Environ Sci Pollut Res (2014) 21:8216–8223 DOI 10.1007/s11356-014-2763-5
RESEARCH ARTICLE
Toxic effect of metal cation binary mixtures to the seaweed Gracilaria 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 important resource for the food, pharmaceutical, cosmetic, and biotechnology industries. G. domingensis is at a part of the food web foundation, providing nutrients and microelements to upper levels. As seaweed storage metals in the vacuoles, they are considered the main vectors to magnify these toxic elements. This work describes the evaluation of the toxicity of binary mixtures of available metal cations based on the growth rates of G. domingensis over a 48-h exposure. The interactive effects of each binary mixture were determined using a toxic unit (TU) concept that was the sum of the relative contribution of each toxicant and calculated using the ratio between the toxicant 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 studied with Cd(II) were antagonistic. Hypotheses that explain the toxicity of binary mixtures at the molecular level are also
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. Colepicolo Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, 26077, 05599-970 São Paulo, SP, Brazil e-mail: [email protected] L. F. Mendes e-mail: [email protected] C. V. Stevani Departamento de Química Fundamental, Instituto de Química, Universidade de São Paulo, São Paulo, SP, Brazil N. S. Yokoya Instituto de Botânica, Núcleo de Pesquisa em Ficologia, São Paulo, SP, Brazil
suggested. These results represent the first effort to characterize the combined effect of available metal cations, based on the TU concept on seaweed in a total controlled medium. The results presented here are invaluable to the understanding of seaweed metal cation toxicity in the marine environment, the mechanism of toxicity action and how the tolerance of the organism. 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 nitrite reductases and therefore are able to reduce nitrate to nitrite and NH3 in order to synthesize amino acids and other nitrogen-containing compounds (Granbom et al. 2004). They are at the foundation of the marine food web and transfer essential compounds such as lipids, aminoacids, minerals, and pigments to upper levels. In addition, algae are becoming important for the pharmaceutical, cosmetic, agricultural, food, and biotechnology industries. They produce large quantities of unique substances with economic importance such as polysaccharides, polyunsaturated fatty acids, and carotenoids (Gressler et al. 2010). The red macroalga Gracilaria domingensis is economically important as a food source and for agar production. This alga is able to store large amounts of metals in vacuoles and therefore is an appropriate target organism to evaluate the toxicity of metallic species in the marine environment (Guaratini et al. 2012; Mendes et al. 2013a). Most of the research conducted on aquatic organism metal toxicity involves the use of bacteria (Fulladosa et al. 2005), crustaceans (Barata et al. 2006), duckweed (Horvat et al.
Environ Sci Pollut Res (2014) 21:8216–8223
2007), and macroalgae (Chaisuksant 2003; Pavasant et al. 2006) by measuring the total uptake concentration of nonspeciated single or binary metal cations by the organism (Pavasant et al. 2006; Collén et al. 2012). The formation of free metal cations in an aqueous solution depends not only on metal 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 metal species, their concentration in a sample represents a more meaningful and consistent parameter for the evaluation of toxic effects (Mendes et al. 2013a; Stevani et al. 2013). Relatively little attention has been given to the study of the toxic effects of mixtures of binary metals on marine organisms (Chaisuksant 2003; Kumar et al. 2008). The toxic unit (TU) metric has become widely used to measure the toxicological effect of binary mixtures of metal cations (Fulladosa et al. 2005; Barata et al. 2006). The TU is the sum of the relative contribution of each toxicant and is calculated using the ratio of the toxicant concentration vs. its endpoint. Initially, the effect is assumed to be additive; the toxic effect is simply the sum of the toxicological contributions of each toxicant. When the calculated value is the same of 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 antagonistic (ANT). In this work, we evaluate the toxic effects of binary mixtures of the speciated metal cations Cd(II), Cu(II), Zn(II), Mg(II), and Ca(II) on the red seaweed G. domingensis in a synthetic seawater medium. The artificial seawater medium was selected to assure a complete control of the culture and assay conditions which are crucial to the accurate determination 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 segments exposed to single and binary metal cation mixtures over 48 h (Mendes et al. 2013a). Several hypotheses that explain the toxicity of binary mixtures at the molecular level are also proposed. To our knowledge, this is the first binary cation toxicological study conducted with relevant seaweed under total controlled medium conditions.
Materials and methods Chemicals and solutions The standard reference materials for the inductively coupled plasma-atomic emission spectroscopy (ICP-AES) determination of metals Cd(II), Cu(II), Zn(II), Mg(II), and Ca(II) were
8217
purchased from PerkinElmer (Table S1). The exact concentrations of several solutions were determined with ICP-AES (Spectro Genesis SOP) using a procedure described in the standard methods by EPA method 6010C. In order to minimize spectral and transport interferences in ICP-AES determination of metal cation concentrations, the standard solutions were matrix-matched and prepared using the same synthetic seawater of the toxicological experiments. The working solutions were prepared using ZnSO4.7H2O, CuSO4.5H2O, 3(CdSO4.8H2O), CaCl2.2H2O, and MgCl2.6H2O purchased from Sigma-Aldrich or Merck (American Chemical Society, 99 % purity) and used without further purification. The stock solutions (mM) of 8.9±0.1 Cd(II), 76±3 Zn(II), 80±1 Cu(II), 953±5 Ca(II), and 2,057±82 Mg(II) were prepared in a sterile synthetic seawater medium. The pH of these stock solutions was adjusted to 7.8± 0.2 using 0.1 mM NaOH (SigmaAldrich) added dropwise and measured with a pH meter Mettler Toledo FE20/EL20. Test solutions containing single and binary metal cation mixtures were prepared by diluting the stock solutions with a sterilized synthetic seawater medium (mM): 0.004–0.085 Cd(II), 0.08–1.53 Zn(II), 0.16–2.36 Cu(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 mM NaOH added dropwise. Toxicological assay The G. domingensis (Kützing) Sonder ex Dickie cultures were provided by the Culture Collection of Algae, Cyanobacteria, and Fungi of Instituto de Botânica (CCIBt), São Paulo, SP, Brazil. The apical segments used in the present work were cultivated in a synthetic seawater medium under the optimal conditions determined as previously described (Mendes et al. 2012). Details regarding the concentration of the components used to prepare the synthetic seawater medium and culture 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 of molybdate) can also be found in Mendes et al. (2012). Briefly, ten apical segments of the algae were cut into small fragments (3 to 4 mm) and added to a freshly prepared 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 days in a climatic chamber, (HotPack, Cambridge, MA, USA) in a 14:10 h (light/dark) regime under illumination by white fluorescent lamps (OSRAM FL 20 W) before the toxicological assay (Mendes et al. 2013a). Three or four apical segments (total weight of 6.0 ± 0.5 mg) in a 150-mL synthetic seawater medium were exposed over 48 h to either single or binary metal cation mixtures. Every experiment was performed in triplicate (n=3).
8218
Environ Sci Pollut Res (2014) 21:8216–8223
Determination of the daily growth rate and IC50 values The daily growth rate (DGR, in μgd−1) values were determined 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 of metal cation concentration plots were fitted using a dose-response sigmoidal function implemented by the Microcal Origin 8.0 software, and the median inhibitory concentration (IC50) were values calculated (Table S2, Table 1).
cation concentration calculated with the MINTEQA2 software (Fig. 2) (ionic strength 0.5–1.0). Therefore, the median inhibitory concentration values determined in Fig. 2 refer directly to the ICF50 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
Metal cation concentration in synthetic seawater A solution containing single or binary metal cation mixtures was prepared in 150 mL of the same synthetic seawater medium and flasks used during the toxicological assay (see above description) and maintained over 48 h in the climatic chamber under the aforementioned illumination regime, according to Mendes et al. (2013a). The only modification was the absence of the algae. The concentration of metal cations in seawater synthetic medium was quantified by ICP-AES (standard methods of the US Environmental Protection Agency, US EPA, method 6010C Table S1). The actual metallic cation concentration was used to plot DGR vs. the logarithm of metal cation concentration (Table S2, Fig. 1). Free metal cation concentration in a solution in synthetic seawater The ratio of hydrated cations ([M(H2O)x]n+) for each specific metal was calculated with the MINTEQA2 software (ionic strength 0.5–0.7) before being used to determine the free ion median inhibitory concentration (ICF50) (Mendes et al. 2013a, b) (Table S3). For the binary metal cation mixtures, the DGR vs. log concentration curves were plotted using the free metal Table 1 Median inhibitory concentrations (IC50 and ICF50) obtained during the 48-h toxicological assay with G. domingensis in synthetic seawater for single metal cations [Cd(II), Cu(II), Zn(II), Ca(II), and Mg(II)] Metals
Nominal IC50 (mM)
ICP-AES actual IC50 (mM)a
Free metal (%)b
Actual ICF50 (M)c
Cd(II) Cu(II) Zn(II) Ca(II) Mg(II)
0.030±0.002 1.0±0.1 0.70±0.05 21±2 126±10
0.030±0.0002 0.90±0.05 0.60±0.01 19±1 118±3
0.2 0.02 9 60 49
55±4×10−9 2.0±0.2×10−6 60±6×10−6 13±1×10−3 62±5×10−3
a
Determined by ICP-AES
b
Estimated using computational chemical equilibrium software (MINTEQA2, US Environmental Protection Agency, Washington, DC, version 3.0); Ionic strength=0.5–0.7
c
Percentage of free metal×ICP-AES IC50
TUmix ¼
C metalA C metalB þ ð1Þ IC50;metalA IC50;metalB
C mix C mix ⇒ IC50;mixC ¼ IC50;mixA TUmix
ð2Þ
where CmetalA, CmetalB, and Cmix are the concentrations of metal cations A, B and A + B; IC50, metalA and IC50, metalB are the experimental median inhibitory concentrations of metal cations A and B; TUmix and IC50, mixC are the calculated binary 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 metal concentration plots using the binary metal cation mixtures (Fig. 2). The IC50, mixC/IC50, mixE ratio reveals the toxicological effect of the binary mixture; specifically, an additive effect can be assigned when the ratio is 1.0 (ADD), a synergistic (or greater than ADD) effect (SYN) is operative when >1.0, and an antagonistic (or less than ADD) effect (ANT) is present when Cu(II) > > Zn(II) > >Ca(II) > >Mg(II) (Table 1). Free cadmium ions are ca. 4- and 1,000-fold more toxic than Cu(II) and Zn(II), respectively. Therefore, Cu(II) is 300-fold more toxic than Zn(II). As expected, the alkaline earth metals, like Ca(II) and Mg(II), are much less toxic than the other metal cations. The experimental median inhibitory concentration values for the binary mixtures of free metal cations (ICF50, mixE) were determined using the same procedure described for the single species (Fig. 2, Table 2, Tables S3 and S4). For each experimental point, the algae were exposed to the sum of the individual concentrations of metal cations.
Toxicological effects DGR vs. the logarithm of the ICP-AES-determined or the MINTEQA2-calculated metal cation concentrations is plotted using the experimental data presented in Tables S2 and S3 (Figs. 1 and 2). In contrast to the ICT50 and ICF50 values for single metal cations, the ICT50, mixE and ICF50, mixE of binary mixtures vary significantly (Table 1 and Table S3). However, the ratio IC50, mixC/IC50, mixE (used to calculate the toxicological effects) remains the same, independent of the individual ICT50, 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) mixtures are additive, based on the 20 % deviation in the IC50, mixC/IC50, mixE ratio. The binary Ca(II)/Mg(II) mixture is synergistic. All other binary mixture combinations with Cd(II) are antagonistic, while the remainder with Cu(II) are
Table 2 Experimental (ICF50, mixE) and calculated (ICF50, mixC) free median inhibitory concentration values for the binary metal cation mixtures obtained with the G. domingensis bioassay Binary mixtures
ICF50mixE, mM (μM)
ICF50,mixC, mMa (μM)
ICF50, mixC/ICF50, mixE
Effectb
Statistical significancec
Cd(II)/Cu(II) Cd(II)/Zn(II) Cd(II)/Mg(II) Cd(II)/Ca(II) Cu(II)/Zn(II)
94±23 0.05±0.01 80±1 14±1 0.02±0.01
75±1 0.36±0.08 22±4 3.8±0.7 0.03±0.01
0.8±0.3 0.1±0.3 0.3±0.2 0.3±0.2 1.5±0.3
ADD ANT ANT ANT SYN
p=0.2263 p=0.0013 p=1), antagonistic effect (ANT 1)), and antagonistic (ANT
RESEARCH ARTICLE
Toxic effect of metal cation binary mixtures to the seaweed Gracilaria 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 important resource for the food, pharmaceutical, cosmetic, and biotechnology industries. G. domingensis is at a part of the food web foundation, providing nutrients and microelements to upper levels. As seaweed storage metals in the vacuoles, they are considered the main vectors to magnify these toxic elements. This work describes the evaluation of the toxicity of binary mixtures of available metal cations based on the growth rates of G. domingensis over a 48-h exposure. The interactive effects of each binary mixture were determined using a toxic unit (TU) concept that was the sum of the relative contribution of each toxicant and calculated using the ratio between the toxicant 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 studied with Cd(II) were antagonistic. Hypotheses that explain the toxicity of binary mixtures at the molecular level are also
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. Colepicolo Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, 26077, 05599-970 São Paulo, SP, Brazil e-mail: [email protected] L. F. Mendes e-mail: [email protected] C. V. Stevani Departamento de Química Fundamental, Instituto de Química, Universidade de São Paulo, São Paulo, SP, Brazil N. S. Yokoya Instituto de Botânica, Núcleo de Pesquisa em Ficologia, São Paulo, SP, Brazil
suggested. These results represent the first effort to characterize the combined effect of available metal cations, based on the TU concept on seaweed in a total controlled medium. The results presented here are invaluable to the understanding of seaweed metal cation toxicity in the marine environment, the mechanism of toxicity action and how the tolerance of the organism. 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 nitrite reductases and therefore are able to reduce nitrate to nitrite and NH3 in order to synthesize amino acids and other nitrogen-containing compounds (Granbom et al. 2004). They are at the foundation of the marine food web and transfer essential compounds such as lipids, aminoacids, minerals, and pigments to upper levels. In addition, algae are becoming important for the pharmaceutical, cosmetic, agricultural, food, and biotechnology industries. They produce large quantities of unique substances with economic importance such as polysaccharides, polyunsaturated fatty acids, and carotenoids (Gressler et al. 2010). The red macroalga Gracilaria domingensis is economically important as a food source and for agar production. This alga is able to store large amounts of metals in vacuoles and therefore is an appropriate target organism to evaluate the toxicity of metallic species in the marine environment (Guaratini et al. 2012; Mendes et al. 2013a). Most of the research conducted on aquatic organism metal toxicity involves the use of bacteria (Fulladosa et al. 2005), crustaceans (Barata et al. 2006), duckweed (Horvat et al.
Environ Sci Pollut Res (2014) 21:8216–8223
2007), and macroalgae (Chaisuksant 2003; Pavasant et al. 2006) by measuring the total uptake concentration of nonspeciated single or binary metal cations by the organism (Pavasant et al. 2006; Collén et al. 2012). The formation of free metal cations in an aqueous solution depends not only on metal 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 metal species, their concentration in a sample represents a more meaningful and consistent parameter for the evaluation of toxic effects (Mendes et al. 2013a; Stevani et al. 2013). Relatively little attention has been given to the study of the toxic effects of mixtures of binary metals on marine organisms (Chaisuksant 2003; Kumar et al. 2008). The toxic unit (TU) metric has become widely used to measure the toxicological effect of binary mixtures of metal cations (Fulladosa et al. 2005; Barata et al. 2006). The TU is the sum of the relative contribution of each toxicant and is calculated using the ratio of the toxicant concentration vs. its endpoint. Initially, the effect is assumed to be additive; the toxic effect is simply the sum of the toxicological contributions of each toxicant. When the calculated value is the same of 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 antagonistic (ANT). In this work, we evaluate the toxic effects of binary mixtures of the speciated metal cations Cd(II), Cu(II), Zn(II), Mg(II), and Ca(II) on the red seaweed G. domingensis in a synthetic seawater medium. The artificial seawater medium was selected to assure a complete control of the culture and assay conditions which are crucial to the accurate determination 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 segments exposed to single and binary metal cation mixtures over 48 h (Mendes et al. 2013a). Several hypotheses that explain the toxicity of binary mixtures at the molecular level are also proposed. To our knowledge, this is the first binary cation toxicological study conducted with relevant seaweed under total controlled medium conditions.
Materials and methods Chemicals and solutions The standard reference materials for the inductively coupled plasma-atomic emission spectroscopy (ICP-AES) determination of metals Cd(II), Cu(II), Zn(II), Mg(II), and Ca(II) were
8217
purchased from PerkinElmer (Table S1). The exact concentrations of several solutions were determined with ICP-AES (Spectro Genesis SOP) using a procedure described in the standard methods by EPA method 6010C. In order to minimize spectral and transport interferences in ICP-AES determination of metal cation concentrations, the standard solutions were matrix-matched and prepared using the same synthetic seawater of the toxicological experiments. The working solutions were prepared using ZnSO4.7H2O, CuSO4.5H2O, 3(CdSO4.8H2O), CaCl2.2H2O, and MgCl2.6H2O purchased from Sigma-Aldrich or Merck (American Chemical Society, 99 % purity) and used without further purification. The stock solutions (mM) of 8.9±0.1 Cd(II), 76±3 Zn(II), 80±1 Cu(II), 953±5 Ca(II), and 2,057±82 Mg(II) were prepared in a sterile synthetic seawater medium. The pH of these stock solutions was adjusted to 7.8± 0.2 using 0.1 mM NaOH (SigmaAldrich) added dropwise and measured with a pH meter Mettler Toledo FE20/EL20. Test solutions containing single and binary metal cation mixtures were prepared by diluting the stock solutions with a sterilized synthetic seawater medium (mM): 0.004–0.085 Cd(II), 0.08–1.53 Zn(II), 0.16–2.36 Cu(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 mM NaOH added dropwise. Toxicological assay The G. domingensis (Kützing) Sonder ex Dickie cultures were provided by the Culture Collection of Algae, Cyanobacteria, and Fungi of Instituto de Botânica (CCIBt), São Paulo, SP, Brazil. The apical segments used in the present work were cultivated in a synthetic seawater medium under the optimal conditions determined as previously described (Mendes et al. 2012). Details regarding the concentration of the components used to prepare the synthetic seawater medium and culture 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 of molybdate) can also be found in Mendes et al. (2012). Briefly, ten apical segments of the algae were cut into small fragments (3 to 4 mm) and added to a freshly prepared 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 days in a climatic chamber, (HotPack, Cambridge, MA, USA) in a 14:10 h (light/dark) regime under illumination by white fluorescent lamps (OSRAM FL 20 W) before the toxicological assay (Mendes et al. 2013a). Three or four apical segments (total weight of 6.0 ± 0.5 mg) in a 150-mL synthetic seawater medium were exposed over 48 h to either single or binary metal cation mixtures. Every experiment was performed in triplicate (n=3).
8218
Environ Sci Pollut Res (2014) 21:8216–8223
Determination of the daily growth rate and IC50 values The daily growth rate (DGR, in μgd−1) values were determined 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 of metal cation concentration plots were fitted using a dose-response sigmoidal function implemented by the Microcal Origin 8.0 software, and the median inhibitory concentration (IC50) were values calculated (Table S2, Table 1).
cation concentration calculated with the MINTEQA2 software (Fig. 2) (ionic strength 0.5–1.0). Therefore, the median inhibitory concentration values determined in Fig. 2 refer directly to the ICF50 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
Metal cation concentration in synthetic seawater A solution containing single or binary metal cation mixtures was prepared in 150 mL of the same synthetic seawater medium and flasks used during the toxicological assay (see above description) and maintained over 48 h in the climatic chamber under the aforementioned illumination regime, according to Mendes et al. (2013a). The only modification was the absence of the algae. The concentration of metal cations in seawater synthetic medium was quantified by ICP-AES (standard methods of the US Environmental Protection Agency, US EPA, method 6010C Table S1). The actual metallic cation concentration was used to plot DGR vs. the logarithm of metal cation concentration (Table S2, Fig. 1). Free metal cation concentration in a solution in synthetic seawater The ratio of hydrated cations ([M(H2O)x]n+) for each specific metal was calculated with the MINTEQA2 software (ionic strength 0.5–0.7) before being used to determine the free ion median inhibitory concentration (ICF50) (Mendes et al. 2013a, b) (Table S3). For the binary metal cation mixtures, the DGR vs. log concentration curves were plotted using the free metal Table 1 Median inhibitory concentrations (IC50 and ICF50) obtained during the 48-h toxicological assay with G. domingensis in synthetic seawater for single metal cations [Cd(II), Cu(II), Zn(II), Ca(II), and Mg(II)] Metals
Nominal IC50 (mM)
ICP-AES actual IC50 (mM)a
Free metal (%)b
Actual ICF50 (M)c
Cd(II) Cu(II) Zn(II) Ca(II) Mg(II)
0.030±0.002 1.0±0.1 0.70±0.05 21±2 126±10
0.030±0.0002 0.90±0.05 0.60±0.01 19±1 118±3
0.2 0.02 9 60 49
55±4×10−9 2.0±0.2×10−6 60±6×10−6 13±1×10−3 62±5×10−3
a
Determined by ICP-AES
b
Estimated using computational chemical equilibrium software (MINTEQA2, US Environmental Protection Agency, Washington, DC, version 3.0); Ionic strength=0.5–0.7
c
Percentage of free metal×ICP-AES IC50
TUmix ¼
C metalA C metalB þ ð1Þ IC50;metalA IC50;metalB
C mix C mix ⇒ IC50;mixC ¼ IC50;mixA TUmix
ð2Þ
where CmetalA, CmetalB, and Cmix are the concentrations of metal cations A, B and A + B; IC50, metalA and IC50, metalB are the experimental median inhibitory concentrations of metal cations A and B; TUmix and IC50, mixC are the calculated binary 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 metal concentration plots using the binary metal cation mixtures (Fig. 2). The IC50, mixC/IC50, mixE ratio reveals the toxicological effect of the binary mixture; specifically, an additive effect can be assigned when the ratio is 1.0 (ADD), a synergistic (or greater than ADD) effect (SYN) is operative when >1.0, and an antagonistic (or less than ADD) effect (ANT) is present when Cu(II) > > Zn(II) > >Ca(II) > >Mg(II) (Table 1). Free cadmium ions are ca. 4- and 1,000-fold more toxic than Cu(II) and Zn(II), respectively. Therefore, Cu(II) is 300-fold more toxic than Zn(II). As expected, the alkaline earth metals, like Ca(II) and Mg(II), are much less toxic than the other metal cations. The experimental median inhibitory concentration values for the binary mixtures of free metal cations (ICF50, mixE) were determined using the same procedure described for the single species (Fig. 2, Table 2, Tables S3 and S4). For each experimental point, the algae were exposed to the sum of the individual concentrations of metal cations.
Toxicological effects DGR vs. the logarithm of the ICP-AES-determined or the MINTEQA2-calculated metal cation concentrations is plotted using the experimental data presented in Tables S2 and S3 (Figs. 1 and 2). In contrast to the ICT50 and ICF50 values for single metal cations, the ICT50, mixE and ICF50, mixE of binary mixtures vary significantly (Table 1 and Table S3). However, the ratio IC50, mixC/IC50, mixE (used to calculate the toxicological effects) remains the same, independent of the individual ICT50, 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) mixtures are additive, based on the 20 % deviation in the IC50, mixC/IC50, mixE ratio. The binary Ca(II)/Mg(II) mixture is synergistic. All other binary mixture combinations with Cd(II) are antagonistic, while the remainder with Cu(II) are
Table 2 Experimental (ICF50, mixE) and calculated (ICF50, mixC) free median inhibitory concentration values for the binary metal cation mixtures obtained with the G. domingensis bioassay Binary mixtures
ICF50mixE, mM (μM)
ICF50,mixC, mMa (μM)
ICF50, mixC/ICF50, mixE
Effectb
Statistical significancec
Cd(II)/Cu(II) Cd(II)/Zn(II) Cd(II)/Mg(II) Cd(II)/Ca(II) Cu(II)/Zn(II)
94±23 0.05±0.01 80±1 14±1 0.02±0.01
75±1 0.36±0.08 22±4 3.8±0.7 0.03±0.01
0.8±0.3 0.1±0.3 0.3±0.2 0.3±0.2 1.5±0.3
ADD ANT ANT ANT SYN
p=0.2263 p=0.0013 p=1), antagonistic effect (ANT 1)), and antagonistic (ANT
