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Effects of interactions between algal densities and cadmium concentrations on Ceriodaphnia dubia fecundity and survival
ARTICLE IN PRESS
Ecotoxicology and Environmental Safety 71 (2008) 765–773 www.elsevier.com/locate/ecoenv
Effects of interactions between algal densities and cadmium concentrations on Ceriodaphnia dubia fecundity and survival Suzelei Rodgher, Evaldo Luiz Gaeta Espı´ ndola Sa˜o Carlos Engineering School, Water Resources and Applied Ecology Center, University of Sa˜o Paulo, Avenida Trabalhador Sa˜o Carlense, 400, C.P 292, Cep 13.560-970 Sa˜o Carlos, SP, Brazil Received 8 November 2006; received in revised form 19 July 2007; accepted 24 August 2007 Available online 23 October 2007
Abstract The influence of different densities of the algae Pseudokirchneriella subcapitata on the chronic toxicity of cadmium to Ceriodaphnia dubia was investigated. The importance of algal cells as a source of metal to zooplankton was studied by exposing P. subcapitata cells to free cadmium ions and supplying the algae as food to C. dubia. The results of a bifactorial analysis (metal versus food levels) showed that metal toxicity to zooplankton was dependent on food level. Significant toxic effects on the fecundity and survival of C. dubia were observed at low metal concentrations with high algal density. Algae contaminated with Cd2+ were less toxic to cladoceran than was the Cd2+ in solution. Green algae retained cadmium and released low metal concentration in the test medium. We concluded that algal cells are an important route of exposure to metal and a factor that has an appreciable influence on the expression of metal toxicity to daphnids. r 2007 Elsevier Inc. All rights reserved. Keywords: Ceriodaphnia dubia; Sources of metal toxicity; Pseudokirchneriella subcapitata; Chronic toxicity
1. Introduction Until a few years ago, feeding activity had been largely ignored in research published on accumulation of heavy metals and their bioavailability to aquatic invertebrates, presumably because feeding activity was not considered an important source of contamination and uptake of metals in previous studies. Recently, many studies have focused on the effects of metals on survival, growth and reproduction of filter-feeding organisms submitted to a variety of food conditions, both in the laboratory and in the field (Reinfelder et al., 1998). The food, represented by the algae, can act as a channel of metal contamination to cladocerans (Hook and Fisher, 2002) and can counteract metal toxicity, due to the algae’s ability to exude organic compounds that complex metals (Lombardi and Vieira, 2000). On the other hand, the algae contribute to the resistance of the organisms to toxic Corresponding author. Fax: +55 16 33738251.
E-mail addresses: [email protected] (S. Rodgher), [email protected] (E. Luiz Gaeta Espı´ ndola). 0147-6513/$ - see front matter r 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.ecoenv.2007.08.012
substances by improving their nutritional state (Chandini, 1989). Since aquatic organisms in their natural environment have food available during toxic exposure, attention should be given to the role of food in toxicity tests (Lanno et al., 1989). A new approach in ecotoxicological studies has focused on the influence of food on the effects of toxic agents on zooplankton. Weltens et al. (2000) verified that particles (sand, clay, algae) contaminated with cadmium are potentially toxic to Daphnia, not only by acting as a source of dissolved metal, but also because the particle-bound fraction of Cd can become free and available within the body of the filter-feeding organism. Barata et al. (2002) confirmed that although water was the major pathway of cadmium uptake for D. magna, a substantial amount of the metal was obtained by the test organisms from the contaminated algae. Dietary exposure to metal of zooplankton may occur because the algae can adsorb and take up dissolved metal from the exposure solution before being ingested by the organisms. However, algae may eliminate metal to the solution during the daphnids’ exposure, which might result
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in significant exposure of organisms to metal via water. The elimination of metals from food in dietary toxicity studies is recognized as an important factor that may confuse the interpretation of the effects of dietary exposure (De Schamphelaere et al., 2004). In this light, the aim of this study was to evaluate the effects of varying the density of the algae Pseudokirchneriella subcapitata on the chronic toxicity of cadmium to the cladoceran Ceriodaphnia dubia, when this organism was exposed to this metal in the water and in contaminated food (cells of P. subcapitata). In these experiments, total dissolved cadmium concentrations and cadmium accumulated by algal cells were measured and free metal ions were estimated in order to distinguish between the effects of aqueous and dietary exposures as sources of metal toxicity to these organisms. Feeding rates of C. dubia were also measured in both treatments. 2. Materials and methods 2.1. Culture of test organisms Cells of P. subcapitata and neonates of C. dubia were obtained from cultures maintained at the Laboratory of Ecotoxicology and Ecophysiology of Aquatic Organisms of the Water Resources and Applied Ecology Center (CRHEA) of the University of Sa˜o Paulo (Sa˜o Paulo State, Brazil). Cultures of C. dubia were maintained in a temperature-controlled chamber at 2571 1C with a 12:12 h light/dark cycle, using reconstituted water as the culture medium (pH 7.2–7.6, conductivity 160 mS/cm and hardness between 42 and 48 mg/L of CaCO3). The water was changed every other day. Reconstituted water consisted of four inorganic salts dissolved in deionized water. Twenty millilitres of a solution (0.01 M of CaSO4.2H2O) and 10 mL of another solution (0.002 M of KCL, 0.06 M of NaHCO3 and 0.04 M of MgSO4.7H2O) were added to deionized water to a final volume of 1 L (ABNT, 2005a). The green algae P. subcapitata were cultivated in L.C. Oligo medium (AFNOR, 1980), which was first autoclaved (121 1C) for 15 min in 2-L Erlenmeyer flasks containing 1 L of the medium. The composition of the culture medium is described in Table 1. This was inoculated with cells to a concentration of around 1 104 cells/mL and the culture was exposed to 100 mE/m2/s2 in 12:12 h light/dark cycle, with constant aeration, at 2472 1C (ABNT, 2005b). Algal cells from exponentially growing
P. subcapitata cultures were used as food for the zooplankton, C. dubia, which was fed every other day with 1 105 cells/mL of the algae and a suspension of yeast and commercial fish food (Vitormonios), in accordance with the procedures set in the Brazilian guidelines ABNT (2005a).
2.2. Toxicity tests 2.2.1. Exposure of algae to metal Cells of P. subcapitata in the exponential growth phase were exposed for 96 h to ascending levels of total dissolved cadmium of 0, 0.63, 1.51, 2.93, 6.85 and 12.60 107 M, corresponding to free cadmium ion concentrations of 0, 0.60, 1.40, 2.70, 6.32 and 11.60 107 M, respectively. Free cadmium ions were estimated using the MINEQL+ model (Schecher and McAvoy, 1991). The test solutions used in this experiment were prepared in volumetric flasks using volumetric pipettes. The stock solution was 8.9 mM Cd(NO3)2.4H2O (solution standard of nitrate of cadmium for atomic absorption, J.T. Backer). The dilution water used in the preparation of the test was LC Oligo medium. After the exposure period, the cells were centrifuged at 1500 rpm for 15 min (FANEM Excelsa centrifuge, model 206 PM), washed three times with reconstituted water and resuspended in the zooplankton culture water (reconstituted water). Finally, suspensions of the cells were stored in polythene bottles in the dark at 4 1C during the chronic toxicity test with zooplankton (De Schamphelaere et al., 2004). Samples (2 mL) from each test flask were taken and fixed with Lugol’s iodine solution to determine the cell density, by counting cells in an Improved Neubauer Bright-Line hemocytometer under optical microscope (Carl Zeiss), standard model 25. The mean number of cells produced at each concentration, after this exposure period, was expressed as a percentage growth reduction with respect to the control. These percentages were used to calculate the IC50 (effective metal concentration giving 50% inhibition of algal growth after 96 h exposure). The dry weight of the algal cells was obtained by filtering a known volume of cells on a pre-weighed filter. The filters with algal cells were dried for 24 h at 60 1C and weighed to determine the cell mass per volume of culture (APHA, 1995). 2.2.2. Chronic toxicity test The test solutions for the chronic toxicity tests with the cladocerans were prepared as in the experiments with the algae and the dilution water used in these preparations was reconstituted water. Acute toxicity tests demonstrated a median effect concentration (EC50, 48 h) of 5.34 107 M Cd to C. dubia (Rodgher, 2005). Based on the result of the acute toxicity
Table 1 Composition of the culture medium L.C. Oligo (AFNOR, 1980) used in the present study Stock solution
Compound
Concentration (M)
Volume (mL) required from stock solution to 1 L of medium
1 2 3 4 5
Ca(NO3)2 4H2O KNO3 MgSO4 7H2O K2HPO4 CuSO4 5H2O (NH4)6Mo7O24 4H2O ZnSO4 7H2O CoCl2 6H2O Mn(NO3)2 4H2O C6H8O2 H2O H3BO3 C6H5FeO7 5H2O FeSO4 7H2O FeCl3 6H2O NaHCO3
0.24 0.99 0.20 0.23 0.00016 0.00005 0.0002 0.0001 0.0002 0.0005 0.001 0.005 0.002 0.002 0.2
1.00 1.00 1.00 1.00 0.50
6
7
0.50
1.00
ARTICLE IN PRESS S. Rodgher, E. Luiz Gaeta Espı´ndola / Ecotoxicology and Environmental Safety 71 (2008) 765–773 tests, we prepared five nominal sublethal metal concentrations. Each successive nominal concentration was about twice the previous one (0.11, 0.22, 0.44, 0.89, 1.78 107 M Cd). Test solution samples from each nominal metal concentration were filtered, acidified and analyzed by graphite-furnace atomic absorption spectrometry to determine the dissolved cadmium concentrations. Considering that free metal ions constitute a metal fraction of higher bioavailability to organisms, we used the MINEQL+ program to calculate Cd2+. The free ion cadmium concentrations were then predicted from the total dissolved cadmium concentrations. The total dissolved cadmium concentrations used in the chronic toxicity tests with zooplankton were 0, 0.11, 0.19, 0.44, 0.87, 1.78 107 M, corresponding to free cadmium ion concentrations of 0, 0.10, 0.17, 0.40, 0.86, 1.60 107 M, respectively. In the chronic toxicity tests, 10 neonates of C. dubia (aged less than 24 h) were exposed, in 10 separate test vessels, to 15 mL of a certain metal concentration and fed with one of three densities of P. subcapitata (low, medium and high: 1 104 cells/mL, 1 105 cells/mL and 1 106 cells/mL, respectively). The organisms were transferred to a freshly prepared test solution every other day and fed with the respective algal concentration daily. The test media were gently stirred using a fine Pasteur pipette twice a day to ensure availability of algal cells to the organisms. A control test without the added metal was also prepared. The toxicity tests lasted seven to eight days, the period needed for production of the third brood, and the survival of adults and number of live neonates per female were recorded (ABNT, 2005a). Two controls were used: a laboratory control (LC) that contained the food concentration normally used in the maintenance of the test organism (1 105 cells/mL plus the suspension of yeast and fish food) and another control (C), containing only the algae at the density being tested. This was a way of testing the physiological condition of the organisms being used in the tests. The chronic toxicity tests were considered valid when a survival of 80% was observed in adult organisms, corresponding to the laboratory control, and when they produced at least 15 neonates per female (ABNT, 2005a). In another experiment, cells of P. subcapitata exposed to free cadmium for 96 h were offered as food to zooplankton. The concentrations of metal used to contaminate P. subcapitata were 0, 0.60, 1.40, 2.70 107 M Cd2+. Neonates of C. dubia were exposed to reconstituted water and fed with three different densities of metal-contaminated P. subcapitata (low, medium and high: 1 104, 1 105 and 1 106 cells/mL, respectively) during seven days. The toxic effects of metal-contaminated algae on fecundity and survival of C. dubia were verified. The feeding and waterchanging procedures for this test were similar to those used when this organism was exposed to different metal concentrations. 2.2.3. Feeding rates of the c. dubia The effects of free cadmium in water and of Cd+2 contaminated algal diet on feeding rates of zooplankton at different algal levels were evaluated using measured filtration and ingestion rates. The feeding experiments with C. dubia were carried out in test vessels containing 50 mL of the medium and 10 individuals under the same condition of the toxicity chronic test. A control test, without the added metal, was also prepared. Test organisms were exposed to two treatments (water and contaminated food) for 24 h, after which the final algal concentration was measured using a hemocytometer (Villarroel et al., 1999). The filtration rate (F) was defined as the volume of medium swept clear per unit of time and the ingestion rate (I) as the number of cells consumed by an animal in a specific time interval. To calculate the average filtration (ml/ind/h) and ingestion rates (cells/ind/h), the equations from Gauld (1951) were used: F¼
V ðln C 0 ln C t Þ A, n t
A¼
ln C 0 ln C 0t , t
I ¼F
pffiffiffiffiffiffiffiffiffiffiffi C0Ct ,
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where C0 and Ct are initial and final food concentrations (cell/mL), t is time (duration of the experiment in hours), and n is the number of daphnids in volume V (mL). A is a correction factor for changespin the ffiffiffiffiffiffiffiffiffiffiffi control with final concentration Ct0 after time t. The expression C 0 C t represents the geometric mean of the food concentration during time t. 2.2.4. Metal analyses At the end of the experiments with P. subcapitata, samples were taken from each treatment to determine the metal accumulated by algal cells. Aliquots of test solutions from experiments with algae and cladocerans were filtered through a 0.45 mm membrane filter The filters with the accumulated fraction were dried and submitted to acid digestion (HNO3 and H2O2) (APHA, 1995). The analysis of metal in the algal cells was carried out without EDTA washing, in order not to remove the metals adsorbed on the cell surface. The measured concentration of metal in algal cells was taken as the total amount of metal accumulated by the cells (i.e., externally and internally bound metal), expressed as mg Cd/mg dry weight of algae. At the end of the toxicity tests with zooplankton, samples also were taken to determine the total dissolved metal and accumulated metal by the cells. These measurements were taken at concentrations where toxic effects to C. dubia could be identified. Filtered samples were preserved by acidifying with concentrated nitric acid, for subsequent determination of total dissolved metal. We used values of total dissolved metal together with the chemical composition of the test medium to compute the concentration of free metal ion using the MINEQL+ program. During the experimental period the metal loss from the algae in the toxicity test with contaminated food was quantified as the total dissolved concentration of metal in the culture medium. For each sample digested, three unused filters were digested and analyzed as blanks (Van Loon, 1985). All the samples were analyzed by graphite-furnace atomic absorption spectrometry (Varian AA 220). The detection limit for Cd, calculated as described in Miller and Miller (1994), was 4.45 1010 M. 2.2.5. Statistical analysis The IC50 value of cadmium for the algae was determined by the trimmed Spearman–Karber method (Hamilton et al., 1977). The algal cell density data obtained from the experiment with the chlorophyte were submitted to tests for normality (Shapiro–Wilk’s test) and homogeneity (Bartlett’s test), and then, to Dunnett’s test (parametric test) to detect significant differences between the controls and each metal treatment. To test the significance of the effect of algal food densities, free metal ion concentration and different densities of metal-contaminated alga on the survival, fecundity and feeding rates of C. dubia, a two-way ANOVA was employed on the chronic test data, using the BioEstat 2.0 program (Ayres et al., 2000). The numbers of neonates produced by C. dubia females and their feeding rates were submitted to tests for normality (Shapiro–Wilk’s test) and homogeneity (Bartlett’s test) and, accordingly, to Dunnett’s test (parametric) to detect significant differences between the controls and each metal treatment. Tukey’s test (parametric) was used in multiple comparisons to detect significant differences among the fecundity obtained in treatments with different algal densities and metal concentrations. Fisher’s exact test was used to distinguish significant differences in the survival of zooplankton between the control and the various treatments at the end of the chronic toxicity tests. The above statistical tests were run using the Toxstat version 3.3 computer package (Gulley et al., 1993).
3. Results 3.1. Exposure of algae to metal The mean value for the IC50 of Cd2+ to P. subcapitata, in a total of 10 tests, was 1.65 107 M Cd2+, with lower and upper limits of 0.70 and 2.60 107 M Cd2+, respectively. Table 2 summarizes the effects of metal on
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the algae and the results of metal analyses. A decrease in the cell density of P. subcapitata was observed with increasing concentrations of cadmium. Concentrations of 2.70 107 M Cd2+ reduced the algal cell density to less than half that of the control (without added metal) and dry weight fell similarly. Metal analyses demonstrated that green algae were able to accumulate cadmium. With increasing free cadmium ions in solution, the metal accumulated by P. subcapitata also increased. For example, when the concentration of Cd2+ increased from 0.57 to 6.32 107 M, the metal concentration accumulated by the green algae increased from 0.001 to 0.01 mmol/mg. 3.2. Chronic toxicity test with zooplankton The fecundity and survival of C. dubia were significantly affected by the food level, the metal ion concentrations, and also by their interactions (Tables 3 and 4). The survival of the cladocerans decreased with increasing Cd2+only at high algal density. At 0.4, 0.86 and 1.60 107 M free Cd2+, the number of neonates per female was higher at the medium food level than at high and low levels. Treatment with 1.60 107 M of free cadmium caused a decrease in the number of neonates produced by C. dubia when medium (105 cells/mL) and high (106 cells/mL) algal densities were supplied as food (Fig. 1). On the other hand, the toxic effects on zooplankton fecundity during exposure to 0.40 and 0.86 107 M of Cd2+ were observed only at high food density. The mean numbers of neonates per female when C. dubia was fed at high algal density and exposed to 0.40 and 1.60 107 M Cd2+ (2.75 and 1.65 neonates/ female, respectively) were significantly lower than those obtained at medium algal density and at the same metal concentrations (7.50 and 4.70 neonates/female, in 0.40 and 1.60 107 M Cd+2, respectively) (Fig. 1). Fig. 2 demonstrates that the test organisms suffered a significant reduction in mean neonate numbers when they were fed with high and medium densities of algae exposed to 1.40 and 2.70 of 107 M Cd2+. The survival of the cladocerans was not significantly affected. The mean number of neonates produced by C. dubia at the high density of algae contaminated with 2.70 107 M Cd2+
(3.33 neonates/female) was significantly lower than that at the medium density (7.29 neonates/female). Considering both chronic toxicity tests, the number of neonates produced per female and survival of C. dubia were lower in the treatment with water contaminated with 0.4, 0.86 and 1.60 107 M Cd2+ at high algal density than were fecundity and survival of C. dubia exposed to a high food level contaminated with 0.6, 1.4 and 2.70 107 M Cd2+ (Tukey’s test, Po0.05). At low food concentrations (104 cells/mL), in both sets of chronic toxicity tests (water and food contaminated with metal), the number of neonates per female was not different to that found in the control (Dunnett’s test, Po0.05). Before the metal effect, there was the effect of low food reducing the fecundity of test organisms, as this was also observed in the control containing 1 104 cells/mL. At exposures of 0.4, 0.86 and 1.6 107 M of free cadmium and in the experiment with food contaminated at 1.4 and 2.7 107 M Cd2+, the fecundity of C. dubia at low food density was equal to that at high food level. The animals might have been affected by metal when fed with high algal densities in both treatments (water and contaminated food), and limited by low food concentration. 3.3. Feeding rates The filtration rates of C. dubia in control test conditions (C) in both treatments (water and contaminated food) were higher at low food concentration than at medium and high food levels, whereas ingestion rates were higher at high than at medium and low algal densities. These results were generally observed during the metal exposures (Figs. 3 and 4). Metal in water did not affect the filtration and ingestion rates of the zooplankton, and the feeding rates only were affected by food levels. In experiments with algal densities contaminated with Cd+2, filtration and ingestion rates of C. dubia were only altered by food levels. 3.4. Metal analyses Concentrations of total dissolved cadmium, free cadmium and cadmium accumulated by algal cells, at the end of toxicity test with C. dubia, are given in Table 5. In the
Table 2 Values of dissolved total metal, free metal, metal accumulated by algal cells, cell density and dry weight in experiments with P. subcapitata algae with cadmium Dissolved total cadmium at start of exposure (107 M) Free cadmium at start of exposure (107 M) Cadmium bound to algal cells (mmol/mg)
C (0) 0 0
Mean cell density after 96 h of exposure (106 cells/mL)
6.39 (1.14) 137.00 (23.36)
Dry weight (mg/L)
Mean values (SD). C, control. a Indicates statistically different from control (C) (Dunnett’s test, Po0.05).
0.63 0.60 0.001 (0.000) 4.91a (1.34) 92.22a (27.34)
1.51 1.40 0.001 (0.000) 3.94a (1.54) 89.91a (31.02)
2.93 2.70 0.004 (0.001) 2.10a (0.65) 64.55a (19.91)
6.85 6.32 0.01 (0.002) 0.77a (0.26) 24.92a (6.31)
12.6 11.6 0.05 (0.001) 0.14a (0.14) 9.70a (2.73)
ARTICLE IN PRESS S. Rodgher, E. Luiz Gaeta Espı´ndola / Ecotoxicology and Environmental Safety 71 (2008) 765–773 Table 3 Results of two-way analyses of variance applied to data on survival, fecundity and feeding rates of C. dubia when exposed to cadmium at several concentrations and fed with algae at various densities d.f.
P
12.60 15.14 2.09
2.16 5.16 10.16
o0.001 o0.001 o0.050
Food Metal Food metal
46.52 11.97 6.02
2.13 5.13 10.13
o0.001 o0.001 o0.001
Filtration rate
Food Metal Food metal
104.22 2.08 1.34
2.36 4.30 8.30
o0.001 NS NS
Ingestion rate
Food Metal Food metal
197.39 2.79 0.66
2.30 4.30 8.30
o0.001 NS NS
Parameter
Source of variation
Survival
Food Metal Food metal
Fecundity
F
d.f.: degree of freedom; P: probability; NS: not significant (P40.05).
Table 4 Results of two-way analyses of variance applied to data on survival, fecundity and feeding rates of C. dubia when this organism was fed with cadmium-contaminated algae at various densities d.f.
P
3.00 3.46 1.56
2.13 4.13 8.13
NS o0.05 NS
Food Metal Food metal
43.78 32.01 11.20
2.90 4.90 8.90
o0.001 o0.001 o0.001
Filtration rate
Food Metal Food metal
25.02 3.92 2.10
2.18 2.18 4.18
o0.001 NS NS
Ingestion rate
Food Metal Food metal
132.50 7.14 6.00
2.18 2.18 4.18
o0.001 NS NS
Parameter
Source of variation
Survival
Food Metal Food metal
Fecundity
F
d.f.: degree of freedom; P: probability; NS: not significant (P40.05).
treatments with 0.40, 0.86 and 1.60 107 M Cd2+, the presence of 106 cells/mL of the green alga P. subcapitata reduced the concentration of free cadmium ions to 0.10, 0.26 and 0.63 107 M, respectively. In the treatment with 1.60 107 M Cd2+, the fraction of accumulated metal (0.020 mmol/mg) at high algal level was higher than that verified at the medium algal concentration (0.012 mmol/mg). Our results demonstrated that metalcontaminated algae released low cadmium concentrations back into the test medium during toxicity tests (Table 6). Algae contaminated with 1.40 and 2.70 107 M Cd2+ offered to zooplankton as food at high density released low cadmium (0.09 and 0.18 107 M Cd, respectively) back into the test medium at end of the test (Tables 5 and 6).
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4. Discussion Metal toxicity to zooplankton can vary with the density of algae offered to the organisms. For example, at low metal concentrations, an increased algal level can enhance the resistance of the species (Sarma et al., 2000). This effect was not observed in the present study, in which toxic effects to C. dubia were demonstrated when exposed to low metal concentrations (0.40 and 0.86 107 M Cd2+) while being fed with a high density of algae. In addition, the mean numbers of neonates produced by C. dubia during exposure to 0.86 and 1.60 107 M Cd+2 in the presence of high algal density were significantly smaller than those obtained at medium algal density. The analysis of the combined effects of food level and metal concentration showed that metal toxicity was dependent on food level. This result agrees with the independent analyses performed with each food level. In general, cladocerans cannot tolerate an algal level higher than 4 106 cells/mL, because such densities can depress their feeding rate (Nandini and Sarma, 2000). In this study, the feeding rate of C. dubia was monitored. At the highest food density used (106 cells/mL), C. dubia filtered less volume of medium and ingested more algal cells. On the other hand, organisms fed with a low algal density showed high filtration and low ingestion rates. The feeding behavior of zooplankton could have consequences for their life cycle (growth, reproduction, survival). Under the control conditions, the fecundity of C. dubia was observed to rise with increasing algal density supplied, and it was significantly reduced by density of 104 cells/mL of P. subcapitata. Algal concentrations of 106 cells/mL probably occur in eutrophic environments, so that studies using this algal density would help in the understanding of metal dynamics in polluted aquatic systems. Algae have been reported to reduce contaminant toxicity to cladocerans. Antunes et al. (2004) observed a decrease in the chronic toxic effect of the pesticide lindane to D. magna when this cladoceran was exposed to a high density of S. capricornutum (6 105 cells/mL). Hauri and Horne (2004) showed that a large amount of algal food reduced the availability of labile copper to C. dubia during chronic toxicity tests, due to complexing of the metal by food particles. The authors reached a general conclusion that a high food level could supply additional energy for growth and reproduction, and at the same time, enhance specific mechanisms of detoxification and of resistance to toxic agents. The results of the present work are consistent with those obtained by Klu¨ttgen and Ratte (1994), who demonstrated inhibited growth and reproduction of D. magna when this organism was exposed to concentrations from 1 to 5 mg/L of Cd, together with a high food concentration of Chlorella vulgaris. According to the authors, the toxic effect of the metal observed in these conditions resulted from the raised Cd uptake by the zooplankton, as a consequence of an increment in their metabolic rate, due to higher availability
ARTICLE IN PRESS S. Rodgher, E. Luiz Gaeta Espı´ndola / Ecotoxicology and Environmental Safety 71 (2008) 765–773
770
high food
medium food
low food Mean number of neonates per female
20 100
% survival
80 #
60 40 20
#
#
a
b a
a
16 c
e
12
0
8
j
gg
e d
o *
m
i
i
f
l
l n
h *
n
*
4
* 0
LC
C 0.1 0.17 0.4 0.86 free cadmium concentration (10-7M)
1.6
0.17 0.4 0.86 C 0.1 free cadmium concentration (10-7M)
LC
1.6
Fig. 1. Percent of survival (A) and mean number of neonates (B) of C. dubia in chronic toxicity tests for cadmium, when fed with various algal densities of P. subcapitata. LC (laboratory control) and C (control). Survival: # indicates statistically different from the control (C) (Fisher’s exact test, Po0.05). Number of neonates per female: * indicates statistically different from the control (C) (Dunnett’s test, Po0.05) and means with different letters are significantly different (Tukey’s test, Po0.05). Error bars denote standard deviation. Each bar for L.C corresponds to one laboratory control of three chronic toxicity tests performed with the three different algal densities.
high food
medium food
low food Mean number of neonates per female
100
% survival
80 60 40 20 0 LC C 0.6 1.4 2.7 free cadmium concentration to which algae were exposed (10-7 M)
20
a
a a
b b d
16 12
c
g, h e
* f
h j * g
i *
i
8 * 4 0 LC C 0.6 2.7 1.4 free cadmium concentration to which algae were exposed (10-7 M)
Fig. 2. Percent of survival (A) and mean number of neonates (B) of C. dubia when fed with various algal densities of P. subcapitata previously exposed to cadmium. LC (laboratory control) and C (control). Survival: # indicates statistically different from the control (C) (Fisher’s exact test, Po0.05). Number of neonates per female: * indicates statistically different from the control (C) (Dunnett’s test, Po0.05) and means with different letters are significantly different (Tukey’s test, Po0.05). Error bars denote standard deviation. Each bar for LC corresponds to one laboratory control of three chronic toxicity tests performed with the three different algal densities.
of food. Similarly, Smolders et al. (2005) also verified that cadmium toxicity to D. magna increased with increasing of food concentration. When the C. dubia females were exposed to free cadmium ion at a high algal density, they filtered less volume of medium and ingested more algal cells with metal bound to the cells, until the toxic effect of the bound metal inhibited their feeding, reproduction and survival. When
the organisms were exposed to 0.4, 0.86 and 1.60 107 M Cd2+ and fed with high algal density, metal analyses demonstrated that algal cells accumulated cadmium. Cadmium associated with algae may form an alternative source of metal contamination to zooplankton, besides the free metal ions from the water. For filter feeding organisms, the digestive system is a considerable contamination route, and a particle-bound toxic substance can
ARTICLE IN PRESS
high food
Filtration rate (µl/ind/h)
1000
c
medium food
f
low food
h
800 o
b e
400 200
l
j
600
a
e
k,l
g
n
i,j
g
k
i
m
0 C
0.1
0.17
0.86
0.4
Ingestion rate ( x 104 cells/indl/h)
S. Rodgher, E. Luiz Gaeta Espı´ndola / Ecotoxicology and Environmental Safety 71 (2008) 765–773
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10 a 8 f
d
6 4
b
k
h d
m
j
h
n
f
2 c
g
i
l
o
0.17
0.4
0.86
1.60
e
0
1.60
C
free cadmium concentration (10-7 M)
0.1
free cadmium concentration (10-7 M)
high food
medium food
low food
Filtration rate (µl/ind/h)
1000 c 800
e
600
g i
d
b 400
h
f
200
f
d
a
h
0 C
0.6
1.4
2.7
Ingestion rate ( x 104 cells/ind/h)
Fig. 3. Filtration and ingestion rates of algal cells by C. dubia when exposed to cadmium and fed with various algal densities of P. subcapitata. Means with the different letters are significantly different (Tukey’s test, Po0.05). Error bars denote standard deviation.
d
10 a 8
g
i
6 b
4 2
e
c
j
h f
j
h
0 C
0.6
1.4
2.7
free cadmium concentration to which algae were exposed (10-7 M)
free cadmium concentration to which algae were exposed (10-7 M)
Fig. 4. Filtration and ingestion rates of algal cells by C. dubia when fed with various algal densities of P. subcapitata previously exposed to cadmium. Means with the different letters are significantly different (Tukey’s test, Po0.05). Error bars denote standard deviation.
Table 5 Total dissolved, free and accumulated metal at the end of chronic toxicity tests with C. dubia to cadmium
Dissolved total metal (107 M) Free metal ions (107 M) Metal in algal cells (m mol/mg)
Control
Water contaminated with metal (107 M Cd2+)
Food contaminated with metal (107 M Cd2+)
0
0.40
0.86
1.60
0.60
1.40
2.70
Medium High food food
Medium High food food
Medium High food food
Medium High food food
Medium High food food
Medium High food food
Medium High food food
0.02 (0.002) 0.02 (0.002) ND
0.21 (0.01) 0.19 (0.01) 0.004 (0.001)
0.58 (0.01) 0.52 (0.03) 0.009 (0.001)
1.11 (0.02) 1.10 (0.02) 0.012 (0.002)
0.02 (0.00) 0.02 (0.01) 0.001 (0.00)
0.03 (0.00) 0.03 (0.01) 0.002 (0.00)
0.05 (0.00) 0.04 (0.00) 0.005 (0.001)
0.02 (0.002) 0.02 (0.002) ND
0.11 (0.02) 0.10 (0.01) 0.002 (0.001)
0.30 (0.01) 0.26 (0.01) 0.005 (0.001)
0.71 (0.01) 0.63 (0.02) 0.020 (0.002)
0.06 (0.01) 0.05 (0.01) 0.001 (0.00)
0.11 (0.01) 0.09 (0.01) 0.001 (0.00)
0.20 (0.00) 0.18 (0.00) 0.003 (0.001)
Mean values (SD). ND: not detected.
readily be filtered from the water and released into their digestive tract (Fliedner, 1997). At low algal density, organisms filtered more water with metal and ingested fewer algal cells than at a high food level. Low acquisition of food by cladocerans would result in less energy for reproduction and for metal toxicity repair mechanisms (Rose et al., 2002).
The importance of algal cells as contamination agents for zooplankton was confirmed by the results obtained in toxicity tests with contaminated food. In this study, a decrease in fecundity of C. dubia was observed when these organisms were fed on P. subcapitata cells exposed to Cd2+. As a result of adding medium and high algal densities contaminated with Cd2+, concentrations between
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Table 6 Dissolved cadmium concentration (107 M) in the culture medium during experiments with C. dubia fed with various algal densities of P. subcapitata previously exposed to cadmium Day 2
Day 4
Day 8
Cadmium concentration to which algal cells were exposed (107 M)
104 cells/ mL
105 cells/ mL
106 cells/ mL
104 cells/ mL
105 cells/ mL
106 cells/ mL
104 cells/ mL
105 cells/ mL
106 cells/ mL
0.60
ND
ND
ND
0.17 (0.02) 0.18 (0.01)
ND
2.70
0.03 (0.00) 0.07 (0.00) 0.09 (0.01)
0.05
ND
0.08 (0.00) 0.12 (0.01) 0.21 (0.01)
ND
1.40
0.02 (0.00) 0.05 (0.00) 0.10 (0.01)
0.02 (0.00) 0.03 (0.00) 0.05 (0.00)
0.06 (0.01) 0.11 (0.01) 0.20 (0.00)
ND ND
ND
Mean values (SD). ND: not detected.
0.03 and 0.20 107 M Cd were released in the test medium. This may occur either via desorption or elimination of metal from algal cells (De Schamphelaere et al., 2004) and from elimination by the daphnids that have incorporated dietary metal in their tissues (Guan and Wang, 2004). Indeed, at such a low cadmium concentration, no toxic effects are expected. We observed effects on the fecundity and survival of C. dubia up to 0.20 107 M Cd2+. These observations suggest that toxic effects in cladocerans given contaminated food can be attributed to cadmium via dietary exposure. Our findings agree with those of De Schamphelaere et al. (2004), who found adverse effects on reproduction of D. magna after exposure to dietary Zn. Sofyan et al. (2006) also demonstrated that Cd in algal cells was available for trophic transfer and capable of producing deleterious effects to C. dubia. In our experiments involving feeding with Cd+2 contaminated algae, C. dubia ingested more algal cells at 106 cells/mL than at 105 and 104 cells/mL. According to Allen et al. (1995) and Taylor et al. (1998), algal cells contaminated with Cd can be collected and ingested normally by daphnids, but the contaminant interferes with digestion, resulting in cells passing through the gut without being digested. This could result in profound changes at the population levels, since the main parameters for population growth and survival are dependent on the energy input from feeding activity. The toxic effects on C. dubia were less pronounced when the zooplankton were exposed to food contaminated with 0.60 and 1.40 107 M Cd2+ than those obtained when the test organisms were exposed to 0.86 and 1.60 107 M Cd2+ in test solution at high algal density. This result implies that free cadmium ions are more bioavailable to cladocerans than metal in food, but a substantial amount of the metal could be obtained from their food. Consequently, it should be noted that metal dietary exposure may be present in treatment with contaminated water. Food quality is at least as important as quantity for the fecundity, population growth and survival of cladocerans
(Kilham et al., 1997). Nishikawa et al. (2003) found that cells of the green algae Chlamydomonas acidophila suffered a reduction in the level of polyphosphate after being exposed to 10 and 20 mM of Cd. Metal could have affected the nutritional value of the algal cells that were exposed to it. Such changes could make the algae poor in nutritional value and unviable as food for C. dubia. In the present study, the cadmium concentrations used to contaminate P. subcapitata were lower than those used by Nishikawa et al. (2003). It is unlikely that the observed reduction in fecundity of C. dubia might result from a dietary quality effect. In general, negative effects may be related to the direct toxic effects of metals on target cells or sensitive tissues responsible for egg production (vitellogenesis) (Hook and Fisher, 2002). Indirect effects of metal, due to a diminution in the process of nutrient assimilation (Munger et al., 1999) have negative repercussions on growth and fecundity of zooplankton. 5. Conclusion Our findings suggested that free cadmium ions were more bioavailable to zooplankton than metal in food. The addition of algae at high density in chronic toxicity tests in treatments with water contaminated with cadmium might have promoted binding sites for the metal and, consequently, generated additional routes for exposure of test organisms to metal. Food should be considered as an additional source of metal exposure to cladocerans. Ecotoxicological studies should consider the influence of varying algal densities on the expression of metal toxicity on daphinids. This would allow a better understanding of the possible relationships between toxicants and aquatic organisms. Acknowledgments We thank the National Research Council (CNPq: Process 140156/2002-0) and the Sa˜o Paulo State Research Support Foundation (FAPESP: Process 10417/2002) for
ARTICLE IN PRESS S. Rodgher, E. Luiz Gaeta Espı´ndola / Ecotoxicology and Environmental Safety 71 (2008) 765–773
financial support. We also express our gratitude to Alessandra Tonietto for assistance with the MINEQL+ program, to Dr. Liane Biehl Printes for valuable comments, and to anonymous reviewers for improving the manuscript. Funding sources: National Research Council (CNPq: Process 140156/2002-0) and the Sa˜o Paulo State Research Support Foundation (FAPESP: Process 10417/2002).
References Allen, Y., Calow, P., Baird, D.J., 1995. A mechanistic model of contaminant-induced feeding inhibition in Daphnia magna. Environ. Toxicol. Chem. 14, 1625–1630. American Public Health Association; American Water Work Association; Water Control Federation, 1995. Standard Methods for the Examination of Water and Wastewater, 19th ed. New York. Antunes, S.C., Castro, B.B., Gonc- alves, F., 2004. Effect of food level on the acute and chronic responses of daphnids to lindane. Environ. Pollut. 127, 367–375. Associac- a˜o Brasileira de Normas Te´cnicas, 2005a. Avaliac- a˜o da toxicidade croˆnica utilizando Ceriodaphnia dubia, Richard, 1894 (Cladocera, Crustacea). NBR13373, Julho, 14p, Rio de Janeiro. Associac- a˜o Brasileira de Normas Te´cnicas, 2005b. Ecotoxicologia aqua´tica—toxicidade croˆnica—Me´todo de ensaio com algas (Chlorophyceae). NBR12468, 27p, Rio de Janeiro. Association Franc- aise de Normalisation Afnor, 1980. Norme experimentale.T90-304. Essais deseaux Determination de I’inhibition de Scenedesmus subspicatus par une substance. Ayres, M., Ayres, M. Jr., Ayres, D.L., Santos, A.S., 2000. BioEstat 2.0: aplicac- o˜es estatı´ stica na a´rea das Cieˆncias Biolo´gicas e Me´dicas. Sociedade Civil Lithera Maciel. Editora Gra´gica Ltda., Mamiraua´. Barata, C., Markich, S.J., Baird, D.J., Soares, A.M.V.M., 2002. The relative importance of water and food as cadmium sources to Daphnia magna Straus. Aquat. Toxicol. 61, 143–154. Chandini, T., 1989. Survival, growth and reproduction of Daphnia carinata (Crustacea: Cladocera) exposed to chronic cadmium stress at different food (Chlorella) levels. Environ. Pollut. 60, 29–45. De Schamphelaere, K.A.C., Canli, M., Lierde, V.V., Forrez, I., Vanhaecke, F., Janssen, C.R., 2004. Reproductive toxicity of dietary zinc to Daphnia magna. Aquat. Toxicol. 70, 233–244. Fliedner, A., 1997. Ecotoxicity of poorly water-soluble substances. Chemosphere 35, 295–305. Gauld, D.T., 1951. The grazing rate of planktonic copepods. J. Mar. Biol. Assoc. UK 29, 695–706. Guan, R., Wang, W.X., 2004. Dietary assimilation and elimination of Cd, Se, and Zn by Daphnia magna at different metal concentrations. Environ. Toxicol. Chem. 23, 2689–2698. Gulley, D.D., Boelter, A.M., Bergman, H.L., 1993. Toxstat Version 3.3. Computer Program. Fish Physiology and Toxicology Laboratory, Department of Zoology and Physiology University of Wyoming, Laramie, WY. Hamilton, M.A., Russo, R.C., Thurston, R.V., 1977. Trimmed Spearman–Karber method for estimating median lethal concentrations in toxicity bioassays. Environ. Sci. Technol. 11, 714–719. Hauri, J.F., Horne, A.J., 2004. Reduction in labile copper in the 7-day Ceriodaphnia dubia toxicity test due to the interaction with zooplankton food. Chemosphere 56, 717–723.
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Hook, S.E., Fisher, N.S., 2002. Relating the reproductive toxicity of five ingested metals in calanoid copepods with sulfur affinity. Mar. Environ. Res. 53, 161–174. Kilham, S.S., Kreeger, D.A., Goulden, C.E., Lynn, S.G., 1997. Effects of algal food quality on fecundity and population growth rates of Daphnia. Freshwater Biol. 38, 639–647. Klu¨ttgen, B., Ratte, H.T., 1994. Effects of different food doses on cadmium toxicity to Daphnia magna. Environ. Toxicol. Chem. 13, 1619–1627. Lanno, R.P., Hickie, B.E., Dixon, D.G., 1989. Feeding and nutritional considerations in aquatic toxicology. Hydrobiologia 188/189, 525–531. Lombardi, A.T., Vieira, A.A.H., 2000. Copper complexation by Cyanophyta and Chlorophyta exudates. Phycologia 39, 118–125. Miller, J.C., Miller, J.N., 1994. Statistics for Analytic Chemistry, third ed. Great Britain. Munger, C., Hare, L., Craig, A., Charest, P.M., 1999. Influence of exposure time on the distribution of cadmium within the cladoceran Ceriodaphnia dubia. Aquat. Toxicol. 44, 195–200. Nandini, S., Sarma, S.S.S., 2000. Lifetable demography of four cladoceran species in relation to algal food (Chlorella vulgaris) density. Hydrobiologia 435, 117–126. Nishikawa, K., Yamakoshi, Y., Uemura, I., Tominga, N., 2003. Ultrastructural changes in Chlamydomonas acidophila (Chlorophyta) induced by heavy metals and polyphosphate metabolism. FEMS Microbiol. Ecol. 44, 253–259. Reinfelder, J.R., Fisher, N.S., Luoma, S.N., Nichols, J.W., Wang, W.X., 1998. Trace element trophic transfer in aquatic organisms: a critique of the kinetic model approach. Sci. Total Environ. 219, 117–135. Rodgher, S., 2005. Avaliac- a˜o dos efeitos ecotoxicolo´gicos dos metais ca´dmio e cromo sobre organismos planctoˆnicos, Sa˜o Carlos. 213p. Tese (Doutorado), Escola de Engenharia de Sa˜o Carlos, Universidade de Sa˜o Paulo. Rose, R.M., Warne, M.S.T.J., Lim, R.P., 2002. Food concentration affects the life history respond of Ceriodaphnia cf. dubia to chemicals with different mechanisms of action. Ecotoxicol. Environ. Saf. 51, 106–114. Sarma, S.S.S., Pe´rez, T.R., Nandini, S., 2000. Comparison of the sensitivity of Brachionus calyciflorus and Brachionus patulus (Rotifera) to selected heavy metals under low and high food (Chlorella vulgaris) levels. Bull. Environ. Contam. Toxicol. 64, 735–739. Schecher, W.D., McAvoy, D.C., 1991. MINEQL+: A Chemical Equilibrium Program for Personal Computers, Version 3.01. Environmental Research Software, Hallowell, ME, USA. Smolders, R., Baillieul, M., Blust, R., 2005. Relationship between the energy status of Daphnia magna and its sensitivity to environmental stress. Aquat. Toxicol. 73, 155–170. Sofyan, A., Shaw, J.R., Birge, W.J., 2006. Metal transfer from algae to cladocerans and the relative importance of dietary metal exposure. Environ. Toxicol. Chem. 25, 1034–1041. Taylor, G., Baird, D.J., Soares, A.M.V.M., 1998. Surface binding contaminants by algae: consequences for lethal toxicity and feeding to Daphnia magna Straus. Environ. Toxicol. Chem. 17, 412–419. Van Loon, J.C., 1985. Select methods of trace metal analysis. Wiley, USA. Villarroel, M.J., Ferrando, M.D., Sancho, E., Andreu, E., 1999. Daphnia magna feeding behavior after exposure to tetradifon and recovery from intoxication. Ecotoxicol. Environ. Saf. 44, 40–46. Weltens, R., Goossens, R., Van Puymbroeck, S., 2000. Ecotoxicity of contaminated suspended solids for filter feeders (Daphnia magna). Arch. Environ. Contam. Toxicol. 39, 315–323.
Ecotoxicology and Environmental Safety 71 (2008) 765–773 www.elsevier.com/locate/ecoenv
Effects of interactions between algal densities and cadmium concentrations on Ceriodaphnia dubia fecundity and survival Suzelei Rodgher, Evaldo Luiz Gaeta Espı´ ndola Sa˜o Carlos Engineering School, Water Resources and Applied Ecology Center, University of Sa˜o Paulo, Avenida Trabalhador Sa˜o Carlense, 400, C.P 292, Cep 13.560-970 Sa˜o Carlos, SP, Brazil Received 8 November 2006; received in revised form 19 July 2007; accepted 24 August 2007 Available online 23 October 2007
Abstract The influence of different densities of the algae Pseudokirchneriella subcapitata on the chronic toxicity of cadmium to Ceriodaphnia dubia was investigated. The importance of algal cells as a source of metal to zooplankton was studied by exposing P. subcapitata cells to free cadmium ions and supplying the algae as food to C. dubia. The results of a bifactorial analysis (metal versus food levels) showed that metal toxicity to zooplankton was dependent on food level. Significant toxic effects on the fecundity and survival of C. dubia were observed at low metal concentrations with high algal density. Algae contaminated with Cd2+ were less toxic to cladoceran than was the Cd2+ in solution. Green algae retained cadmium and released low metal concentration in the test medium. We concluded that algal cells are an important route of exposure to metal and a factor that has an appreciable influence on the expression of metal toxicity to daphnids. r 2007 Elsevier Inc. All rights reserved. Keywords: Ceriodaphnia dubia; Sources of metal toxicity; Pseudokirchneriella subcapitata; Chronic toxicity
1. Introduction Until a few years ago, feeding activity had been largely ignored in research published on accumulation of heavy metals and their bioavailability to aquatic invertebrates, presumably because feeding activity was not considered an important source of contamination and uptake of metals in previous studies. Recently, many studies have focused on the effects of metals on survival, growth and reproduction of filter-feeding organisms submitted to a variety of food conditions, both in the laboratory and in the field (Reinfelder et al., 1998). The food, represented by the algae, can act as a channel of metal contamination to cladocerans (Hook and Fisher, 2002) and can counteract metal toxicity, due to the algae’s ability to exude organic compounds that complex metals (Lombardi and Vieira, 2000). On the other hand, the algae contribute to the resistance of the organisms to toxic Corresponding author. Fax: +55 16 33738251.
E-mail addresses: [email protected] (S. Rodgher), [email protected] (E. Luiz Gaeta Espı´ ndola). 0147-6513/$ - see front matter r 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.ecoenv.2007.08.012
substances by improving their nutritional state (Chandini, 1989). Since aquatic organisms in their natural environment have food available during toxic exposure, attention should be given to the role of food in toxicity tests (Lanno et al., 1989). A new approach in ecotoxicological studies has focused on the influence of food on the effects of toxic agents on zooplankton. Weltens et al. (2000) verified that particles (sand, clay, algae) contaminated with cadmium are potentially toxic to Daphnia, not only by acting as a source of dissolved metal, but also because the particle-bound fraction of Cd can become free and available within the body of the filter-feeding organism. Barata et al. (2002) confirmed that although water was the major pathway of cadmium uptake for D. magna, a substantial amount of the metal was obtained by the test organisms from the contaminated algae. Dietary exposure to metal of zooplankton may occur because the algae can adsorb and take up dissolved metal from the exposure solution before being ingested by the organisms. However, algae may eliminate metal to the solution during the daphnids’ exposure, which might result
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in significant exposure of organisms to metal via water. The elimination of metals from food in dietary toxicity studies is recognized as an important factor that may confuse the interpretation of the effects of dietary exposure (De Schamphelaere et al., 2004). In this light, the aim of this study was to evaluate the effects of varying the density of the algae Pseudokirchneriella subcapitata on the chronic toxicity of cadmium to the cladoceran Ceriodaphnia dubia, when this organism was exposed to this metal in the water and in contaminated food (cells of P. subcapitata). In these experiments, total dissolved cadmium concentrations and cadmium accumulated by algal cells were measured and free metal ions were estimated in order to distinguish between the effects of aqueous and dietary exposures as sources of metal toxicity to these organisms. Feeding rates of C. dubia were also measured in both treatments. 2. Materials and methods 2.1. Culture of test organisms Cells of P. subcapitata and neonates of C. dubia were obtained from cultures maintained at the Laboratory of Ecotoxicology and Ecophysiology of Aquatic Organisms of the Water Resources and Applied Ecology Center (CRHEA) of the University of Sa˜o Paulo (Sa˜o Paulo State, Brazil). Cultures of C. dubia were maintained in a temperature-controlled chamber at 2571 1C with a 12:12 h light/dark cycle, using reconstituted water as the culture medium (pH 7.2–7.6, conductivity 160 mS/cm and hardness between 42 and 48 mg/L of CaCO3). The water was changed every other day. Reconstituted water consisted of four inorganic salts dissolved in deionized water. Twenty millilitres of a solution (0.01 M of CaSO4.2H2O) and 10 mL of another solution (0.002 M of KCL, 0.06 M of NaHCO3 and 0.04 M of MgSO4.7H2O) were added to deionized water to a final volume of 1 L (ABNT, 2005a). The green algae P. subcapitata were cultivated in L.C. Oligo medium (AFNOR, 1980), which was first autoclaved (121 1C) for 15 min in 2-L Erlenmeyer flasks containing 1 L of the medium. The composition of the culture medium is described in Table 1. This was inoculated with cells to a concentration of around 1 104 cells/mL and the culture was exposed to 100 mE/m2/s2 in 12:12 h light/dark cycle, with constant aeration, at 2472 1C (ABNT, 2005b). Algal cells from exponentially growing
P. subcapitata cultures were used as food for the zooplankton, C. dubia, which was fed every other day with 1 105 cells/mL of the algae and a suspension of yeast and commercial fish food (Vitormonios), in accordance with the procedures set in the Brazilian guidelines ABNT (2005a).
2.2. Toxicity tests 2.2.1. Exposure of algae to metal Cells of P. subcapitata in the exponential growth phase were exposed for 96 h to ascending levels of total dissolved cadmium of 0, 0.63, 1.51, 2.93, 6.85 and 12.60 107 M, corresponding to free cadmium ion concentrations of 0, 0.60, 1.40, 2.70, 6.32 and 11.60 107 M, respectively. Free cadmium ions were estimated using the MINEQL+ model (Schecher and McAvoy, 1991). The test solutions used in this experiment were prepared in volumetric flasks using volumetric pipettes. The stock solution was 8.9 mM Cd(NO3)2.4H2O (solution standard of nitrate of cadmium for atomic absorption, J.T. Backer). The dilution water used in the preparation of the test was LC Oligo medium. After the exposure period, the cells were centrifuged at 1500 rpm for 15 min (FANEM Excelsa centrifuge, model 206 PM), washed three times with reconstituted water and resuspended in the zooplankton culture water (reconstituted water). Finally, suspensions of the cells were stored in polythene bottles in the dark at 4 1C during the chronic toxicity test with zooplankton (De Schamphelaere et al., 2004). Samples (2 mL) from each test flask were taken and fixed with Lugol’s iodine solution to determine the cell density, by counting cells in an Improved Neubauer Bright-Line hemocytometer under optical microscope (Carl Zeiss), standard model 25. The mean number of cells produced at each concentration, after this exposure period, was expressed as a percentage growth reduction with respect to the control. These percentages were used to calculate the IC50 (effective metal concentration giving 50% inhibition of algal growth after 96 h exposure). The dry weight of the algal cells was obtained by filtering a known volume of cells on a pre-weighed filter. The filters with algal cells were dried for 24 h at 60 1C and weighed to determine the cell mass per volume of culture (APHA, 1995). 2.2.2. Chronic toxicity test The test solutions for the chronic toxicity tests with the cladocerans were prepared as in the experiments with the algae and the dilution water used in these preparations was reconstituted water. Acute toxicity tests demonstrated a median effect concentration (EC50, 48 h) of 5.34 107 M Cd to C. dubia (Rodgher, 2005). Based on the result of the acute toxicity
Table 1 Composition of the culture medium L.C. Oligo (AFNOR, 1980) used in the present study Stock solution
Compound
Concentration (M)
Volume (mL) required from stock solution to 1 L of medium
1 2 3 4 5
Ca(NO3)2 4H2O KNO3 MgSO4 7H2O K2HPO4 CuSO4 5H2O (NH4)6Mo7O24 4H2O ZnSO4 7H2O CoCl2 6H2O Mn(NO3)2 4H2O C6H8O2 H2O H3BO3 C6H5FeO7 5H2O FeSO4 7H2O FeCl3 6H2O NaHCO3
0.24 0.99 0.20 0.23 0.00016 0.00005 0.0002 0.0001 0.0002 0.0005 0.001 0.005 0.002 0.002 0.2
1.00 1.00 1.00 1.00 0.50
6
7
0.50
1.00
ARTICLE IN PRESS S. Rodgher, E. Luiz Gaeta Espı´ndola / Ecotoxicology and Environmental Safety 71 (2008) 765–773 tests, we prepared five nominal sublethal metal concentrations. Each successive nominal concentration was about twice the previous one (0.11, 0.22, 0.44, 0.89, 1.78 107 M Cd). Test solution samples from each nominal metal concentration were filtered, acidified and analyzed by graphite-furnace atomic absorption spectrometry to determine the dissolved cadmium concentrations. Considering that free metal ions constitute a metal fraction of higher bioavailability to organisms, we used the MINEQL+ program to calculate Cd2+. The free ion cadmium concentrations were then predicted from the total dissolved cadmium concentrations. The total dissolved cadmium concentrations used in the chronic toxicity tests with zooplankton were 0, 0.11, 0.19, 0.44, 0.87, 1.78 107 M, corresponding to free cadmium ion concentrations of 0, 0.10, 0.17, 0.40, 0.86, 1.60 107 M, respectively. In the chronic toxicity tests, 10 neonates of C. dubia (aged less than 24 h) were exposed, in 10 separate test vessels, to 15 mL of a certain metal concentration and fed with one of three densities of P. subcapitata (low, medium and high: 1 104 cells/mL, 1 105 cells/mL and 1 106 cells/mL, respectively). The organisms were transferred to a freshly prepared test solution every other day and fed with the respective algal concentration daily. The test media were gently stirred using a fine Pasteur pipette twice a day to ensure availability of algal cells to the organisms. A control test without the added metal was also prepared. The toxicity tests lasted seven to eight days, the period needed for production of the third brood, and the survival of adults and number of live neonates per female were recorded (ABNT, 2005a). Two controls were used: a laboratory control (LC) that contained the food concentration normally used in the maintenance of the test organism (1 105 cells/mL plus the suspension of yeast and fish food) and another control (C), containing only the algae at the density being tested. This was a way of testing the physiological condition of the organisms being used in the tests. The chronic toxicity tests were considered valid when a survival of 80% was observed in adult organisms, corresponding to the laboratory control, and when they produced at least 15 neonates per female (ABNT, 2005a). In another experiment, cells of P. subcapitata exposed to free cadmium for 96 h were offered as food to zooplankton. The concentrations of metal used to contaminate P. subcapitata were 0, 0.60, 1.40, 2.70 107 M Cd2+. Neonates of C. dubia were exposed to reconstituted water and fed with three different densities of metal-contaminated P. subcapitata (low, medium and high: 1 104, 1 105 and 1 106 cells/mL, respectively) during seven days. The toxic effects of metal-contaminated algae on fecundity and survival of C. dubia were verified. The feeding and waterchanging procedures for this test were similar to those used when this organism was exposed to different metal concentrations. 2.2.3. Feeding rates of the c. dubia The effects of free cadmium in water and of Cd+2 contaminated algal diet on feeding rates of zooplankton at different algal levels were evaluated using measured filtration and ingestion rates. The feeding experiments with C. dubia were carried out in test vessels containing 50 mL of the medium and 10 individuals under the same condition of the toxicity chronic test. A control test, without the added metal, was also prepared. Test organisms were exposed to two treatments (water and contaminated food) for 24 h, after which the final algal concentration was measured using a hemocytometer (Villarroel et al., 1999). The filtration rate (F) was defined as the volume of medium swept clear per unit of time and the ingestion rate (I) as the number of cells consumed by an animal in a specific time interval. To calculate the average filtration (ml/ind/h) and ingestion rates (cells/ind/h), the equations from Gauld (1951) were used: F¼
V ðln C 0 ln C t Þ A, n t
A¼
ln C 0 ln C 0t , t
I ¼F
pffiffiffiffiffiffiffiffiffiffiffi C0Ct ,
767
where C0 and Ct are initial and final food concentrations (cell/mL), t is time (duration of the experiment in hours), and n is the number of daphnids in volume V (mL). A is a correction factor for changespin the ffiffiffiffiffiffiffiffiffiffiffi control with final concentration Ct0 after time t. The expression C 0 C t represents the geometric mean of the food concentration during time t. 2.2.4. Metal analyses At the end of the experiments with P. subcapitata, samples were taken from each treatment to determine the metal accumulated by algal cells. Aliquots of test solutions from experiments with algae and cladocerans were filtered through a 0.45 mm membrane filter The filters with the accumulated fraction were dried and submitted to acid digestion (HNO3 and H2O2) (APHA, 1995). The analysis of metal in the algal cells was carried out without EDTA washing, in order not to remove the metals adsorbed on the cell surface. The measured concentration of metal in algal cells was taken as the total amount of metal accumulated by the cells (i.e., externally and internally bound metal), expressed as mg Cd/mg dry weight of algae. At the end of the toxicity tests with zooplankton, samples also were taken to determine the total dissolved metal and accumulated metal by the cells. These measurements were taken at concentrations where toxic effects to C. dubia could be identified. Filtered samples were preserved by acidifying with concentrated nitric acid, for subsequent determination of total dissolved metal. We used values of total dissolved metal together with the chemical composition of the test medium to compute the concentration of free metal ion using the MINEQL+ program. During the experimental period the metal loss from the algae in the toxicity test with contaminated food was quantified as the total dissolved concentration of metal in the culture medium. For each sample digested, three unused filters were digested and analyzed as blanks (Van Loon, 1985). All the samples were analyzed by graphite-furnace atomic absorption spectrometry (Varian AA 220). The detection limit for Cd, calculated as described in Miller and Miller (1994), was 4.45 1010 M. 2.2.5. Statistical analysis The IC50 value of cadmium for the algae was determined by the trimmed Spearman–Karber method (Hamilton et al., 1977). The algal cell density data obtained from the experiment with the chlorophyte were submitted to tests for normality (Shapiro–Wilk’s test) and homogeneity (Bartlett’s test), and then, to Dunnett’s test (parametric test) to detect significant differences between the controls and each metal treatment. To test the significance of the effect of algal food densities, free metal ion concentration and different densities of metal-contaminated alga on the survival, fecundity and feeding rates of C. dubia, a two-way ANOVA was employed on the chronic test data, using the BioEstat 2.0 program (Ayres et al., 2000). The numbers of neonates produced by C. dubia females and their feeding rates were submitted to tests for normality (Shapiro–Wilk’s test) and homogeneity (Bartlett’s test) and, accordingly, to Dunnett’s test (parametric) to detect significant differences between the controls and each metal treatment. Tukey’s test (parametric) was used in multiple comparisons to detect significant differences among the fecundity obtained in treatments with different algal densities and metal concentrations. Fisher’s exact test was used to distinguish significant differences in the survival of zooplankton between the control and the various treatments at the end of the chronic toxicity tests. The above statistical tests were run using the Toxstat version 3.3 computer package (Gulley et al., 1993).
3. Results 3.1. Exposure of algae to metal The mean value for the IC50 of Cd2+ to P. subcapitata, in a total of 10 tests, was 1.65 107 M Cd2+, with lower and upper limits of 0.70 and 2.60 107 M Cd2+, respectively. Table 2 summarizes the effects of metal on
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the algae and the results of metal analyses. A decrease in the cell density of P. subcapitata was observed with increasing concentrations of cadmium. Concentrations of 2.70 107 M Cd2+ reduced the algal cell density to less than half that of the control (without added metal) and dry weight fell similarly. Metal analyses demonstrated that green algae were able to accumulate cadmium. With increasing free cadmium ions in solution, the metal accumulated by P. subcapitata also increased. For example, when the concentration of Cd2+ increased from 0.57 to 6.32 107 M, the metal concentration accumulated by the green algae increased from 0.001 to 0.01 mmol/mg. 3.2. Chronic toxicity test with zooplankton The fecundity and survival of C. dubia were significantly affected by the food level, the metal ion concentrations, and also by their interactions (Tables 3 and 4). The survival of the cladocerans decreased with increasing Cd2+only at high algal density. At 0.4, 0.86 and 1.60 107 M free Cd2+, the number of neonates per female was higher at the medium food level than at high and low levels. Treatment with 1.60 107 M of free cadmium caused a decrease in the number of neonates produced by C. dubia when medium (105 cells/mL) and high (106 cells/mL) algal densities were supplied as food (Fig. 1). On the other hand, the toxic effects on zooplankton fecundity during exposure to 0.40 and 0.86 107 M of Cd2+ were observed only at high food density. The mean numbers of neonates per female when C. dubia was fed at high algal density and exposed to 0.40 and 1.60 107 M Cd2+ (2.75 and 1.65 neonates/ female, respectively) were significantly lower than those obtained at medium algal density and at the same metal concentrations (7.50 and 4.70 neonates/female, in 0.40 and 1.60 107 M Cd+2, respectively) (Fig. 1). Fig. 2 demonstrates that the test organisms suffered a significant reduction in mean neonate numbers when they were fed with high and medium densities of algae exposed to 1.40 and 2.70 of 107 M Cd2+. The survival of the cladocerans was not significantly affected. The mean number of neonates produced by C. dubia at the high density of algae contaminated with 2.70 107 M Cd2+
(3.33 neonates/female) was significantly lower than that at the medium density (7.29 neonates/female). Considering both chronic toxicity tests, the number of neonates produced per female and survival of C. dubia were lower in the treatment with water contaminated with 0.4, 0.86 and 1.60 107 M Cd2+ at high algal density than were fecundity and survival of C. dubia exposed to a high food level contaminated with 0.6, 1.4 and 2.70 107 M Cd2+ (Tukey’s test, Po0.05). At low food concentrations (104 cells/mL), in both sets of chronic toxicity tests (water and food contaminated with metal), the number of neonates per female was not different to that found in the control (Dunnett’s test, Po0.05). Before the metal effect, there was the effect of low food reducing the fecundity of test organisms, as this was also observed in the control containing 1 104 cells/mL. At exposures of 0.4, 0.86 and 1.6 107 M of free cadmium and in the experiment with food contaminated at 1.4 and 2.7 107 M Cd2+, the fecundity of C. dubia at low food density was equal to that at high food level. The animals might have been affected by metal when fed with high algal densities in both treatments (water and contaminated food), and limited by low food concentration. 3.3. Feeding rates The filtration rates of C. dubia in control test conditions (C) in both treatments (water and contaminated food) were higher at low food concentration than at medium and high food levels, whereas ingestion rates were higher at high than at medium and low algal densities. These results were generally observed during the metal exposures (Figs. 3 and 4). Metal in water did not affect the filtration and ingestion rates of the zooplankton, and the feeding rates only were affected by food levels. In experiments with algal densities contaminated with Cd+2, filtration and ingestion rates of C. dubia were only altered by food levels. 3.4. Metal analyses Concentrations of total dissolved cadmium, free cadmium and cadmium accumulated by algal cells, at the end of toxicity test with C. dubia, are given in Table 5. In the
Table 2 Values of dissolved total metal, free metal, metal accumulated by algal cells, cell density and dry weight in experiments with P. subcapitata algae with cadmium Dissolved total cadmium at start of exposure (107 M) Free cadmium at start of exposure (107 M) Cadmium bound to algal cells (mmol/mg)
C (0) 0 0
Mean cell density after 96 h of exposure (106 cells/mL)
6.39 (1.14) 137.00 (23.36)
Dry weight (mg/L)
Mean values (SD). C, control. a Indicates statistically different from control (C) (Dunnett’s test, Po0.05).
0.63 0.60 0.001 (0.000) 4.91a (1.34) 92.22a (27.34)
1.51 1.40 0.001 (0.000) 3.94a (1.54) 89.91a (31.02)
2.93 2.70 0.004 (0.001) 2.10a (0.65) 64.55a (19.91)
6.85 6.32 0.01 (0.002) 0.77a (0.26) 24.92a (6.31)
12.6 11.6 0.05 (0.001) 0.14a (0.14) 9.70a (2.73)
ARTICLE IN PRESS S. Rodgher, E. Luiz Gaeta Espı´ndola / Ecotoxicology and Environmental Safety 71 (2008) 765–773 Table 3 Results of two-way analyses of variance applied to data on survival, fecundity and feeding rates of C. dubia when exposed to cadmium at several concentrations and fed with algae at various densities d.f.
P
12.60 15.14 2.09
2.16 5.16 10.16
o0.001 o0.001 o0.050
Food Metal Food metal
46.52 11.97 6.02
2.13 5.13 10.13
o0.001 o0.001 o0.001
Filtration rate
Food Metal Food metal
104.22 2.08 1.34
2.36 4.30 8.30
o0.001 NS NS
Ingestion rate
Food Metal Food metal
197.39 2.79 0.66
2.30 4.30 8.30
o0.001 NS NS
Parameter
Source of variation
Survival
Food Metal Food metal
Fecundity
F
d.f.: degree of freedom; P: probability; NS: not significant (P40.05).
Table 4 Results of two-way analyses of variance applied to data on survival, fecundity and feeding rates of C. dubia when this organism was fed with cadmium-contaminated algae at various densities d.f.
P
3.00 3.46 1.56
2.13 4.13 8.13
NS o0.05 NS
Food Metal Food metal
43.78 32.01 11.20
2.90 4.90 8.90
o0.001 o0.001 o0.001
Filtration rate
Food Metal Food metal
25.02 3.92 2.10
2.18 2.18 4.18
o0.001 NS NS
Ingestion rate
Food Metal Food metal
132.50 7.14 6.00
2.18 2.18 4.18
o0.001 NS NS
Parameter
Source of variation
Survival
Food Metal Food metal
Fecundity
F
d.f.: degree of freedom; P: probability; NS: not significant (P40.05).
treatments with 0.40, 0.86 and 1.60 107 M Cd2+, the presence of 106 cells/mL of the green alga P. subcapitata reduced the concentration of free cadmium ions to 0.10, 0.26 and 0.63 107 M, respectively. In the treatment with 1.60 107 M Cd2+, the fraction of accumulated metal (0.020 mmol/mg) at high algal level was higher than that verified at the medium algal concentration (0.012 mmol/mg). Our results demonstrated that metalcontaminated algae released low cadmium concentrations back into the test medium during toxicity tests (Table 6). Algae contaminated with 1.40 and 2.70 107 M Cd2+ offered to zooplankton as food at high density released low cadmium (0.09 and 0.18 107 M Cd, respectively) back into the test medium at end of the test (Tables 5 and 6).
769
4. Discussion Metal toxicity to zooplankton can vary with the density of algae offered to the organisms. For example, at low metal concentrations, an increased algal level can enhance the resistance of the species (Sarma et al., 2000). This effect was not observed in the present study, in which toxic effects to C. dubia were demonstrated when exposed to low metal concentrations (0.40 and 0.86 107 M Cd2+) while being fed with a high density of algae. In addition, the mean numbers of neonates produced by C. dubia during exposure to 0.86 and 1.60 107 M Cd+2 in the presence of high algal density were significantly smaller than those obtained at medium algal density. The analysis of the combined effects of food level and metal concentration showed that metal toxicity was dependent on food level. This result agrees with the independent analyses performed with each food level. In general, cladocerans cannot tolerate an algal level higher than 4 106 cells/mL, because such densities can depress their feeding rate (Nandini and Sarma, 2000). In this study, the feeding rate of C. dubia was monitored. At the highest food density used (106 cells/mL), C. dubia filtered less volume of medium and ingested more algal cells. On the other hand, organisms fed with a low algal density showed high filtration and low ingestion rates. The feeding behavior of zooplankton could have consequences for their life cycle (growth, reproduction, survival). Under the control conditions, the fecundity of C. dubia was observed to rise with increasing algal density supplied, and it was significantly reduced by density of 104 cells/mL of P. subcapitata. Algal concentrations of 106 cells/mL probably occur in eutrophic environments, so that studies using this algal density would help in the understanding of metal dynamics in polluted aquatic systems. Algae have been reported to reduce contaminant toxicity to cladocerans. Antunes et al. (2004) observed a decrease in the chronic toxic effect of the pesticide lindane to D. magna when this cladoceran was exposed to a high density of S. capricornutum (6 105 cells/mL). Hauri and Horne (2004) showed that a large amount of algal food reduced the availability of labile copper to C. dubia during chronic toxicity tests, due to complexing of the metal by food particles. The authors reached a general conclusion that a high food level could supply additional energy for growth and reproduction, and at the same time, enhance specific mechanisms of detoxification and of resistance to toxic agents. The results of the present work are consistent with those obtained by Klu¨ttgen and Ratte (1994), who demonstrated inhibited growth and reproduction of D. magna when this organism was exposed to concentrations from 1 to 5 mg/L of Cd, together with a high food concentration of Chlorella vulgaris. According to the authors, the toxic effect of the metal observed in these conditions resulted from the raised Cd uptake by the zooplankton, as a consequence of an increment in their metabolic rate, due to higher availability
ARTICLE IN PRESS S. Rodgher, E. Luiz Gaeta Espı´ndola / Ecotoxicology and Environmental Safety 71 (2008) 765–773
770
high food
medium food
low food Mean number of neonates per female
20 100
% survival
80 #
60 40 20
#
#
a
b a
a
16 c
e
12
0
8
j
gg
e d
o *
m
i
i
f
l
l n
h *
n
*
4
* 0
LC
C 0.1 0.17 0.4 0.86 free cadmium concentration (10-7M)
1.6
0.17 0.4 0.86 C 0.1 free cadmium concentration (10-7M)
LC
1.6
Fig. 1. Percent of survival (A) and mean number of neonates (B) of C. dubia in chronic toxicity tests for cadmium, when fed with various algal densities of P. subcapitata. LC (laboratory control) and C (control). Survival: # indicates statistically different from the control (C) (Fisher’s exact test, Po0.05). Number of neonates per female: * indicates statistically different from the control (C) (Dunnett’s test, Po0.05) and means with different letters are significantly different (Tukey’s test, Po0.05). Error bars denote standard deviation. Each bar for L.C corresponds to one laboratory control of three chronic toxicity tests performed with the three different algal densities.
high food
medium food
low food Mean number of neonates per female
100
% survival
80 60 40 20 0 LC C 0.6 1.4 2.7 free cadmium concentration to which algae were exposed (10-7 M)
20
a
a a
b b d
16 12
c
g, h e
* f
h j * g
i *
i
8 * 4 0 LC C 0.6 2.7 1.4 free cadmium concentration to which algae were exposed (10-7 M)
Fig. 2. Percent of survival (A) and mean number of neonates (B) of C. dubia when fed with various algal densities of P. subcapitata previously exposed to cadmium. LC (laboratory control) and C (control). Survival: # indicates statistically different from the control (C) (Fisher’s exact test, Po0.05). Number of neonates per female: * indicates statistically different from the control (C) (Dunnett’s test, Po0.05) and means with different letters are significantly different (Tukey’s test, Po0.05). Error bars denote standard deviation. Each bar for LC corresponds to one laboratory control of three chronic toxicity tests performed with the three different algal densities.
of food. Similarly, Smolders et al. (2005) also verified that cadmium toxicity to D. magna increased with increasing of food concentration. When the C. dubia females were exposed to free cadmium ion at a high algal density, they filtered less volume of medium and ingested more algal cells with metal bound to the cells, until the toxic effect of the bound metal inhibited their feeding, reproduction and survival. When
the organisms were exposed to 0.4, 0.86 and 1.60 107 M Cd2+ and fed with high algal density, metal analyses demonstrated that algal cells accumulated cadmium. Cadmium associated with algae may form an alternative source of metal contamination to zooplankton, besides the free metal ions from the water. For filter feeding organisms, the digestive system is a considerable contamination route, and a particle-bound toxic substance can
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high food
Filtration rate (µl/ind/h)
1000
c
medium food
f
low food
h
800 o
b e
400 200
l
j
600
a
e
k,l
g
n
i,j
g
k
i
m
0 C
0.1
0.17
0.86
0.4
Ingestion rate ( x 104 cells/indl/h)
S. Rodgher, E. Luiz Gaeta Espı´ndola / Ecotoxicology and Environmental Safety 71 (2008) 765–773
771
10 a 8 f
d
6 4
b
k
h d
m
j
h
n
f
2 c
g
i
l
o
0.17
0.4
0.86
1.60
e
0
1.60
C
free cadmium concentration (10-7 M)
0.1
free cadmium concentration (10-7 M)
high food
medium food
low food
Filtration rate (µl/ind/h)
1000 c 800
e
600
g i
d
b 400
h
f
200
f
d
a
h
0 C
0.6
1.4
2.7
Ingestion rate ( x 104 cells/ind/h)
Fig. 3. Filtration and ingestion rates of algal cells by C. dubia when exposed to cadmium and fed with various algal densities of P. subcapitata. Means with the different letters are significantly different (Tukey’s test, Po0.05). Error bars denote standard deviation.
d
10 a 8
g
i
6 b
4 2
e
c
j
h f
j
h
0 C
0.6
1.4
2.7
free cadmium concentration to which algae were exposed (10-7 M)
free cadmium concentration to which algae were exposed (10-7 M)
Fig. 4. Filtration and ingestion rates of algal cells by C. dubia when fed with various algal densities of P. subcapitata previously exposed to cadmium. Means with the different letters are significantly different (Tukey’s test, Po0.05). Error bars denote standard deviation.
Table 5 Total dissolved, free and accumulated metal at the end of chronic toxicity tests with C. dubia to cadmium
Dissolved total metal (107 M) Free metal ions (107 M) Metal in algal cells (m mol/mg)
Control
Water contaminated with metal (107 M Cd2+)
Food contaminated with metal (107 M Cd2+)
0
0.40
0.86
1.60
0.60
1.40
2.70
Medium High food food
Medium High food food
Medium High food food
Medium High food food
Medium High food food
Medium High food food
Medium High food food
0.02 (0.002) 0.02 (0.002) ND
0.21 (0.01) 0.19 (0.01) 0.004 (0.001)
0.58 (0.01) 0.52 (0.03) 0.009 (0.001)
1.11 (0.02) 1.10 (0.02) 0.012 (0.002)
0.02 (0.00) 0.02 (0.01) 0.001 (0.00)
0.03 (0.00) 0.03 (0.01) 0.002 (0.00)
0.05 (0.00) 0.04 (0.00) 0.005 (0.001)
0.02 (0.002) 0.02 (0.002) ND
0.11 (0.02) 0.10 (0.01) 0.002 (0.001)
0.30 (0.01) 0.26 (0.01) 0.005 (0.001)
0.71 (0.01) 0.63 (0.02) 0.020 (0.002)
0.06 (0.01) 0.05 (0.01) 0.001 (0.00)
0.11 (0.01) 0.09 (0.01) 0.001 (0.00)
0.20 (0.00) 0.18 (0.00) 0.003 (0.001)
Mean values (SD). ND: not detected.
readily be filtered from the water and released into their digestive tract (Fliedner, 1997). At low algal density, organisms filtered more water with metal and ingested fewer algal cells than at a high food level. Low acquisition of food by cladocerans would result in less energy for reproduction and for metal toxicity repair mechanisms (Rose et al., 2002).
The importance of algal cells as contamination agents for zooplankton was confirmed by the results obtained in toxicity tests with contaminated food. In this study, a decrease in fecundity of C. dubia was observed when these organisms were fed on P. subcapitata cells exposed to Cd2+. As a result of adding medium and high algal densities contaminated with Cd2+, concentrations between
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Table 6 Dissolved cadmium concentration (107 M) in the culture medium during experiments with C. dubia fed with various algal densities of P. subcapitata previously exposed to cadmium Day 2
Day 4
Day 8
Cadmium concentration to which algal cells were exposed (107 M)
104 cells/ mL
105 cells/ mL
106 cells/ mL
104 cells/ mL
105 cells/ mL
106 cells/ mL
104 cells/ mL
105 cells/ mL
106 cells/ mL
0.60
ND
ND
ND
0.17 (0.02) 0.18 (0.01)
ND
2.70
0.03 (0.00) 0.07 (0.00) 0.09 (0.01)
0.05
ND
0.08 (0.00) 0.12 (0.01) 0.21 (0.01)
ND
1.40
0.02 (0.00) 0.05 (0.00) 0.10 (0.01)
0.02 (0.00) 0.03 (0.00) 0.05 (0.00)
0.06 (0.01) 0.11 (0.01) 0.20 (0.00)
ND ND
ND
Mean values (SD). ND: not detected.
0.03 and 0.20 107 M Cd were released in the test medium. This may occur either via desorption or elimination of metal from algal cells (De Schamphelaere et al., 2004) and from elimination by the daphnids that have incorporated dietary metal in their tissues (Guan and Wang, 2004). Indeed, at such a low cadmium concentration, no toxic effects are expected. We observed effects on the fecundity and survival of C. dubia up to 0.20 107 M Cd2+. These observations suggest that toxic effects in cladocerans given contaminated food can be attributed to cadmium via dietary exposure. Our findings agree with those of De Schamphelaere et al. (2004), who found adverse effects on reproduction of D. magna after exposure to dietary Zn. Sofyan et al. (2006) also demonstrated that Cd in algal cells was available for trophic transfer and capable of producing deleterious effects to C. dubia. In our experiments involving feeding with Cd+2 contaminated algae, C. dubia ingested more algal cells at 106 cells/mL than at 105 and 104 cells/mL. According to Allen et al. (1995) and Taylor et al. (1998), algal cells contaminated with Cd can be collected and ingested normally by daphnids, but the contaminant interferes with digestion, resulting in cells passing through the gut without being digested. This could result in profound changes at the population levels, since the main parameters for population growth and survival are dependent on the energy input from feeding activity. The toxic effects on C. dubia were less pronounced when the zooplankton were exposed to food contaminated with 0.60 and 1.40 107 M Cd2+ than those obtained when the test organisms were exposed to 0.86 and 1.60 107 M Cd2+ in test solution at high algal density. This result implies that free cadmium ions are more bioavailable to cladocerans than metal in food, but a substantial amount of the metal could be obtained from their food. Consequently, it should be noted that metal dietary exposure may be present in treatment with contaminated water. Food quality is at least as important as quantity for the fecundity, population growth and survival of cladocerans
(Kilham et al., 1997). Nishikawa et al. (2003) found that cells of the green algae Chlamydomonas acidophila suffered a reduction in the level of polyphosphate after being exposed to 10 and 20 mM of Cd. Metal could have affected the nutritional value of the algal cells that were exposed to it. Such changes could make the algae poor in nutritional value and unviable as food for C. dubia. In the present study, the cadmium concentrations used to contaminate P. subcapitata were lower than those used by Nishikawa et al. (2003). It is unlikely that the observed reduction in fecundity of C. dubia might result from a dietary quality effect. In general, negative effects may be related to the direct toxic effects of metals on target cells or sensitive tissues responsible for egg production (vitellogenesis) (Hook and Fisher, 2002). Indirect effects of metal, due to a diminution in the process of nutrient assimilation (Munger et al., 1999) have negative repercussions on growth and fecundity of zooplankton. 5. Conclusion Our findings suggested that free cadmium ions were more bioavailable to zooplankton than metal in food. The addition of algae at high density in chronic toxicity tests in treatments with water contaminated with cadmium might have promoted binding sites for the metal and, consequently, generated additional routes for exposure of test organisms to metal. Food should be considered as an additional source of metal exposure to cladocerans. Ecotoxicological studies should consider the influence of varying algal densities on the expression of metal toxicity on daphinids. This would allow a better understanding of the possible relationships between toxicants and aquatic organisms. Acknowledgments We thank the National Research Council (CNPq: Process 140156/2002-0) and the Sa˜o Paulo State Research Support Foundation (FAPESP: Process 10417/2002) for
ARTICLE IN PRESS S. Rodgher, E. Luiz Gaeta Espı´ndola / Ecotoxicology and Environmental Safety 71 (2008) 765–773
financial support. We also express our gratitude to Alessandra Tonietto for assistance with the MINEQL+ program, to Dr. Liane Biehl Printes for valuable comments, and to anonymous reviewers for improving the manuscript. Funding sources: National Research Council (CNPq: Process 140156/2002-0) and the Sa˜o Paulo State Research Support Foundation (FAPESP: Process 10417/2002).
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