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Daphnia magna Feeding Behavior after Exposure to Tetradifon and Recovery from Intoxication
Ecotoxicology and Environmental Safety 44, 40}46 (1999) Environmental Research, Section B Article ID eesa.1999.1817, available online at http://www.idealibrary.com on
Daphnia magna Feeding Behavior after Exposure to Tetradifon and Recovery from Intoxication M. J. Villarroel, M. D. Ferrando, E. Sancho, and E. Andreu Laboratory for Ecotoxicology, Department of Animal Biology (Animal Physiology), Faculty of Biological Sciences, University of Valencia, Dr. Moliner 50, E-46100 Burjasot, Valencia, Spain Received July 15, 1998
The "ltration of food particles from water by freshwater species of zooplankton, the injestion of "ltered particles, and the assimilation or absorption of ingested material from the digestive tract are subjects that have been researched extensively from an ecological point of view (Hirayama and Ogawa, 1972; Schlosser and Anger, 1982). Several researchers (Flickinger et al., 1982; Day and Kaushik, 1987; Fernandez}Casalderrey et al., 1994) have suggested that the feeding behavior of zooplankton is a physiological function. This could be considered an important factor in studies dealing with the toxicity of pollutants to aquatic organisms. Thus, the impairment of biochemical and physiological functions of an organism may be taken as the "rst indication of the e!ect of an environmental perturbation. This impairment could result in measurable changes in the behavior of the organism (Sprague, 1976). These changes may be used as rapid and sensitive indicators of toxic stress and may help to explain other observed changes in the survival, growth, and reproduction of nontarget organisms. Rates of "ltration of particles by zooplankton have been observed to decrease following exposure to various chemicals under laboratory conditions (Day and Kaushik, 1987; FernaH ndez}Casalderrey et al., 1992). However, no data are available which re#ect the in#uence of tetradifon during exposure and recovery over several generations of the investigated animals. The objective of this study was to assess the e!ects of low levels of the acaricide tetradifon (4-chlorophenyl 2,4,5-trichlorophenyl sulfone) on the feeding behavior of three generations of the cladocera D. magna and their recovery from intoxication, using a short-term toxicity test as sublethal test criterion.
The feeding behavior of the cladocera Daphnia magna subjected to a short-term exposure to the acaricide tetradifon (4-chlorophenyl 2,4,5-trichlorophenyl sulfone) was studied. The experiments were performed using the unicellular algae Nannochloris oculata at a density of 5ⴛ105 cells/ml as food for the organisms. In a 5rst experiment, three generations (F0, F1, and F3) of the daphnids were exposed to sublethal levels of tetradifon (0.1, 0.18, 0.22, and 0.44 mg/l) and the e4ect of the toxicant on 5ltration and ingestion rates was determined. Rates of 5ltration and ingestion of D. magna declined in the three generations studied with increasing toxicant concentrations; however, toxicant e4ect was greater in daphnids from generations F1 and F3 than in those from the parental generation F0. A second experiment was conducted in order to evaluate whether animals of a 5rst (1) or third (F3) generation coming from parental daphnids (F0) previously exposed to those pesticide concentrations exhibited any alteration in feeding behavior when transferred to clean water (recovery period). The results indicated that the feeding rates of D. magna generations F1 and F3 were still a4ected during the recovery period but to a less degree. The e4ective tetradifon concentrations D. magna at which feeding rates were reduced to 50% that of controls (EC50) were also calculated. 1999 Academic Press Key Words: tetradifon; feeding; recovery; multigeneration; Daphnia magna.
INTRODUCTION
Acute toxicity tests using Daphnia magna require an exposure time of 24 h (or 48 h) and use mortality (immobilization) as an endpoint. The use of alternative endpoints in invertebrate bioassays that would allow shorter-term tests has been little explored (Ferrando et al., 1993; Janssen et al., 1994; Bitton et al., 1996). Behavioral changes have been used successfully as rapid and sensitive indicators of toxic stress in "sh (Little and Finger, 1990) but little has been done to develop behavioral indices with zooplankton (Ferrando and Andreu, 1993; Villarroel et al., 1998).
MATERIAL AND METHODS
Test Species D. magna were obtained from continuous cultures maintained in this laboratory in 6-L aquaria with dechlorinated tap water (total hardness 180}200 mg/L as CaCO ; pH 40
0147-6513/99 $30.00 Copyright 1999 by Academic Press All rights of reproduction in any form reserved.
D. magna FEEDING BEHAVIOR
41
7.9#0.2; alkalinity 4.1 mmol/L). The medium was renewed two times weekly. Culture densities were kept below 50 animals/L, and the daphnids were fed daily ad libitum with the unicellular algae Nannochloris oculata. The aquaria were placed in a temperature-controlled room (22$13C) with a 12 : 12-hr light}dark cycle at 1000 lux light intensity at water level. Daphnids, less than 24 h old (neonates), were used for the experiments. The microalgae N. oculata, which was used as food in all experiments, was cultured in BBM medium (Bischo! and Bold, 1983). The log phase algae were harvested, centrifuged, washed, and resuspended in water. Concentration of algae was measured by counting with a hemocytometer. The suspension was then diluted to produce the desired concentration, Nannochloris density chosen for all the experiments was 5;10 cells/ml (Ferrand et al., 1993).
where Co and Ct are initial and "nal food concentrations (cell/ll), t is time (duration of the experiment in hours), and n is the number of daphnids in volume < (ll). A is a correction factor for changes in the control with "nal concentrations C t after time t. The expression Co ) Ct represents the geometric mean of food concentration during time t.
Test Chemical
Multigeneration Experiments
Tetradifon (4-chlorophenyl 2,4,5-trichlorophenyl sulfone) used on this study was 99% pure as assayed by AFRASA Co. (Spain). Stock solutions were prepared by dissolving the toxicant in acetone immediately prior to each experiment. Since acetone was required as a carrier, a control composed of acetone only (0.25 ml/L) was also included.
This study was conducted for three generations of the daphnids, and it was divided in two di!erent expressions. In the "rst experiment (Fig. 1), the parental generation (F0) and generations F1 and F3 were continuously exposed to the selected tetradifon concentrations, plus the blank and acetone control. Feeding rates were calculated as indicated before. A second experiment was performed in order to evaluate if there was a toxicant transfer from the parental individuals to their progeny. This test was also conducted for three generations of the daphnids, but only the parental generation (F0) was continuously exposed to tetradifon. The other generations (F1 and F3) were chosen from Fo-exposed animals and transferred to clean water (with food but without toxicant); it was called recovery phase (Fig. 2).
Feeding Experiments Based on the results from previous acute toxicity tests (LC 24 h"8.9 mg/L; Ferrando et al., 1996), four tet radifon concentrations were chosen for the feeding study. Daphnids were exposed to 0.10, 0.18, 0.22, and 0.44 mg/L of tetradifon. Each treatment consisted of "ve replicates. Filtration and ingestion rates were used as measures of feeding behavior. Feeding experiments with D. magna were carried out in 60-ml glass beakers containing 50 ml of the medium and 10 daphnids ((24 h old). The vials were placed in a temperature-controlled room at 223C under darkness and static conditions (Ferrando et al., 1993). Test organisms were exposed to the test solution containing food for 5 h, after which the "nal food concentration was measured using a hemocytometer. Filtration rate (F) is de"ned 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 speci"c interval time. For the calculation of average "ltration (ll ) ind\ ) h\) and ingestion rate (cells ) ind\ ) h\), the equations from Gauld (1951) were used,
FIG. 1. Test design for multigeneration feeding study during exposure to tetradifon (Experiment 1).
Statistical Analysis Data for feeding experiments were compared by analysis of variance (ANOVA), in case of signi"cant di!erences (P(0.05); the mean values for exposed populations were compared to those of the control by Duncan's multiple range test. The EC (the concentration of the toxicant that reduces feeding rate to 50%) were calculated using linear regression analysis.
< (ln Co!ln Ct) F" !A n t ln Co!ln C t A" t I"F(Co Ct ,
FIG. 2. Test design for multigeneration feeding study during the recovery period (Experiment 2).
42
VILLARROEL ET AL.
RESULTS
The e!ect of tetradifon on "ltration and ingestion rates of D. magna parental generation (F0) is provided in Fig. 3 (Villarroel et al., 1998). Filtration rates were signi"cantly reduced from 512 ll/animal/h (control) to 211, 166, 193, and 116 ll/animal/h at 0.10, 0.18, 0.22, and 0.44 mg/l of pesticide, respectively. Filtration rate in acetone controls was 514 lL/animal/h ($54.7). Ingestion rates were also reduced at those toxicant concentrations from 10;10 cells/animal/h in the control to 62;10, 54;10, 69;10, and 50;10 cells/animal/h. However, ingestion rate in those animals exposed to the solvent (acetone) was 102.4
FIG. 4. Rates of "ltration and ingestion of N. oculata by D. magna generation (F1) after exposure to tetradifon. *P(0.05.
FIG. 3. Rates of "ltration and ingestion of N. oculata by D. magna parental generation (F0) after exposure to tetradifon. *P(0.05.
($5.2);10 cells/animal/h. The highest concentration of tetradifon used (0.44 mg/L) produced a reduction of 77 and 54% in "ltration and ingestion rates, respectively, compared with control values. The e!ect of tetradifon on the feeding rates of generations F1 and F3 (Figs. 4 and 5) was greater than those from generation F0. Tetradifon concentration of 0.44 mg/L reduced "ltration rates 78% in both F1 and F3 generations. On the other hand, ingestion rates were reduced 82% in F1 and F3 generations exposed to 0.44 mg/L of tetradifon. Statistically signi"cant di!erences between "ltration rates in controls (F1"415 ll/ind/h, F3"419 ll/ind/h) and acetone controls (F1"392.5 ll/ind/h, F3"411.25 ll/ind/h)
D. magna FEEDING BEHAVIOR
43
were not found. Ingestion rates in daphnids exposed to the solvent (F1"160.67;10 cells/animal/h, F3"143;10 cells/animal/h were no di!erent (P'0.05) from those of the blank controls (F1"172;10cells/animal/h, F3"152; 10cells/animal/h). In a second experiment, F1 and F3 generations (neonates) from parentals (F0) preexposed to the same tetradifon concentrations were transferred to clean water (recovery period) and the feeding rates were also evaluated. The results from these experiments are presented in Figs. 6 and 7. As can be seen in Fig. 6 "ltration rates of F1 generation animals from parentals exposed to 0.10 and 0.18 mg/L were no di!erent from control values, while those from parentals exposed to
FIG. 6. Rates of "ltration and ingestion of N. oculata by D. magna generation (F1) after the recovery period. *P(0.05.
FIG. 5. Rates of "ltration and ingestion of N. oculata by D. magna generation (F3) after exposure to tetradifon. *P(0.05.
0.22 and 0.44 mg/L were statistically di!erent. A reduction of 67% was observed in the "ltration rate of the animals (F1) from parentals (F0) exposed to 0.44 mg/L, even during the recovery period. So, it is assumed that there was a small recuperation in the "ltration rates of F1 generation daphnids but animals from parentals exposed to the highest pesticide concentrations were still a!ected. However, the e!ect was even greater on the ingestion rates (Fig. 6). F1 generation daphnids exhibited a reduction in this parameter during the recovery period, especially those daphnids
44
VILLARROEL ET AL.
coming from parentals exposed to 0.44 mg/L tetradifon (82% reduction). Figure 7 presents the e!ect of a recovery period in the feeding rates of F3 generation daphnids. It seems that F3 generation was more a!ected by the toxicant which was transferred from their mothers (F0). The parental generation (F0) previously exposed to tetradifon had the "rst progeny (F1) the 7th day of pesticide treatment, while the third generation of daphnids (F3) was obtained the 11th day, so F3 generation animals were coming from mothers who were exposed to the pesticide for 4 more days. So, when F3
neonates were transferred to clean water (recovery period), they did not recuperate completely and their feeding rates were still reduced. Statistically di!erences were found in both "ltration and ingestion rates of F3 generation daphnids from parentals exposed to all the pesticide concentrations tested. Feeding rates of animals from parentals exposed to the highest concentration (0.44 mg/L) were reduced a 65 and 85% for "ltration and ingestion rates, respectively. From these results it could be assumed that it is possible the pesticide tetradifon was bioaccumulated and transferred from the mothers (F0) to the neonates (F1 and F3), and then the highest toxicant e!ect was found in F1}F3 generations exposed to the pesticide compared with F0 parentals, but there was also a strong e!ect in nonexposed F1}F3 neonates (recovery period). DISCUSSION
FIG. 7. Rates of "ltration and ingestion of N. oculata by D. magna generation (F3) after the recovery period. *P(0.05.
The e!ects of xenobiotics on feeding rates of some species of cladocerans have been investigated by several workers. Kersting and Van der Honing (1981) pointed out that the "ltering rates of daphnids might be used as very sensitive toxicity parameters. Day and Kaushik (1987) found that the "ltration rate of the cladocera Daphnia galeata mendotae and Ceriodaphnia lacustris and of the calanoid Diaptomus oregonensis was decreased by sublethal concentrations (0.01}0.1 lg/L) of fenvalerate. It induced the adhesion of particulate material (algae) and detritus) onto the various setate appendages of D. galeata mendotae. The degree of adhesion was greater as the concentration of fenvalerate increased. Such adhesion impeded daphnid mobility and animals had di$culty swimming above the bottom of the beaker. This resulted in a decreased feeding rate. The "ltration rate of D. magna was reduced signi"cantly after a 5-h exposure to 0.062, 0.077, and 0.15 ng/L methylparathion (FernaH ndez}Casalderrey et al., 1993). Feeding rates were also reduced in D. magna after exposure to endosulfan (0.31, 0.41, and 0.62 mg/L) and diazinon (0.45, 060, and 0.90 lg/L) (FernaH ndez}Casalderrey et al., 1994). The "ltration of food by "lter-feeding zooplankton requires movement of appendages and coordination of the nervous system. Therefore, those toxicants (as the acaricides) that a!ect nervous system (Ware, 1983) will cause loss of coordination and/or paralysis and will reduce rates of "ltration. A useful quantitative parameter analogous to the LC derived from mortality data in acute toxicity tests is the EC . This is the median e!ective concentration of the toxicant at which the value of a given parameter is reduced to 50% of that in controls. The EC values for "ltration and ingestion rates were derived for the toxicant tested using the regression equations given in Table 1. These regression equations as well as their correlation coe$cients
45
D. magna FEEDING BEHAVIOR
TABLE 1 Regression Equations and Correlation Coe7cients (r2) Describing the Relation between Tetradifon Concentrations (x) and Filtration and Ingestion Rates (y) of D. magna Exposed to the Pesticide (Generations F0, F1, F3) Generation
Parameter
Regression equations
Correlation coe$cient (r)
F0
Filtration Ingestion Filtration Ingestion Filtration Ingestion
y"19.3#160x y"17.8#99x y"24#127x y"25.3#134x y"25.6#150x y"21.5#162x
0.86 0.90 0.93 0.90 0.83 0.86
F1 F3
Note. EC
EC
0.19 0.32 0.20 0.18 0.16 0.17
e!ect criterion for rapid toxicity screening of xenobiotics. The results from the &&feeding'' toxicity test, as described in this study, also indicated that behavioral indices can re#ect the potential for subsequent mortality (acute mortality tests) at lowest toxicant concentrations. On the other hand, the e!ect of tetradifon on feeding rates increased over the di!erent generations studied. Thus F3 generation was more sensitive to the pesticide than the parental generation, F0, and a transfer of the bioaccumulated pesticide from the mothers to the neonates could take place. Recovery experiments carried out on F1 and F3 generations determined the need for longer periods in clean water to eliminate the toxicant and recover the a!ected feeding rates.
(mg/L) was also calculated.
ACKNOWLEDGMENT
(r) indicate the relationship between the pesticide concentrations used and the rates of "ltration and ingestion obtained in the present study. The results indicate that the observed e!ect in the feeding behavior of D. magna will be greater when the amount of pesticide used in the medium is increased. The EC for "ltration and ingestion rates (Table 1) on D. magna parental generation (F0) indicated values of 0.19 and 0.16 and 0.32 mg/L for both rates while the EC s for the same parameters in generation F3 were 0.16 and 0.17 mg/L. This means that animals from the third generation (F3) were more sensitive than the parentals (F0) and less pesticide in the water will reduce their feeding rates 50%. The EC values calculated in this study were much lower than the 24-h LC (Ferrando et al., 1996) for this species (8.9 mg/L). Thus, toxicant levels below the 24-h LC value are su$cient to reduce feeding rates to 50 within 5 h. EC values for the feeding rates of animals during the recovery experiment were not calculated, those animals (generations F1 and F3) were not exposed to the pesticide. There are no data in the literature about the e!ect of pesticides on the feeding rates of a species through several generations. Multigeneration experiments carried out in D. magna demonstrated the e!ects of di!erent pollutants such as nickel (MuK nzinger, 1990), chromium (MuK nzinger and Monicelli, 1992), and CaCl (Bervoets et al., 1996)in the survival, reproduction and growth of di!erent generations. It is thought that the evaluation of the feeding rates in zooplankton species could be a useful parameter to determine the e!ect of xenobiotics in a short period of time through several generations of the same species.
CONCLUSIONS
The results indicate that the feeding behavior of the freshwater cladocera D. magna might be an interesting new
This work was supported by a grant (AMB96:11188-CO1) from the Comision Interministerial de Ciencia y TecnologmH a (I#D) (CICYT) del Ministerio de EducacioH n y Ciencia, Spain.
REFERENCES Bervoets, L., Baillieul, M., Blust, R., and Verheyen, R. (1996). Evaluation of e%uent toxicity and ambient toxicity in a polluted lowland river. Environ. Pollut. 91, 333}341. Bischo!, H. W., and Bold, H. C.(1983). Phycological studies. IV. Some algae from enchanted rock and related algae species. ;niv. ¹exas Publ. 6318. Bitton, G., Rhodes, K., and Koopman, B. (1996). Ceriofast: An acute toxicity test based on Ceriodaphnia dubia feeding behavior. Environ. ¹oxicol. Chem. 15, 123}125. Day, K., and Kaushik, N. K. (1987). An assessment of the chronic toxicity of synthetic pyrethroid, fenvalerate, to Daphnia galeata mendotae, using life tables. Arch. Environ. Contam. ¹oxicol. 16, 423}432. Fernandez-Casalderrey, A., Ferrando, M. D., and Andreu, E. (1992). Filtration and ingestion rates of Brachionus calyci-orus after exposure to endosulfan and diazinon. Comp. Biochem. Physiol. C 103(2), 357}361. Fernandez-Casalderrey, A., Ferrando, M. D. and Andreu, E. (1993). E!ect of the insecticide methylparathion on "ltration and ingestion rates of Brachionus calyci-orus and Daphnia magna. Sci. ¹otal. Environ (Suppl.), 867}876. Fernandez}Casalderrey. A. Ferrando. M. D., and Andreu, E. (1994). E!ect of sublethal concentrations of pesticides on the feeding behavior of Daphnia magna. Ecotoxicol. Environ. Saf. 27, 82}89. Ferrando, M. D., and Andreu, E. (1993). Feeding behavior as an index of copper stress in Daphnia magna and Brachionus calyci-orus. Comp. Biochem. Physiol. 106(2), 327}331. Ferrando, M. D., Janssen, C., Andreu, E., and Persoone, G. (1993). Ecotoxicological studies with the freshwater rotifer Brachionus calyci-orus. III The e!ects of chemicals on the feeding behavior. Ecotoxicol. Environ. Saf. 26, 1}9. Ferrando, M. D., Sancho, E., and Andreu, E. (1996). Accumulation of tetradifon in an algae (Nannochloris oculata) and the cladoceran, Daphnia magna. Bull. Environ. Contam. ¹oxicol. 57, 139}145. Flickinger, A. L., Bruins, R. J. F., Winner, R. W., and Skillings, J. H. (1982). Filtration and phototactic behavior as indices of chronic copper stress Daphnia magna. Arch. Environ. Contam. ¹oxicol. 11, 457}463.
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Gauld, T. (1951). The grazing rate of marine copepods. J. Mar. Biol. Assoc. 26, 695}706. Hirayama, K., and Ogawa, S. (1972). Fundamental studies on physiology of rotifer for its mass culture. I. Filter feeding of roti"er. Bull. Japn. Soc. Sci. Fish. 38, 1207}1214. Janssen, C. R., Ferrando, M. D., and Persoone, G. (1994). Ecotoxicological studies with the freshwater roti"er Brachionus calyci-orus. IV. Rotier behavior as a sensitive and rapid sublethal test criterion. Ecotoxicol. Environ. Saf. 28, 244}255. Kersting, K., and Van der Honing, H. (1981). E!ect of the herbicide dichlobenil on the feeding and "ltering rate of Daphnia magna.
MuK nzinger, A. (1990). E!ects of nikel on Daphnia magna during chronic exposure and alterations in the toxicity to generations pre-exposed to nickel. =at. Res. 24, 845}852. MuK nzinger, A., and Monicelli, F. (1992). Heavy metal co-tolerance in a chromium tolerant strain of Dophina magna. Aquat. ¹oxicol. 23, 203}216. Schlosser, K. J., and Anger, K. (1982). The signi"cance of some methodological e!ects of "ltration and ingestion rates of the rotifer Brachionus plicatilis. Helgol Meeresun tersuch 35, 215}225. Sprague, J. B. (1976). Current status of sublethal tests of pollutants on aquatic organisms. J. Fish. Res. Board Can. 33, 1988}1992. Villarroel, M.J., Ferrando, M. D., and Andreu. E. (1998). Toxic anorexia as a sensitive endpoint in Daphnia magna. J. Environ. Sci. Health. B 33, 151}160. Ware, G. W. (1983). Pesticides. ¹heory and Application. Freeman, New York.
Daphnia magna Feeding Behavior after Exposure to Tetradifon and Recovery from Intoxication M. J. Villarroel, M. D. Ferrando, E. Sancho, and E. Andreu Laboratory for Ecotoxicology, Department of Animal Biology (Animal Physiology), Faculty of Biological Sciences, University of Valencia, Dr. Moliner 50, E-46100 Burjasot, Valencia, Spain Received July 15, 1998
The "ltration of food particles from water by freshwater species of zooplankton, the injestion of "ltered particles, and the assimilation or absorption of ingested material from the digestive tract are subjects that have been researched extensively from an ecological point of view (Hirayama and Ogawa, 1972; Schlosser and Anger, 1982). Several researchers (Flickinger et al., 1982; Day and Kaushik, 1987; Fernandez}Casalderrey et al., 1994) have suggested that the feeding behavior of zooplankton is a physiological function. This could be considered an important factor in studies dealing with the toxicity of pollutants to aquatic organisms. Thus, the impairment of biochemical and physiological functions of an organism may be taken as the "rst indication of the e!ect of an environmental perturbation. This impairment could result in measurable changes in the behavior of the organism (Sprague, 1976). These changes may be used as rapid and sensitive indicators of toxic stress and may help to explain other observed changes in the survival, growth, and reproduction of nontarget organisms. Rates of "ltration of particles by zooplankton have been observed to decrease following exposure to various chemicals under laboratory conditions (Day and Kaushik, 1987; FernaH ndez}Casalderrey et al., 1992). However, no data are available which re#ect the in#uence of tetradifon during exposure and recovery over several generations of the investigated animals. The objective of this study was to assess the e!ects of low levels of the acaricide tetradifon (4-chlorophenyl 2,4,5-trichlorophenyl sulfone) on the feeding behavior of three generations of the cladocera D. magna and their recovery from intoxication, using a short-term toxicity test as sublethal test criterion.
The feeding behavior of the cladocera Daphnia magna subjected to a short-term exposure to the acaricide tetradifon (4-chlorophenyl 2,4,5-trichlorophenyl sulfone) was studied. The experiments were performed using the unicellular algae Nannochloris oculata at a density of 5ⴛ105 cells/ml as food for the organisms. In a 5rst experiment, three generations (F0, F1, and F3) of the daphnids were exposed to sublethal levels of tetradifon (0.1, 0.18, 0.22, and 0.44 mg/l) and the e4ect of the toxicant on 5ltration and ingestion rates was determined. Rates of 5ltration and ingestion of D. magna declined in the three generations studied with increasing toxicant concentrations; however, toxicant e4ect was greater in daphnids from generations F1 and F3 than in those from the parental generation F0. A second experiment was conducted in order to evaluate whether animals of a 5rst (1) or third (F3) generation coming from parental daphnids (F0) previously exposed to those pesticide concentrations exhibited any alteration in feeding behavior when transferred to clean water (recovery period). The results indicated that the feeding rates of D. magna generations F1 and F3 were still a4ected during the recovery period but to a less degree. The e4ective tetradifon concentrations D. magna at which feeding rates were reduced to 50% that of controls (EC50) were also calculated. 1999 Academic Press Key Words: tetradifon; feeding; recovery; multigeneration; Daphnia magna.
INTRODUCTION
Acute toxicity tests using Daphnia magna require an exposure time of 24 h (or 48 h) and use mortality (immobilization) as an endpoint. The use of alternative endpoints in invertebrate bioassays that would allow shorter-term tests has been little explored (Ferrando et al., 1993; Janssen et al., 1994; Bitton et al., 1996). Behavioral changes have been used successfully as rapid and sensitive indicators of toxic stress in "sh (Little and Finger, 1990) but little has been done to develop behavioral indices with zooplankton (Ferrando and Andreu, 1993; Villarroel et al., 1998).
MATERIAL AND METHODS
Test Species D. magna were obtained from continuous cultures maintained in this laboratory in 6-L aquaria with dechlorinated tap water (total hardness 180}200 mg/L as CaCO ; pH 40
0147-6513/99 $30.00 Copyright 1999 by Academic Press All rights of reproduction in any form reserved.
D. magna FEEDING BEHAVIOR
41
7.9#0.2; alkalinity 4.1 mmol/L). The medium was renewed two times weekly. Culture densities were kept below 50 animals/L, and the daphnids were fed daily ad libitum with the unicellular algae Nannochloris oculata. The aquaria were placed in a temperature-controlled room (22$13C) with a 12 : 12-hr light}dark cycle at 1000 lux light intensity at water level. Daphnids, less than 24 h old (neonates), were used for the experiments. The microalgae N. oculata, which was used as food in all experiments, was cultured in BBM medium (Bischo! and Bold, 1983). The log phase algae were harvested, centrifuged, washed, and resuspended in water. Concentration of algae was measured by counting with a hemocytometer. The suspension was then diluted to produce the desired concentration, Nannochloris density chosen for all the experiments was 5;10 cells/ml (Ferrand et al., 1993).
where Co and Ct are initial and "nal food concentrations (cell/ll), t is time (duration of the experiment in hours), and n is the number of daphnids in volume < (ll). A is a correction factor for changes in the control with "nal concentrations C t after time t. The expression Co ) Ct represents the geometric mean of food concentration during time t.
Test Chemical
Multigeneration Experiments
Tetradifon (4-chlorophenyl 2,4,5-trichlorophenyl sulfone) used on this study was 99% pure as assayed by AFRASA Co. (Spain). Stock solutions were prepared by dissolving the toxicant in acetone immediately prior to each experiment. Since acetone was required as a carrier, a control composed of acetone only (0.25 ml/L) was also included.
This study was conducted for three generations of the daphnids, and it was divided in two di!erent expressions. In the "rst experiment (Fig. 1), the parental generation (F0) and generations F1 and F3 were continuously exposed to the selected tetradifon concentrations, plus the blank and acetone control. Feeding rates were calculated as indicated before. A second experiment was performed in order to evaluate if there was a toxicant transfer from the parental individuals to their progeny. This test was also conducted for three generations of the daphnids, but only the parental generation (F0) was continuously exposed to tetradifon. The other generations (F1 and F3) were chosen from Fo-exposed animals and transferred to clean water (with food but without toxicant); it was called recovery phase (Fig. 2).
Feeding Experiments Based on the results from previous acute toxicity tests (LC 24 h"8.9 mg/L; Ferrando et al., 1996), four tet radifon concentrations were chosen for the feeding study. Daphnids were exposed to 0.10, 0.18, 0.22, and 0.44 mg/L of tetradifon. Each treatment consisted of "ve replicates. Filtration and ingestion rates were used as measures of feeding behavior. Feeding experiments with D. magna were carried out in 60-ml glass beakers containing 50 ml of the medium and 10 daphnids ((24 h old). The vials were placed in a temperature-controlled room at 223C under darkness and static conditions (Ferrando et al., 1993). Test organisms were exposed to the test solution containing food for 5 h, after which the "nal food concentration was measured using a hemocytometer. Filtration rate (F) is de"ned 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 speci"c interval time. For the calculation of average "ltration (ll ) ind\ ) h\) and ingestion rate (cells ) ind\ ) h\), the equations from Gauld (1951) were used,
FIG. 1. Test design for multigeneration feeding study during exposure to tetradifon (Experiment 1).
Statistical Analysis Data for feeding experiments were compared by analysis of variance (ANOVA), in case of signi"cant di!erences (P(0.05); the mean values for exposed populations were compared to those of the control by Duncan's multiple range test. The EC (the concentration of the toxicant that reduces feeding rate to 50%) were calculated using linear regression analysis.
< (ln Co!ln Ct) F" !A n t ln Co!ln C t A" t I"F(Co Ct ,
FIG. 2. Test design for multigeneration feeding study during the recovery period (Experiment 2).
42
VILLARROEL ET AL.
RESULTS
The e!ect of tetradifon on "ltration and ingestion rates of D. magna parental generation (F0) is provided in Fig. 3 (Villarroel et al., 1998). Filtration rates were signi"cantly reduced from 512 ll/animal/h (control) to 211, 166, 193, and 116 ll/animal/h at 0.10, 0.18, 0.22, and 0.44 mg/l of pesticide, respectively. Filtration rate in acetone controls was 514 lL/animal/h ($54.7). Ingestion rates were also reduced at those toxicant concentrations from 10;10 cells/animal/h in the control to 62;10, 54;10, 69;10, and 50;10 cells/animal/h. However, ingestion rate in those animals exposed to the solvent (acetone) was 102.4
FIG. 4. Rates of "ltration and ingestion of N. oculata by D. magna generation (F1) after exposure to tetradifon. *P(0.05.
FIG. 3. Rates of "ltration and ingestion of N. oculata by D. magna parental generation (F0) after exposure to tetradifon. *P(0.05.
($5.2);10 cells/animal/h. The highest concentration of tetradifon used (0.44 mg/L) produced a reduction of 77 and 54% in "ltration and ingestion rates, respectively, compared with control values. The e!ect of tetradifon on the feeding rates of generations F1 and F3 (Figs. 4 and 5) was greater than those from generation F0. Tetradifon concentration of 0.44 mg/L reduced "ltration rates 78% in both F1 and F3 generations. On the other hand, ingestion rates were reduced 82% in F1 and F3 generations exposed to 0.44 mg/L of tetradifon. Statistically signi"cant di!erences between "ltration rates in controls (F1"415 ll/ind/h, F3"419 ll/ind/h) and acetone controls (F1"392.5 ll/ind/h, F3"411.25 ll/ind/h)
D. magna FEEDING BEHAVIOR
43
were not found. Ingestion rates in daphnids exposed to the solvent (F1"160.67;10 cells/animal/h, F3"143;10 cells/animal/h were no di!erent (P'0.05) from those of the blank controls (F1"172;10cells/animal/h, F3"152; 10cells/animal/h). In a second experiment, F1 and F3 generations (neonates) from parentals (F0) preexposed to the same tetradifon concentrations were transferred to clean water (recovery period) and the feeding rates were also evaluated. The results from these experiments are presented in Figs. 6 and 7. As can be seen in Fig. 6 "ltration rates of F1 generation animals from parentals exposed to 0.10 and 0.18 mg/L were no di!erent from control values, while those from parentals exposed to
FIG. 6. Rates of "ltration and ingestion of N. oculata by D. magna generation (F1) after the recovery period. *P(0.05.
FIG. 5. Rates of "ltration and ingestion of N. oculata by D. magna generation (F3) after exposure to tetradifon. *P(0.05.
0.22 and 0.44 mg/L were statistically di!erent. A reduction of 67% was observed in the "ltration rate of the animals (F1) from parentals (F0) exposed to 0.44 mg/L, even during the recovery period. So, it is assumed that there was a small recuperation in the "ltration rates of F1 generation daphnids but animals from parentals exposed to the highest pesticide concentrations were still a!ected. However, the e!ect was even greater on the ingestion rates (Fig. 6). F1 generation daphnids exhibited a reduction in this parameter during the recovery period, especially those daphnids
44
VILLARROEL ET AL.
coming from parentals exposed to 0.44 mg/L tetradifon (82% reduction). Figure 7 presents the e!ect of a recovery period in the feeding rates of F3 generation daphnids. It seems that F3 generation was more a!ected by the toxicant which was transferred from their mothers (F0). The parental generation (F0) previously exposed to tetradifon had the "rst progeny (F1) the 7th day of pesticide treatment, while the third generation of daphnids (F3) was obtained the 11th day, so F3 generation animals were coming from mothers who were exposed to the pesticide for 4 more days. So, when F3
neonates were transferred to clean water (recovery period), they did not recuperate completely and their feeding rates were still reduced. Statistically di!erences were found in both "ltration and ingestion rates of F3 generation daphnids from parentals exposed to all the pesticide concentrations tested. Feeding rates of animals from parentals exposed to the highest concentration (0.44 mg/L) were reduced a 65 and 85% for "ltration and ingestion rates, respectively. From these results it could be assumed that it is possible the pesticide tetradifon was bioaccumulated and transferred from the mothers (F0) to the neonates (F1 and F3), and then the highest toxicant e!ect was found in F1}F3 generations exposed to the pesticide compared with F0 parentals, but there was also a strong e!ect in nonexposed F1}F3 neonates (recovery period). DISCUSSION
FIG. 7. Rates of "ltration and ingestion of N. oculata by D. magna generation (F3) after the recovery period. *P(0.05.
The e!ects of xenobiotics on feeding rates of some species of cladocerans have been investigated by several workers. Kersting and Van der Honing (1981) pointed out that the "ltering rates of daphnids might be used as very sensitive toxicity parameters. Day and Kaushik (1987) found that the "ltration rate of the cladocera Daphnia galeata mendotae and Ceriodaphnia lacustris and of the calanoid Diaptomus oregonensis was decreased by sublethal concentrations (0.01}0.1 lg/L) of fenvalerate. It induced the adhesion of particulate material (algae) and detritus) onto the various setate appendages of D. galeata mendotae. The degree of adhesion was greater as the concentration of fenvalerate increased. Such adhesion impeded daphnid mobility and animals had di$culty swimming above the bottom of the beaker. This resulted in a decreased feeding rate. The "ltration rate of D. magna was reduced signi"cantly after a 5-h exposure to 0.062, 0.077, and 0.15 ng/L methylparathion (FernaH ndez}Casalderrey et al., 1993). Feeding rates were also reduced in D. magna after exposure to endosulfan (0.31, 0.41, and 0.62 mg/L) and diazinon (0.45, 060, and 0.90 lg/L) (FernaH ndez}Casalderrey et al., 1994). The "ltration of food by "lter-feeding zooplankton requires movement of appendages and coordination of the nervous system. Therefore, those toxicants (as the acaricides) that a!ect nervous system (Ware, 1983) will cause loss of coordination and/or paralysis and will reduce rates of "ltration. A useful quantitative parameter analogous to the LC derived from mortality data in acute toxicity tests is the EC . This is the median e!ective concentration of the toxicant at which the value of a given parameter is reduced to 50% of that in controls. The EC values for "ltration and ingestion rates were derived for the toxicant tested using the regression equations given in Table 1. These regression equations as well as their correlation coe$cients
45
D. magna FEEDING BEHAVIOR
TABLE 1 Regression Equations and Correlation Coe7cients (r2) Describing the Relation between Tetradifon Concentrations (x) and Filtration and Ingestion Rates (y) of D. magna Exposed to the Pesticide (Generations F0, F1, F3) Generation
Parameter
Regression equations
Correlation coe$cient (r)
F0
Filtration Ingestion Filtration Ingestion Filtration Ingestion
y"19.3#160x y"17.8#99x y"24#127x y"25.3#134x y"25.6#150x y"21.5#162x
0.86 0.90 0.93 0.90 0.83 0.86
F1 F3
Note. EC
EC
0.19 0.32 0.20 0.18 0.16 0.17
e!ect criterion for rapid toxicity screening of xenobiotics. The results from the &&feeding'' toxicity test, as described in this study, also indicated that behavioral indices can re#ect the potential for subsequent mortality (acute mortality tests) at lowest toxicant concentrations. On the other hand, the e!ect of tetradifon on feeding rates increased over the di!erent generations studied. Thus F3 generation was more sensitive to the pesticide than the parental generation, F0, and a transfer of the bioaccumulated pesticide from the mothers to the neonates could take place. Recovery experiments carried out on F1 and F3 generations determined the need for longer periods in clean water to eliminate the toxicant and recover the a!ected feeding rates.
(mg/L) was also calculated.
ACKNOWLEDGMENT
(r) indicate the relationship between the pesticide concentrations used and the rates of "ltration and ingestion obtained in the present study. The results indicate that the observed e!ect in the feeding behavior of D. magna will be greater when the amount of pesticide used in the medium is increased. The EC for "ltration and ingestion rates (Table 1) on D. magna parental generation (F0) indicated values of 0.19 and 0.16 and 0.32 mg/L for both rates while the EC s for the same parameters in generation F3 were 0.16 and 0.17 mg/L. This means that animals from the third generation (F3) were more sensitive than the parentals (F0) and less pesticide in the water will reduce their feeding rates 50%. The EC values calculated in this study were much lower than the 24-h LC (Ferrando et al., 1996) for this species (8.9 mg/L). Thus, toxicant levels below the 24-h LC value are su$cient to reduce feeding rates to 50 within 5 h. EC values for the feeding rates of animals during the recovery experiment were not calculated, those animals (generations F1 and F3) were not exposed to the pesticide. There are no data in the literature about the e!ect of pesticides on the feeding rates of a species through several generations. Multigeneration experiments carried out in D. magna demonstrated the e!ects of di!erent pollutants such as nickel (MuK nzinger, 1990), chromium (MuK nzinger and Monicelli, 1992), and CaCl (Bervoets et al., 1996)in the survival, reproduction and growth of di!erent generations. It is thought that the evaluation of the feeding rates in zooplankton species could be a useful parameter to determine the e!ect of xenobiotics in a short period of time through several generations of the same species.
CONCLUSIONS
The results indicate that the feeding behavior of the freshwater cladocera D. magna might be an interesting new
This work was supported by a grant (AMB96:11188-CO1) from the Comision Interministerial de Ciencia y TecnologmH a (I#D) (CICYT) del Ministerio de EducacioH n y Ciencia, Spain.
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