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Phototactic behavior of Daphnia as a tool in the continuous monitoring of water quality: Experiments with a positively phototactic Daphnia magna clone
PII: S0043-1354(98)00213-9
Wat. Res. Vol. 33, No. 2, pp. 401±408, 1999 # 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0043-1354/98 $19.00 + 0.00
PHOTOTACTIC BEHAVIOR OF DAPHNIA AS A TOOL IN THE CONTINUOUS MONITORING OF WATER QUALITY: EXPERIMENTS WITH A POSITIVELY PHOTOTACTIC DAPHNIA MAGNA CLONE M E. MICHELS*, M. LEYNEN* , C. COUSYN, L. DE MEESTER and F. OLLEVIER
Laboratory of Ecology and Aquaculture, Katholieke Universiteit Leuven, Naamsestraat 59, 3000 Leuven, Belgium (First received October 1997; accepted in revised form April 1998) AbstractÐWe tested the hypothesis that the use of a positively phototactic Daphnia magna clone can reduce the background noise and thereby increases the sensitivity of a continuous biomonitor that uses changes in phototactic behavior as a variable for the detection of stress caused by pollutants. We selected D. magna genotypes with a stable and predictable phototactic behavior in presence and absence of ®sh kairomones. Two clones were positively phototactic in absence of ®sh kairomones (clone C134 and C1242), and two clones (C1242 and C131) showed no signi®cant change in behavior in response to ®sh kairomones. Using a simple experimental set-up, we quanti®ed the changes in phototactic behavior of adult animals of the positive phototactic clone C134 in response to two standard pollutants, Cu2+ and pentachlorophenol (PCP). Sublethal concentrations of Cu2+ and PCP in ISO standard medium resulted in a linear decrease of the phototactic behavior of clone C134 after 3.30 h. The detection limit for changes in phototactic behavior of clone C134 was 0.045 mg lÿ1 for Cu2+ and 0.8 mg lÿ1 for PCP. # 1998 Elsevier Science Ltd. All rights reserved Key wordsÐbiomonitoring, water quality, phototaxis, Daphnia, copper, pentachlorophenol, behaviour, ®sh kairomones
INTRODUCTION
A well designed program for the prevention of surface water pollution contains two aspects of ecotoxicological research (Calow, 1993). Whereas anticipating tests such as single-species and multiple species tests are necessary to asses the degree to which chemical compounds may aect ecological systems, there is also a need for the assessment of the current pollution level of a system. Continuous monitoring of water quality has recently attracted growing attention because it provides a way to continuously assess the overall water quality. As such, it may be vital to protect amongst others, water supplies. Continuous monitoring can be realized by means of biomonitors, in which a physiological or behavioral response of a living organism is used as a variable to estimate the overall water quality (Kramer and Botterweg, 1991). Contrary to semicontinuous chemical monitoring, the response of living organisms employed in biomonitoring integrates global water quality, since living organisms react to a multitude of chemical compounds. In addition, the use of biological responses has the ad*Author to whom all correspondence should be addressed. [Tel: +32-16-323966; Fax: +32-16-324575; E-mail: [email protected]]. 401
vantage that bioavailability is taken into consideration. The eectiveness of a biomonitor for the detection of stress caused by pollutants depends on the intrinsic variability of the response that is monitored and the degree to which the response is in¯uenced by other stress factors. In addition, the kind of test organism used is of high importance since the biology of the test organism determines the ecological conditions under which a measurable response to pollutants can be observed. Daphnia has been shown to be very sensitive to the presence of chemicals in the environment, and to respond to the presence of pollutants with a multitude of traits (e.g. Dodson and Hanazato, 1995). To evaluate the sensitivity of a given biological response to stress caused by pollutants, a solid knowledge of the changes in response caused by other environmental factors is necessary. During the last decades, several biological monitoring systems have been developed based on behavioral responses of organisms of dierent trophic levels (Borcherding and Volpers, 1994). A number of studies has suggested the use of phototactic behavior of Daphnia for the detection of sub-lethal concentrations of toxic compounds (Flickinger et al., 1982; Di Delupis and Rotondo, 1988). Daphnia (Crustacea: Anomopoda; Fryer, 1987) are a major
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component of freshwater ecosystems. Since Daphnia species and D. magna in particular are standard organisms in ecotoxicological research, toxicity data for Daphnia to all kind of toxic compounds are abundant (Baudo, 1987; Persoone and Janssen, 1993). So far, two continuous monitors using Daphnia have already been developed: a monitor based on the swimming activity of D. magna (Knie, 1978) and a monitor based on changes in phototactic behavior of D. magna (Kerren, 1991). The latter was recently improved using video registration and digital image processing (Van Hoof et al., 1994). Technological optimalization can, however, only lead to an eective and operational monitor if background variability in the behavior can be minimized and interferences from relevant ecological factors on the behavior can be taken into account. A phototactic response is an orientated reaction to light stimuli (Ringelberg, 1964, 1987). Using a quantitative genetic approach, De Meester (1991) showed that primary phototaxis (the response to a constant gradient in light intensity) is a heritable trait in Daphnia magna, and distinguished between positively, intermediately and negatively phototactic genotypes. The behavior of juveniles is less clone speci®c than that of adults (De Meester, 1992). Although phototactic behavior is a highly heritable trait under standardized conditions, the behavior can be modi®ed by environmental factors. Temperature can have an important eect on phototactic behavior and the vertical distribution of Daphnia (Calaban and Makarewicz, 1982), but the behavior of negatively and positively phototactic clones is less sensitive to changes in temperature than that of intermediately phototactic clones (Van Uytvanck and De Meester, 1990). Van Uytvanck and De Meester (1990) also showed that Daphnia magna exhibits a more positive phototactic response when the pH of the medium is raised to pH = 9. Food concentration does not in¯uence phototactic behavior over a wide range (5104±5105 Scenedesmus acutus cells mlÿ1), but the animals become less positively phototactic when food level is dropped below 5104 cells mlÿ1 (De Meester and Dumont, 1988). In addition, it has recently become clear that Daphnia is able to change its morphology, life history and behavior in response to the presence of infochemicals produced by their predators (reviewed by Larsson and Dodson, 1993; Dodson and Hanazato, 1995). Daphnia may become negatively phototactic in presence of ®sh kairomones and more positively phototactic in presence of kairomones produced by invertebrate predators such as Chaoborus larvae (Dodson, 1988; Ringelberg, 1991; De Meester, 1993a; Nesbitt et al., 1996). The behavioral changes induced by the presence of predator kairomones are often very pronounced (see De Meester, 1993a; Loose, 1993), but De Meester (1993a, 1996a) has shown that there are interclonal dierences with respect to these phenotypic plas-
ticity changes. Whereas some D. magna clones show a striking change in phototactic behavior in presence of ®sh kairomones, others do not alter their behavior in presence of ®sh kairomones. Environmental factors (temperature, food concentration, the presence of vertebrate and invertebrate predators) are liable to continuous variation in nature and their in¯uence on phototactic behavior should be taken into account in the development of a continuous monitor that is meant to be used for the monitoring of surface waters. There are basically three ways to accomplish this. First, one can employ dynamic modelling to take the environmental variability into account when analyzing the data (Berckmans et al., in prep.). If one knows which environmental factors interfere with the variable measured, one can also alter the monitor in such a way that the natural variation in this variable is reduced. For instance, given that low oxygen concentration induces a slight change in phototactic behavior (Cousyn and De Meester, in prep), one can aerate the water prior to its passage through the biomonitor, such that the water is always saturated with oxygen. The third option is that one selects genotypes that are rather insensitive to changing environmental conditions, without being less sensitive to stress caused by pollutants. Daphnia magna reproduces by cyclical parthenogenesis (Hebert, 1987; De Meester, 1996b), and under favorable conditions, females reproduce by amictic parthenogenesis, resulting in the formation of clones. Under unfavorable environmental conditions (e.g. low food concentration, high population density, presence of ®sh kairomones, changes in photoperiod) the animals reproduce sexually (Hebert, 1987, Zaagnini, 1987). Clonal test organisms create the opportunity for supplementary standardization of ecotoxicological tests (Baird et al., 1989, 1990). With respect to the development of a biomonitor using a behavioral response, the clonal reproduction of Daphnia also creates the opportunity to select for clones with a relatively low sensitivity to environmental changes other than the concentration of pollutants. Positively phototactic D. magna clones exhibit a highly repeatable behavior that is rather insensitive to a variety of environmental factors (De Meester and Dumont, 1988; Van Uytvanck and De Meester, 1990; De Meester, 1991, 1993b), indicating that the use of positively phototactic clones may reduce background noise interfering with the detection of pollutants in a biomonitor. Although it has been shown that positively phototactic Daphnia clones may be highly sensitive to the presence of ®sh kairomones (De Meester and Cousyn, 1997), the presence of genetic variation in the response to ®sh kairomones (De Meester, 1993a, 1996a) indicates that clones may be selected for that do not change their phototactic behavior dramatically in the presence of ®sh kairomones. We here report on experiments
Phototactic behavior of Daphnia
designed to reduce the intrinsic variability of the behavior of the test animals. The present study has two aims: (1) the selection of Daphnia magna genotypes with stable and predictable behavior in the presence and absence of ®sh kairomones, and (2) the determination of the sensitivity of the positively phototactic clone C134 to two standard pollutants, Cu2+ and pentachlorophenol (PCP).
MATERIAL AND METHODS
Selection of genotypes with repeatable phototactic behavior We choose to work with positively phototactic Daphnia magna clones because their behavior is highly repeatable under a variety of environmental conditions (temperature, acidity of the medium, food concentration; De Meester and Dumont, 1988; Van Uytvanck and De Meester, 1990; De Meester, 1993b), and because the presence of pollutants is expected to result in a less rather than in a more positively phototactic behavior. Previous work has shown that clone C134 shows a relatively weak change in phototactic behavior in the presence of ®sh kairomones (De Meester, 1993a). We screened six sister clones of C134 for their phototactic behavior in the absence and presence of ®sh kairomones to check whether we could detect clones that showed an even more stable and repeatable behavior than clone C134. C134 and its sister clones (C131, C1149, C155, C1158, C1240 and C1242) were obtained through intraclonal mixes of clone C1, which was isolated in 1989 as a single female from a small ®shless city pond in Gent (Citadelpark, Small Pond). The sister clones resulted from the hatching of resting eggs that were collected after sexual reproduction within cultures of clone C1. From a genetical point of view, intraclonal mixes in Daphnia is equivalent to sel®ng, as males and parthenogenetic females are genetically identical (Hebert, 1987). For more details on the methods to obtain intraclonal ospring, we refer to De Meester (1991). For comparison, we included the positively phototactic clone P132,85 in our analysis. Clone P132,85 was obtained from two generations of intraclonal mixes within clone P1, isolated from a small pond which contained ®sh (Driehoeksvijver, Heusden, isolated in august 1986). Clone P132,85 is known to become negatively phototactic in the presence of ®sh chemicals (De Meester, 1993a). There were two treatments: animals cultured in the absence and in the presence of ®sh kairomones. For each clone-treatment combination, at least four experiments were carried out. Each experiment was carried out with animals from a dierent generation or culture, to obviate common environment eects. All animals were cultured at 208C (218) and in a long-day photoperiod (14 h light±10 h dark).
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Animals were cultured in one liter jars. Density was kept relatively low: 20±30 individuals lÿ1. Food concentration was daily restored to approximately 2105 Scenedesmus acutus cells mlÿ1, which is above the incipient limiting level (Lampert, 1987). Daily, one fourth of the culture medium was replaced by an equivalent volume of fresh medium (either dechlorinated tap water or ®ltered ®sh conditioned medium). Daily refreshment of the medium was necessary, as ®sh chemicals may loose their activity in 24 h due to bacterial degradation (Larsson and Dodson, 1993). Fish conditioned medium was prepared by allowing two Leuciscus idus of approximate 8 cm length, to swim in a 60 l aquarium ®lled with dechlorinated tap water. Each day, 12 l of the medium was ®ltered over 40 mm. The density of ®sh in the ®sh conditioned medium was very high, corresponding to about one ®sh in 120 l (only one fourth of the medium was refreshed daily). To exclude maternal eects, all clones were cultured under the experimental conditions for two generations prior to the experiment (Lynch and Ennis, 1983). Dose±response experiments All dose±response experiments were carried out with animals of the positively phototactic D. magna clone C134. We used adults of 10±15 d old, carrying their second clutch. Culture conditions were identical to those described above for the screening of the dierent clones. Changes in phototactic behavior of clone C134 where tested after 3.30 hours exposure to sub-lethal concentrations of the test-pollutants (Cu2+ and PCP) in arti®cial medium without food. All experiments were conducted in arti®cial medium (ISO, 1989) with the following composition: 0.294 g CaCl22H2O, 0.123 g MgSO47H2O, 0.065 g NaHCO3 and 0.006 g KCl pro liter, pH adjusted to 7.9 with 1 N NaOH or 1 N HCl. Experiments were carried out in the absence of food because this made the analysis of the dose±response relationship more straightforward, since the bioavailability of the pollutants was not aected by adsorption to food particles. In an operational biomonitor, however, the behavioral responses will be determined by ambient bioavailability of the pollutants, and will thus be in¯uenced by the adsorption of the pollutants to suspended particles. The absence of food has no eect on the phototactic behavior of Daphnia magna in the present set-up, at least not as long as experiments do not last long (>5 h; De Meester, pers. obs.). The following nominal copper concentrations were applied: 0; 0.005; 0.01; 0.02; 0.04 mg lÿ1 Cu2+. All test solutions were made in ISO arti®cial medium out of a stock solution with a 1000-fold pollutant concentration. Stock-solutions for copper were prepared daily by adding the required amount of CuSO45H2O into 1 l demineralized water (Millipore). The exact copper concen-
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Fig. 1. Phototactic behavior (mean value I22 SE) of Daphnia magna clones cultured for one generation in the absence and presence of ®sh kairomones. All clones except P132,85 are sister-clones produced by intraclonal reproduction from clone C1.
tration was veri®ed by atomic absorption on samples taken after the experiments. Stock-solutions of PCP were prepared in acetone (400 mg PCP in 4 ml acetone). Final PCP test solutions were prepared in 1 l arti®cial medium and adjusted to pH 7.9 (Liber and Solomon, 1994). The test solutions in the PCP control experiments contained 4 ml of acetone to verify for any eect of acetone on phototactic behavior. Nominal concentrations of 0, 0.2, 0.4, 0.8 and 1.6 mg lÿ1 PCP were applied. Three to four experiments, each with ten animals, were carried out for every pollutant concentration. Dierent animals from dierent cultures or generations were used in the replicates to minimize common environment eects and thus prevent pseudoreplication (Lynch and Ennis, 1983; Hurlbert, 1984). Experimental set-up and protocol The experimental set-up is basically the same as described in De Meester (1991, 1993a), though the experimental cuvet is somewhat modi®ed. The experimental cuvet consisted of two glass columns, the inner of which was 10 cm high and had an internal cross-section of 5 cm. It had a bottom and top made of plankton gauze and functioned as the test chamber. The test chamber 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 column is placed in a light-tight PVC box (16 28 35 cm, provided with a peep-hole of b 1 cm for observations) and illuminated from above with a ®ber light (150 W halogen light source) positioned two cm above the water surface in the outer column. An experiment lasted 10 min. At one minute intervals, the number
of animals in each compartment was counted by looking through the small peep-hole in the PVC-box. The phototactic behavior of the test population was characterized by the index I = (U ÿ L)/(U + M1+M2+L). I was averaged over the last ®ve minutes of the experiment to minimize in¯uences of the initial ¯ight reaction (De Meester, 1993b). Values for the phototactic index can range from 1 for extremely positive behavior to ÿ1 for extremely negative phototactic behavior. All experiments were done in a temperature-buered room (208C 218C), in early afternoon (start 13.00±15.00 h) to minimize possible eects of an endogenous rhythm (though the eect of an endogenous rhythm was found to be negligible in positive phototactic clones; De Meester, 1993b). Prior to an experiment, the animals were adapted for three hours to the test solution. They were then pipetted into the cuvet and given a dark adaptation of 20 min. Subsequently, the light was switched on for the ®rst observation period of 10 min. After the ®rst observation period, the animals were again dark adapted for 20 min and subjected to light for 10 min. As such, a 10 min observation period was given every half an hour for a period of two hours. Each experiment thus consisted of four repeated observations.
Statistical analysis Data on the response of dierent clones to the presence of ®sh kairomones were analyzed by two-way ANOVA, with both clone and treatment as ®xed eects. One-way ANOVA and linear regression was carried out to test for signi®cant eects of pollutant concentration on phototactic behavior (Sokal and Rohlf, 1995). Only the data of the ®rst observation period were used in the one-way ANOVA. Homogeneity of variance was tested using Bartlett's test. Detection limits were determined by Tukey multiple comparisons after one-way ANOVA. All statistical analyses were preformed using the statistical computing package STATISTICA (Statsoft, 1994).
Phototactic behavior of Daphnia
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Table 1. Two-way ANOVA table with ®xed eects of the presence of ®sh kairomones on the phototactic behavior of D. magna clones C134; C1149; C1155; C1158; C1240; P132,85 Eect
df
MS eect
df error
MS error
F
p-level
Clone Treatment Clone*treatment
5 1 5
1.32 9.72 1.01
49 49 49
0.41 0.41 0.41
3.21 23.57 2.44
0.014 0.000 0.047
RESULTS
The eect of ®sh kairomones The phototactic behavior of clone C134, clone P132,85 and six sister clones of C134 in the presence and absence of ®sh chemicals is plotted in Fig. 1. All clones are characterized by an intermediately to positively phototactic behavior in the absence of ®sh kairomones. The presence of ®sh kairomones induced a more negatively phototactic response in all D. magna clones tested except clone C131 and C1242. Two way ANOVA showed a signi®cant clone by treatment interaction (Table 1). Dierences in phototactic behavior of each clone in control vs ®sh conditioned medium were tested by means of contrast analysis. Clones C134, C1149, C1155, C1158, C1240 and P132,85 showed a signi®cant change in phototactic behavior in the presence of ®sh chemicals (p < 0.05), whereas clone C131 (intermediately phototactic) and C1242 (positively phototactic) showed no signi®cant change in behavior in response to the presence of ®sh kairomones (Fig. 1). The in¯uence of pollutants Figure 2 presents the linear regression between the values for the phototactic index (I) and the Cu2+ concentrations in the test solution (as determined by atomic absorption), of animals that were exposed for 3 h to the pollutant prior to the experiment. Clone C134 shows a clear response to sublethal concentrations of Cu2+, and values for I are
decreasing linearly with increasing Cu2+ concentration. To check whether the response is repeatable upon repeated stimulation by light, we also analyzed the relation between the phototactic behavior and the concentration of copper after 4 h, 4.30 h, and 5 h exposure. Regression coecients are given in Table 2. The slopes of all regression lines are signi®cantly dierent from zero. The relationship is strongest after 4 hours of exposure (R2=0.64). On average, there is a decrease of the value I with 0.11 for every increase of 10 ppm Cu2+ in the test solution. In the absence of pollutants, clone C134 was positively phototactic. In order to determine the resolution with which Cu2+ can be detected using phototactic behavior of clone C134, and because the veri®ed concentrations of Cu2+ in the samples did not correspond completely with the nominal Cu2+ concentrations, we grouped our 3.30 h exposure data into four concentration intervals (0.005±0.015; 0.016±0.025; 0.026±0.035; 0.036±0.05 mg lÿ1 Cu2+) and performed a one way ANOVA. There is a signi®cant eect of pollutant on the phototactic behavior of clone C134 (df: 3; MS group: 0.145; MS error: 0.018; F: 7.966; P < 0.01). Tukey post hoc comparisons show that the animals exhibited a signi®cantly less positively phototactic behavior at a concentration of 0.036±0.05 mg Cu2+ lÿ1 compared to lower concentrations. The in¯uence of sub-lethal concentrations of PCP on the phototactic behavior of D. magna clone C134 was investigated after 3.30 hours adaptation to the pollutant. Figure 3 shows that the index I is
Fig. 2. Phototactic behavior (value I) of adult D. magna clone C134, after 3.30 h exposure to Cu2. Regression line and 95% con®dence limits of the value I in relation to the exact Cu2+ concentration (veri®ed by atomic absorption). LC50 (48 h) = 0.054 mg Cu2+ lÿ1 (Mount and Norberg, 1984).
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Table 2. Regression analysis of dose response experiments with clone C1 34 being exposed during dierent periods (3.30 h, 4 h, 4.30 h and 5 h) to a given Cu2+ concentration (nominal concentrations: control, 0.01, 0.02, 0.04 mg lÿ1 Cu2+) or PCP concentration (nominal concentrations: control (4 ml acetone), 0.2, 0.4, 0.8, 1.6 mg lÿ1 PCP). N = number of experiments
Cu
2+
PCP
Exposure time
N
R2
p-level
3.30 h 4h 4.30 h 15 h 3.30 h
18 18 18 18 20
0.51 0.64 0.49 0.39 0.67
<0.001 <0.001 <0.00 <0.001 <0.001
decreasing linearly with increasing PCP concentration. The slope of the regression line of the phototactic behavior against PCP-concentration is signi®cantly dierent from zero and explains 67% of the variance (Table 1). One-way ANOVA indicates that there is a signi®cant eect of PCP on the phototactic behavior of clone C134 (df: 4; MS group: 1.365; MS error: 0.105; F: 13.032; p < 0.001). Tukey pairwise comparisons indicate that clone C134 behaves signi®cantly more negatively phototactic at a concentration of 0.8 mg lÿ1 PCP or more compared to lower concentrations. The presence of 4 ml acetone lÿ1 does not in¯uence the phototactic behavior of D. magna clone C134, as the animals were positively phototactic in the control experiment. DISCUSSION
Our results con®rm the stable and predictable positively phototactic behavior of clone C134 in the absence of pollutants and ®sh chemicals (see De Meester, 1993a). A survey of sister clones of C134 yielded two clones that showed no signi®cant change in phototactic behavior in presence of ®sh kairomones. The use of a standard positively phototactic clone may improve the potential of a biomonitor based on phototactic behavior of Daphnia considerably. First, some positively phototactic ani-
mals have a stable and repeatable behavior under a wide range of ecological conditions in the absence of pollutants (De Meester and Dumont, 1988; Van Uytvanck and De Meester, 1990; De Meester, 1993b; present study). Secondly, the fact that their behavior is extremely positive phototactic under standard conditions makes it easier to detect stress caused by pollutants. The present study indeed shows that, at least for clone C134, the presence of two standard pollutants induces a more negatively phototactic behavior. As an additional advantage, working with one genotype results in a standardized sensitivity to pollutants (Baird et al., 1989, 1990). The short 10 min visual experiments reported in the present study indicate that the sensitivity of a test system using phototactic behavior of Daphnia is rather high. The LC50 (48 h) value for Cu2+ is reported to be 0.054 mg lÿ1 (Mount and Norberg, 1984) and that for PCP 1.23 mg lÿ1 (Liber and Solomon, 1994), whereas we were able to detect lower concentrations (65±83%) after 3.30 hours of exposure to the pollutant. The dynamic Daphnia monitor of Knie (1978) is able to detect pesticides at concentrations around LC50 (24 h) (see Matthias and Puzichia, 1990). Although juveniles are more sensitive to toxic compounds than adults (Persoone and Janssen, 1993), it may be dicult to work with juveniles in a biomonitor using phototactic behavior because the phototactic behavior of juveniles is less
Fig. 3. Phototactic behavior (value I) of adult D. magna clone C1 34, after 3.30 h exposure to a certain PCP-concentration (nominal concentrations: 0 (4 ml acetone lÿ1), 0.2, 0.4, 0.8, 1.6 mg lÿ1). Regression curve and 95% con®dence limits of the value I in relation to the PCP concentration. LC50 (48 h) = 1.23 mg PCP lÿ1 (Liber and Solomon, 1994).
Phototactic behavior of Daphnia
clear-cut positively phototactic than that of adults (De Meester, 1992). Technically, the method used is very crude. As our experiments are simple and short, we were able to carry out a reasonably large number of experiments to verify the repeatability of the phototactic behavior under standardized conditions and under pollutant stress. Our visual counting method has, however, a limited resolution. Image-analysis using video cameras may detect dierences in vertical position of the animals with a resolution of less than 0.1 mm (J. M. Aerts, pers. comm.). This may potentially further increase the detection capacity of the biomonitor. AcknowledgementsÐWe thank Dr. F. Van Hoof and the Antwerpse Water Werken for the veri®cation of the copper concentration in the test medium. This study was ®nancially supported by the Flemish government (VLIM project H/9410). EM is a fellow of the ¯emish Institute for the promotion of Scienti®c±Technological Research in industry (I. W. T.). Part of this study was carried out while LDM and CC were associated with the Laboratory of Animal Ecology, University of Gent. We thank F. Van Hoof and three anonymous reviewers for constructive comments on an earlier version of the manuscript.
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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, eds H. Lokke, H. Tyle and F. Bro-Rasmussen, pp. 204± 207, Technical University Lynby. 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 eects. Funct. Ecol. 4, 399±407. Baudo, R. (1987) Ecotoxicological testing with Daphnia. In Daphnia, eds R. H. Peters and R. de Bernardi, Mem Ist. Idrobiol. Ital., Pallanza, 45, 461±482. Borcherding J. and Volpers M. (1994) The Dreissenamonitor: First results on the application of this biological warning system in continuos monitoring of water quality. Water Sci. Tech. 29, 199±201. Calaban M. J. and Makarewicz J. C. (1982) The eect of temperature and density on the amplitude of vertical migration of Daphnia magna. Limnol. Oceanogr. 27, 262± 271. Calow P. (1993) General principles an overview. In Handbook of ecotoxicology, ed. P. Calow, pp. 1±15. Blackwell Scienti®c Publ., London. De Meester L. (1991) An analysis of the phototactic behaviour of Daphnia magna clones and their sexual descendants. Hydrobiologia 225, 217±227. De Meester L. (1992) The phototactic behaviour of male and female Daphnia magna. Anim. Behav. 43, 696±698. De Meester L. (1993a) Genotype, ®sh-mediated chemicals, and phototactic behaviour in Daphnia magna. Ecology 74, 1467±1474. De Meester L. (1993b) Phototactic behaviour in Daphnia magna. Academiae Analecta 55, 145±187. De Meester L. (1996a) Evolutionary potential and local genetic dierentiation in phenotypically plastic trait of cyclical parthenogen Daphnia magna. Evolution 50, 1293±1298.
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De Meester L. (1996b) Local genetic dierentiation and adaptation in freshwater zooplanktonpopulations: Patterns and processes. Ecoscience 3, 385±399. De Meester L. and Cousyn C. (1997) The change in phototactic behaviour of a Daphia magna clone in the presence of ®sh kairomones: The eect 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. Ecotox. Environ. Safety 16, 189±193. Dodson S. I. (1988) The ecological role of chemical stimuli for the zooplankton: Predator-avoidance behavior in Daphnia. Limnol. Oceanogr. 33, 1431±1439. Dodson S. I. and Hanazato T. (1995) Commentary on eects of anthropogenic and natural organic chemicals on development, swimming behaviour, and reproduction of Daphnia, a key member of aquatic ecosystems. Environ. Health Persp. 103(Supplement 4), 7±11. Flickinger A. L., Bruins R. J. F., Winner R. W. and Skillings J. H. (1982) Filtration and phototactic behaviour as indices of chronic copper stress in Daphnia magna Straus. Arch. Environ. Toxicol. 11, 457±463. Fryer G. (1987) Morphology and classi®cation of the socalled Cladocera. Hydrobiologia 145, 19±28. Hebert, P. D. N. (1987) Genetics in Daphnia. In Daphnia, eds R. H. Peters and R. de Bernardi, Mem. Ist. Idrobiol. Ital., Pallanza, 45, 439±460. Hurlbert S. H. (1984) Pseudoreplication and the design of ecological ®eld experiments. Ecol. Mon. 54, 187±211. ISO (1989) Water quality ± Determination of the inhibition of the mobility of Daphnia magna Straus (Cladocera, Crustacea). ISO 6341, 01.10.1986. Kerren, K. (1991) Aqua-Tox-Control-Daphnia. Kerren Umwelttechnik, Viersen. Knie J. (1978) Der Dynamischen Daphnientest ± ein automatischer Biomonitor zur Uberwachtung von GewaÈssern und AbwaÈssern. Wasser und Boden 12, 310± 312. Kramer, K. J. M. and Botterweg, J. (1991) Aquatic biological early warning systems: An overview. In Bioindicators and environmental management, eds D. W. Jerey and B. Madden, pp. 95±126, Academic Press, London. Lampert, W. (1987) Feeding and nutrition in Daphnia. In Daphnia, eds R. H. Peters and R. de Bernardi. Mem Ist. Idrobiol ital., Pallanza, 45, 143±192. Larsson P. and Dodson S. (1993) Chemical communication in planktonic animals. Arch. Hydrobiol. 129, 129±155. Liber K. and Solomon K. R. (1994) Acute and chronic toxicity of 2,3,4,6-tetrachlorophenol and pentachlorophenol to Daphnia and rotifers. Arch. Environ. Contam. Toxicol. 26, 212±221. Loose C. (1993) Daphnia diel vertical migration behavior: Response to vertebrate predator abundance. Arch. Hydrobiol. Ergebn. Limnol. 39, 129±155. Lynch M. and Ennis R. (1983) Resource availability, maternal eects and longevity. Exp. Geront. 18, 147±165. Matthias U. and Puzichia H. (1990) Erfahrungen mit den Dynamischen Daphnientest ± Ein¯uss von Pestiziden auf das Swimmverhalten von Daphnia magna under Labor- und Praxisbedingungen. Z. Wasser-Forsch. 23, 193±198. Mount D. I. and Norberg T. J. (1984) A seven day lifecycle cladoceran toxicity test. Environ. Toxicol. Chem. 3, 425±434. Nesbitt L. M., Riessen H. P. and Ramcharan C. W. (1996) Opposing predation pressures and induced vertical mi-
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gration responses in Daphnia. Limnol. Oceanogr. 41, 1306±1311. Persoone, G. and Janssen, C. R. (1993) Freshwater invertebrate toxicity tests. In Handbook of ecotoxicology, ed. P. Calow, pp. 51±65. Blackwell Scienti®c Publ., London. Ringelberg J. (1964) The positively phototactic reaction of Daphnia magna Straus. ± A contribution to the understanding of diurnal migration. Neth. J. Sea Res. 2, 319± 406. Ringelberg, J. (1987) Light induced behaviour in Daphnia. In Daphnia, eds R. H. Peters and R. de Bernardi, Mem Ist. Idrobiol. Ital. Pallanza, 45, 285±323. Ringelberg J. (1991) Enhancement of the phototactic behaviour in Daphnia by a chemical mediated by juvenile prech (Perca ¯uviatilis). J. Plankton Res. 12, 17±25.
Sokal, R. and Rohlf, F. J. (1995) Biometry, third edition. W. H. Freeman and Company, New York. Statsoft (1994) STATISTICA for the windows operating system. Statsoft inc. Tulsa. Van Hoof F., Sluyts H., Paulussen J., Berckmans D. and Bloemen H. (1994) Evaluation of a biomonitor based on the phototactic behaviour 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 in¯uence of temperature and acidity on the phototactic behaviour of Daphnia genotypes. J. Plankton Res. 12, 1089±1097. Zaagnini, J. (1987) Reproduction in Daphnia. In Daphnia, eds R. H. Peters and R. de Bernardi, Mem. Ist. Idrobiol. Ital., Pallanza, 45, 245±284.
Wat. Res. Vol. 33, No. 2, pp. 401±408, 1999 # 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0043-1354/98 $19.00 + 0.00
PHOTOTACTIC BEHAVIOR OF DAPHNIA AS A TOOL IN THE CONTINUOUS MONITORING OF WATER QUALITY: EXPERIMENTS WITH A POSITIVELY PHOTOTACTIC DAPHNIA MAGNA CLONE M E. MICHELS*, M. LEYNEN* , C. COUSYN, L. DE MEESTER and F. OLLEVIER
Laboratory of Ecology and Aquaculture, Katholieke Universiteit Leuven, Naamsestraat 59, 3000 Leuven, Belgium (First received October 1997; accepted in revised form April 1998) AbstractÐWe tested the hypothesis that the use of a positively phototactic Daphnia magna clone can reduce the background noise and thereby increases the sensitivity of a continuous biomonitor that uses changes in phototactic behavior as a variable for the detection of stress caused by pollutants. We selected D. magna genotypes with a stable and predictable phototactic behavior in presence and absence of ®sh kairomones. Two clones were positively phototactic in absence of ®sh kairomones (clone C134 and C1242), and two clones (C1242 and C131) showed no signi®cant change in behavior in response to ®sh kairomones. Using a simple experimental set-up, we quanti®ed the changes in phototactic behavior of adult animals of the positive phototactic clone C134 in response to two standard pollutants, Cu2+ and pentachlorophenol (PCP). Sublethal concentrations of Cu2+ and PCP in ISO standard medium resulted in a linear decrease of the phototactic behavior of clone C134 after 3.30 h. The detection limit for changes in phototactic behavior of clone C134 was 0.045 mg lÿ1 for Cu2+ and 0.8 mg lÿ1 for PCP. # 1998 Elsevier Science Ltd. All rights reserved Key wordsÐbiomonitoring, water quality, phototaxis, Daphnia, copper, pentachlorophenol, behaviour, ®sh kairomones
INTRODUCTION
A well designed program for the prevention of surface water pollution contains two aspects of ecotoxicological research (Calow, 1993). Whereas anticipating tests such as single-species and multiple species tests are necessary to asses the degree to which chemical compounds may aect ecological systems, there is also a need for the assessment of the current pollution level of a system. Continuous monitoring of water quality has recently attracted growing attention because it provides a way to continuously assess the overall water quality. As such, it may be vital to protect amongst others, water supplies. Continuous monitoring can be realized by means of biomonitors, in which a physiological or behavioral response of a living organism is used as a variable to estimate the overall water quality (Kramer and Botterweg, 1991). Contrary to semicontinuous chemical monitoring, the response of living organisms employed in biomonitoring integrates global water quality, since living organisms react to a multitude of chemical compounds. In addition, the use of biological responses has the ad*Author to whom all correspondence should be addressed. [Tel: +32-16-323966; Fax: +32-16-324575; E-mail: [email protected]]. 401
vantage that bioavailability is taken into consideration. The eectiveness of a biomonitor for the detection of stress caused by pollutants depends on the intrinsic variability of the response that is monitored and the degree to which the response is in¯uenced by other stress factors. In addition, the kind of test organism used is of high importance since the biology of the test organism determines the ecological conditions under which a measurable response to pollutants can be observed. Daphnia has been shown to be very sensitive to the presence of chemicals in the environment, and to respond to the presence of pollutants with a multitude of traits (e.g. Dodson and Hanazato, 1995). To evaluate the sensitivity of a given biological response to stress caused by pollutants, a solid knowledge of the changes in response caused by other environmental factors is necessary. During the last decades, several biological monitoring systems have been developed based on behavioral responses of organisms of dierent trophic levels (Borcherding and Volpers, 1994). A number of studies has suggested the use of phototactic behavior of Daphnia for the detection of sub-lethal concentrations of toxic compounds (Flickinger et al., 1982; Di Delupis and Rotondo, 1988). Daphnia (Crustacea: Anomopoda; Fryer, 1987) are a major
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component of freshwater ecosystems. Since Daphnia species and D. magna in particular are standard organisms in ecotoxicological research, toxicity data for Daphnia to all kind of toxic compounds are abundant (Baudo, 1987; Persoone and Janssen, 1993). So far, two continuous monitors using Daphnia have already been developed: a monitor based on the swimming activity of D. magna (Knie, 1978) and a monitor based on changes in phototactic behavior of D. magna (Kerren, 1991). The latter was recently improved using video registration and digital image processing (Van Hoof et al., 1994). Technological optimalization can, however, only lead to an eective and operational monitor if background variability in the behavior can be minimized and interferences from relevant ecological factors on the behavior can be taken into account. A phototactic response is an orientated reaction to light stimuli (Ringelberg, 1964, 1987). Using a quantitative genetic approach, De Meester (1991) showed that primary phototaxis (the response to a constant gradient in light intensity) is a heritable trait in Daphnia magna, and distinguished between positively, intermediately and negatively phototactic genotypes. The behavior of juveniles is less clone speci®c than that of adults (De Meester, 1992). Although phototactic behavior is a highly heritable trait under standardized conditions, the behavior can be modi®ed by environmental factors. Temperature can have an important eect on phototactic behavior and the vertical distribution of Daphnia (Calaban and Makarewicz, 1982), but the behavior of negatively and positively phototactic clones is less sensitive to changes in temperature than that of intermediately phototactic clones (Van Uytvanck and De Meester, 1990). Van Uytvanck and De Meester (1990) also showed that Daphnia magna exhibits a more positive phototactic response when the pH of the medium is raised to pH = 9. Food concentration does not in¯uence phototactic behavior over a wide range (5104±5105 Scenedesmus acutus cells mlÿ1), but the animals become less positively phototactic when food level is dropped below 5104 cells mlÿ1 (De Meester and Dumont, 1988). In addition, it has recently become clear that Daphnia is able to change its morphology, life history and behavior in response to the presence of infochemicals produced by their predators (reviewed by Larsson and Dodson, 1993; Dodson and Hanazato, 1995). Daphnia may become negatively phototactic in presence of ®sh kairomones and more positively phototactic in presence of kairomones produced by invertebrate predators such as Chaoborus larvae (Dodson, 1988; Ringelberg, 1991; De Meester, 1993a; Nesbitt et al., 1996). The behavioral changes induced by the presence of predator kairomones are often very pronounced (see De Meester, 1993a; Loose, 1993), but De Meester (1993a, 1996a) has shown that there are interclonal dierences with respect to these phenotypic plas-
ticity changes. Whereas some D. magna clones show a striking change in phototactic behavior in presence of ®sh kairomones, others do not alter their behavior in presence of ®sh kairomones. Environmental factors (temperature, food concentration, the presence of vertebrate and invertebrate predators) are liable to continuous variation in nature and their in¯uence on phototactic behavior should be taken into account in the development of a continuous monitor that is meant to be used for the monitoring of surface waters. There are basically three ways to accomplish this. First, one can employ dynamic modelling to take the environmental variability into account when analyzing the data (Berckmans et al., in prep.). If one knows which environmental factors interfere with the variable measured, one can also alter the monitor in such a way that the natural variation in this variable is reduced. For instance, given that low oxygen concentration induces a slight change in phototactic behavior (Cousyn and De Meester, in prep), one can aerate the water prior to its passage through the biomonitor, such that the water is always saturated with oxygen. The third option is that one selects genotypes that are rather insensitive to changing environmental conditions, without being less sensitive to stress caused by pollutants. Daphnia magna reproduces by cyclical parthenogenesis (Hebert, 1987; De Meester, 1996b), and under favorable conditions, females reproduce by amictic parthenogenesis, resulting in the formation of clones. Under unfavorable environmental conditions (e.g. low food concentration, high population density, presence of ®sh kairomones, changes in photoperiod) the animals reproduce sexually (Hebert, 1987, Zaagnini, 1987). Clonal test organisms create the opportunity for supplementary standardization of ecotoxicological tests (Baird et al., 1989, 1990). With respect to the development of a biomonitor using a behavioral response, the clonal reproduction of Daphnia also creates the opportunity to select for clones with a relatively low sensitivity to environmental changes other than the concentration of pollutants. Positively phototactic D. magna clones exhibit a highly repeatable behavior that is rather insensitive to a variety of environmental factors (De Meester and Dumont, 1988; Van Uytvanck and De Meester, 1990; De Meester, 1991, 1993b), indicating that the use of positively phototactic clones may reduce background noise interfering with the detection of pollutants in a biomonitor. Although it has been shown that positively phototactic Daphnia clones may be highly sensitive to the presence of ®sh kairomones (De Meester and Cousyn, 1997), the presence of genetic variation in the response to ®sh kairomones (De Meester, 1993a, 1996a) indicates that clones may be selected for that do not change their phototactic behavior dramatically in the presence of ®sh kairomones. We here report on experiments
Phototactic behavior of Daphnia
designed to reduce the intrinsic variability of the behavior of the test animals. The present study has two aims: (1) the selection of Daphnia magna genotypes with stable and predictable behavior in the presence and absence of ®sh kairomones, and (2) the determination of the sensitivity of the positively phototactic clone C134 to two standard pollutants, Cu2+ and pentachlorophenol (PCP).
MATERIAL AND METHODS
Selection of genotypes with repeatable phototactic behavior We choose to work with positively phototactic Daphnia magna clones because their behavior is highly repeatable under a variety of environmental conditions (temperature, acidity of the medium, food concentration; De Meester and Dumont, 1988; Van Uytvanck and De Meester, 1990; De Meester, 1993b), and because the presence of pollutants is expected to result in a less rather than in a more positively phototactic behavior. Previous work has shown that clone C134 shows a relatively weak change in phototactic behavior in the presence of ®sh kairomones (De Meester, 1993a). We screened six sister clones of C134 for their phototactic behavior in the absence and presence of ®sh kairomones to check whether we could detect clones that showed an even more stable and repeatable behavior than clone C134. C134 and its sister clones (C131, C1149, C155, C1158, C1240 and C1242) were obtained through intraclonal mixes of clone C1, which was isolated in 1989 as a single female from a small ®shless city pond in Gent (Citadelpark, Small Pond). The sister clones resulted from the hatching of resting eggs that were collected after sexual reproduction within cultures of clone C1. From a genetical point of view, intraclonal mixes in Daphnia is equivalent to sel®ng, as males and parthenogenetic females are genetically identical (Hebert, 1987). For more details on the methods to obtain intraclonal ospring, we refer to De Meester (1991). For comparison, we included the positively phototactic clone P132,85 in our analysis. Clone P132,85 was obtained from two generations of intraclonal mixes within clone P1, isolated from a small pond which contained ®sh (Driehoeksvijver, Heusden, isolated in august 1986). Clone P132,85 is known to become negatively phototactic in the presence of ®sh chemicals (De Meester, 1993a). There were two treatments: animals cultured in the absence and in the presence of ®sh kairomones. For each clone-treatment combination, at least four experiments were carried out. Each experiment was carried out with animals from a dierent generation or culture, to obviate common environment eects. All animals were cultured at 208C (218) and in a long-day photoperiod (14 h light±10 h dark).
403
Animals were cultured in one liter jars. Density was kept relatively low: 20±30 individuals lÿ1. Food concentration was daily restored to approximately 2105 Scenedesmus acutus cells mlÿ1, which is above the incipient limiting level (Lampert, 1987). Daily, one fourth of the culture medium was replaced by an equivalent volume of fresh medium (either dechlorinated tap water or ®ltered ®sh conditioned medium). Daily refreshment of the medium was necessary, as ®sh chemicals may loose their activity in 24 h due to bacterial degradation (Larsson and Dodson, 1993). Fish conditioned medium was prepared by allowing two Leuciscus idus of approximate 8 cm length, to swim in a 60 l aquarium ®lled with dechlorinated tap water. Each day, 12 l of the medium was ®ltered over 40 mm. The density of ®sh in the ®sh conditioned medium was very high, corresponding to about one ®sh in 120 l (only one fourth of the medium was refreshed daily). To exclude maternal eects, all clones were cultured under the experimental conditions for two generations prior to the experiment (Lynch and Ennis, 1983). Dose±response experiments All dose±response experiments were carried out with animals of the positively phototactic D. magna clone C134. We used adults of 10±15 d old, carrying their second clutch. Culture conditions were identical to those described above for the screening of the dierent clones. Changes in phototactic behavior of clone C134 where tested after 3.30 hours exposure to sub-lethal concentrations of the test-pollutants (Cu2+ and PCP) in arti®cial medium without food. All experiments were conducted in arti®cial medium (ISO, 1989) with the following composition: 0.294 g CaCl22H2O, 0.123 g MgSO47H2O, 0.065 g NaHCO3 and 0.006 g KCl pro liter, pH adjusted to 7.9 with 1 N NaOH or 1 N HCl. Experiments were carried out in the absence of food because this made the analysis of the dose±response relationship more straightforward, since the bioavailability of the pollutants was not aected by adsorption to food particles. In an operational biomonitor, however, the behavioral responses will be determined by ambient bioavailability of the pollutants, and will thus be in¯uenced by the adsorption of the pollutants to suspended particles. The absence of food has no eect on the phototactic behavior of Daphnia magna in the present set-up, at least not as long as experiments do not last long (>5 h; De Meester, pers. obs.). The following nominal copper concentrations were applied: 0; 0.005; 0.01; 0.02; 0.04 mg lÿ1 Cu2+. All test solutions were made in ISO arti®cial medium out of a stock solution with a 1000-fold pollutant concentration. Stock-solutions for copper were prepared daily by adding the required amount of CuSO45H2O into 1 l demineralized water (Millipore). The exact copper concen-
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Fig. 1. Phototactic behavior (mean value I22 SE) of Daphnia magna clones cultured for one generation in the absence and presence of ®sh kairomones. All clones except P132,85 are sister-clones produced by intraclonal reproduction from clone C1.
tration was veri®ed by atomic absorption on samples taken after the experiments. Stock-solutions of PCP were prepared in acetone (400 mg PCP in 4 ml acetone). Final PCP test solutions were prepared in 1 l arti®cial medium and adjusted to pH 7.9 (Liber and Solomon, 1994). The test solutions in the PCP control experiments contained 4 ml of acetone to verify for any eect of acetone on phototactic behavior. Nominal concentrations of 0, 0.2, 0.4, 0.8 and 1.6 mg lÿ1 PCP were applied. Three to four experiments, each with ten animals, were carried out for every pollutant concentration. Dierent animals from dierent cultures or generations were used in the replicates to minimize common environment eects and thus prevent pseudoreplication (Lynch and Ennis, 1983; Hurlbert, 1984). Experimental set-up and protocol The experimental set-up is basically the same as described in De Meester (1991, 1993a), though the experimental cuvet is somewhat modi®ed. The experimental cuvet consisted of two glass columns, the inner of which was 10 cm high and had an internal cross-section of 5 cm. It had a bottom and top made of plankton gauze and functioned as the test chamber. The test chamber 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 column is placed in a light-tight PVC box (16 28 35 cm, provided with a peep-hole of b 1 cm for observations) and illuminated from above with a ®ber light (150 W halogen light source) positioned two cm above the water surface in the outer column. An experiment lasted 10 min. At one minute intervals, the number
of animals in each compartment was counted by looking through the small peep-hole in the PVC-box. The phototactic behavior of the test population was characterized by the index I = (U ÿ L)/(U + M1+M2+L). I was averaged over the last ®ve minutes of the experiment to minimize in¯uences of the initial ¯ight reaction (De Meester, 1993b). Values for the phototactic index can range from 1 for extremely positive behavior to ÿ1 for extremely negative phototactic behavior. All experiments were done in a temperature-buered room (208C 218C), in early afternoon (start 13.00±15.00 h) to minimize possible eects of an endogenous rhythm (though the eect of an endogenous rhythm was found to be negligible in positive phototactic clones; De Meester, 1993b). Prior to an experiment, the animals were adapted for three hours to the test solution. They were then pipetted into the cuvet and given a dark adaptation of 20 min. Subsequently, the light was switched on for the ®rst observation period of 10 min. After the ®rst observation period, the animals were again dark adapted for 20 min and subjected to light for 10 min. As such, a 10 min observation period was given every half an hour for a period of two hours. Each experiment thus consisted of four repeated observations.
Statistical analysis Data on the response of dierent clones to the presence of ®sh kairomones were analyzed by two-way ANOVA, with both clone and treatment as ®xed eects. One-way ANOVA and linear regression was carried out to test for signi®cant eects of pollutant concentration on phototactic behavior (Sokal and Rohlf, 1995). Only the data of the ®rst observation period were used in the one-way ANOVA. Homogeneity of variance was tested using Bartlett's test. Detection limits were determined by Tukey multiple comparisons after one-way ANOVA. All statistical analyses were preformed using the statistical computing package STATISTICA (Statsoft, 1994).
Phototactic behavior of Daphnia
405
Table 1. Two-way ANOVA table with ®xed eects of the presence of ®sh kairomones on the phototactic behavior of D. magna clones C134; C1149; C1155; C1158; C1240; P132,85 Eect
df
MS eect
df error
MS error
F
p-level
Clone Treatment Clone*treatment
5 1 5
1.32 9.72 1.01
49 49 49
0.41 0.41 0.41
3.21 23.57 2.44
0.014 0.000 0.047
RESULTS
The eect of ®sh kairomones The phototactic behavior of clone C134, clone P132,85 and six sister clones of C134 in the presence and absence of ®sh chemicals is plotted in Fig. 1. All clones are characterized by an intermediately to positively phototactic behavior in the absence of ®sh kairomones. The presence of ®sh kairomones induced a more negatively phototactic response in all D. magna clones tested except clone C131 and C1242. Two way ANOVA showed a signi®cant clone by treatment interaction (Table 1). Dierences in phototactic behavior of each clone in control vs ®sh conditioned medium were tested by means of contrast analysis. Clones C134, C1149, C1155, C1158, C1240 and P132,85 showed a signi®cant change in phototactic behavior in the presence of ®sh chemicals (p < 0.05), whereas clone C131 (intermediately phototactic) and C1242 (positively phototactic) showed no signi®cant change in behavior in response to the presence of ®sh kairomones (Fig. 1). The in¯uence of pollutants Figure 2 presents the linear regression between the values for the phototactic index (I) and the Cu2+ concentrations in the test solution (as determined by atomic absorption), of animals that were exposed for 3 h to the pollutant prior to the experiment. Clone C134 shows a clear response to sublethal concentrations of Cu2+, and values for I are
decreasing linearly with increasing Cu2+ concentration. To check whether the response is repeatable upon repeated stimulation by light, we also analyzed the relation between the phototactic behavior and the concentration of copper after 4 h, 4.30 h, and 5 h exposure. Regression coecients are given in Table 2. The slopes of all regression lines are signi®cantly dierent from zero. The relationship is strongest after 4 hours of exposure (R2=0.64). On average, there is a decrease of the value I with 0.11 for every increase of 10 ppm Cu2+ in the test solution. In the absence of pollutants, clone C134 was positively phototactic. In order to determine the resolution with which Cu2+ can be detected using phototactic behavior of clone C134, and because the veri®ed concentrations of Cu2+ in the samples did not correspond completely with the nominal Cu2+ concentrations, we grouped our 3.30 h exposure data into four concentration intervals (0.005±0.015; 0.016±0.025; 0.026±0.035; 0.036±0.05 mg lÿ1 Cu2+) and performed a one way ANOVA. There is a signi®cant eect of pollutant on the phototactic behavior of clone C134 (df: 3; MS group: 0.145; MS error: 0.018; F: 7.966; P < 0.01). Tukey post hoc comparisons show that the animals exhibited a signi®cantly less positively phototactic behavior at a concentration of 0.036±0.05 mg Cu2+ lÿ1 compared to lower concentrations. The in¯uence of sub-lethal concentrations of PCP on the phototactic behavior of D. magna clone C134 was investigated after 3.30 hours adaptation to the pollutant. Figure 3 shows that the index I is
Fig. 2. Phototactic behavior (value I) of adult D. magna clone C134, after 3.30 h exposure to Cu2. Regression line and 95% con®dence limits of the value I in relation to the exact Cu2+ concentration (veri®ed by atomic absorption). LC50 (48 h) = 0.054 mg Cu2+ lÿ1 (Mount and Norberg, 1984).
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Table 2. Regression analysis of dose response experiments with clone C1 34 being exposed during dierent periods (3.30 h, 4 h, 4.30 h and 5 h) to a given Cu2+ concentration (nominal concentrations: control, 0.01, 0.02, 0.04 mg lÿ1 Cu2+) or PCP concentration (nominal concentrations: control (4 ml acetone), 0.2, 0.4, 0.8, 1.6 mg lÿ1 PCP). N = number of experiments
Cu
2+
PCP
Exposure time
N
R2
p-level
3.30 h 4h 4.30 h 15 h 3.30 h
18 18 18 18 20
0.51 0.64 0.49 0.39 0.67
<0.001 <0.001 <0.00 <0.001 <0.001
decreasing linearly with increasing PCP concentration. The slope of the regression line of the phototactic behavior against PCP-concentration is signi®cantly dierent from zero and explains 67% of the variance (Table 1). One-way ANOVA indicates that there is a signi®cant eect of PCP on the phototactic behavior of clone C134 (df: 4; MS group: 1.365; MS error: 0.105; F: 13.032; p < 0.001). Tukey pairwise comparisons indicate that clone C134 behaves signi®cantly more negatively phototactic at a concentration of 0.8 mg lÿ1 PCP or more compared to lower concentrations. The presence of 4 ml acetone lÿ1 does not in¯uence the phototactic behavior of D. magna clone C134, as the animals were positively phototactic in the control experiment. DISCUSSION
Our results con®rm the stable and predictable positively phototactic behavior of clone C134 in the absence of pollutants and ®sh chemicals (see De Meester, 1993a). A survey of sister clones of C134 yielded two clones that showed no signi®cant change in phototactic behavior in presence of ®sh kairomones. The use of a standard positively phototactic clone may improve the potential of a biomonitor based on phototactic behavior of Daphnia considerably. First, some positively phototactic ani-
mals have a stable and repeatable behavior under a wide range of ecological conditions in the absence of pollutants (De Meester and Dumont, 1988; Van Uytvanck and De Meester, 1990; De Meester, 1993b; present study). Secondly, the fact that their behavior is extremely positive phototactic under standard conditions makes it easier to detect stress caused by pollutants. The present study indeed shows that, at least for clone C134, the presence of two standard pollutants induces a more negatively phototactic behavior. As an additional advantage, working with one genotype results in a standardized sensitivity to pollutants (Baird et al., 1989, 1990). The short 10 min visual experiments reported in the present study indicate that the sensitivity of a test system using phototactic behavior of Daphnia is rather high. The LC50 (48 h) value for Cu2+ is reported to be 0.054 mg lÿ1 (Mount and Norberg, 1984) and that for PCP 1.23 mg lÿ1 (Liber and Solomon, 1994), whereas we were able to detect lower concentrations (65±83%) after 3.30 hours of exposure to the pollutant. The dynamic Daphnia monitor of Knie (1978) is able to detect pesticides at concentrations around LC50 (24 h) (see Matthias and Puzichia, 1990). Although juveniles are more sensitive to toxic compounds than adults (Persoone and Janssen, 1993), it may be dicult to work with juveniles in a biomonitor using phototactic behavior because the phototactic behavior of juveniles is less
Fig. 3. Phototactic behavior (value I) of adult D. magna clone C1 34, after 3.30 h exposure to a certain PCP-concentration (nominal concentrations: 0 (4 ml acetone lÿ1), 0.2, 0.4, 0.8, 1.6 mg lÿ1). Regression curve and 95% con®dence limits of the value I in relation to the PCP concentration. LC50 (48 h) = 1.23 mg PCP lÿ1 (Liber and Solomon, 1994).
Phototactic behavior of Daphnia
clear-cut positively phototactic than that of adults (De Meester, 1992). Technically, the method used is very crude. As our experiments are simple and short, we were able to carry out a reasonably large number of experiments to verify the repeatability of the phototactic behavior under standardized conditions and under pollutant stress. Our visual counting method has, however, a limited resolution. Image-analysis using video cameras may detect dierences in vertical position of the animals with a resolution of less than 0.1 mm (J. M. Aerts, pers. comm.). This may potentially further increase the detection capacity of the biomonitor. AcknowledgementsÐWe thank Dr. F. Van Hoof and the Antwerpse Water Werken for the veri®cation of the copper concentration in the test medium. This study was ®nancially supported by the Flemish government (VLIM project H/9410). EM is a fellow of the ¯emish Institute for the promotion of Scienti®c±Technological Research in industry (I. W. T.). Part of this study was carried out while LDM and CC were associated with the Laboratory of Animal Ecology, University of Gent. We thank F. Van Hoof and three anonymous reviewers for constructive comments on an earlier version of the manuscript.
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