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Interactive toxic effects of heavy metals and humic acids on Vibrio fischeri
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Ecotoxicology and Environmental Safety 63 (2006) 158–167 www.elsevier.com/locate/ecoenv
Interactive toxic effects of heavy metals and humic acids on Vibrio fischeri V. Tsiridisa,b, M. Petalaa,b, P. Samarasc, S. Hadjispyroud, G. Sakellaropoulosa,b, A. Kungolose, a
Chemical Process Engineering Laboratory, Department of Chemical Engineering, Aristotle University of Thessaloniki, 54006 Thessaloniki, Greece b Chemical Process Engineering Research Institute, 6th km Harilaou Thermi Road, 57001 Thessaloniki, Greece c Department of Pollution Control Technologies, Technological Education Institution of West Macedonia, 50100 Kozani, Greece d Laboratory of Inorganic Chemistry, Department of Chemical Engineering, Aristotle University of Thessaloniki, 54006 Thessaloniki, Greece e Department of Planning and Regional Development, University of Thessaly, 38334 Volos, Greece Received 29 September 2004; received in revised form 12 April 2005; accepted 18 April 2005 Available online 4 June 2005
Abstract The effect of humic acids (HAs) on the toxicity of copper, zinc, and lead was investigated using the photobacterium Vibrio fischeri (Microtox test) as a test organism. The effects of HAs on metal toxicity were evaluated as functions of time and concentration in pure compound solutions. The toxicities of copper and lead were generally comparable, while the toxicity of zinc was lower than those of the other two metals. The toxicity of copper decreased with the addition of HAs, while the toxicity of zinc remained almost constant. On the other hand, the toxicity of lead increased, depending on the concentration of HAs. The interactive effects between copper and zinc and between lead and zinc were synergistic, while the interactive effect between copper and lead on the bioluminescence of V. fischeri was additive. The presence of HAs caused relatively high toxicity reduction in the binary mixtures of zinc and copper or zinc and lead, while the toxicity reduction in the case of the binary mixture of copper and lead was negligible. r 2005 Elsevier Inc. All rights reserved. Keywords: Microtox; Heavy metals; Copper; Zinc; Lead; Interactive effect; Humic acids; Bioavailability; Toxicity
1. Introduction The presence of heavy metals in solid and liquid wastes is an important issue related to the pollution of the environment. It is generally accepted that the solubility, bioavailability, and toxicity of heavy metals are dependent on various physicochemical parameters such as pH, hardness, interactive effects, and presence of natural organic matter (Janssen et al., 2003; Heijerick et al., 2003; Hadjispyrou et al., 2001; Peijnenburg and Jager, 2003; Manusadzianas et al., 2003). The pH affects the solubility, speciation, and transportation of metals Corresponding author. Fax: +30 24210 74380
E-mail address: [email protected] (A. Kungolos). 0147-6513/$ - see front matter r 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ecoenv.2005.04.005
from solid to liquid phase. Additionally, heavy metals are often present in the environment in mixtures, making the assessment of environmental hazards even more difficult due to the antagonistic or synergistic actions that may occur. The investigation of the joint toxic effects of chemicals in a mixture is generally based on comparison of the actual toxic effect of the mixture with the theoretically expected toxic effect deduced by a statistical model, using the toxic effects of the individual chemicals (Aoyama et al., 1987; Kungolos et al., 1999; Mowat and Bundy, 2002) The bioavailability of metals may be affected by the presence of natural organic matter, such as humic acids (HAs), which are produced by the degradation of dead organic materials. HAs consist of a variety of
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molecular structures such as alcylaromatic, quinoid, and aliphatic structures in the core and amino-acid-like or carbohydrate-like structures and carbonyl, carboxyl, phenyl, and hydroxyl groups in the periphery (Meems et al., 2004). HAs are able to bind a variety of metals at their carboxylic groups, altering the bioavailability and consequently the toxicity of these compounds. Many studies on the effect of complexation on the toxicity of heavy metals have been conducted using various complexing agents, such as ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), and various humic and fulvic acids. Sillanpaa and Oikari (1996) studied the impact of complexation by EDTA and DTPA on the toxicity of various heavy metals using the photobacterium Vibrio fischeri. The results indicated that the complexation did not significantly influence the toxicity of copper, cadmium, and mercury and reduced the toxicity of zinc and lead. Sorvari and Sillanpaa (1996) investigated the toxic effects of heavy metals with the presence of EDTA and DTPA on the crustacean Daphnia magna, concluding that the toxicity of copper, cadmium, and zinc decreased, while the toxicity of mercury slightly increased. Among the complexing agents, HAs are of major concern for many studies because they consist of natural organic matter ubiquitous in the environment. The assessment of the toxic effects of cadmium and chromium on the crustacean Daphnia pulex in the presence of various concentrations of HAs from 0.5 to 50 mg/L showed that the toxicity of cadmium depended on the concentrations of HAs, while the toxicity of chromium was not so influenced (Stackhouse and Benson, 1988). Furthermore, Winner (1984) studied the toxicity and bioaccumulation of cadmium and chromium in the presence of HAs using the crustacean D. magna. The results obtained from this study indicated that the presence of HAs decreased the toxicity of copper and increased the toxicity of cadmium, while there was no effect on the bioaccumulation for both metals tested. The effect of HAs on the toxicity of copper has been studied extensively compared with other heavy metals, due to the strong complexation capacity of this metal with HAs (Lubal et al., 1998; Pandey et al., 2000), resulting in most cases in a toxicity reduction (Winner, 1984; Kim et al., 1999; Alberts et al., 2000). The interpretation of the complexation mechanisms of heavy metals in the presence of HAs and the consequent changes in their toxicity and bioaccumulation are very complicated issues. Therefore, various models have been developed for the identification of complexation mechanisms and of interactions of heavy metals with HAs (Tipping, 1998). Recently, the biotic ligand models (BLMs) providing a quantity framework for the assessment of metal toxicity over a range of metal speciation, pH, hardness, and inorganic and
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organic complexing agents levels have been developed (Paquin et al., 2000). The BLMs have been successfully applied for the prediction of the toxic effects of heavy metals, such as copper, silver, and zinc, on the crustacean D. magna, the microalgae Selenastrum capricornutum, and the fathead minnow Pimephales promelas (Janssen et al., 2003; Heijerick et al., 2002; De Schamphelaere et al., 2002; Di Toro et al., 2001). Although many studies on the influence of complexation on the toxicity of heavy metals on various test organisms and on the interactive toxic effects of chemicals have been reported, there is a lack of available data on the effect of HAs on the toxicity of metal mixtures. Furthermore, few studies have dealt with the effect of HAs on the toxicity of metals in V. fischeri (Alberts et al., 2000), which does not exhibit high sensitivity to heavy metal toxicity in comparison to other test species, such as D. magna or Lemna minor (Ince et al., 1999; Kungolos et al., 2004). Still, V. fischeri is widely used because of its simplicity and rapid response, while at the same time it is considered a good indicator of cytotoxicity of compounds. The objective of this study was the evaluation of the effects of HAs on the toxicity of copper, zinc, and lead and on their binary mixtures, using V. fischeri as a test organism.
2. Materials and methods 2.1. Preparation of metal and HA solutions The solutions with initial concentrations of 10 mg/L of the tested metals were freshly prepared by dissolving the following chloride salts in deionized water: zinc chloride (ZnCl2) provided by Merck (Germany) and copper chloride dehydrated (CuCl2.2H2O) and lead chloride (PbCl2) provided by J.T. Baker (The Netherlands). The effect of HA on the toxicity of the tested heavy metals was evaluated using 1, 10, and 20 mg/L HAs that were prepared by a stock HA (Fluka, Germany) solution of 100 mg/L. The stock HA solution was prepared at pH 10, with continuous stirring to achieve a complete dissolution. All chemicals used were of analytical grade and the concentrations of the metals were expressed as mg/L of metal ions. Furthermore, the pH value of all metal solutions was adjusted to 770.2, prior to the toxicity tests, by the addition of 0.1 N HCl or 0.1 N NaOH solutions. 2.2. Toxicity assessment of heavy metals The toxicity of heavy metals was evaluated using the bioluminescence bacteria V. fischeri (Microtox test) that were in freeze-dried form (SDI, USA) and activated prior to use by a reconstitution solution. Since V. fischeri is a marine organism, an adjustment of the osmotic
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pressure of the samples was applied to obtain samples with 2% salinity, using a concentrated salt solution (solution containing 22% NaCl in deionized water). The light emission of the test organisms obtained by their direct contact with the samples was measured using the Microtox 500 analyzer (SDI) within a short exposure time of 15 min. The data processing was performed using the Microtox Omni software (SDI). The EC50 values (metal ion concentration that caused 50% inhibition on the bioluminescence of V. fischeri; expressed as mg/L of Cu, Zn, or Pb metal ion) of the heavy metals in the presence and in the absence of HA was evaluated using the 45% basic test (Microbics Corp., 1992), while the effect of HA on the toxicity of heavy metals was assessed with contact times of 0.5, 2, and 4 h. The tested concentrations of HA in heavy metal solutions were 1, 10, and 20 mg/L. Additionally, the toxicity of HA was measured in the applied concentration range for the determination of a potential toxic effect that may be due to the action of HA. 2.3. Toxicity assessment of binary mixtures The toxicities of certain concentrations of heavy metals and corresponding binary mixtures were evaluated to determine their interactive toxic effects. The effect of HA on the toxicity of the binary mixtures of heavy metals was evaluated in the presence of 10 mg/L HA. The toxicity of the metal mixture containing 10 mg/ L HA was evaluated after 3 h contact time and the results were compared with the corresponding values obtained without the addition of HA. The toxicities of the solutions containing one metal and those of the binary mixtures in the presence and in the absence of 10 mg/L HA, expressed as percent effect, were evaluated using the 45% screening test (Microbics Corp., 1992). The theoretically expected effect of the binary mixtures was evaluated using a simple mathematical model based on the theory of probabilities (Kungolos et al., 1999). According to this model, if P1 is the inhibition caused by a certain concentration of chemical A1 and P2 the inhibition caused by a certain concentration of chemical A2, then the theoretically expected additive inhibition PðEÞ, when those concentrations of the two chemicals are applied together, is given by the following equation: PðEÞ ¼ P1 þ P2 P1 P2 =100.
(1)
The null hypotheses were that the observed values were higher or lower than the theoretically predicted values, for synergistic and antagonistic effects, respectively. The result was considered to be antagonistic or synergistic only if the observed effect was significantly lower or higher, respectively, than the theoretically predicted value at the 0.05 level of significance.
3. Results 3.1. Effect of HA on the toxicity of heavy metals The toxicity measurements of the pure compounds showed that Cu and Pb exhibited similar toxic effects and the toxic effect of Zn was lower than that of the other metals. The EC50 values for Cu and Pb were 0.25 mg/L (confidence range 0.18–0.30 mg/L) and 0.48 mg/L (confidence range 0.37–0.63 mg/L), respectively, while the corresponding value for Zn was 1.50 mg/L (confidence range 1.36–1.59 mg/L). Similar results were found by Mowat and Bundy (2002) and Utkigar et al. (2004). On the other hand, for concentrations between 1 and 20 mg/L, the observed effect of HA on the bioluminescence of V. fischeri was negligible, varying from 10% to 10%. The effect of color of tested HA solutions on the reduction of light emission was also estimated using the Microtox Omni software. According to the procedure, absorbance of the tested solutions at a wavelength of 490 nm was recorded and introduced to the software. The absorbances for solutions of 20, 10, and 1 mg/L HA were 0.05, 0.025, and 0.002 cm1, respectively, resulting in minor alterations in the toxic effect values. Therefore, it was assumed that color correction did not influence the results and this negligible effect was not taken into account. The effect of HA on the toxicity of Cu, Zn, and Pb as a function of contact time with HA is shown in Table 1. The EC50 values and the corresponding 95% confidence ranges are expressed as mg/L of the total metal ion concentration that may be present in the solution as free ions or bound to HA. As presented in Table 1, the toxicity of Cu decreased with increasing HA concentration and contact time. It was observed that the addition of 1 mg/L HA was not sufficient to alter the toxicity of Cu, since the EC50 value was approximately the same as compared to that of Cu without the addition of HA, even after 4 h of contact time. The toxic effect of Cu with the addition of 10 mg/L HA was slightly reduced after 2 h of contact time, while no further significant reduction was recorded after 4 h of contact time. The higher toxicity reduction, equal to 55%, was observed for HA concentration of 20 mg/L after 4 h contact time. This result could be attributed to the increase of Cu complexation with HA, which, according to Kim et al. (1999), is time and HA dose dependent. Alberts et al. (2000) found that the reduction of toxic effect of Cu on V. fischeri varied between 44% and 100%, depending on the type of HA used. On the other hand, the presence of HA did not exhibit any significant effect on the toxicity of Zn, while a maximum 81% increase of the toxic effect was observed in the case of Pb. Furthermore, it is clear that contact time had a small effect on Cu toxicity, while the
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Table 1 Effect of HA on the toxicity of Cu, Zn, and Pb as a function of contact time with HA Heavy metal
HA concentration (mg/L)
15 min EC50 (95% confidence range) Contact time 0.5 h
Contact time 2 h
Contact time 4 h
Cu
0 1 10 20
0.20 0.21 0.25 0.31
(0.17–0.24) (0.17–0.25) (0.22–0.28) (0.28–0.33)
0.22 0.24 0.32 0.42
(0.17–0.28) (0.19–0.29) (0.28–0.37) (0.41–0.43)
0.23 0.25 0.34 0.46
(0.18–0.29) (0.22–0.29) (0.27–0.43) (0.39–0.54)
Zn
0 1 10 20
1.49 1.53 1.58 1.51
(1.37–1.59) (1.52–1.55) (1.37–1.85) (1.13–1.96)
1.42 1.49 1.43 1.32
(1.31–1.55) (1.20–1.79) (1.26–1.63) (1.24–1.39)
1.51 1.58 1.51 1.60
(1.40–1.61) (1.37–1.81) (1.15–1.93) (1.34–1.98)
Pb
0 1 10 20
0.50 0.33 0.31 0.14
(0.38–0.65) (0.23–0.47) (0.19–0.47) (0.09–0.25)
0.49 0.36 0.16 0.22
(0.38–0.63) (0.33–0.39) (0.11–0.22) (0.06–0.26)
0.44 0.38 0.15 0.09
(0.36–0.52) (0.34–0.43) (0.14–0.16) (0.04–0.23)
Fig. 1. Comparison of dose response curves for Cu and Cu/HA solutions after 2 h contact time.
toxicities of Zn and Pb were not affected with contact time increase. The dose response curves of Cu, Zn, and Pb for single-metal solutions and in the presence of 10 and 20 mg/L HA after 2 h contact time are illustrated in Figs. 1–3, respectively. As shown in Fig. 1, the toxicity of Cu in the absence of HA increased with concentration, reaching up to 100% for Cu concentrations higher than 0.8 mg/L, while a decrease of toxic effect was observed with the addition of HA at almost all dilutions. On the other hand, the addition of HA in Zn solution (Fig. 2) did not affect the dose response curve of Zn pure Zn solution and the solutions containing 10 and 20 mg/L HA showed the same patterns. In the case of Pb (Fig. 3), the dose response curve and the effect of HA on the metal toxicity were completely different. As shown in Fig. 3, a steep increase of metal toxicity was observed for pure Pb concentrations up to 1 mg/L, while for higher concentrations the toxicity was almost constant, reaching up to 90%. This result
Fig. 2. Comparison of dose response curves for Zn and Zn/HA solutions after 2 h contact time.
Fig. 3. Comparison of dose response curves for Pb and Pb/HA solutions after 2 h contact time.
could be attributed to the saturation of Pb transport sites through the biological membrane of V. fischeri (Slaveykova et al., 2004). The presence of HA in Pb
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Fig. 4. Comparison between theoretically expected and observed inhibitions for the combined effect of Cu and Zn on the bioluminescence of V. fischeri.
Fig. 5. Comparison between theoretically expected and observed inhibitions for the combined effect of Cu and Pb on the bioluminescence of V. fischeri.
solution caused a relatively high increase of metal toxicity at low concentrations, while for Pb concentrations higher than 1 mg/L the toxicity was almost constant, reaching up to 95%. 3.2. Toxicity of binary mixtures of heavy metals The interactive effects between Cu and Zn on the bioluminescence of V. fischeri for some concentration combinations without the addition of HA are illustrated in Fig. 4. As shown in Fig. 4, the effect was synergistic at all the concentration combinations tested. The gray bars corresponding to the observed effects in A, B, and C indicate that the increase of Zn concentration from 0.43 to 1.73 mg/L induces an increase of toxic effect from about 40% to about 80%, while the concentration of Cu was maintained constant and equal to 0.12 mg/L. In general, the differences between the observed interactive toxic effects and the theoretically expected effects for binary mixtures were greater in the mixtures with
relatively low Cu concentrations than in those with higher Cu concentrations. The observed bioluminescence inhibition for the mixtures with 0.12 mg/L Cu was about 45% higher than the theoretically expected inhibition. On the other hand, for the mixtures containing 0.25 mg/L Cu, the differences between the observed and the expected effects were smaller, reaching up to 25%. Still, it is important to point out that the differences between theoretically expected and observed values were significant at all concentration combinations tested. The toxic responses of the binary mixtures of Cu and Pb were found to be close to the theoretically calculated values, as shown in Fig. 5. The expected toxic effects varied from 20% to 60%, while the observed values did not differ significantly. Therefore, an additive interactive mode of action can be assumed for the effect of Cu and Pb on V. fischeri. The interactive effect between Zn and Pb on the bioluminescence of V. fischeri for some concentration
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Fig. 6. Comparison between theoretically expected and observed inhibitions for the combined effect of Zn and Pb on the bioluminescence of V. fischeri.
Fig. 7. Comparison between observed effects of combined concentrations of Cu and Zn on the bioluminescence of V. fischeri, without the addition of HA and with the addition of 10 mg/L HA after 3 h contact time.
combinations is illustrated in Fig. 6. The theoretically expected values ranged between 28% and 52% and the observed values between 41% and 87%. The observed values were significantly higher than the theoretically predicted values for all concentration combinations, confirming synergistic action between Zn and Pb. 3.3. Effect of HA on the toxicity of binary mixtures of heavy metals The toxicities of the binary mixtures of heavy metals that were tested for the investigation of the interactive effects were also measured in the presence of 10 mg/L HA for contact time 3 h. The effect of HA on the toxicity of some binary mixtures of Cu and Zn is depicted in Fig. 7. It was observed that, in all tested metal concentration combinations, the addition of HA caused a significant reduction in the toxic effect of metals after 3 h contact time. The toxicity reduction with the addition of HA varied between 40% and 90%.
Higher reductions were observed for the mixtures with relatively low Zn concentrations. Thus, for the mixture A (0.12 mg/L Cu and 0.43 mg/L Zn) the bioluminescence inhibition decreased from 40% to 5% with the addition of 10 mg/L HA. Fig. 8 shows the effect of HA on the toxicity of different binary mixtures of Cu and Pb. The presence of 10 mg/L HA in the binary mixtures of Cu and Pb did not significantly influence the toxicity of mixtures compared with the corresponding values in the absence of HA. Although different metal concentration combinations were tested, in most cases the bioluminescence inhibition did not significantly vary in the presence of HA. The effect of HA on the toxicity of some binary mixtures of Zn and Pb is depicted in Fig. 9. As shown in Fig. 9, in most metal concentration combinations tested the toxicity decreased in the presence of HA. The range of toxicity reduction was between 28% and 45% for Zn concentrations up to 0.86 mg/L, while for Zn concentration of 1.73 mg/L, the toxicity reduction was about 15%.
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Fig. 8. Comparison between observed effects of combined concentrations of Cu and Pb on the bioluminescence of V. fischeri, without the addition of HA and with the addition of 10 mg/L HA after 3 h contact time.
Fig. 9. Comparison between the observed effects of combined concentrations of Zn and Pb on the bioluminescence of V. fischeri, without the addition of HA and with the addition of 10 mg/L HA after 3 h contact time.
4. Discussion The investigation of the influence of HA on the toxicity of Cu, Zn, and Pb showed that the toxic response varied depending on the type of heavy metals and the concentration of HA. The toxicity of Zn was not influenced by the presence of HA, while the toxicity of the other heavy metals was influenced by HA concentrations higher than 1 mg/L. A decrease of toxic effect due to complexation was observed only in the case of Cu, whereas the complexation of Pb resulted in an increase of toxic effect. Based on complexation kinetics of the tested heavy metals, reported in the literature, Cu has the greatest binding efficiency, while the binding efficiency of Zn is the lowest compared with the other metals of concern (Lubal et al., 1998; Pandey et al., 2000). As a result of Cu complexation, a toxicity reduction was observed and it could be assumed that Cu toxicity is dominated by the
free ion activity. These results are consistent with results by other researchers reporting that Cu complexation with organic ligands results in a decrease of toxic effect on various test organisms (Campbell et al., 2000; Winner, 1984; Kim et al., 1999; Alberts et al., 2000). Furthermore, Cheng and Allen (2001) observed bioavailability reduction of copper to plants, when complexed with organic ligands, resulting in toxicity decrease. With regarding to Pb toxicity, although Pb forms relatively strong complexes with HA, an increase of the toxic effect was observed in the presence of HA (Fig. 3). This could be attributed to potential interactions between the Pb complexes and the transport sites of biological membrane of V. fischeri (Campbell et al., 2000; Slaveykova et al., 2004). It can be assumed that the Pb complexes with HAs are bioavailable to a higher extent than the soluble phases of Pb. A different pattern of Pb toxicity is shown while comparing the dose response curves of the tested heavy metals. The toxicity
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of Pb remained almost constant for concentrations higher than 1 mg/L (Fig. 6), whereas the toxicity of the other heavy metals increased steeply with concentration, reaching up to 100% (Figs. 4 and 5). Considering that, in the first place, Pb forms complexes with organic ligands and, in the second place, the toxicity after complexation increases, it is clear that the free ions of Pb did not determine the degree of the toxic effect. Although the free metal ions are considered the most bioavailable form, the metal toxicity may be affected by various interactions between living cells and ligands and the by physiological status of the organism (Campbell et al., 2000; Simkiss and Taylor, 2001). For example, free metal ions may attach to binding sites and induce changes in cellular behavior. Furthermore, there are organisms that can modify their extracellular sites in powerful ways (Simkiss and Taylor, 2001). The interactive effects between Cu and Zn were synergistic for all concentration combinations tested (Fig. 4). This result is in accordance with results presented in a study conducted by Utkigar et al. (2004). Preston et al. (2000) also found that the combined solutions of Zn with Cu or Cd caused synergistic interactions on Escherichia coli, using genetically modified luminescence-based microbial biosensors. On the other hand, Cu in combination with Pb generally presented an additive mode of interaction on V. fischeri (Fig. 5). Ren et al. (2004), using the Shk1 assay, found that the additive, synergistic, or antagonistic effects of various concentration combinations between Cu and Pb depended on the concentration of each metal in the binary mixture. Such results were not found in our study, where in all concentration combinations tested the difference between the theoretically expected and the observed result was not significant when Cu was applied together with Pb. Furthermore, in our study it was found that the binary mixtures of Zn and Pb showed a synergistic effect for all concentration combinations tested (Fig. 6). The interactive effect of all binary mixtures that were examined varied between additive and synergistic, while an antagonistic effect was not observed in any case. In addition, it can be concluded that the interactive effect depended on the type of the pair of metals contained in the binary mixture, as Cu in the presence of Zn showed synergism, while the same metal in the presence of Pb exhibited an additive effect. Furthermore, the concentrations of the metals in the binary mixtures did not affect the type of interaction, at least for all the concentration combinations tested in this study. However, it has been shown in other studies that the mode of interaction may be dependent on metal concentrations (Hadjispyrou et al., 2001; Ren et al., 2004). The addition of HAs in the binary metal mixtures resulted in a toxicity reduction in most of the cases studied. Comparing the toxic effects of the binary
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mixtures with the corresponding values in the presence of 10 mg/L HA, it is clear that relatively high toxicity reduction was observed for the binary mixtures of Cu/ Zn and Zn/Pb (Figs. 7 and 9), while the toxicity reduction of the binary mixtures between Cu and Pb was generally low (Fig. 8). The high toxicity reduction of the Cu/Zn mixture could be attributed to the complexation of Cu with HA that may be assumed to eliminate the bioavailable phase of Cu. On the other hand, Cu complexation seemed to cause a relatively small effect on the toxicity of Cu/Pb mixtures, possibly due to the antagonistic action of complexation as both metals form moderate to strong complexes with HA. Furthermore, it can be assumed that the failure of HA to reduce the toxicity of Cu in the presence of Pb may be linked with the inability of HA to decrease the toxicity of Pb, another result that has been obtained in the current study. On the contrary, although the presence of HA caused an increase in Pb toxicity and did not affect the toxicity of Zn, the toxicity of Zn/Pb mixtures decreased in the presence of 10 mg/L HA. It can be assumed that the reduction in Pb toxicity in the presence of Zn can be attributed to the formation of complexes between Pb and HA, because the Pb concentrations in this experiment were relatively low (0.27–0.67 mg/L). On the other hand, in the experiments where HA failed to reduce the toxicity of Pb applied alone the tested concentrations were quite higher, ranging up to 3.5 mg/L. Moreover, with regard to the complexation capacity of the metals with HA in the binary mixtures, the molecular relative ratios should also be considered. It should be noted that the metal/HA ratio in single-metal solutions was significantly lower than the ratios examined in the binary mixtures. The HA concentration of 10 mg/L was chosen as a representative concentration of natural organic matter contained in the aquatic environment (Stackhouse and Benson, 1988). Furthermore, the addition of NaCl for the adjustment of the osmotic pressure of the solutions prior to the toxicity test includes another parameter that may affect the complexation of heavy metals and as a result their toxicity. This addition may cause an increase in the ionic strength of the solutions that in many cases reduces the binding capacity of heavy metals to HA (HamiltonTaylor et al., 2002). Furthermore, it should be noticed that the tested metal concentrations were significantly higher than the water quality criteria posed by several countries. For example, the continuous concentration criteria in freshwater for dissolved copper are 9 mg/L in the US (recommended), 5 mg/L in Canada, and 2 mg/L in Switzerland, while the criterion for lead is 2.5 mg/L in the US, and the criteria for zinc are 120 mg/L in the US and 5 mg/L in Switzerland (USEPA, 2002; Xue et al., 2003; Nriagu et al., 1998). There were basically two reasons that relatively high metal concentrations were used in this study. First, as explained in the introduction, V.
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fischeri does not exhibit high sensitivity to heavy metal toxicity. Second, the high metal concentrations used assisted in investigating the potential detoxification effects of HAs. As an extension to this work, further studies investigating both the toxic effects of heavy metals at concentrations as low as those found in surface waters, and the impact of HAs on the toxicity of combined metal solutions on test organisms that exhibit high sensitivity could be conducted.
5. Conclusions The complexation of heavy metals with HAs is an important parameter that may alter the toxicity and bioavailability of heavy metals in aquatic systems. The examination of the complexation of heavy metals with HAs showed that the toxicity of Cu decreased with the addition of HAs while the toxicity of Zn remained almost constant and the toxicity of Pb increased. The interactive effects of Cu and Zn mixtures and Pb and Zn mixtures on V. fischeri were found to be synergistic, while the interactive effect of Cu and Pb mixtures was additive for all concentration combinations tested in this study. It was found that the mode of interaction depended on the type of the pair of metals and not on the concentrations tested. The presence of HA in the binary mixtures caused relatively high toxicity reduction mainly in the cases where synergistic action occurred. It was found that the presence of HAs reduced both the toxicity of Cu and Zn applied together and that of Pb and Zn applied together. On the contrary the toxicity of Cu and Pb applied together on V. fischeri was not much altered by the presence of HA. It can also be concluded that, for the proper estimation of the environmental hazard caused by a chemical, the presence of other constituents that may interact with the chemical of concern should be evaluated.
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acute copper toxicity to Daphnia magna. Comp. Biochem. Phys. C 133, 243–258. Di Toro, D.M., Allen, H.E., Bergman, H.L., Meyer, J.S., Paquin, P.R., Santore, R.C., 2001. Biotic ligand model of the acute toxicity of metals. 1. Technical basis. Environ. Toxicol. Chem. 20 (10), 2383–2396. Hadjispyrou, S., Kungolos, A., Anagnostopoulos, A., 2001. Toxicity, bioaccumulation, and interactive effects of organotin, cadmium, and chromium on Artemia franciscana. Ecotoxicol. Environ. Saf. 49, 179–186. Hamilton-Taylor, J., Postill, A.S., Tipping, E., Harper, M.P., 2002. Laboratory measurements and modeling of metal–humic interactions under estuarine conditions. Geochim. Cosmochim. Acta 66 (3), 403–415. Heijerick, D.G., De Schamphelaere, K.A.C., Janssen, C.R., 2002. Biotic ligand model development predicting Zn toxicity to the alga Pseudokirchneriella subcapitata: possibilities and limitations. Comp. Biochem. Phys. C 133, 207–218. Heijerick, D.G., Janssen, C.R., De Coen, W.M., 2003. The combined effects of hardness, pH, and dissolved organic carbon on the chronic toxicity of Zn to D. magna: development of a surface response model. Arch. Environ. Contam. Toxicol. 44, 210–217. Ince, N.H., Dirilgen, N., Apikyan, I.G., Tezcanli, G., Ustun, B., 1999. Assessment of toxic interactions of heavy metals in binary mixtures: a statistical approach. Arch. Environ. Contam. Toxicol. 36, 365–372. Janssen, C.R., Heijerick, D.G., De Schamphelaere, K.A.C., Allen, H.E., 2003. Environmental risk assessment of metals: tools for incorporating bioavailability. Environ. Int. 28, 793–800. Kim, S.D., Ma, H., Allen, H.E., Cha, D.K., 1999. Influence of dissolved organic matter on the toxicity of copper to Ceriodaphnia dubia: effect of complexation kinetics. Environ. Toxicol. Chem. 18 (11), 2433–2437. Kungolos, A., Samaras, P., Kipopoulou, A.M., Zoumboulis, A., Sakellaropoulos, G.P., 1999. Interactive toxic effects of agrochemicals on aquatic organisms. Water Sci. Technol. 40 (1), 357–364. Kungolos, A., Hadjispyrou, S., Petala, M., Tsiridis, V., Samaras, P., Sakellaropoulos, G.P., 2004. Toxic properties of metals and organotin compounds and their interactions on Daphnia magna and Vibrio fischeri. Water Air Soil Pollut. Focus 4, 101–110. Lubal, P., Siroky, D., Fetsch, D., Havel, J., 1998. The acidobasic and complexation properties of humic acids: study of complexation of Czech humic acids with metal ions. Talanta 47, 401–412. Manusadzianas, L., Balkelytey, L., Sadauskas, K., Blinova, I., Pollumaa, L., Kahru, A., 2003. Ecotoxicological study of Lithuanian and Estonian wastewaters: selection of the biotests, and correspondence between toxicity and chemical-based indices. Aquat. Toxicol. 63, 27–41. Meems, N., Steinberg, C.E.W., Wiegand, C., 2004. Direct and interacting toxicological effects on the waterflea (Daphnia magna) by natural organic matter, synthetic humic substances and cypermethrin. Sci. Total Environ. 319, 123–136. Microbics Corp., 1992. Microtox Manual, A Toxicity Testing Handbook, vol. 1–5. Microbics Corp., Carlsbad, CA, USA. Mowat, F.S., Bundy, K.J., 2002. Experimental and mathematical/ computational assessment of the acute toxicity of chemical mixtures from the Microtox assay. Adv. Environ. Res. 6, 547–558. Nriagu, J.O., Wong, H.K.T., Lawson, G., Daniel, P., 1998. Saturation of ecosystems with toxic metals in Sudbury basin, Ontario, Canada. Sci. Total Environ. 223, 99–117. Pandey, A.K., Pandey, S.D., Misra, V., 2000. Stability constants of metal-humic acid complexes and its role in environmental detoxification. Ecotoxicol. Environ. Saf. 47, 195–200. Paquin, P.R., Santore, R.C., Wu, K.B., Kavvadas, C.D., Di Toro, D.M., 2000. The biotic ligand model: a model of the acute toxicity of metals to aquatic life. Environ. Sci. Policy 3, 175–182.
ARTICLE IN PRESS V. Tsiridis et al. / Ecotoxicology and Environmental Safety 63 (2006) 158–167 Peijnenburg, W.J.G.M., Jager, T., 2003. Monitoring approaches to assess bioaccessibility and bioavailability of metals: matrix issues. Ecotoxicol. Environ. Saf. 56, 63–77. Preston, S., Coad, N., Townend, J., Killham, K., Graeme, I.P., 2000. Biosensing the acute toxicity of metal interactions: are they additive, synergistic, or antagonistic? Environ. Toxicol. Chem. 19 (3), 775–780. Ren, S., Mee, R.W., Frymier, P.D., 2004. Using factorial experiments to study the toxicity of metal mixtures. Ecotoxicol. Environ. Saf. 59, 38–43. Sillanpaa, M., Oikari, A., 1996. Assessing the impact of complexation by EDTA and DTPA on heavy metal toxicity using Microtox bioassay. Chemosphere 32 (8), 1485–1497. Simkiss, K., Taylor, M.G., 2001. Trace element speciation at cell membranes: aqueous, solid and lipid phase effects. J. Environ. Monitor. 3, 15–21. Slaveykova, V.I., Parthasarathy, N., Buffle, J., Wilkinson, K.J., 2004. Permeation liquid membrane as a tool for monitoring bioavailable Pb in natural waters. Sci. Total Environ. 328, 55–68. Sorvari, J., Sillanpaa, M., 1996. Influence of metal complex formation on heavy metals and free EDTA and DTPA acute
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toxicity determined by Daphnia magna. Chemosphere 33 (6), 1119–1127. Stackhouse, A., Benson, W., 1988. The influence of humic acid on the toxicity and bioavailability of selected trace metals. Aquat. Toxicol. 13, 99–108. Tipping, E., 1998. Humic ion-binding model VI: an improved description of the interactions of protons and metal ions with humic substances. Aquat. Geochem. 4, 3–48. USEPA, 2002. National Recommended Water Quality Criteria: 2002. US Environmental Protection Agency, Washington, DC EPA/822R-02-047. Utkigar, V.P., Chaudhary, N., Koeniger, A., Tabak, H.H., Haines, J.R., Govind, R., 2004. Toxicity of metals and metal mixtures: analysis of concentration and time dependence for zinc and copper. Water Res. 38, 3651–3658. Winner, R.W., 1984. The toxicity and bioaccumulation of cadmium and copper as affected by humic acid. Aquat. Toxicol. 5, 267–274. Xue, H., Nhat, P.H., Gachter, R., Hooda, P.S., 2003. The transport of Cu and Zn from agricultural soils to surface water in a small catchment. Adv. Environ. Res. 8, 69–76.
Ecotoxicology and Environmental Safety 63 (2006) 158–167 www.elsevier.com/locate/ecoenv
Interactive toxic effects of heavy metals and humic acids on Vibrio fischeri V. Tsiridisa,b, M. Petalaa,b, P. Samarasc, S. Hadjispyroud, G. Sakellaropoulosa,b, A. Kungolose, a
Chemical Process Engineering Laboratory, Department of Chemical Engineering, Aristotle University of Thessaloniki, 54006 Thessaloniki, Greece b Chemical Process Engineering Research Institute, 6th km Harilaou Thermi Road, 57001 Thessaloniki, Greece c Department of Pollution Control Technologies, Technological Education Institution of West Macedonia, 50100 Kozani, Greece d Laboratory of Inorganic Chemistry, Department of Chemical Engineering, Aristotle University of Thessaloniki, 54006 Thessaloniki, Greece e Department of Planning and Regional Development, University of Thessaly, 38334 Volos, Greece Received 29 September 2004; received in revised form 12 April 2005; accepted 18 April 2005 Available online 4 June 2005
Abstract The effect of humic acids (HAs) on the toxicity of copper, zinc, and lead was investigated using the photobacterium Vibrio fischeri (Microtox test) as a test organism. The effects of HAs on metal toxicity were evaluated as functions of time and concentration in pure compound solutions. The toxicities of copper and lead were generally comparable, while the toxicity of zinc was lower than those of the other two metals. The toxicity of copper decreased with the addition of HAs, while the toxicity of zinc remained almost constant. On the other hand, the toxicity of lead increased, depending on the concentration of HAs. The interactive effects between copper and zinc and between lead and zinc were synergistic, while the interactive effect between copper and lead on the bioluminescence of V. fischeri was additive. The presence of HAs caused relatively high toxicity reduction in the binary mixtures of zinc and copper or zinc and lead, while the toxicity reduction in the case of the binary mixture of copper and lead was negligible. r 2005 Elsevier Inc. All rights reserved. Keywords: Microtox; Heavy metals; Copper; Zinc; Lead; Interactive effect; Humic acids; Bioavailability; Toxicity
1. Introduction The presence of heavy metals in solid and liquid wastes is an important issue related to the pollution of the environment. It is generally accepted that the solubility, bioavailability, and toxicity of heavy metals are dependent on various physicochemical parameters such as pH, hardness, interactive effects, and presence of natural organic matter (Janssen et al., 2003; Heijerick et al., 2003; Hadjispyrou et al., 2001; Peijnenburg and Jager, 2003; Manusadzianas et al., 2003). The pH affects the solubility, speciation, and transportation of metals Corresponding author. Fax: +30 24210 74380
E-mail address: [email protected] (A. Kungolos). 0147-6513/$ - see front matter r 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ecoenv.2005.04.005
from solid to liquid phase. Additionally, heavy metals are often present in the environment in mixtures, making the assessment of environmental hazards even more difficult due to the antagonistic or synergistic actions that may occur. The investigation of the joint toxic effects of chemicals in a mixture is generally based on comparison of the actual toxic effect of the mixture with the theoretically expected toxic effect deduced by a statistical model, using the toxic effects of the individual chemicals (Aoyama et al., 1987; Kungolos et al., 1999; Mowat and Bundy, 2002) The bioavailability of metals may be affected by the presence of natural organic matter, such as humic acids (HAs), which are produced by the degradation of dead organic materials. HAs consist of a variety of
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molecular structures such as alcylaromatic, quinoid, and aliphatic structures in the core and amino-acid-like or carbohydrate-like structures and carbonyl, carboxyl, phenyl, and hydroxyl groups in the periphery (Meems et al., 2004). HAs are able to bind a variety of metals at their carboxylic groups, altering the bioavailability and consequently the toxicity of these compounds. Many studies on the effect of complexation on the toxicity of heavy metals have been conducted using various complexing agents, such as ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), and various humic and fulvic acids. Sillanpaa and Oikari (1996) studied the impact of complexation by EDTA and DTPA on the toxicity of various heavy metals using the photobacterium Vibrio fischeri. The results indicated that the complexation did not significantly influence the toxicity of copper, cadmium, and mercury and reduced the toxicity of zinc and lead. Sorvari and Sillanpaa (1996) investigated the toxic effects of heavy metals with the presence of EDTA and DTPA on the crustacean Daphnia magna, concluding that the toxicity of copper, cadmium, and zinc decreased, while the toxicity of mercury slightly increased. Among the complexing agents, HAs are of major concern for many studies because they consist of natural organic matter ubiquitous in the environment. The assessment of the toxic effects of cadmium and chromium on the crustacean Daphnia pulex in the presence of various concentrations of HAs from 0.5 to 50 mg/L showed that the toxicity of cadmium depended on the concentrations of HAs, while the toxicity of chromium was not so influenced (Stackhouse and Benson, 1988). Furthermore, Winner (1984) studied the toxicity and bioaccumulation of cadmium and chromium in the presence of HAs using the crustacean D. magna. The results obtained from this study indicated that the presence of HAs decreased the toxicity of copper and increased the toxicity of cadmium, while there was no effect on the bioaccumulation for both metals tested. The effect of HAs on the toxicity of copper has been studied extensively compared with other heavy metals, due to the strong complexation capacity of this metal with HAs (Lubal et al., 1998; Pandey et al., 2000), resulting in most cases in a toxicity reduction (Winner, 1984; Kim et al., 1999; Alberts et al., 2000). The interpretation of the complexation mechanisms of heavy metals in the presence of HAs and the consequent changes in their toxicity and bioaccumulation are very complicated issues. Therefore, various models have been developed for the identification of complexation mechanisms and of interactions of heavy metals with HAs (Tipping, 1998). Recently, the biotic ligand models (BLMs) providing a quantity framework for the assessment of metal toxicity over a range of metal speciation, pH, hardness, and inorganic and
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organic complexing agents levels have been developed (Paquin et al., 2000). The BLMs have been successfully applied for the prediction of the toxic effects of heavy metals, such as copper, silver, and zinc, on the crustacean D. magna, the microalgae Selenastrum capricornutum, and the fathead minnow Pimephales promelas (Janssen et al., 2003; Heijerick et al., 2002; De Schamphelaere et al., 2002; Di Toro et al., 2001). Although many studies on the influence of complexation on the toxicity of heavy metals on various test organisms and on the interactive toxic effects of chemicals have been reported, there is a lack of available data on the effect of HAs on the toxicity of metal mixtures. Furthermore, few studies have dealt with the effect of HAs on the toxicity of metals in V. fischeri (Alberts et al., 2000), which does not exhibit high sensitivity to heavy metal toxicity in comparison to other test species, such as D. magna or Lemna minor (Ince et al., 1999; Kungolos et al., 2004). Still, V. fischeri is widely used because of its simplicity and rapid response, while at the same time it is considered a good indicator of cytotoxicity of compounds. The objective of this study was the evaluation of the effects of HAs on the toxicity of copper, zinc, and lead and on their binary mixtures, using V. fischeri as a test organism.
2. Materials and methods 2.1. Preparation of metal and HA solutions The solutions with initial concentrations of 10 mg/L of the tested metals were freshly prepared by dissolving the following chloride salts in deionized water: zinc chloride (ZnCl2) provided by Merck (Germany) and copper chloride dehydrated (CuCl2.2H2O) and lead chloride (PbCl2) provided by J.T. Baker (The Netherlands). The effect of HA on the toxicity of the tested heavy metals was evaluated using 1, 10, and 20 mg/L HAs that were prepared by a stock HA (Fluka, Germany) solution of 100 mg/L. The stock HA solution was prepared at pH 10, with continuous stirring to achieve a complete dissolution. All chemicals used were of analytical grade and the concentrations of the metals were expressed as mg/L of metal ions. Furthermore, the pH value of all metal solutions was adjusted to 770.2, prior to the toxicity tests, by the addition of 0.1 N HCl or 0.1 N NaOH solutions. 2.2. Toxicity assessment of heavy metals The toxicity of heavy metals was evaluated using the bioluminescence bacteria V. fischeri (Microtox test) that were in freeze-dried form (SDI, USA) and activated prior to use by a reconstitution solution. Since V. fischeri is a marine organism, an adjustment of the osmotic
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pressure of the samples was applied to obtain samples with 2% salinity, using a concentrated salt solution (solution containing 22% NaCl in deionized water). The light emission of the test organisms obtained by their direct contact with the samples was measured using the Microtox 500 analyzer (SDI) within a short exposure time of 15 min. The data processing was performed using the Microtox Omni software (SDI). The EC50 values (metal ion concentration that caused 50% inhibition on the bioluminescence of V. fischeri; expressed as mg/L of Cu, Zn, or Pb metal ion) of the heavy metals in the presence and in the absence of HA was evaluated using the 45% basic test (Microbics Corp., 1992), while the effect of HA on the toxicity of heavy metals was assessed with contact times of 0.5, 2, and 4 h. The tested concentrations of HA in heavy metal solutions were 1, 10, and 20 mg/L. Additionally, the toxicity of HA was measured in the applied concentration range for the determination of a potential toxic effect that may be due to the action of HA. 2.3. Toxicity assessment of binary mixtures The toxicities of certain concentrations of heavy metals and corresponding binary mixtures were evaluated to determine their interactive toxic effects. The effect of HA on the toxicity of the binary mixtures of heavy metals was evaluated in the presence of 10 mg/L HA. The toxicity of the metal mixture containing 10 mg/ L HA was evaluated after 3 h contact time and the results were compared with the corresponding values obtained without the addition of HA. The toxicities of the solutions containing one metal and those of the binary mixtures in the presence and in the absence of 10 mg/L HA, expressed as percent effect, were evaluated using the 45% screening test (Microbics Corp., 1992). The theoretically expected effect of the binary mixtures was evaluated using a simple mathematical model based on the theory of probabilities (Kungolos et al., 1999). According to this model, if P1 is the inhibition caused by a certain concentration of chemical A1 and P2 the inhibition caused by a certain concentration of chemical A2, then the theoretically expected additive inhibition PðEÞ, when those concentrations of the two chemicals are applied together, is given by the following equation: PðEÞ ¼ P1 þ P2 P1 P2 =100.
(1)
The null hypotheses were that the observed values were higher or lower than the theoretically predicted values, for synergistic and antagonistic effects, respectively. The result was considered to be antagonistic or synergistic only if the observed effect was significantly lower or higher, respectively, than the theoretically predicted value at the 0.05 level of significance.
3. Results 3.1. Effect of HA on the toxicity of heavy metals The toxicity measurements of the pure compounds showed that Cu and Pb exhibited similar toxic effects and the toxic effect of Zn was lower than that of the other metals. The EC50 values for Cu and Pb were 0.25 mg/L (confidence range 0.18–0.30 mg/L) and 0.48 mg/L (confidence range 0.37–0.63 mg/L), respectively, while the corresponding value for Zn was 1.50 mg/L (confidence range 1.36–1.59 mg/L). Similar results were found by Mowat and Bundy (2002) and Utkigar et al. (2004). On the other hand, for concentrations between 1 and 20 mg/L, the observed effect of HA on the bioluminescence of V. fischeri was negligible, varying from 10% to 10%. The effect of color of tested HA solutions on the reduction of light emission was also estimated using the Microtox Omni software. According to the procedure, absorbance of the tested solutions at a wavelength of 490 nm was recorded and introduced to the software. The absorbances for solutions of 20, 10, and 1 mg/L HA were 0.05, 0.025, and 0.002 cm1, respectively, resulting in minor alterations in the toxic effect values. Therefore, it was assumed that color correction did not influence the results and this negligible effect was not taken into account. The effect of HA on the toxicity of Cu, Zn, and Pb as a function of contact time with HA is shown in Table 1. The EC50 values and the corresponding 95% confidence ranges are expressed as mg/L of the total metal ion concentration that may be present in the solution as free ions or bound to HA. As presented in Table 1, the toxicity of Cu decreased with increasing HA concentration and contact time. It was observed that the addition of 1 mg/L HA was not sufficient to alter the toxicity of Cu, since the EC50 value was approximately the same as compared to that of Cu without the addition of HA, even after 4 h of contact time. The toxic effect of Cu with the addition of 10 mg/L HA was slightly reduced after 2 h of contact time, while no further significant reduction was recorded after 4 h of contact time. The higher toxicity reduction, equal to 55%, was observed for HA concentration of 20 mg/L after 4 h contact time. This result could be attributed to the increase of Cu complexation with HA, which, according to Kim et al. (1999), is time and HA dose dependent. Alberts et al. (2000) found that the reduction of toxic effect of Cu on V. fischeri varied between 44% and 100%, depending on the type of HA used. On the other hand, the presence of HA did not exhibit any significant effect on the toxicity of Zn, while a maximum 81% increase of the toxic effect was observed in the case of Pb. Furthermore, it is clear that contact time had a small effect on Cu toxicity, while the
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Table 1 Effect of HA on the toxicity of Cu, Zn, and Pb as a function of contact time with HA Heavy metal
HA concentration (mg/L)
15 min EC50 (95% confidence range) Contact time 0.5 h
Contact time 2 h
Contact time 4 h
Cu
0 1 10 20
0.20 0.21 0.25 0.31
(0.17–0.24) (0.17–0.25) (0.22–0.28) (0.28–0.33)
0.22 0.24 0.32 0.42
(0.17–0.28) (0.19–0.29) (0.28–0.37) (0.41–0.43)
0.23 0.25 0.34 0.46
(0.18–0.29) (0.22–0.29) (0.27–0.43) (0.39–0.54)
Zn
0 1 10 20
1.49 1.53 1.58 1.51
(1.37–1.59) (1.52–1.55) (1.37–1.85) (1.13–1.96)
1.42 1.49 1.43 1.32
(1.31–1.55) (1.20–1.79) (1.26–1.63) (1.24–1.39)
1.51 1.58 1.51 1.60
(1.40–1.61) (1.37–1.81) (1.15–1.93) (1.34–1.98)
Pb
0 1 10 20
0.50 0.33 0.31 0.14
(0.38–0.65) (0.23–0.47) (0.19–0.47) (0.09–0.25)
0.49 0.36 0.16 0.22
(0.38–0.63) (0.33–0.39) (0.11–0.22) (0.06–0.26)
0.44 0.38 0.15 0.09
(0.36–0.52) (0.34–0.43) (0.14–0.16) (0.04–0.23)
Fig. 1. Comparison of dose response curves for Cu and Cu/HA solutions after 2 h contact time.
toxicities of Zn and Pb were not affected with contact time increase. The dose response curves of Cu, Zn, and Pb for single-metal solutions and in the presence of 10 and 20 mg/L HA after 2 h contact time are illustrated in Figs. 1–3, respectively. As shown in Fig. 1, the toxicity of Cu in the absence of HA increased with concentration, reaching up to 100% for Cu concentrations higher than 0.8 mg/L, while a decrease of toxic effect was observed with the addition of HA at almost all dilutions. On the other hand, the addition of HA in Zn solution (Fig. 2) did not affect the dose response curve of Zn pure Zn solution and the solutions containing 10 and 20 mg/L HA showed the same patterns. In the case of Pb (Fig. 3), the dose response curve and the effect of HA on the metal toxicity were completely different. As shown in Fig. 3, a steep increase of metal toxicity was observed for pure Pb concentrations up to 1 mg/L, while for higher concentrations the toxicity was almost constant, reaching up to 90%. This result
Fig. 2. Comparison of dose response curves for Zn and Zn/HA solutions after 2 h contact time.
Fig. 3. Comparison of dose response curves for Pb and Pb/HA solutions after 2 h contact time.
could be attributed to the saturation of Pb transport sites through the biological membrane of V. fischeri (Slaveykova et al., 2004). The presence of HA in Pb
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Fig. 4. Comparison between theoretically expected and observed inhibitions for the combined effect of Cu and Zn on the bioluminescence of V. fischeri.
Fig. 5. Comparison between theoretically expected and observed inhibitions for the combined effect of Cu and Pb on the bioluminescence of V. fischeri.
solution caused a relatively high increase of metal toxicity at low concentrations, while for Pb concentrations higher than 1 mg/L the toxicity was almost constant, reaching up to 95%. 3.2. Toxicity of binary mixtures of heavy metals The interactive effects between Cu and Zn on the bioluminescence of V. fischeri for some concentration combinations without the addition of HA are illustrated in Fig. 4. As shown in Fig. 4, the effect was synergistic at all the concentration combinations tested. The gray bars corresponding to the observed effects in A, B, and C indicate that the increase of Zn concentration from 0.43 to 1.73 mg/L induces an increase of toxic effect from about 40% to about 80%, while the concentration of Cu was maintained constant and equal to 0.12 mg/L. In general, the differences between the observed interactive toxic effects and the theoretically expected effects for binary mixtures were greater in the mixtures with
relatively low Cu concentrations than in those with higher Cu concentrations. The observed bioluminescence inhibition for the mixtures with 0.12 mg/L Cu was about 45% higher than the theoretically expected inhibition. On the other hand, for the mixtures containing 0.25 mg/L Cu, the differences between the observed and the expected effects were smaller, reaching up to 25%. Still, it is important to point out that the differences between theoretically expected and observed values were significant at all concentration combinations tested. The toxic responses of the binary mixtures of Cu and Pb were found to be close to the theoretically calculated values, as shown in Fig. 5. The expected toxic effects varied from 20% to 60%, while the observed values did not differ significantly. Therefore, an additive interactive mode of action can be assumed for the effect of Cu and Pb on V. fischeri. The interactive effect between Zn and Pb on the bioluminescence of V. fischeri for some concentration
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Fig. 6. Comparison between theoretically expected and observed inhibitions for the combined effect of Zn and Pb on the bioluminescence of V. fischeri.
Fig. 7. Comparison between observed effects of combined concentrations of Cu and Zn on the bioluminescence of V. fischeri, without the addition of HA and with the addition of 10 mg/L HA after 3 h contact time.
combinations is illustrated in Fig. 6. The theoretically expected values ranged between 28% and 52% and the observed values between 41% and 87%. The observed values were significantly higher than the theoretically predicted values for all concentration combinations, confirming synergistic action between Zn and Pb. 3.3. Effect of HA on the toxicity of binary mixtures of heavy metals The toxicities of the binary mixtures of heavy metals that were tested for the investigation of the interactive effects were also measured in the presence of 10 mg/L HA for contact time 3 h. The effect of HA on the toxicity of some binary mixtures of Cu and Zn is depicted in Fig. 7. It was observed that, in all tested metal concentration combinations, the addition of HA caused a significant reduction in the toxic effect of metals after 3 h contact time. The toxicity reduction with the addition of HA varied between 40% and 90%.
Higher reductions were observed for the mixtures with relatively low Zn concentrations. Thus, for the mixture A (0.12 mg/L Cu and 0.43 mg/L Zn) the bioluminescence inhibition decreased from 40% to 5% with the addition of 10 mg/L HA. Fig. 8 shows the effect of HA on the toxicity of different binary mixtures of Cu and Pb. The presence of 10 mg/L HA in the binary mixtures of Cu and Pb did not significantly influence the toxicity of mixtures compared with the corresponding values in the absence of HA. Although different metal concentration combinations were tested, in most cases the bioluminescence inhibition did not significantly vary in the presence of HA. The effect of HA on the toxicity of some binary mixtures of Zn and Pb is depicted in Fig. 9. As shown in Fig. 9, in most metal concentration combinations tested the toxicity decreased in the presence of HA. The range of toxicity reduction was between 28% and 45% for Zn concentrations up to 0.86 mg/L, while for Zn concentration of 1.73 mg/L, the toxicity reduction was about 15%.
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Fig. 8. Comparison between observed effects of combined concentrations of Cu and Pb on the bioluminescence of V. fischeri, without the addition of HA and with the addition of 10 mg/L HA after 3 h contact time.
Fig. 9. Comparison between the observed effects of combined concentrations of Zn and Pb on the bioluminescence of V. fischeri, without the addition of HA and with the addition of 10 mg/L HA after 3 h contact time.
4. Discussion The investigation of the influence of HA on the toxicity of Cu, Zn, and Pb showed that the toxic response varied depending on the type of heavy metals and the concentration of HA. The toxicity of Zn was not influenced by the presence of HA, while the toxicity of the other heavy metals was influenced by HA concentrations higher than 1 mg/L. A decrease of toxic effect due to complexation was observed only in the case of Cu, whereas the complexation of Pb resulted in an increase of toxic effect. Based on complexation kinetics of the tested heavy metals, reported in the literature, Cu has the greatest binding efficiency, while the binding efficiency of Zn is the lowest compared with the other metals of concern (Lubal et al., 1998; Pandey et al., 2000). As a result of Cu complexation, a toxicity reduction was observed and it could be assumed that Cu toxicity is dominated by the
free ion activity. These results are consistent with results by other researchers reporting that Cu complexation with organic ligands results in a decrease of toxic effect on various test organisms (Campbell et al., 2000; Winner, 1984; Kim et al., 1999; Alberts et al., 2000). Furthermore, Cheng and Allen (2001) observed bioavailability reduction of copper to plants, when complexed with organic ligands, resulting in toxicity decrease. With regarding to Pb toxicity, although Pb forms relatively strong complexes with HA, an increase of the toxic effect was observed in the presence of HA (Fig. 3). This could be attributed to potential interactions between the Pb complexes and the transport sites of biological membrane of V. fischeri (Campbell et al., 2000; Slaveykova et al., 2004). It can be assumed that the Pb complexes with HAs are bioavailable to a higher extent than the soluble phases of Pb. A different pattern of Pb toxicity is shown while comparing the dose response curves of the tested heavy metals. The toxicity
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of Pb remained almost constant for concentrations higher than 1 mg/L (Fig. 6), whereas the toxicity of the other heavy metals increased steeply with concentration, reaching up to 100% (Figs. 4 and 5). Considering that, in the first place, Pb forms complexes with organic ligands and, in the second place, the toxicity after complexation increases, it is clear that the free ions of Pb did not determine the degree of the toxic effect. Although the free metal ions are considered the most bioavailable form, the metal toxicity may be affected by various interactions between living cells and ligands and the by physiological status of the organism (Campbell et al., 2000; Simkiss and Taylor, 2001). For example, free metal ions may attach to binding sites and induce changes in cellular behavior. Furthermore, there are organisms that can modify their extracellular sites in powerful ways (Simkiss and Taylor, 2001). The interactive effects between Cu and Zn were synergistic for all concentration combinations tested (Fig. 4). This result is in accordance with results presented in a study conducted by Utkigar et al. (2004). Preston et al. (2000) also found that the combined solutions of Zn with Cu or Cd caused synergistic interactions on Escherichia coli, using genetically modified luminescence-based microbial biosensors. On the other hand, Cu in combination with Pb generally presented an additive mode of interaction on V. fischeri (Fig. 5). Ren et al. (2004), using the Shk1 assay, found that the additive, synergistic, or antagonistic effects of various concentration combinations between Cu and Pb depended on the concentration of each metal in the binary mixture. Such results were not found in our study, where in all concentration combinations tested the difference between the theoretically expected and the observed result was not significant when Cu was applied together with Pb. Furthermore, in our study it was found that the binary mixtures of Zn and Pb showed a synergistic effect for all concentration combinations tested (Fig. 6). The interactive effect of all binary mixtures that were examined varied between additive and synergistic, while an antagonistic effect was not observed in any case. In addition, it can be concluded that the interactive effect depended on the type of the pair of metals contained in the binary mixture, as Cu in the presence of Zn showed synergism, while the same metal in the presence of Pb exhibited an additive effect. Furthermore, the concentrations of the metals in the binary mixtures did not affect the type of interaction, at least for all the concentration combinations tested in this study. However, it has been shown in other studies that the mode of interaction may be dependent on metal concentrations (Hadjispyrou et al., 2001; Ren et al., 2004). The addition of HAs in the binary metal mixtures resulted in a toxicity reduction in most of the cases studied. Comparing the toxic effects of the binary
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mixtures with the corresponding values in the presence of 10 mg/L HA, it is clear that relatively high toxicity reduction was observed for the binary mixtures of Cu/ Zn and Zn/Pb (Figs. 7 and 9), while the toxicity reduction of the binary mixtures between Cu and Pb was generally low (Fig. 8). The high toxicity reduction of the Cu/Zn mixture could be attributed to the complexation of Cu with HA that may be assumed to eliminate the bioavailable phase of Cu. On the other hand, Cu complexation seemed to cause a relatively small effect on the toxicity of Cu/Pb mixtures, possibly due to the antagonistic action of complexation as both metals form moderate to strong complexes with HA. Furthermore, it can be assumed that the failure of HA to reduce the toxicity of Cu in the presence of Pb may be linked with the inability of HA to decrease the toxicity of Pb, another result that has been obtained in the current study. On the contrary, although the presence of HA caused an increase in Pb toxicity and did not affect the toxicity of Zn, the toxicity of Zn/Pb mixtures decreased in the presence of 10 mg/L HA. It can be assumed that the reduction in Pb toxicity in the presence of Zn can be attributed to the formation of complexes between Pb and HA, because the Pb concentrations in this experiment were relatively low (0.27–0.67 mg/L). On the other hand, in the experiments where HA failed to reduce the toxicity of Pb applied alone the tested concentrations were quite higher, ranging up to 3.5 mg/L. Moreover, with regard to the complexation capacity of the metals with HA in the binary mixtures, the molecular relative ratios should also be considered. It should be noted that the metal/HA ratio in single-metal solutions was significantly lower than the ratios examined in the binary mixtures. The HA concentration of 10 mg/L was chosen as a representative concentration of natural organic matter contained in the aquatic environment (Stackhouse and Benson, 1988). Furthermore, the addition of NaCl for the adjustment of the osmotic pressure of the solutions prior to the toxicity test includes another parameter that may affect the complexation of heavy metals and as a result their toxicity. This addition may cause an increase in the ionic strength of the solutions that in many cases reduces the binding capacity of heavy metals to HA (HamiltonTaylor et al., 2002). Furthermore, it should be noticed that the tested metal concentrations were significantly higher than the water quality criteria posed by several countries. For example, the continuous concentration criteria in freshwater for dissolved copper are 9 mg/L in the US (recommended), 5 mg/L in Canada, and 2 mg/L in Switzerland, while the criterion for lead is 2.5 mg/L in the US, and the criteria for zinc are 120 mg/L in the US and 5 mg/L in Switzerland (USEPA, 2002; Xue et al., 2003; Nriagu et al., 1998). There were basically two reasons that relatively high metal concentrations were used in this study. First, as explained in the introduction, V.
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fischeri does not exhibit high sensitivity to heavy metal toxicity. Second, the high metal concentrations used assisted in investigating the potential detoxification effects of HAs. As an extension to this work, further studies investigating both the toxic effects of heavy metals at concentrations as low as those found in surface waters, and the impact of HAs on the toxicity of combined metal solutions on test organisms that exhibit high sensitivity could be conducted.
5. Conclusions The complexation of heavy metals with HAs is an important parameter that may alter the toxicity and bioavailability of heavy metals in aquatic systems. The examination of the complexation of heavy metals with HAs showed that the toxicity of Cu decreased with the addition of HAs while the toxicity of Zn remained almost constant and the toxicity of Pb increased. The interactive effects of Cu and Zn mixtures and Pb and Zn mixtures on V. fischeri were found to be synergistic, while the interactive effect of Cu and Pb mixtures was additive for all concentration combinations tested in this study. It was found that the mode of interaction depended on the type of the pair of metals and not on the concentrations tested. The presence of HA in the binary mixtures caused relatively high toxicity reduction mainly in the cases where synergistic action occurred. It was found that the presence of HAs reduced both the toxicity of Cu and Zn applied together and that of Pb and Zn applied together. On the contrary the toxicity of Cu and Pb applied together on V. fischeri was not much altered by the presence of HA. It can also be concluded that, for the proper estimation of the environmental hazard caused by a chemical, the presence of other constituents that may interact with the chemical of concern should be evaluated.
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