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Effect of Sublethal Doses of Cadmium on the Phototactic Behavior of Daphnia magna
Ecotoxicology and Environmental Safety 47, 261–265 (2000) Environmental Research, Section B doi:10.1006/eesa.2000.1962, available online at http://www/idealibrary.com on
E¡ect of Sublethal Doses of Cadmium on the Phototactic Behavior of Daphnia magna E. Michels,*,1 S. Semsari,{ C. Bin,{ and L. De Meester* *Laboratory of Aquatic Ecology, K. U. Leuven, Ch. De Be´riotstraat 32, 3000 Leuven, Belgium; {Universite´ de Blida, Institut de Chemie Industrielle, B. P. 270 Blida, Alge´rie; and {Institute of Terrestrial Ecology, Swiss Federal Institute of Technology, Grabenstrasse 3, 8952 Schlieren, Switzerland Received November 26, 1999
The effect of a sublethal concentration of cadmium (0.06 mg/ L) was tested on the phototactic behavior of a positively phototactic Daphnia magna clone. In experiments lasting
10 min, using animals that had been exposed to cadmium for 1 to 6 h, it was observed that the animals became significantly less positively phototactic after 4 h of exposure to 0.06 mg/L cadmium compared to control animals that had not been exposed to cadmium. In flow-through experiments that lasted for 6 h and during which there were repeated measurements, there was again a significant effect of cadmium exposure on the phototactic behavior of the animals. Irrespective of treatment, time had a significant effect. Results suggest that phototactic behavior can be used to detect sublethal concentrations of pollutant within a few hours, in short-term as well as in longer-lasting experiments with continuous flowthrough and repeated stimulation of the animals. # 2000 Academic Press
Key Words: phototactic behavior Daphnia; continuous biomonitoring; early warning systems; cadmium.
INTRODUCTION
Recently, continuous monitoring of water quality has attracted growing attention, as it provides an early warning system (Chapman and Jackson, 1996). Several continuous biomonitors have been developed in which a physiological or behavioral response of a living organism is used as a variable to estimate overall water quality (Kramer and Botterweg, 1991; Schmitz et al., 1994; Chapman and Jackson, 1996). If rigorously standardized, behavioral criteria can be more sensitive and rapid indicators of stress than the morphological or life-history criteria used in 1
To Whom correspondence should be addressed. Fax: 00 32 16 32 45 75. E-mail: [email protected].
traditional ecotoxicological tests (Charoy et al., 1995). Contrary to (semi) continuous chemical monitoring, the response of living organisms integrates overall water quality, since living organisms react to a multitude of chemical compounds at different levels (Chapman and Jackson, 1996). In addition, in using a biological response, bioavailability is automatically accounted for. Daphnia has been found to be very sensitive to the presence of a large number of chemicals in the environment, and to respond to the presence of pollutants with a multitude of traits (e.g., Dodson and Hanazato, 1995). A number of authors have suggested the use of phototactic behavior of Daphnia for the detection of sublethal concentrations of toxic compounds (Flickinger et al., 1982; Di Delupis and Rotondo, 1988). Three biomonitors using behavioral traits in Daphnia have so far been developed. The monitor of Knie (1978) uses the swimming activity of D. magna to assess stress and the monitor developed by Kerren (1991) uses changes in phototactic behavior of D. magna. More recently, the Daphnia Toximeter, manufactured by BBE Moldaenke (1997) was developed. It uses an alarm analysis based on swimming velocity, swimming behavior, and growth observation for continuous detection of hazardous compounds. The Kerren (1991) monitor has been found to exhibit a very variable response, which reduces its reliability (Van Hoof et al., 1994). In a previous study (Michels et al., 1999), data were presented that indicated that the sensitivity of a biomonitor using phototactic behavior can be increased considerably by the use of specific clones of D. magna. The use of clonal test organisms increases standardization of ecotoxicity tests, because there is genetic variation in the sensitivity of D. magna to a number of pollutants (Baird et al., 1989, 1990). Michels et al. (1999) suggested the use of extremely positively phototactic clones of D. magna in a monitor, as these animals exhibit very typical behavior in the absence
261 0147-6513/00 $35.00 Copyright # 2000 by Academic Press All rights of reproduction in any form reserved.
262
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of pollutants and a low sensitivity to changes in environmental conditions such as changes in pH, oxygen concentration, food concentration, and temperature (see De Meester and Dumont, 1988; Van Uytvanck and De Meester, 1990; De Meester, 1991; Michels and De Meester, 1998). In addition, Michels et al. (1999) selected clones that have only a weak response to the presence of fish kairomones, thus further standardizing the behavior in the absence of pollutants. They found that the animals demonstrate a significantly less positively phototactic behavior in the presence of sublethal concentrations of copper and pentachlorophenol. The study was done using short-term exposure experiments, in which the behavior of animals was monitored for a 10-min observation period following exposure to the pollutant for a variable period (up to several hours), using different animals for each experiment. In an operational biomonitor, however, the animals will be monitored continuously or semicontinuously, and the same animals will thus be exposed to repeated cycles of light stimuli. In the present study, the object was to test whether repeated exposure to experimental light stimuli alters the phototactic response of Daphnia in relation to pollutant stress. Cadmium ions were used as a standard pollutant, and the results of short experiments (10-min observation period, the animals being exposed to pollutants for 0 to 6 h prior to the experiments) were compared with the results obtained from experiments in which experiments animals were subjected to cadmium stress for 5 h in an experimental set-up that enabled a continuous flow-through of fresh medium. During the latter series of experiments, the animals were exposed to light stimuli (10-min observation period) every half-hour. MATERIALS AND METHODS
All experiments were carried out with adult females of the positively phototactic D. magna clone C134. This clone is an intraclonal offspring of clone C1, which was isolated from a fishless city pond in Gent (Belgium). Animals 10–15 days old, carrying their second clutch, were used. All animals were cultured at 208C (+18) in a long day photoperiod (14 h light–10 h dark). Animals were cultured in 1-L jars. Density was kept relatively low: 20–30 individuals per liter. Food concentration was restored daily up to approximately 2.56105 Scenedemus acutus cells/ml. One-fourth of the culture medium was replaced daily by an equivalent of fresh medium. Changes in phototactic behavior of clone C134 were tested after exposure to a constant concentration of 0.06 mg/L Cd2þ (added as CdSO48H2O) in an artificial medium (ISO, 1989) prepared from demineralized water (Milli Ro). The ISO (1989) medium had the following composition: 0.294 g CaCl22H2O, 0.123 g MgSO47H2, 0.065 g NaHCO3, and 0.006 g KCl per liter,
pH adjusted to 7.9 with 1 N NaOH or 1 N HCl (see Michels et al., 1999), without additional food. The concentration of Cd2þ that was applied is approximately one-fifth of the LC50 (24 h) concentration (estimated to be 0.31 mg/L by Dave et al., 1980). The experiments were carried out in the absence of food in order to prevent the bioavailability of the pollutant being affected by adsorption of the metal to food particles. Previous long-term experiments (up to 10 h) have found that the phototactic behavior is not dramatically altered by the absence of food (De Meester and Cousyn, 1997). The experimental set-up and the flow-through system were the same as those used by De Meester and Cousyn (1997); the flow-through rate was set at 10 ml/min and a concentration of 0.06 mg/L Cd2þ. The test medium was prepared daily to prevent loss of Cd2þ through adsorption. Cd2þ concentrations were verified by inductively coupled plasma mass spectrometry (PQ2þ, VG-Elemental) on samples taken after the experiments. Verified concentrations were all in correspondence with nominal concentrations. In the repeated light stimulation experimental series, six experiments were carried out in both the control and the Cd2þ exposure treatment. In the 10-min experiments, during which there was no flowthrough of medium, four replicate experiments were done for each treatment. In the latter experimental series, the time of exposure to cadmium varied from 1 to 6 h, with hourly intervals (seven treatments). All experiments were carried out with 10 animals each. For each replicate, different animals of different cultures or generations were used to minimize common environmental effects and thus to prevent pseudoreplication (Lynch and Ennis, 1983; Hurlbert, 1984). The experimental set-up to quantify the phototactic behavior has been described by De Meester and Cousyn (1997) and Michels et al. (1999). The experimental cuvet consisted of two glass columns that were placed concentrically into each other (see Fig. 1). The outer column can be connected to a flow-through system, whereas the inner column (10 cm height; Ø 5 cm) represents the observation chamber in which the animals were placed. The inner column had a bottom and top made of plankton gauze to prevent the animals from swimming out of the observation chamber, and was externally divided into four compartments of 2.5 cm length each: an upper compartment U, two middle compartments M1 and M2, and a lower compartment L. During an experiment, the cuvet was placed in a light-tight PVC box (16628635 cm, provided with a peephole of Ø 1 cm for observations) and illuminated from above with a fiber light source (150 W) of which the fiber ending was positioned 2 cm above the water surface in the outer column. Each light exposure period lasted 10 min. At 1-min intervals, the number of animals in each compartment was counted. The phototactic behavior of the test population was characterized by the index I = (U7L)/
EFFECTS OF Cd ON PHOTOTACTIC BEHAVIOR OF D. magna
263
observations. The 10-min experiments followed the standard procedure adopted by Michels et al. (1999), with a 5min dark adaptation followed by a 10-min observation period. In this experimental series, all animals were adapted for 6 h to ISO medium, and the medium was replaced by ISO medium containing cadmium for one to several hours (just prior to the actual experiment), depending on the treatment. During the dark adaptation and the experiment itself, the animals were also exposed to cadmium. The data of the 10-min experiments were anlyzed by oneway ANOVA followed by contrast analysis to test for the effect of varying exposure times on phototactic behavior. The data of the experiments with repeated light stimuli were analyzed using repeated-measures ANOVA, testing for an effect of time, treatment, and a Time 6 Treatment interaction (Sokal and Rohlf, 1995). Homogeneity of variance was tested using Bartlett’s test. All statistical analyses were performed using STATISTICA (Statsoft, 1994). RESULTS
FIG. 1. Experimental cuvet for the quantification of phototactic behavior. Test animals (10) are placed in the central cylinder, which is externally divided into four compartments (U, M1, M2, and L) of 2.5 cm. Top (1) and bottom (2) of the central cylinder are closed with plankton gauze. During an experiment the cuvet is placed in a light-tight PVC box and illuminated from above with a fiber light source.
(UþM1þM2þL), averaged over the last 5 min of the experiment to minimize influences of the initial flight reaction. Values of the phototactic index can range from þ1 for extremely positive to 71 for extremely negative phototactic behavior. All experiments were done in a constant temperature room (20+18C), in early afternoon (start 1300–1500 h) to minimize possible effects of an endogenous rhythm (the effect of an endogenous rhythm was, however, found to be negligible in positive phototactic clones; see De Meester, 1993). In the repeated light exposure experiment, the animals were adapted for 3 h to the ISO medium, without food or pollutants added, prior to the experiment. They were then pipetted into the cuvet, and the flow-through was switched on, gradually replacing the old medium in the cuvet with fresh test medium. Before the first observation period of 10 min, the animals were given a dark adaptation of 20 min. Subsequently, the light was switched on and observations were made. After 10 min, the animals were again given a dark adaptation for 20 min. In this way, a 10-min observation period was given every half-hour for a period of 6 h. Each experiment thus consisted of 12 repeated
Figure 2 presents the changes in phototactic behavior of D. magna of clone C134 after exposure to 0.06 mg/L Cd2þ for a total period ranging from 0 to 6 h prior to the experiment. The phototactic response of clone C134 can be used to detect sublethal concentrations of cadmium: the animals reveal a highly significant response to the sublethal concentration of Cd2þ, with the values of phototactic index I decreasing with increasing exposure time to the pollutant (df = 6,21; MS effect = 0.0896; MS error = 0.009; F = 9.913; P50.0001). Contrast analysis after one-way ANOVA indicates that there is a significant difference in phototactic behavior of animals exposed to 0.06 mg/L
FIG. 2. Phototactic behavior (mean value of I+2 SE; n=4) of adult Daphnia magna clone C134 exposed for different periods (1–6 h) to 0.06 mg/L Cd2þ (LC50 24 h)= 0.31; Dave et al., 1980).
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Cd2þ with the control treatment (0 h exposure) after 4 h exposure to the pollutant. Figure 3 presents the results of the experiments with repeated light stimulation. Repeated-measures ANOVA indicates that there is a highly significant effect of treatment (presence or absence of cadmium; P = 0.026) and time (P50.001) on the phototactic behavior of the animals, but no Time6Treatment interaction effect (P = 0.164). In the absence of cadmium, the phototactic behavior of clone C134 remains fairly positively phototactic after repeated light stimulation (the value of I remains higher than 0.6 for the first 4.5 h). The phototactic index in the control treatment varied from 0.76 (0.5 h) to 0.5 (6 h), whereas the value of the phototactic index in the cadmium treatment ranged from 0.60 (0.5 h) to 0.08 (6 h). The absence of a significant Treatment6Time interaction effect indicates that time has a similar effect on both the control and the pollutant treatment: although the effect of the presence of pollutant is highly significant, the difference between the control and the pollutant treatment does not increase with exposure time to the pollutant.
DISCUSSION
Results confirm the stable and predictable phototactic behavior of clone C134 in the absence of pollutants and fish chemicals (see De Meester, 1993; Michels et al., 1999) and confirm the potential to use phototactic behavior of this clone to detect sublethal levels of pollutant (Michels et al., 1999). Michels et al. (1999) reported that the detection limit for Cu2þ and pentachlorophenol using changes in phototactic behavior of clone C134 was 0.045 mg/L for Cu2þ and 0.8 mg/L for PCP, both values being lower than the LC50 (24 h) Michels et al., 1999). The cadmium
concentration used in the present experiments (0.06 mg/L Cd2þ) is only about one-fifth of the LC50 (24 h) value reported by Dave et al. (1980) and is thus clearly sublethal for D. magna. Present results, therefore, indicate that the clone used in the present study might be suitable for use in a continuous biomonitor, since its phototactic behavior under standard conditions is stable enough to have a reasonably low intrinsic variability in behavior during the course of time. Both in the 10-min experiments and in the repeated light stimuli experiment, a highly significant effect was observed of the presence of pollutant on the phototactic behavior of the animals. In the 10-min experiments, this effect was significant after 4 h of exposure to the pollutant, whereas the difference was observed almost immediately (within 1 h) in the experiments with continuous flow-through and repeated light stimuli. Strikingly, a dosage effect was not observed in the repeated light stimuli experiment, as there is no different effect of time (exposure time) on the phototactic behavior of the animals exposed to cadmium. This result cannot be explained, but it is likely that the phototactic behavior of the control animals became slightly less positively phototactic at the end of the experimental period due to a hunger effect. At the end of the experiment, the animals had been without food for 9 h. In a real biomonitor, it might be advisable to feed the animals during the assessment periods, even though the addition of food may change the bioavailability of pollutants in the medium. Alternatively, one could feed the animals daily for a limited period of time. During that period, no readings can be taken from the particular set-up, but continuous monitoring can be ensured by having two set-ups run in parallel, and feeding the animals at different times of the day.
CONCLUSION
FIG. 3. Phototactic behavior (mean value of I + 2 SE; n = 6) of adult Daphnia magna clone C134 after different exposure times to 0.06 mg/ L Cd2þ (open symbols) and under control conditions (solid symbols) in the experiments with repeated light stimulation.
After 4 h of exposure to 0.06 mg/L cadmium, the phototactic behavior of clone C134 became significantly less positively phototactic, compared to animals in the control condition (not exposed to cadmium). In absence of pollutants and fish chemicals, the phototactic behavior of clone C134 is stable and predicable. In longer experiments with continuous flow-through and repeated stimulation of the animals, the phototactic behavior of clone C134 was significantly different from those of animals in control conditions within 1 h of exposure to 0.06 mg/L cadmium. The results from this study suggest that changes in the phototactic behavior of D. magna clone C134 can be used to detect sublethal concentrations (0.06 mg/L) of cadmium within a few hours, in short term as well as in experiments with repeated light stimuli, similar to the conditions in an operational biomonitor.
EFFECTS OF Cd ON PHOTOTACTIC BEHAVIOR OF D. magna
ACKNOWLEDGMENTS The authors thank Prof. C. Vandecasteele for verifications of the cadmium concentrations in the test medium and J. Vingerhoeds for help with Fig. 1. E. Michels is a fellow of the Flemish Institute for the promotion of Scientific–Technological Research in Industry (I.W.T.). The authors also thank F. Van Hoof, M. Leynen, and an anonymous referee for constructive comments on an earlier version the manuscript.
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ming behavior, and reproduction of Daphnia, a key member of aquatic ecosystems. Environ. Health Perspect. 103, (Suppl. 4), 7–11. 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 in Daphnia magna Straus. Arch. Environ. Toxicol. 11, 457–463. Hurlbert, S. H. (1984). Pseudoreplication and the design of ecological field experiments. Ecol. Mon. 54, 187–211.
REFERENCES
ISO (1989). Water quality—Determination of the Inhibition of the Mobility of Daphnia magna Straus (Cladocera, Crustacea). ISO 6341, 01.10.1986.
Baird, D. J., Barber, I., and Calow, P. (1990). Clonal variation in general responses of Daphnia magna Straus to toxic stress. I. Chronic life-history effects. Funct. Ecol. 4, 399–407.
Kerren, K. (1991). ‘‘Aqua-Tox-Control-Daphnia. ’’ Kerren Umwelttechnik, Viersen.
Baird, D. J., Soares, A. M. V. M., Girling, I., Barber, I., Bradley, M. C., and Calow, P. (1989). The long term maintenance of Daphnia magna Straus for use in ecotoxicity tests: Problems and prospects. In ‘‘Proceedings of the 1st European Conference on Ecotoxicology, SETOX’’ (H. Lokke, H. Tyle, and F. Bro-Rasmussen, Eds.), pp. 204–207. Technical University, Lynby. Chapman, D., and Jackson, J. (1996). Biological monitoring. In ‘‘Water Quality Monitoring’’ J. Bartram and R. Ballance, Eds.), pp. 263–299. Chapman & Hall, London. Charoy, C. P., Janssen, C. R., Persoone, G., and Clement, P. (1995). The swimming behavior of Branchionus calyciflorus (rotifer) under toxic stress. I. The use of automated trajectometry for determing sublethal effects of chemicals. Aquat. Toxicol. 32, 271–282. Dave, G., Andersson, K., Berglind, R., and Hasselrot, B. (1980). Toxicity of eight solvent extraction chemicals and of cadmium to water fleas, Daphnia magna, rainbow trout, Salmo gairderi, and zebrafish, Brachydanio rerio. Comp Biochem. Physiol. 69, 11–20. De Meester, L. (1991). An analysis of the phototactic behavior of Daphnia magna clones and their sexual descendants. Hydrobiologia 225, 217–227. De Meester, L. (1993). Genotype, fish-mediated chemicals, and phototactic behavior in Daphnia magna. Ecology 74, 1467–1474. De Meester, L., and Cousyn, C. (1997). The change in phototactic behavior of a Daphnia magna clone in the presence of fish kairomones: The effect of exposure time. Hydrobiologia 360, 169–175. De Meester, L., and Dumont, H. J. (1988). The genetics of phototaxis in Daphnia magna: Existence of three phenotypes for vertical migration among parthenogenetic females. Hydrobiologia 162, 47–55. Di Delupis, G. D., and Rotondo, V. (1988). Phototaxis in aquatic invertebrates: Possible use in ecotoxicity tests. Ecotoxicol Environ. Saf. 16, 189–193. Dodson, S. I., and Hanazato, T. (1995). Commentary on effects of anthropogenic and natural organic chemicals on development, swim-
Knie, J. (1978). Der Dynamischen Daphnientest-ein automatischer Biomonitor zur Uberwachtung von Gewa¨ssern und Abwa¨ssern. Wasser Boden 12, 310–312. Kramer, K. J. M., and Botterweg, J. (1991). Aquatic biological early warning systems: An overview. In ‘‘Bioindicators and Environmental Management’’ (D. W. Jeffrey and B. Madden, Eds.), pp. 95–176. Academic Press, London. Lynch, M., and Ennis, R. (1983). Resource availability, maternal effects and longevity. Exp. Gerontol. 18, 147–165. Michels, E., and De Meester, L. (1998). The influence of food quality on the phototactic behavior of Daphnia magna Straus. Hydrobiologia 379, 199–206. Michels, E., Leynen, M., Cousyn, C., De Meester, L., Ollevier, F. (1999). Phototactic behavior of Daphnia as a tool in the continuous monitoring of water quality: Experiments with a positively phototatic Daphnia magna clone. Water Res. 33, 401–408. Moldaenke (1997). ‘‘Daphnia Toximeter.’’ BBE und J. Uley Software entwicklung. Schmitz, P., Krebs, F., and Irmer, U. (1994). Development, testing and implementation of automated biotests for the monitoring of the River Rhine, demonstrated by bacteria and algae tests. Water Sci. Tech. 29, 215–221. Sokal, R., and Rohlf, F. J. (1995). ‘‘Biometry,’’ 3rd ed. Freeman, New York. Statsoft (1994). ‘‘STATISTICA for the Windows Operating System.’’ Statsoft, Inc., Tulsa, UK. Van Hoof, F., Sluyts, H., Paulussen, J., Berckmans, D., and Bloemen, H. (1994). Evaluation of a biomonitor based on the phototactic behavior of Daphnia magna using infra-red detection and digital image processing. Water Sci. Tech. 30, 79–86. Van Uytvanck, J., and De Meester, L. (1990). Phototaxis in Daphnia magna: The influence of temperature and acidity on the phototactic behavior of Daphnia genotypes. J. Plankton Res. 12, 1089–1097.
E¡ect of Sublethal Doses of Cadmium on the Phototactic Behavior of Daphnia magna E. Michels,*,1 S. Semsari,{ C. Bin,{ and L. De Meester* *Laboratory of Aquatic Ecology, K. U. Leuven, Ch. De Be´riotstraat 32, 3000 Leuven, Belgium; {Universite´ de Blida, Institut de Chemie Industrielle, B. P. 270 Blida, Alge´rie; and {Institute of Terrestrial Ecology, Swiss Federal Institute of Technology, Grabenstrasse 3, 8952 Schlieren, Switzerland Received November 26, 1999
The effect of a sublethal concentration of cadmium (0.06 mg/ L) was tested on the phototactic behavior of a positively phototactic Daphnia magna clone. In experiments lasting
10 min, using animals that had been exposed to cadmium for 1 to 6 h, it was observed that the animals became significantly less positively phototactic after 4 h of exposure to 0.06 mg/L cadmium compared to control animals that had not been exposed to cadmium. In flow-through experiments that lasted for 6 h and during which there were repeated measurements, there was again a significant effect of cadmium exposure on the phototactic behavior of the animals. Irrespective of treatment, time had a significant effect. Results suggest that phototactic behavior can be used to detect sublethal concentrations of pollutant within a few hours, in short-term as well as in longer-lasting experiments with continuous flowthrough and repeated stimulation of the animals. # 2000 Academic Press
Key Words: phototactic behavior Daphnia; continuous biomonitoring; early warning systems; cadmium.
INTRODUCTION
Recently, continuous monitoring of water quality has attracted growing attention, as it provides an early warning system (Chapman and Jackson, 1996). Several continuous biomonitors have been developed in which a physiological or behavioral response of a living organism is used as a variable to estimate overall water quality (Kramer and Botterweg, 1991; Schmitz et al., 1994; Chapman and Jackson, 1996). If rigorously standardized, behavioral criteria can be more sensitive and rapid indicators of stress than the morphological or life-history criteria used in 1
To Whom correspondence should be addressed. Fax: 00 32 16 32 45 75. E-mail: [email protected].
traditional ecotoxicological tests (Charoy et al., 1995). Contrary to (semi) continuous chemical monitoring, the response of living organisms integrates overall water quality, since living organisms react to a multitude of chemical compounds at different levels (Chapman and Jackson, 1996). In addition, in using a biological response, bioavailability is automatically accounted for. Daphnia has been found to be very sensitive to the presence of a large number of chemicals in the environment, and to respond to the presence of pollutants with a multitude of traits (e.g., Dodson and Hanazato, 1995). A number of authors have suggested the use of phototactic behavior of Daphnia for the detection of sublethal concentrations of toxic compounds (Flickinger et al., 1982; Di Delupis and Rotondo, 1988). Three biomonitors using behavioral traits in Daphnia have so far been developed. The monitor of Knie (1978) uses the swimming activity of D. magna to assess stress and the monitor developed by Kerren (1991) uses changes in phototactic behavior of D. magna. More recently, the Daphnia Toximeter, manufactured by BBE Moldaenke (1997) was developed. It uses an alarm analysis based on swimming velocity, swimming behavior, and growth observation for continuous detection of hazardous compounds. The Kerren (1991) monitor has been found to exhibit a very variable response, which reduces its reliability (Van Hoof et al., 1994). In a previous study (Michels et al., 1999), data were presented that indicated that the sensitivity of a biomonitor using phototactic behavior can be increased considerably by the use of specific clones of D. magna. The use of clonal test organisms increases standardization of ecotoxicity tests, because there is genetic variation in the sensitivity of D. magna to a number of pollutants (Baird et al., 1989, 1990). Michels et al. (1999) suggested the use of extremely positively phototactic clones of D. magna in a monitor, as these animals exhibit very typical behavior in the absence
261 0147-6513/00 $35.00 Copyright # 2000 by Academic Press All rights of reproduction in any form reserved.
262
MICHELS ET AL.
of pollutants and a low sensitivity to changes in environmental conditions such as changes in pH, oxygen concentration, food concentration, and temperature (see De Meester and Dumont, 1988; Van Uytvanck and De Meester, 1990; De Meester, 1991; Michels and De Meester, 1998). In addition, Michels et al. (1999) selected clones that have only a weak response to the presence of fish kairomones, thus further standardizing the behavior in the absence of pollutants. They found that the animals demonstrate a significantly less positively phototactic behavior in the presence of sublethal concentrations of copper and pentachlorophenol. The study was done using short-term exposure experiments, in which the behavior of animals was monitored for a 10-min observation period following exposure to the pollutant for a variable period (up to several hours), using different animals for each experiment. In an operational biomonitor, however, the animals will be monitored continuously or semicontinuously, and the same animals will thus be exposed to repeated cycles of light stimuli. In the present study, the object was to test whether repeated exposure to experimental light stimuli alters the phototactic response of Daphnia in relation to pollutant stress. Cadmium ions were used as a standard pollutant, and the results of short experiments (10-min observation period, the animals being exposed to pollutants for 0 to 6 h prior to the experiments) were compared with the results obtained from experiments in which experiments animals were subjected to cadmium stress for 5 h in an experimental set-up that enabled a continuous flow-through of fresh medium. During the latter series of experiments, the animals were exposed to light stimuli (10-min observation period) every half-hour. MATERIALS AND METHODS
All experiments were carried out with adult females of the positively phototactic D. magna clone C134. This clone is an intraclonal offspring of clone C1, which was isolated from a fishless city pond in Gent (Belgium). Animals 10–15 days old, carrying their second clutch, were used. All animals were cultured at 208C (+18) in a long day photoperiod (14 h light–10 h dark). Animals were cultured in 1-L jars. Density was kept relatively low: 20–30 individuals per liter. Food concentration was restored daily up to approximately 2.56105 Scenedemus acutus cells/ml. One-fourth of the culture medium was replaced daily by an equivalent of fresh medium. Changes in phototactic behavior of clone C134 were tested after exposure to a constant concentration of 0.06 mg/L Cd2þ (added as CdSO48H2O) in an artificial medium (ISO, 1989) prepared from demineralized water (Milli Ro). The ISO (1989) medium had the following composition: 0.294 g CaCl22H2O, 0.123 g MgSO47H2, 0.065 g NaHCO3, and 0.006 g KCl per liter,
pH adjusted to 7.9 with 1 N NaOH or 1 N HCl (see Michels et al., 1999), without additional food. The concentration of Cd2þ that was applied is approximately one-fifth of the LC50 (24 h) concentration (estimated to be 0.31 mg/L by Dave et al., 1980). The experiments were carried out in the absence of food in order to prevent the bioavailability of the pollutant being affected by adsorption of the metal to food particles. Previous long-term experiments (up to 10 h) have found that the phototactic behavior is not dramatically altered by the absence of food (De Meester and Cousyn, 1997). The experimental set-up and the flow-through system were the same as those used by De Meester and Cousyn (1997); the flow-through rate was set at 10 ml/min and a concentration of 0.06 mg/L Cd2þ. The test medium was prepared daily to prevent loss of Cd2þ through adsorption. Cd2þ concentrations were verified by inductively coupled plasma mass spectrometry (PQ2þ, VG-Elemental) on samples taken after the experiments. Verified concentrations were all in correspondence with nominal concentrations. In the repeated light stimulation experimental series, six experiments were carried out in both the control and the Cd2þ exposure treatment. In the 10-min experiments, during which there was no flowthrough of medium, four replicate experiments were done for each treatment. In the latter experimental series, the time of exposure to cadmium varied from 1 to 6 h, with hourly intervals (seven treatments). All experiments were carried out with 10 animals each. For each replicate, different animals of different cultures or generations were used to minimize common environmental effects and thus to prevent pseudoreplication (Lynch and Ennis, 1983; Hurlbert, 1984). The experimental set-up to quantify the phototactic behavior has been described by De Meester and Cousyn (1997) and Michels et al. (1999). The experimental cuvet consisted of two glass columns that were placed concentrically into each other (see Fig. 1). The outer column can be connected to a flow-through system, whereas the inner column (10 cm height; Ø 5 cm) represents the observation chamber in which the animals were placed. The inner column had a bottom and top made of plankton gauze to prevent the animals from swimming out of the observation chamber, and was externally divided into four compartments of 2.5 cm length each: an upper compartment U, two middle compartments M1 and M2, and a lower compartment L. During an experiment, the cuvet was placed in a light-tight PVC box (16628635 cm, provided with a peephole of Ø 1 cm for observations) and illuminated from above with a fiber light source (150 W) of which the fiber ending was positioned 2 cm above the water surface in the outer column. Each light exposure period lasted 10 min. At 1-min intervals, the number of animals in each compartment was counted. The phototactic behavior of the test population was characterized by the index I = (U7L)/
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observations. The 10-min experiments followed the standard procedure adopted by Michels et al. (1999), with a 5min dark adaptation followed by a 10-min observation period. In this experimental series, all animals were adapted for 6 h to ISO medium, and the medium was replaced by ISO medium containing cadmium for one to several hours (just prior to the actual experiment), depending on the treatment. During the dark adaptation and the experiment itself, the animals were also exposed to cadmium. The data of the 10-min experiments were anlyzed by oneway ANOVA followed by contrast analysis to test for the effect of varying exposure times on phototactic behavior. The data of the experiments with repeated light stimuli were analyzed using repeated-measures ANOVA, testing for an effect of time, treatment, and a Time 6 Treatment interaction (Sokal and Rohlf, 1995). Homogeneity of variance was tested using Bartlett’s test. All statistical analyses were performed using STATISTICA (Statsoft, 1994). RESULTS
FIG. 1. Experimental cuvet for the quantification of phototactic behavior. Test animals (10) are placed in the central cylinder, which is externally divided into four compartments (U, M1, M2, and L) of 2.5 cm. Top (1) and bottom (2) of the central cylinder are closed with plankton gauze. During an experiment the cuvet is placed in a light-tight PVC box and illuminated from above with a fiber light source.
(UþM1þM2þL), averaged over the last 5 min of the experiment to minimize influences of the initial flight reaction. Values of the phototactic index can range from þ1 for extremely positive to 71 for extremely negative phototactic behavior. All experiments were done in a constant temperature room (20+18C), in early afternoon (start 1300–1500 h) to minimize possible effects of an endogenous rhythm (the effect of an endogenous rhythm was, however, found to be negligible in positive phototactic clones; see De Meester, 1993). In the repeated light exposure experiment, the animals were adapted for 3 h to the ISO medium, without food or pollutants added, prior to the experiment. They were then pipetted into the cuvet, and the flow-through was switched on, gradually replacing the old medium in the cuvet with fresh test medium. Before the first observation period of 10 min, the animals were given a dark adaptation of 20 min. Subsequently, the light was switched on and observations were made. After 10 min, the animals were again given a dark adaptation for 20 min. In this way, a 10-min observation period was given every half-hour for a period of 6 h. Each experiment thus consisted of 12 repeated
Figure 2 presents the changes in phototactic behavior of D. magna of clone C134 after exposure to 0.06 mg/L Cd2þ for a total period ranging from 0 to 6 h prior to the experiment. The phototactic response of clone C134 can be used to detect sublethal concentrations of cadmium: the animals reveal a highly significant response to the sublethal concentration of Cd2þ, with the values of phototactic index I decreasing with increasing exposure time to the pollutant (df = 6,21; MS effect = 0.0896; MS error = 0.009; F = 9.913; P50.0001). Contrast analysis after one-way ANOVA indicates that there is a significant difference in phototactic behavior of animals exposed to 0.06 mg/L
FIG. 2. Phototactic behavior (mean value of I+2 SE; n=4) of adult Daphnia magna clone C134 exposed for different periods (1–6 h) to 0.06 mg/L Cd2þ (LC50 24 h)= 0.31; Dave et al., 1980).
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Cd2þ with the control treatment (0 h exposure) after 4 h exposure to the pollutant. Figure 3 presents the results of the experiments with repeated light stimulation. Repeated-measures ANOVA indicates that there is a highly significant effect of treatment (presence or absence of cadmium; P = 0.026) and time (P50.001) on the phototactic behavior of the animals, but no Time6Treatment interaction effect (P = 0.164). In the absence of cadmium, the phototactic behavior of clone C134 remains fairly positively phototactic after repeated light stimulation (the value of I remains higher than 0.6 for the first 4.5 h). The phototactic index in the control treatment varied from 0.76 (0.5 h) to 0.5 (6 h), whereas the value of the phototactic index in the cadmium treatment ranged from 0.60 (0.5 h) to 0.08 (6 h). The absence of a significant Treatment6Time interaction effect indicates that time has a similar effect on both the control and the pollutant treatment: although the effect of the presence of pollutant is highly significant, the difference between the control and the pollutant treatment does not increase with exposure time to the pollutant.
DISCUSSION
Results confirm the stable and predictable phototactic behavior of clone C134 in the absence of pollutants and fish chemicals (see De Meester, 1993; Michels et al., 1999) and confirm the potential to use phototactic behavior of this clone to detect sublethal levels of pollutant (Michels et al., 1999). Michels et al. (1999) reported that the detection limit for Cu2þ and pentachlorophenol using changes in phototactic behavior of clone C134 was 0.045 mg/L for Cu2þ and 0.8 mg/L for PCP, both values being lower than the LC50 (24 h) Michels et al., 1999). The cadmium
concentration used in the present experiments (0.06 mg/L Cd2þ) is only about one-fifth of the LC50 (24 h) value reported by Dave et al. (1980) and is thus clearly sublethal for D. magna. Present results, therefore, indicate that the clone used in the present study might be suitable for use in a continuous biomonitor, since its phototactic behavior under standard conditions is stable enough to have a reasonably low intrinsic variability in behavior during the course of time. Both in the 10-min experiments and in the repeated light stimuli experiment, a highly significant effect was observed of the presence of pollutant on the phototactic behavior of the animals. In the 10-min experiments, this effect was significant after 4 h of exposure to the pollutant, whereas the difference was observed almost immediately (within 1 h) in the experiments with continuous flow-through and repeated light stimuli. Strikingly, a dosage effect was not observed in the repeated light stimuli experiment, as there is no different effect of time (exposure time) on the phototactic behavior of the animals exposed to cadmium. This result cannot be explained, but it is likely that the phototactic behavior of the control animals became slightly less positively phototactic at the end of the experimental period due to a hunger effect. At the end of the experiment, the animals had been without food for 9 h. In a real biomonitor, it might be advisable to feed the animals during the assessment periods, even though the addition of food may change the bioavailability of pollutants in the medium. Alternatively, one could feed the animals daily for a limited period of time. During that period, no readings can be taken from the particular set-up, but continuous monitoring can be ensured by having two set-ups run in parallel, and feeding the animals at different times of the day.
CONCLUSION
FIG. 3. Phototactic behavior (mean value of I + 2 SE; n = 6) of adult Daphnia magna clone C134 after different exposure times to 0.06 mg/ L Cd2þ (open symbols) and under control conditions (solid symbols) in the experiments with repeated light stimulation.
After 4 h of exposure to 0.06 mg/L cadmium, the phototactic behavior of clone C134 became significantly less positively phototactic, compared to animals in the control condition (not exposed to cadmium). In absence of pollutants and fish chemicals, the phototactic behavior of clone C134 is stable and predicable. In longer experiments with continuous flow-through and repeated stimulation of the animals, the phototactic behavior of clone C134 was significantly different from those of animals in control conditions within 1 h of exposure to 0.06 mg/L cadmium. The results from this study suggest that changes in the phototactic behavior of D. magna clone C134 can be used to detect sublethal concentrations (0.06 mg/L) of cadmium within a few hours, in short term as well as in experiments with repeated light stimuli, similar to the conditions in an operational biomonitor.
EFFECTS OF Cd ON PHOTOTACTIC BEHAVIOR OF D. magna
ACKNOWLEDGMENTS The authors thank Prof. C. Vandecasteele for verifications of the cadmium concentrations in the test medium and J. Vingerhoeds for help with Fig. 1. E. Michels is a fellow of the Flemish Institute for the promotion of Scientific–Technological Research in Industry (I.W.T.). The authors also thank F. Van Hoof, M. Leynen, and an anonymous referee for constructive comments on an earlier version the manuscript.
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