich or Merck (American Chemical Society,99 % purity) and used without further purification. The stocksolutions (mM) of 8.9±0.1 Cd(II), 76±3 Zn(II), 80±1 Cu(II),953±5 Ca(II), and 2,057±82Mg(II) were prepared in a sterilesynthetic seawater medium. The pH of these stock solutionswas adjusted to 7.8±0.2 using 0.1 mM NaOH (Sigma-Aldrich) added dropwise and measured with a pH meterMettler Toledo FE20/EL20. Test solutions containing singleand binary metal cation mixtures were prepared by dilutingthe stock solutions with a sterilized synthetic seawater medi-um (mM): 0.004–0.085 Cd(II), 0.08–1.53 Zn(II), 0.16–2.36Cu(II), 5.00–99.80 Ca(II), and 12.34–740.43 Mg(II)(Table S2). The pH was adjusted to 7.8±0.3 with 0.1 mMNaOH added dropwise.

Toxicological assay

The G. domingensis (Kützing) Sonder ex Dickie cultureswere provided by the Culture Collection of Algae,Cyanobacteria, and Fungi of Instituto de Botânica(CCIBt), São Paulo, SP, Brazil. The apical segmentsused in the present work were cultivated in a syntheticseawater medium under the optimal conditions deter-mined as previously described (Mendes et al. 2012).Details regarding the concentration of the componentsused to prepare the synthetic seawater medium andculture conditions (e.g., temperature at 26.0±0.5 °C,photon flux density of 74±10 μM photons m−2 s−1,80 μM of nitrate, 8 μM of phosphate, and 1 nM ofmolybdate) can also be found in Mendes et al. (2012).Briefly, ten apical segments of the algae were cut intosmall fragments (3 to 4 mm) and added to a freshlyprepared synthetic seawater at pH 7.8±0.2 and 33.5±0.5‰ salinity (measured with a salinometer, Biobrix,model 211). The samples were maintained for 3 daysin a climatic chamber, (HotPack, Cambridge, MA, USA)in a 14:10 h (light/dark) regime under illumination bywhite fluorescent lamps (OSRAM FL 20 W) before thetoxicological assay (Mendes et al. 2013a). Three or fourapical segments (total weight of 6.0±0.5 mg) in a150-mL synthetic seawater medium were exposed over48 h to either single or binary metal cation mixtures.Every experiment was performed in triplicate (n=3).

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Determination of the daily growth rate and IC50 values

The daily growth rate (DGR, in μgd−1) values were deter-mined using the ratio of fresh mass variation (mfinal−minitial)over time (tfinal−tinitial); DGR=Δ(m/t) (Mendes et al. 2013a).The DGR vs. the logarithm ofmetal cation concentration plotswere fitted using a dose-response sigmoidal function imple-mented by the Microcal Origin 8.0 software, and the medianinhibitory concentration (IC50) were values calculated(Table S2, Table 1).

Metal cation concentration in synthetic seawater

A solution containing single or binary metal cation mixtureswas prepared in 150 mL of the same synthetic seawatermedium and flasks used during the toxicological assay (seeabove description) and maintained over 48 h in the climaticchamber under the aforementioned illumination regime, ac-cording to Mendes et al. (2013a). The only modification wasthe absence of the algae. The concentration of metal cations inseawater synthetic medium was quantified by ICP-AES (stan-dard methods of the US Environmental Protection Agency,US EPA, method 6010C Table S1). The actual metallic cationconcentration was used to plot DGR vs. the logarithm ofmetalcation concentration (Table S2, Fig. 1).

Free metal cation concentration in a solution in syntheticseawater

The ratio of hydrated cations ([M(H2O)x]n+) for each specific

metal was calculated with the MINTEQA2 software (ionicstrength 0.5–0.7) before being used to determine the free ionmedian inhibitory concentration (ICF

50) (Mendes et al. 2013a,b) (Table S3). For the binary metal cation mixtures, the DGRvs. log concentration curves were plotted using the free metal

cation concentration calculated with the MINTEQA2 soft-ware (Fig. 2) (ionic strength 0.5–1.0). Therefore, the medianinhibitory concentration values determined in Fig. 2 referdirectly to the ICF

50 in this case (Fig. 2, Table S3).

Mathematical modeling

The toxicity of metal cations was expressed in toxic units (TU)(Bliss 1939):

TUmix ¼ TUmetalA þ TUmetalB ¼ CmetalA

IC50;metalAþ CmetalB

IC50;metalBð1Þ

TUmix ¼ Cmix

IC50;mixA⇒ IC50;mixC ¼ Cmix

TUmixð2Þ

where CmetalA, CmetalB, and Cmix are the concentrations ofmetal cations A, B and A + B; IC50, metalA and IC50, metalB

are the experimental median inhibitory concentrations of met-al cations A and B; TUmix and IC50, mixC are the calculatedbinary mixture toxic unit values and the median inhibitory,respectively, assuming there is an additive effect.

The experimental median inhibitory concentration (IC50,

mixE) was obtained from the DGR vs. the logarithm of metalconcentration plots using the binary metal cation mixtures(Fig. 2). The IC50, mixC/IC50, mixE ratio reveals the toxicolog-ical effect of the binarymixture; specifically, an additive effectcan be assigned when the ratio is 1.0 (ADD), a synergistic (orgreater than ADD) effect (SYN) is operative when >1.0, andan antagonistic (or less than ADD) effect (ANT) is presentwhen <1.0 within experimental error (Fulladosa et al. 2005).

Results and discussion

Toxicological assay

Using the synthetic seawater medium enabled the accuratedistribution of chemical species present in the solution to becalculated using an equilibrium speciation model, such asMINTEQA2. Conventionally, aqueous complexes or freemetal cations, specifically the hydrated cations [M(H2O)x]

n+,are considered the most toxic species present in the solution(Mendes and Stevani 2010; Mendes et al. 2010, 2013a, b).However, using the concentration of the free metal cation thatthe algae is exposed to as the use of nominal concentrationdoes not reflect the concentration of the most toxic metallicspecies in the solution (Mendes et al. 2013a). For the binarymixtures, the interaction between the metallic species and theanions, as well as the influence of ionic strength, could further

Table 1 Median inhibitory concentrations (IC50 and ICF50) obtained

during the 48-h toxicological assay with G. domingensis in syntheticseawater for single metal cations [Cd(II), Cu(II), Zn(II), Ca(II), andMg(II)]

Metals Nominal IC50

(mM)ICP-AES actualIC50 (mM)a

Free metal(%)b

Actual ICF50

(M)c

Cd(II) 0.030±0.002 0.030±0.0002 0.2 55±4×10−9

Cu(II) 1.0±0.1 0.90±0.05 0.02 2.0±0.2×10−6

Zn(II) 0.70±0.05 0.60±0.01 9 60±6×10−6

Ca(II) 21±2 19±1 60 13±1×10−3

Mg(II) 126±10 118±3 49 62±5×10−3

a Determined by ICP-AESb Estimated using computational chemical equilibrium software(MINTEQA2, US Environmental Protection Agency, Washington, DC,version 3.0); Ionic strength=0.5–0.7c Percentage of free metal×ICP-AES IC50

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alter the actual concentration of free metal cations in thesolution.

The median inhibitory concentrations for the individualmetal cations (Cd, Cu, Zn, Mg, and Ca) were determined from

the DGR vs. log concentration curves (Mendes et al. 2013a).The metal cation concentration was determined by ICP-AES(ICT

50, Table S2, Fig. 1), and the free ion fraction was calcu-lated using the MINTEQA2 software (ICF

50, Table 1). Every

Fig. 1 DGR vs. log metal concentration plots obtained from the toxico-logical assay using G. domingensis in a synthetic seawater medium atpH=7.5±0.3 over 48 h. a filled circle Cd(II), filled star Cu(II), and filleddiamondCd(II) + Cu(II); b filled circleCd(II), filled star Zn(II), and filleddiamond Cd(II)+Zn(II); c filled circle Cd(II), filled starMg(II), and filleddiamond Cd(II)+Mg(II); d filled circleCd(II), filled starCa(II), and filleddiamond Cd(II)+Ca(II); e filled circle Cu(II), filled star Zn(II), and filled

diamond Cd(II)+Zn(II); f filled circle Cu(II), filled starMg(II), and filleddiamond Cu(II)+Mg(II); g filled circle Cu(II), filled starCa(II), and filleddiamond Cu(II)+Ca(II); h filled circle Zn(II), filled starMg(II), and filleddiamond Zn(II)+Mg(II); i filled circle Zn(II), filled star Ca(II), and filleddiamond Zn(II)+Ca(II); and j filled circle Ca(II), filled star Mg(II), andfilled diamond Ca(II)+Mg(II)

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type of median inhibitory concentration (i.e., IC50, ICT50,

and ICF50) determined in this work was within the stan-

dard deviation, similar to the values reported previously(Mendes et al. 2013a, b). Free single metal cations can beranked in the following order based on their ICF

50 (M):Cd(II)>Cu(II) >>Zn(II) >>Ca(II) >>Mg(II) (Table 1).Free cadmium ions are ca. 4- and 1,000-fold more toxicthan Cu(II) and Zn(II), respectively. Therefore, Cu(II) is300-fold more toxic than Zn(II). As expected, the alkalineearth metals, like Ca(II) and Mg(II), are much less toxicthan the other metal cations.

The experimental median inhibitory concentration valuesfor the binary mixtures of free metal cations (ICF

50, mixE) weredetermined using the same procedure described for the singlespecies (Fig. 2, Table 2, Tables S3 and S4). For each experi-mental point, the algae were exposed to the sum of theindividual concentrations of metal cations.

Toxicological effects

DGR vs. the logarithm of the ICP-AES-determined or theMINTEQA2-calculated metal cation concentrations is plottedusing the experimental data presented in Tables S2 and S3(Figs. 1 and 2). In contrast to the ICT

50 and ICF50 values for

single metal cations, the ICT50, mixE and ICF

50, mixE of binarymixtures vary significantly (Table 1 and Table S3). However,the ratio IC50, mixC/IC50, mixE (used to calculate the toxicolog-ical effects) remains the same, independent of the individualICT

50, mixE or ICF50, mixE values (Figs. 1 and 2, Tables S5 and

S6).Only the free binary Cd(II)/Cu(II) and Zn(II)/Ca(II) mix-

tures are additive, based on the 20 % deviation in the IC50,

mixC/IC50, mixE ratio. The binary Ca(II)/Mg(II) mixture issynergistic. All other binary mixture combinations withCd(II) are antagonistic, while the remainder with Cu(II) are

Fig. 2 DGR vs. log free metal concentration (determined by ICP-AESusing the MINTEQA2 software, version 3) plots obtained from thetoxicological data from G. domingensis in a synthetic seawater mediumat pH=7.5±0.3 over 48 h. a filled circle Cd(II)+Cu(II), filled diamondCd(II)+Zn(II), filled star Cd(II)+Mg(II), filled triangle Cd(II) and

Cd(II)+Ca(II); b filled circle Cu(II)+Zn(II), filled diamond Cu(II)+Ca(II), and filled star Cu(II)+Mg(II); c filled circle Zn(II)+Ca(II), filleddiamond Mg(II)+Ca(II), and filled star Zn(II)+Mg(II). Ionic strength=0.55–1.0

Table 2 Experimental (ICF50, mixE) and calculated (IC

F50, mixC) freemedian inhibitory concentration values for the binarymetal cationmixtures obtained

with the G. domingensis bioassay

Binary mixtures ICF50mixE, mM ICF

50,mixC, mMa ICF50, mixC/IC

F50, mixE Effectb Statistical significancec

(μM) (μM)

Cd(II)/Cu(II) 94±23 75±1 0.8±0.3 ADD p=0.2263

Cd(II)/Zn(II) 0.05±0.01 0.36±0.08 0.1±0.3 ANT p=0.0013

Cd(II)/Mg(II) 80±1 22±4 0.3±0.2 ANT p=<0.0001

Cd(II)/Ca(II) 14±1 3.8±0.7 0.3±0.2 ANT p=0.0020

Cu(II)/Zn(II) 0.02±0.01 0.03±0.01 1.5±0.3 SYN p=0.2879

Cu(II)/Mg(II) 13±2 42±13 3.3±0.3 SYN p=0.0156

Cu(II)/Ca(II) 3±1 9±3 3.2±0.5 SYN p=0.0201

Zn(II)/Mg(II) 32±5 45±19 1.4±0.3 SYN p=0.3128

Zn(II)/Ca(II) 9±1 9±3 1.0±0.3 ADD p=0.7670

Ca(II)/Mg(II) 23±1 40±6 1.6±0.2 SYN p=0.0204

a The ICF50, mixC values were obtained using Eqs. 1 and 2 and the actual IC

F50 values depicted in Table 1 (see calculations for the TUmodels in Table S3)

b Additive effect (ADD ∼1), synergistic (greater than additive) effect (SYN >1), antagonistic effect (ANT <1)CUnpaired t test (two-tailed) for the EC50, mixE vs. IC50, mixC. Ionic strength=0.5–1.0

8220 Environ Sci Pollut Res (2014) 21:8216–8223

synergistic (Table 2, Fig. 3). Many studies have described theuptake of metal cations by macroalgae (Chaisuksant 2003);however, this work represents the first effort to characterizethe combined effects of essential and nonessential free metalcations on red seaweed under controlled conditions.Considering that an additive effect indicates that there is nointeraction between the metallic species, only the antagonisticand synergistic effects [e.g., Cd(II) and Zn(II), Mg(II) orCa(II); Cu(II) and Zn(II), Mg(II) or Ca(II); Zn(II) andMg(II); Ca(II) and Mg(II)] will be discussed further in thecontext of putative ion-biomolecule interactions.

Antagonistic effects

Cd(II) is the most toxic of the five metals tested, and thistoxicity can be rationalized based on its tendency to formcomplexes with sulfur-containing biomolecules, such as pro-teins, amino acids, and glutathione as well as its ability toinhibit photosystem II by competing with calcium sites and tohinder chlorophyll synthesis while increasing oxidative stress(Faller et al. 2005). However, when Zn(II) toxicity occurs, themitochondrial electron transport chain is uncoupled, and thecalcium uptake related to the Ca-ATPase activity decreases(Mendes et al. 2013a). Moreover, both cations can also inducethe synthesis of phytochelatins (Tsuji et al. 2003); these com-pounds are important for detoxifying the organism by chelat-ing metallic species.

For the binary Cd(II) and Zn(II) mixture, Cd(II) can replacethe latter in zinc-containing proteins, such as superoxide dis-mutase (Stohs and Bagchi 1995). Therefore, under high Zn(II)concentrations, the Cd(II) toxicity decreases, most likely dueto the competition for binding sites in the target proteins.Pellegrini et al. (1993) studied the effect of binary Cd(II)/Zn(II) mixtures on weight-growth and chlorophyll and

carotenoid syntheses using a brown macroalga (Cystoseirabarbata). According to the authors, the effect was antagonisticon carotenoid synthesis depending on the Zn(II) concentra-tion, promoting an antagonistic effect on weight-growth.Romera et al. (2008) investigated the biosorption of Cd(II)by Codium vermilara, Chondrus crispus, and Ascophyllumnodosum (seaweeds) in the presence of Zn(II), concluding thatthe zinc ions are protective during the absorption of cadmiumions. Pavasant et al. (2006) also observed competitive adsorp-tion effects for this binary mixture with Caulerpa lentillifera(green algae).

Nonessential Cd(II) ions can also replace essential Ca(II)andMg(II) ions in several physiological activities (Davis et al.2003; Faller et al. 2005). Mixed combinations are consideredsynergistic or antagonistic, whether one metal species assiststhe uptake of the other, or they compete for the same transportsites on the cell membrane, respectively (Franklin et al. 2002).High Ca(II) concentrations in the solution decrease the toxic-ity of Cd(II), consequently enhancing growth and chlorophylland carotenoid syntheses in Cystoseira barbata (Pellegriniet al. 1993). Brown and Beckett (1985) also observed anantagonistic effect for binary Cd(II)/Ca(II) and Cd(II)/Mg(II)mixtures on the sorption of cadmium ions by a moss(Rhytidiadelphus squarrosus). However, another author didnot observe any significant difference in Cd(II) uptake whenNa(I), K(I), Ca(II), and Mg(II) ions were present for the redseaweed Gracilaria fisheri (Chaisuksant 2003). In summary,the decrease in Cd(II) toxicity in the presence of either Ca(II)or Mg(II) can be partially explained by the decrease of Cd(II)uptake.

Synergistic effects

Copper ions are required to synthesize chlorophyll in plants;however, cellular copper imbalance triggers the production ofhydroxyl radicals via the Fenton and/or Haber-Weiss reaction,inducing an increase in the algal oxidative stress (Pinto et al.2003; Mendes et al. 2013a). At low concentrations, Cu(II) andZn(II) are essential micronutrients that are required by severalmetabolic processes in algae and higher plants (Mendes et al.2013a). The binary Cu(II)/Zn(II) mixture exerted a synergisticeffect on G. domingensis due to the increased membranepermeability, as well as the decreased cell-surface potentialor adenosine triphosphate production by mitochondria, pro-ducing reactive oxygen species (Franklin et al. 2002).

The effect observed for the binary mixture of Ca(II)/Mg(II)could involve electrolyte redox imbalance, membrane depo-larization, and even cell rupture by osmosis due to the highconcentration of both ions required to induce an observabletoxicological effect (Mendes et al. 2013a, b).

A pronounced synergistic effect was observed for the bi-nary Cu(II)/Mg(II) and Cu(II)/Ca(II) mixtures (ICF

50, mixC/ICF

50, mixE=3.3±0.3 and 3.2±0.5, respectively; Table 2),

Fig. 3 Ratio of the experimental and calculated values for the freemedian inhibitory concentrations (ICF

50, mixE/ICF50, mixC) of the binary

metal cation mixtures obtained during the G. domingensis bioassay. Thetoxicological effects may be additive (ADD ∼1), synergistic (greater thanadditive (SYN >1)), and antagonistic (ANT <1)

Environ Sci Pollut Res (2014) 21:8216–8223 8221

unlike the antagonistic effects observed with the binary Cd(II)mixtures. In contrast to Cd(II), neither Mg(II) nor Ca(II)competes with the algal Cu(II) uptake by G. fisheri at theconcentrations (10 mM) used by the authors (Chaisuksant2003). In our case, when using much higher concentrationsof both Mg(II) (12–740 mM) or Ca(II) (5–100 mM), thesemetal cations might damage the algal cell membranes andalter the ability of the algae to control their Cu(II) uptake orincrease membrane permeability. The same argumentationmight be used for the binary Zn(II)/Mg(II) mixture.

Environmental relevance

Macroalgae contribute to the foundation of the food chain andare highly nutritive with regard to vitamin, protein, organiccompound, pigment, mineral, fiber, and essential fatty acidcontents (Gressler et al. 2011). Algae from the genusGracilaria are cosmopolitan organisms that may be foundworldwide; they grow primarily in the intertidal zones ofrocky shores (Plastino and Oliveira 2002). Therefore, it isreasonable to assume they are exposed to higher concentra-tions of micronutrients from the sedimentary pore water. Infact, the metal cation concentrations inside these algae aremuch higher (mgL−1) than that commonly found in the ocean(Tonon et al. 2011); this value is in the nanomolar (nM) rangeon non-polluted marine coasts (Ansari et al. 2004). Coastalland mining may impact the marine environment by raisingthe concentration of metal cations in water column or sedi-mentary plumes from tailings and discharges (Lee et al. 2002).In the areas with these activities, the metal concentration in thesediment can exceed the amount normally observed in coastalseawater, endangering marine organisms.

Conclusion

This study has focused on the acute toxic effect of binarymetal cation mixtures to a specific macroalga. It is toxicolog-ically important to understand how metals can mutually in-crease or decrease their toxicity to seaweeds, despite the highmetal concentrations needed to cause an observable inhibitoryeffect. Results also showed that the alga G. domingensis isnotably tolerant to metal cations. Moreover, high metal con-centrations can be found naturally at some specific coastalareas in addition to locations with anthropogenic activities. Ingeneral, there is still a lack of information describing the long-term (chronic) effects of either binary or higher order metalcationmixtures to seaweeds. The intrinsic (physical-chemical)properties of the free metal ions in the binary mixtures can beused for the prediction of metal activity trends in biologicalsystems. In addition, the use of natural seawater in the con-duction of toxicological assays, whose concentration of ions

depends on environmental factors, can lead to the overestima-tion of metal toxicity.

Acknowledgments The authors are grateful for the financial and tech-nical support during this study from the following institutions and people:Ministério de Ciência, Tecnologia e Inovação, CNPq, NAP-Biodiversidade Marinha, INCT-Redoxoma, Fundação de Amparo àPesquisa do Estado de São Paulo-FAPESP: 09/54718-4 (L.F.M), andNAP-PhotoTech (the USP Research Consortium for PhotochemicalTechnology) from C. V. Stevani.

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