Effects of aqueous, dietary and combined exposures of cadmium to Ceriodaphnia dubia

Science of the Total Environment 385 (2007) 108 – 116 www.elsevier.com/locate/scitotenv

Effects of aqueous, dietary and combined exposures of cadmium to Ceriodaphnia dubia Agus Sofyan a,b,⁎, David J. Price a , Wesley J. Birge a,c b

a Department of Biology, University of Kentucky, Lexington, KY 40506, USA College of Mathematics and Natural Sciences, Lampung University, Bandar Lampung 35145, Indonesia c Graduate Center for Toxicology, University of Kentucky, Lexington, KY 40506, USA

Received 7 February 2006; received in revised form 10 May 2006; accepted 2 July 2006 Available online 4 August 2006

Abstract While effects of water-borne metal exposures on freshwater animals have been well documented, the effect of dietary metal exposure is less understood but is gaining importance. However, little attention has been given to the importance of combining both exposure pathways. In this study, we compared effects of aqueous (‘water only’), dietary (‘food only’) and combined (‘water + food’) exposures of cadmium to the freshwater cladocerans, Ceriodaphnia dubia. Major test endpoints included survival, feeding rate and reproduction. The C. dubia three-brood reproduction tests were conducted according to the United States Environmental Protection Agency (U.S. EPA) methods. Three exposure scenarios were used: aqueous, dietary, and combined aqueous and dietary exposures. Results showed that all three exposures affected survival, feeding rate and reproduction of C. dubia. Interestingly, combined exposure showed contribution effects of aqueous and dietary exposures. Lower cadmium concentrations were needed in combined exposure to produce effects as compared to higher concentrations in aqueous or dietary exposure alone. These results demonstrated the potential importance of dietary and combined exposures for consideration of cadmium regulation and risk assessment of metals. © 2006 Elsevier B.V. All rights reserved. Keywords: Metal; Cadmium; Ceriodaphnia dubia; Exposure pathways; Toxicity

1. Introduction Aquatic animals are exposed to pollutants from both dissolved and particle associated forms. Dissolved metals are accumulated directly from water, while particle-associated forms mostly are assimilated via the diet following ingestion and digestion (Wang and Fisher, 1999). While ingestion of contaminated food in terrestrial organisms is considered as the primary route of metal intoxication, ⁎ Corresponding author. Department of Biology University of Kentucky, Lexington, KY 405066, USA. Tel.: +1 859 257 5800; fax: +1 859 257 1717. E-mail address: [email protected] (A. Sofyan). 0048-9697/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.scitotenv.2006.07.003

branchial (i.e., respiratory) uptake of metals is generally considered the primary route of exposure in aquatic organisms, at least for acute toxicity. Therefore, the primary target organ for metal toxicity in aquatic animals (i.e., fish) is the gill system (Wood et al., 1999, 2002). For many decades, toxicity and biological effects of metals on aquatic organisms have been mostly assessed by aqueous exposure in the absence of metal-contaminated food (Roy and Hare, 1999; Munger et al., 1999). This type of exposure assumes that metal uptake from food is negligible (Luoma, 1995; Birge et al., 1998). Because of that, dermal absorption and oral uptake of metals in aquatic organisms (i.e., fish) are for the most part unknown. Nevertheless, there are some evidence that

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dietary metals can play a crucial role in aquatic systems (Allen et al., 1995; Taylor et al., 1998; Hook and Fisher, 2001; De Schamphelaere et al., 2004; Sofyan et al., 2006). Several investigations have reported that water is the important source of metals for filter feeders such as bivalves (Wang and Fisher, 1996), while food is the important source for predatory arthropods (Hare, 1992; Munger and Hare, 1997; Roy and Hare, 1999). However, other studies have found that toxicity effects of metals are not solely due to uptake from aqueous phase. For example, Taylor et al. (1998) observed toxic effects (i.e., feeding inhibition) of cadmium on Daphnia magna (a filter feeder) due almost entirely to cadmium bound to the food (i.e., algae). Furthermore, Barata et al. (2002) observed that, in D. magna, cadmium uptake was independent of the source and the effect was contributed from both sources. These discrepancies show that multiple exposure pathways are important for aquatic animals and additional research is needed to identify and clarify the main targets and the effects of each exposure type. Laboratory confirmations with aqueous, dietary, and combined exposures are a key in determining the importance of different combinations. This study was designed to distinguish effects of different types of exposures on Ceriodaphnia dubia. A trophic relationship between Pseudokirchniriella subcapitata and C. dubia was selected for dietary and combined exposures due to their importance in many toxicity tests and risk assessments (U.S. EPA, 2002). Furthermore, C. dubia has been known for its sensitivity to many types of pollutants including metals (U.S. EPA, 2002). The primary objective in this study was to examine and compare different types of cadmium exposures (aqueous, dietary and combined exposures) on C. dubia survival, feeding rate and reproduction. 2. Materials and methods 2.1. Test organisms Unicellular green algae, P. subcapitata, were obtained from the Culture Collection at the University of Texas at Austin, TX (Starr and Zeikus, 1993), whereas the cladocerans, C. dubia, were obtained from Aquatic Biosystems, Fort Collins, CO. Both species were cultured according to U.S. EPA methods (U.S. EPA, 2002). 2.2. C. dubia survival, reproduction and feeding rates C. dubia three-brood toxicity tests were performed according to U.S. EPA Method 1002.0 (U.S. EPA, 2002) for the three types of exposures. All tests were performed

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in American Society for Testing and Materials (ASTM) water with alkalinity of 60 mg CaCO3/l and hardness of 100 mg CaCO3/l (U.S. EPA, 2002). These tests were conducted in a controlled environmental room maintained at 25 ± 0.5 °C with a 16-h:8-h light/dark photoperiod. During the light period (16 h), the light intensity was maintained at a low intensity (i.e., 2.80 ± 0.60 μEinstein/m2/s) to reduce potential algal growth in the test medium. In the aqueous experiment, C. dubia was exposed to six different concentrations of cadmium (0, 5, 10, 20, 40 and 80 μg/l) in ASTM water for 7 days. In the dietary experiment, C. dubia was exposed to six different dietary concentrations of cadmium (0.02, 0.16, 0.33, 0.56, 3.11 and 5.60 μg/g dry wt). Concentration of cadmium that was assimilated daily by each C. dubia was calculated as the dietary dose (dd). This was calculated by multiplying number of algae consumed by C. dubia, cadmium concentration in the algae (μg/g dry wt) and algal cell dry wt (i.e., 6.29 ± 1.76 ng/cell, n = 10). Rates of algal consumption were determined by subtracting number of algae at the beginning with number algae at the end of the 24-h observation period. Microscopical cell counts with a hemocytometer were used for counting the algae (ASTM, 1989). After calculation, the dd for the six dietary cadmium concentrations were 0.004, 0.029, 0.056, 0.090, 0.130 and 0.179 ng/day per daphnid. In the combined exposure experiment, metal concentration in the food (P. subcapitata) was fixed and selected at concentration of 0.26 μg/g dry wt, which was between IC10 (0.17 μg/g dry wt) and IC25 (0.33 μg/g dry wt) values for C. dubia reproduction. This concentration should be high enough to exert some effects on C. dubia, but relatively low to avoid great reduction of neonate production or affect survival. The dietary concentration of 0.26 μg/g dry wt then was combined with each of five different cadmium concentrations in the ASTM water (0, 2, 5, 10 and 20 μg/l). Neonates less than 24 h of age were used to initiate tests. One neonate was placed in each chamber. Thirty replicates for a total of 30 organisms were employed for each test concentration. Test organisms (C. dubia) were individually housed in a 30 ml polystyrene container (Plastics, St. Paul, MN, USA). Each contained 20 ml of test solution and 1 × 106 P. subcapitata cells. Test solutions and feeding were renewed daily. Test endpoints included survival, feeding rate (number of P. subcapitata cells consumed daily) and reproduction (number of neonates per adult). These endpoints were measured each day for 7 days (i.e., through three broods). Tests were deemed acceptable if control survival was at or above 80% and the average reproductive output within control groups was ≥15 neonates (U.S. EPA, 2002).

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Table 1 Cadmium dosing and concentrations in the algae P. subcapitata after grown in different cadmium concentrations in the algal test media for 4 daysa Control

Cadmium concentrations in algal medium 5 μg/l

10 μg/l

20 μg/l

40 μg/l

80 μg/l

0.02 ± 0.02

0.16 ± 0.09

0.33 ± 0.09

0.56 ± 0.14

3.11 ± 2.47

5.60 ± 3.56

These cadmium-treated algae then used for food in the dietary experiment. a Values given as means of three replications ± standard deviation (S.D.).

2.3. Dosing and cadmium analysis of algae Cadmium dosing for the algal diet was conducted according to the U.S. EPA method 1003.0 (U.S. EPA, 2002). The exposures were carried out in Erlenmeyer

Fig. 1. Cadmium whole-body burdens from aqueous (A), dietary (B) and combined exposure (C). Exposures were 7 days. Results are given as means and are shown in solid lines. Asterisks denote statistically significant differences from control at p b 0.05.

flasks. Each flask contained 1 l of test solution, 1 × 106 cells of P. subcapitata and different concentrations of cadmium (5, 10, 20, 40 and 80 μg Cd/l). Following 4 days, samples were centrifuged at 1000×g at 4 °C and washed three times for 15 min each with moderately hard water containing 0.1 mM ethylenediamine tetraacetate (EDTA) to remove sorbed metals and then resuspended (ASTM, 1989; Munger et al., 1999). A 50 ml subsample was taken from each test concentration. These subsamples were filtered through 0.45 μm biological membranes (Pall Gelman Laboratory, Ann Arbor, MI, USA). Both the membranes and the algae were dried, weighed and then digested in 0.5% (volume/ volume) HNO3 (trace metal grade Fisher Scientific). This was followed by addition of H2O2 (Mallinckrodt Baker, Paris, KY, USA) after which the samples were reconstituted in 5 ml 0.5% (v/v) HNO3 (Hogstrand et al., 1996; Shaw et al., 1998). Digested samples and filtrates were analyzed for metals using graphite furnace atomic spectrophotometry (Varian Atomic Absorption Spectrometer, Model Spectra AA-20, Varian Instruments, Santa Clarita, CA, USA). The cadmium-treated algae were stored at 4 °C in regular culture water prior to and during the feeding experiments with C. dubia.

Fig. 2. Effects of waterborne cadmium to C. dubia survival, feeding rate and reproduction. Exposures were 7 days with daily renewal of test solution and feeding. Results are given as means and are shown in solid lines. Asterisks denote statistically significant differences from control at p b 0.05.

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2.4. Sample digestion and cadmium analysis in C. dubia Following 7 days of exposure, C. dubia were rinsed for 5 min in 0.1 mM EDTA solution to remove sorbed metals (Munger et al., 1999) and digested in 0.5% (v/v) HNO3 (trace metal grade Fisher Scientific, New Jersey, NJ). This was followed by addition of H2O2 (Mallinckrodt Baker, Paris, KY), after which the samples were reconstituted in 3 ml 0.5% (v/v) HNO3 (Hogstrand et al., 1996; Shaw et al., 1998). As before, digested samples were analyzed for metals using graphite furnace atomic absorption spectrophotometer (Varian AAS, Model Spectra AA-20, Santa Clarita, CA). 2.5. General water quality Water samples were monitored for temperature, pH, conductivity, hardness and alkalinity. Measurements of the first three parameters were conducted with a digital thermometer (CheckTemp, Hanna Instruments, Woonsocket, RI, USA), pH meter (pH Stick, Hanna Instruments Model 8414) and conductivity meter (Amber Science Model 604, Eugene, OR, USA). Water hardness and alkalinity were analyzed with the EDTA and the bromocresol green-methyl red titrimetric procedures, respectively (Clesceri et al., 1995). 2.6. Data analysis Concentrations resulting in 50% (IC50), 25% (IC25) and 10% (IC10) inhibition of reproduction and resulting in 50% mortality (LC50) were calculated using interpolation p-percent inhibition concentration (ICp) software version 2.0, 1993 (U.S. EPA Environmental Research Laboratory, Duluth, MN). Significant differences in survival between control and treatment groups were analyzed with Fisher's exact test (U.S. EPA, 2002). Significant differences between feeding rate and neonate production between control and treatment groups were tested with analysis of variance (ANOVA), followed by

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the Tukey honest significant difference (HSD) test using Statistica® software (StatSoft, Tulsa, OK, USA). 3. Results 3.1. Cadmium concentrations in algae and C. dubia Cadmium concentrations in the algae used for dietary exposures ranged from 0.02 to 5.60 μg/g dry wt (Table 1). While cadmium in the water increased its whole-body burdens in C. dubia dose dependently (Fig. 1A), dietary cadmium only increased whole-body burdens at the lower dietary exposure concentrations (i.e., ≤0.56 μg Cd/g algal dry wt). Cadmium body burden then decreased at the higher dietary metal exposures (Fig. 1B). In the combined exposures, cadmium whole-body burdens in C. dubia increased dose-dependently as the water concentrations increased (Fig. 1C). Cadmium whole-body burdens in C. dubia from combined exposures showed an uptake from both aqueous and dietary phases (Fig. 1A,B,C). 3.2. Effects of aqueous cadmium to C. dubia Cadmium in the water significantly ( p b 0.05) reduced C. dubia survival, feeding rate and reproduction (Fig. 2, Table 2). Reproduction was observed to be the most sensitive endpoint while feeding rate was the least sensitive endpoint. The significant inhibition of neonate production was observed to begin at the lowest concentration used (i.e., 5 μg/l), while significant reduction on survival and feeding rates began at 10 and 20 μg/l, respectively (Fig. 2, Table 2). The calculated 7-day IC50, IC25 and IC10 for reproduction were 7.24, 4.74 and 1.98 μg/l, while the 7-day LC50 was 10.67 μg/l (Table 3). 3.3. Effects of dietary cadmium to C. dubia In the dietary exposure, reproduction was also observed to be the most sensitive endpoint compared

Table 2 The lowest observed effect concentration (LOEC) of waterborne, dietary and combined exposures of cadmium on C. dubia survival (S), feeding rate (F) and reproduction (R) at p b 0.05 Dietary (μg/g dry wt)a

Waterborne (μg/l)

Combination (μg/l)b

S

F

R

S

F

R

S

F

R

10

20

5

3.11 [0.13]

3.11 [0.13]

0.56 [0.09]

10

5

2

Exposure times were 7 days with daily renewal of test medium and feeding. a Values in the brackets are daily doses (dd) of cadmium, which was calculated as a dose of metal given to a daphnid per day (ng/day per daphnid). b The LOEC for combined exposure was calculated only based on cadmium concentration in the water. The ‘food’ concentration in the combined exposure was fixed at 0.26 μg/g dry wt.

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Table 3 Comparison of 7-day IC10, IC25 and IC50 for reproduction, and 7-day LC50 for survival between waterborne, dietary and combined exposures of cadmium on C. dubia.a

7-day IC10 7-day IC25 7-day IC50 7-day LC50

Waterborne (μg/l)

Dietary (μg/g dry wt)b

Combination (μg/l)c

1.98 ± 0.24 4.78 ± 0.43 7.24 ± 0.19 10.67 ± 1.02

0.17 ± 0.02 [0.031 ± 0.003] 0.33 ± 0.02 [0.057 ± 0.003] 0.94 ± 0.06 [0.096 ± 0.001] 3.42 ± 0.24 [0.136 ± 0.005]

0.78 ± 0.21 2.32 ± 0.19 5.37 ± 0.32 9.13 ± 0.68

a

Bootstraps estimates mean ± S.D. (95% confidence intervals). Values in the brackets are daily doses (dd) of cadmium, which was calculated as a dose of metal given to a daphnid per day (ng/d per daphnid). c The IC and LC values were calculated based only on cadmium concentration in the water. The 'food' concentration in the combined exposure was fixed at 0.26 μg/g dry wt. b

to feeding rate and survival. Dietary cadmium significantly ( p b 0.05) reduced C. dubia reproduction at ≥ 0.56 μg/g dry wt (i.e., a dd of 0.09 ng/day), while significant reduction on feeding rate and survival was observed at 3.11 μg/g dry wt (a dd of 0.13 ng/day) or above (Fig. 3, Table 2). The calculated 7-day IC50, IC25 and IC10 for reproduction were 0.94, 0.33 and 0.17 μg/g dry wt, while the 7-day LC50 was 3.42 μg/g dry wt (Table 3). 3.4. Effects of combined exposures of cadmium to C. dubia In the combined exposure, reproduction also was the most sensitive endpoint. A concentration of 2 μg/l + 0.26 μg/g dry wt in food significantly reduced C. dubia reproduction, whereas higher combined concentrations were needed to significantly affect survival and feeding rate (Fig. 4, Table 2). Compared to individual aqueous and dietary exposures, the effects of combined exposures were due to both avenues of exposure (Fig. 4, Table 2). For example, in the aqueous and dietary

Fig. 3. Effects of dietary cadmium to C. dubia survival, feeding rate and reproduction. Exposures were 7 days with daily renewal of test solution and feeding. Results are given as means and are shown in solid lines. Asterisks denote statistically significant differences from control at p b 0.05.

exposures alone, the cadmium concentrations needed to significantly ( p b 0.05) affect reproduction were 5 μg/ l and 0.56 μg/g dry wt, respectively. In comparison, a cadmium concentration of 2 μg/l + 0.26 μg/g dry wt was needed to significantly reduce C. dubia reproduction (Fig. 4, Table 2). Contribution effect of combined exposure also was observed on survival and feeding rate. The water and dietary concentrations affecting survival and feeding rates were ≥ 10 μg/l and 3.11 μg/g dry wt, respectively, while a combination of only ≤10 μg/l + 0.26 μg/g dry wt was needed to significantly reduce both C. dubia survival and feeding rate (Fig. 4, Table 2). Furthermore, the deleterious effects of combined exposures were indicated by the lower concentration of dissolved cadmium required to produce toxicity to C. dubia when combined with cadmium-contaminated food, as compared to water only exposures (Table 3).

Fig. 4. Effects of combined exposure of cadmium to C. dubia survival, feeding rate and reproduction. Cadmium in the algae (dietary) was fixed at 0.26 μg/g dry wt. Exposures were 7 days with daily renewal of test solution and feeding. Results are given as means and are shown in solid lines. Asterisks denote statistically significant differences from control at p b 0.05. Statistics was calculated based only on cadmium concentration in the water with the assumption that cadmium from dietary (0.26 μg/g dry wt) did not change significantly to all the experimental groups.

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4. Discussion Toxicity of aqueous and dietary metal exposures has been studied more than combined exposures. Even though combined exposure is the most likely pathway in natural systems, very few studies have explored this alternative. This study demonstrates that combined exposures were more toxic to C. dubia than either aqueous or dietary exposures. Cadmium whole-body burdens in C. dubia from different route of exposures indicated different pattern of uptake (Fig. 1). Furthermore, compared to aqueous and dietary exposures, the combined exposures showed a contribution of cadmium uptake from both the aqueous and dietary phases. It was shown that lower concentrations of cadmium in the water + food combined exposures significantly increased body burden in C. dubia as compared to cadmium in the water-only exposures (Fig. 1A,C). These results supported Barata et al. (2002) who reported independent uptake of cadmium from water plus food by another cladoceran (D. magna). These uptake patterns and effects could be indicative of two separate kinetic compartments for assimilation, one driven by aqueous and the other driven by dietary exposure (Baudin and Fritsch, 1989; Miller et al., 1993). Effects of cadmium on survival may be related to its toxicity to the main targets, which were the calcium channels located at the apical membrane and the CaATPase located at the basolateral membrane of absorptive cells (Playle and Dixon, 1993; Bondgaard and Bjerregaard, 2005). Aqueous cadmium mainly disturbed calcium uptake on the gills (Playle and Dixon, 1993; Bondgaard and Bjerregaard, 2005), while dietary cadmium mainly disturbed the calcium uptake in the middiverticula of the gut (Taylor et al., 1998; Munger et al., 1999). Lower concentration of cadmium in the water + food in combined exposures compared to aqueous and dietary exposure alone (Fig. 4, Table 2) could indicate different effect from the two exposure pathways. Smaller amounts of cadmium in the water or in the food that did not affect survival when given alone were observed to be toxic to C. dubia when the two exposure pathways were combined (Fig. 4, Tables 2 and 3). Feeding reduction observed in this study might be caused by several factors. For example, in the aqueous exposure, feeding reduction could be caused by systemic (i.e., internal effects) or behavioral responses (i.e., food avoidance or reduced filtration rates). Several studies have reported reduction of D. magna feeding after exposure to aqueous cadmium (Allen et al., 1995; Baillieul and Blust, 1999). Both of these studies indicated that reduced D. magna feeding was merely considered as behavioral

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responses rather than systemic or physiological effects. However, results of this study showed that high concentrations were required (i.e., ≥20 μg/l) to significantly reduce C. dubia feeding (Table 2). This concentration was higher than the concentration needed to affect survival (i.e., 10 μg/l). This could indicate that effects on physiology also played an important role in reducing food consumption. In the dietary exposure, feeding reduction could be caused by avoidance or by toxic impairment of digestion. Taylor et al. (1998) reported that the primary cause of feeding reduction in D. magna, after being exposed to dietary cadmium, was an impairment of its digestive system. In the combined exposures, both systemic (e.g., impairment of digestion) and behavioral (i.e., food avoidance) factors might have worked together to reduce C. dubia feeding rates as noted above. C. dubia feeding was significantly reduced by low concentrations of cadmium in water + food (i.e., 5 μg/l + 0.26 μg/g dry wt). This cadmium combination was lower than the cadmium concentration in either aqueous or dietary exposure (i.e., 20 μg/l and 3.11 μg/g dry wt, respectively). No matter the exposure pathways and the mechanisms, the overall effect was a reduction of food intake which could have some consequences in C. dubia populations, such as reduced reproduction or vulnerability to predation (Allen et al., 1995; Taylor et al., 1998; Baillieul and Blust, 1999). The present study showed that effects on feeding occurred only in cadmium treatment levels which also caused significant effects on survival and reproduction (Table 2). Reduced C. dubia reproduction could be caused by systemic (i.e., biochemical, physiological) or behavioral responses (i.e., feeding inhibition). For the aqueous exposure, cadmium taken up from the gills could go to C. dubia reproductive organs such as the fat cells and oocytes. The fat cells are known to be the site for vitellogenin (precursor for the yolk protein) synthesis while oocytes are the sites for yolk protein (lipovitellin) synthesis (Bodar et al., 1990). A disturbance caused by cadmium to each or both sites could reduce the quantity or quality of the eggs. Ghosh and Thomas (1995) reported a binding of cadmium to vitellogenin and a transfer of this cadmium-vitellogenin complex to ovaries in the red drum (Sciaenops ocellatus) and the Atlantic croaker (Micropogonias undulatus). Furthermore, Hwang et al. (2000) reported reduced vitellogenin production in rainbow trout (Oncohrynchus mykiss) after aluminum and cadmium exposures. In addition to the effects on reproductive organs, cadmium also could affect neonate production indirectly through feeding reduction. Several studies have reported reduction of growth and reproduction of D. magna after exposure to aqueous cadmium (Taylor et al., 1998; Baillieul and

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Blust, 1999). Dietary exposure also could impair reproduction through direct effects on the reproductive system or indirectly through feeding reduction. Lee and Noone (1995) observed reduction on lipovitellin accumulation in the blue crab (Callinectes sapidus) after dietary cadmium exposure. Also, Hook and Fisher (2001) observed reduced egg protein production in marine copepods after dietary silver exposure. Our previous report showed that while reduced neonate production in C. dubia strongly correlated (r2 = 0.95) with reduced feeding, it did not correlate well (r2 = 0.05) with cadmium whole-body burden (Sofyan et al., 2006). This could indicate that the accumulation at specific sites (i.e., fat cells and oocytes) was more important than whole-body burden in affecting reproduction, as suggested by De Schamphelaere et al. (2004). For the combined exposures, both systemic and behavioral responses could contribute to reduce C. dubia reproduction. Both responses (i.e., systemic and behavioral) could be caused by contributions of cadmium in water and in food administered together. The values in Table 3 indicate that, when combined with cadmium-contaminated food, lower amounts of dissolved cadmium in water were required to affect reproduction, while higher aqueous cadmium exposures alone were required to produce a similar effect. The findings that combined exposures produced deleterious effects on survival, feeding rate and reproduction could have relevance on exposure scenarios in natural aquatic systems, as well as consequences on cadmium water quality criteria (WQC). Metal can be found in many different forms or species in natural aquatic systems. According to Mota and dos Santos (1995), only small concentrations of metals were present as free hydrated ions because of the formation of stable complexes with a large variety of inorganic and organic ligands, including algae. Therefore, it is very likely that aquatic organisms are exposed to both free ions and particle-associated metal (i.e., food) and consequently are affected by both pathways of exposure. The present study has shown that all exposure pathways caused significant toxicity to C. dubia, but the effect from water + food in combined exposures was most severe. Some studies reported that in several contaminated sections of U.S. natural waters, concentration of cadmium were higher than 10 μg/l (Kopp and Kroner, 1967; Birge et al., 1981). Because of that, cladocerans (i.e., C. dubia) living in such contaminated natural waters may be in a dangerous condition. This study indicated that the 7-day IC50 for C. dubia reproduction was only 7.24 μg/l. This value was even lower (i.e., 5.37 μg/l) when aqueous exposure was linked with cadmium-contaminated food (algae). The 7-day IC50

from dietary exposure was 0.94 μg/g algal dry wt which was produced when algae were exposed to cadmium at concentrations less than 40 μg/l (Sofyan et al., 2006). Fortunately, these values were higher than the current water quality criteria for cadmium (i.e., 2.00 μg/l for acute and 0.25 μg/l for chronic effects) at a water hardness of 100 mg/l (U.S. EPA, 2004). However, U.S. EPA also reported that the cadmium Genus Mean Chronic Value (GMCV) for C. dubia was 27.17 μg/l (U.S. EPA, 2001). This value is considered to be high and may not protect C. dubia or other species from the chronic effects of cadmium, particularly when both avenues of exposure are active. However, some care should be taken when comparing laboratory results and natural water systems. The latter are far more complex and contain many more organic and inorganic substances that can reduce metal bioavailabity and toxicity to aquatic biota (Mota and dos Santos, 1995; Bianchini and Wood, 2002). Because few studies have been completed on combined exposures, more investigations are needed to focus on risk assessment or WQC development. Furthermore, due to the difficulty in distinguishing and characterizing which pathway was more toxic, it is difficult to indicate which type of exposure was more important. Nevertheless, this study observed toxicity from all three exposures. This could indicate that dietary exposure could be as important as aqueous exposure. These results also suggested that responses in specific target organs were independent of the exposure pathway, and both aqueous and dietary cadmium contributed to the overall toxic effects on C. dubia. 5. Conclusions The present study illustrates that aqueous, dietary and combined exposures of cadmium are toxic to freshwater animals. More specifically, the results indicate that both aqueous and dietary pathways are toxic to C. dubia survival, feeding rate and reproduction. However, this study could not indicate whether cadmium from water or from the food is the most significant cause for toxicity in C. dubia. Nevertheless, these results show that dietary and combined exposures are toxicologically relevant and can be as important as water exposure. Furthermore, contribution effects of combined exposures indicate that the responses may be independent of the route and both routes can be similarly important. This study may be reliable as to predict metal dynamics in the field, including the uptake and toxicity of different types of metal exposure. These results underline that dietary and combined exposures have some potential important consequences for the interpretation in a regulatory assessment for cadmium.

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