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The swimming behaviour of Brachionus calyciflorus (rotifer) under toxic stress. I. The use of automated trajectometry for determining sublethal effects of chemicals
Aquatic
Toxicology
32 (1995) 271-282
The swimming behaviour of Brachionus caZyczj2oru.s(rotifer) under toxic stress. I. The use of automated trajectometry for determining sublethal effects of chemicals C.P. Charoy”,*, C.R. Janssenb, G. Persooneb, P. Ckment” “Lahoratoire de TrajectomPtrie, Comportemrnts et Connai~ssances, Brit. 403 Universitk C. Berncrrd Leon I. 43. Boulevard du II Novembre 1918, 69 622 ViNeurbanne Cedex, France bLaborator_v,for Biological Resenrch in Aquatic Pollution, University of Ghent. J. Plateaustruat 22. 9000 Ghent. Belgium Received
14 September
1994; revised 28 November
1994; accepted
I I December
1994
Abstract Changes in the locomotory behaviour of the freshwater rotifer Brachionus culyctjkrus were used as sublethal indicators of toxic stress. To that end, the swimming behaviour of this rotifer was analysed using an automated tracking system. The swimming speed (temporal factor), the swimming sinuosity (spatial factor), and the periods of swimming were measured and the influence of four chemicals, each representing a distinct chemical class (copper, pentachlorophenol, lindane and 3,4-dichloroaniline), on the rotifer’s swimming characteristics were examined. The three test parameters exhibit different sensitivities depending on the chemical tested. The 2-h EC&s obtained with the behavioural test were of the same order of magnitude as the 24-h LC,,s resulting from conventional acute toxicity tests with the same test species. This potential use of behavioural test criteria for sublethal toxicity testing with rotifers is briefly discussed. Brachionus calycl$orus; Swimming Pentachlorophenol; Lindane; Dichloroaniline
Kqword.~:
behaviour;
Behavioural
toxicology;
Copper;
1. Introduction Although the use of invertebrates as biological models for testing the potential toxicity of wastewaters and chemicals is universally accepted, the test criteria used in such tests are usually restricted to survival, growth and/or reproduction. However, several authors have suggested that behavioural criteria may be more sensitive and rapid indicators of toxic stress than conventional criteria. Modifications of the feed*Corresponding
author.
Tel. (+33-72)
43 10 95; Fax (+33-72)
43 12 26.
0166-445X/95/$09.500 1995 Elsevier Science B.V. All rights reserved SSDI 0166-445X(94)00098-0
212
C. P. Charoy et al. I Aquatic Toxicology 32 (1995) 271-282
ing behaviour of various invertebrate species exposed to sublethal concentrations of chemicals have been reported by, amongst others, Geiger and Buikoma (1981), Flickinger et al. (1982) Day and Kaushik (1987) Jones et al. (1991) and FerandezCasalderrey et al. (1994). Changes in the locomotory characteristics of invertebrates were suggested as ecotoxicological criteria by Borlakoglu and Kickuth (1990) and Goodrich and Lech (1990). Nevertheless, to date, most ecotoxicological behavioural studies with invertebrates have been performed with respresentatives of the genus Duphniu. Other important components of freshwater communities such as rotifers have been rarely used in ecotoxicology in general, and in behavioural toxicology more specifically. A review of the use of rotifers as test organisms for toxicity testing purposes is given by Snell and Janssen (in press). Rotifers, however, especially those belonging to the genus Brachionus, are ideal biological test models because of their small size, short generation time, ease of culturing in the laboratory and the availability of resting eggs (test organisms available on demand) (Snell and Persoone, 1989a,b). In addition to these advantages, the selection of Brachionus caZyciJlorus for toxicological studies is ecologically well justified as this species is not only very abundant and plays a major role in several ecological processes in freshwater communities, but also has a cosmopolitan distribution. Furthermore, for pelagic rotifers like B. cuIyc@orus, the locomotory activity is essential to their survival as it enables the rotifers to stay in a favourable environment (Gilbert, 1963; Charoy and Clement, 1993). Swimming activity of this species is performed by the coordinated beat of the cingulum cilia, and is controlled (Clement, 1977a) by two innerved muscles inserted on the infraciliature (Clement, 1977b, 1987; Clement and Wurdak, 1991). Changes in the swimming characteristics can thus result from modifications of the beating ability of the cilia (Luciani, 1982) and/or from the independent contractions of the innerved muscles (Clement, 1987). In the present study we have examined the influence of four chemicals on different aspects of the locomotory behaviour of B. calyczjlorus using an automated video tracking system. The sensitivity of the different behavioural criteria was compared with the results of acute toxicity tests obtained with the same species.
2. Materials and methods The rotifer species Brachionus calyciJiorus used in the experiments was originally collected (1983) in the wild in Gainesville (Florida, USA) (Snell et al., 1991). Since then the rotifers have been cultured in the laboratory and were induced to produce resting eggs (cysts) at regular intervals. Details of the cyst characteristics are given in Snell and Persoone (1989b) and Snell et al. (1991). All test animals were obtained by hatching cysts. The hatching procedure consisted of transferring the dry cysts into a synthetic, moderately hard, freshwater medium (EPA, 1986) and incubating them in light (6000 lux, fluorescent tubes) at 25°C. Under these conditions, hatching was synchronous and the first neonates appeared after 16 to 18 h, at which time they were collected and used in the tests.
C.P. Charoy et al. IAquatic Toxico1og.v32 (1995) 271 282 INCUBATION MEDIUM
OF CYSTS IN THE SYNTHETIC
213
FRESHWATER
HATCHING, EXPOSURE OF NEONATES TO TOXICANTS
END OF EXPOSURE TIME, TRANSFER OF EACH INDIVIDUAL
TO THE RECORDING
CHAMBER
DATA PROCESSING Smoothing (mobile mean of 5 successiw
points)
Sampling (kept 1 point from every 5)
+
J Periods of swimming
Speed and sinuosity Fig.
FILE PROCESSING
I,
Experimental
design
The swimming behaviour of the neonates exposed to a range of toxicant concentrations was examined using an automatic tracking system (Coulon et al., 1983; Clement et al., 1988). Prior to the actual observations of the locomotory behaviour, the neonates were placed individually in small petridishes and exposed to the respective toxicant concentrations for a period of 2 h. The 2-h exposure period used in this study has been selected on the basis of preliminary experiments in which the swimming behaviour of B.calyc$orus exposed for 5 min, 2 and 6 h to the chemicals was examined. These results show that with the chemicals tested, the 2-h EC,,s obtained using effects on swimming behaviour as criteria are of the same order of magnitude as the 24-h LC,,s obtained in acute toxicity tests (manuscript in prep.). The experimental procedures involved in the observation and the analysis of the behaviour with this system are represented in Fig. 1. In the first data-aquisition phase, each animal is placed individually into a flat glass chamber (20 x 20 x 0.5 mm) containing the same
C. P. Charoy et al. I Aquatic
274
Toxicology 32 (1995) 271-282
test medium as that used during the 2-h exposure period. The behaviour of the rotifer was video-taped during 10 min, using a fixed camera placed above the test chamber. Recordings were performed at 23 ? 1°C red light was used in order to avoid possible phototactic effects (Cornillac et al., 1983). In the second data-processing phase, 25 XY coordinates of the barycenter of the animal were recorded per second, and stored in a computer. In the final phase of the procedure, the data files were analysed and the swimming parameters, speed (mm s-i) and sinuosity (rad mm-“‘) (Bovet and Benhamou, 1988), were calculated using the smoothed (mobile mean of 5 sucessive points) and the sampled (1 of every 5 points retained) rotifer trajectories. The last two procedures are aimed at removing computer-associated artifacts (non-biological phenomena) (Clement et al., 1987). The use of the sinuosity of the rotifer behaviour in addition to the speed makes it possible to describe the displacements in spatial and temporal terms separately. During the analysis, periods of rotifer immobility were timed and recorded. This latter factor was excluded from the files which were used to calculate the rotifer’s speed and sinuosity. The third test parameter, periods of swimming (s), was calculated from duration and frequency of rotifer immobility. Four chemical compounds were tested: copper (CuSO,.5H,O), sodium pentachlorophenol (PCP), lindane and 3,4-dichloroaniline (DCA). For lindane and DCA, acetone was used as solvent (6.32 g 1-l and 3.16 g 1-l for DCA and lindane respectively, according to the highest concentration used in the experiment). All chemical concentrations were nominal. For each toxicant, four toxicant concentrations were tested (Table 1). Per treatment, the swimming behaviour of 8 rotifers was recorded. Significant differences between the controls and the toxicant treatments were determined with the aid of a one-way ANOVA (P < 0.05). For each toxicant, a polynomial regression model was applied to determine the EC,,+ for the behavioural criteria. A polynomial regression model of the second order 0, = ax2 + bx + c) was used as it resulted in a better fit to the experimental data than commonly used linear models.
3. Results For both lindane and DCA, acetone was used as solvent, consequently
acetone-
Table I Test groups
Controls
(EPA ml)
Acetone
controls
Concentrations of test compound (mg I-‘)
CU
PCP
Lindane
3,4 DCA
1
1
1
1
Acetone 0.40% (3.16 g 1-l)
Acetone 0.80% (6.32 g 1-l)
5 10 20 40
20 40 60 80
2.5 6.2 12.5 25.
10-2 lO-3 1o-x lo-’
1 2.5 5 10
C. P. Charoy et al. I Aquatic Toxicology 32 (I 995) 2 71-282
Speed (mm/s)
1.0
275
a
0.8 0.6
I
2.0 Sinuosity (radlmm?
b
1.6
1.2
I
0.8
64xlPeriods of swimming (s)‘ 400-
Fig. 2. Effects of copper on the swimming behaviour of B. calyciJorus neonates. Swimming criteria (a, speed; b, sinuosity; and c, periods of swimming) as function of toxicant concentration. C: control. Values are means with 1 se., **O.Ol 2 P > 0.001, ***O.OOl 2 P.
control treatments were performed concurrently in each toxicity test. For all parameters measured in this study, no significant effect of the acetone was noted. Within the range of Cu concentrations tested, a gradual decrease of the linear speed with increasing concentrations was observed. Compared to the speed in the control treatment (0.472 mm s-‘) a decrease of 5.3, 16.3 and 41.5% was noted for 6.2, 12.5 and
216
C. P. Charoy et al. IAquatic Toxicology 32 (1995) 271-282
25 ,ug 1-l Cu, respectively (Fig. 2a). Only at this latter concentration, however, was the decrease in speed statistically significant. Moreover, copper induced a dose-dependent effect on the periods of swimming (Fig. 2~). Despite a small increase, the rotifers’ sinuosity was not significantly affected by Cu (Fig. 2b). An overview of the EC,& NOECs and LOECs for the different test endpoints is given in Table 2. Significant reductions in the swimming speed were noted at concentrations ~2.5 mg 1-l PCP (Fig. 3a). The mean swimming speed decreased from 0.532 mm s-l in the controls to 0.425, 0.379, 0.325 and 0.218 mm s-’ in the 1, 2.5, 5 and 10 mg 1-l PCP, respectively. The sinuosity of rotifers exposed to 2.5 mg 1-l PCP and higher was significantly affected. The gradual decrease of this parameter is presented in Fig. 3b in which a reduction of 25 to 66% within the PCP concentration range was noted. Furthermore, at 10 mg PCP l-l, the time of swimming decreased significantly. Compared to control treatment, the periods of swimming decreased by 47% at the highest concentration (Fig. 3~). Lindane did not, within the concentration range tested, significantly affect the rotifers’ speed and total periods of swimming (Fig. 4a,c). The sinuosity, however, was adversely affected by lindane and exhibited a gradual decrease with increasing toxicant concentrations. At 40 mg 1-l lindane the sinuosity of the B. calyczjlo~u~neonates was reduced by 41% compared to that of the control animals (Fig. 4b). Neither speed nor sinuosity was significantly affected by 3,4-DCA (concentration range: 20-80 mg 1-l DCA) (Fig. 5a,b). However, the periods of swimming of rotifers exposed to 80 mg 1-l DCA decreased drastically (Fig. 5~).
Table 2 Comparison between 2-h EC,,s obtained with behavioural toxicity tests and 24-h LC,, values based on acute toxicity tests using the same test species. a, from Janssen et al. (1994); B. from Ferrando et al. (1992) Acute test
Behavioural
tests
Swimming Speed activity’
Sinuosity
Period of swimming
2-h EC,,
2-h EC,, LOEC
NOEC
2-h EC& LOEC
16.0”
28.16
25.0
12.50
3.90”
6.52
2.50
1.o
Lindane 22.50” (mg 1-l) 22.5@
15.0”
61.50” DCA (mg I-‘) 61.508
63.50”
24-h LC,,
PCP
0.92”
NOEC
2-h EC,,, LOEC
NOEC
No significant effect
20.82
12.50
6.20
3.59
5.0
2.50
9.59
10.0
5.0
No significant effect
47.88
40.00
20.0
No significant effect
No significant effect
No significant effect
(mg I-‘) 2.168
102.64
80.0
60.0
C. P. Charoy et al. IAqualic Toxicology 32 (1995) 271-282
271
a Speed (mm/s)
0.6
*** I
I
C
I
2
4
6
PCP
2.0-
Sinuosity (rad/mmT
8
10
12
(mg/l)
b
_
1.6. 1.2 0.8 *** IA
0.4 -
o.o!
I
C
I
2
1
I
I
I
1
4
6
a
10
12
c
i‘/-200-
0.0:
I
C
I
I
2
4
.
I
I
I
6
8
10
Fig. 3. Effects of PCP on the swimming behaviour of B. caIyc$orus b, sinuosity; and c, periods of swimming) as function of toxicant means with 1 s.e., **O.Ol 2 P > 0.001. ***O.OOl 2 P.
1
12
neonates. Swimming criteria (a, speed; concentration. C: control. Values are
4. Discussion After a 2-h exposure, for all four chemicals, quantitative changes in the locomotory parameters of B. caZycz$orus neonates were noted; a gradual decrease of the values of
C.P. Charoy et al. I Aquatic Toxicology 32 (1995) 271-282
218
1
.o
a
Speed (mm/s) 0.8
0.6
I
Lindane
(mg/l)
b
2.0 Sinuosity (radlmm”l) 1.6 -
IIll
C
10
20
30
40
50
30
40
50
600 Periods of swimming
I (s
400
200
0.0
C
10
20
I
Fig. 4. Effects of lindane on the swimming behaviour of B. calycijlorus neonates. Swimming criteria (a, speed; b, sinuosity; and c, periods of swimming) as function of toxicant concentration. C: control. Values are means with 1 se.. *0.05 > P > 0.01.
the swimming criteria was observed. Rotifers swam less, and the characteristics of the locomotion were clearly affected. In Table 2, the results of the present study are compared with those reported by Janssen et al. (1994). These authors examined the effects of the same four chemicals on the swimming behaviour of B. cuZyczj7orus, but used the ‘swimming activity’ crite-
779
C. P. Charoy et al. IAquatic Toxicology 32 (1995) 271-282
1.0
Speed (mm/s)
0.8
1
0.6-
a
I
’
0.4
-
0.2
-
o.oI .
I
C
I
10
,
20
I
30
I.
40
DCA
I
50
I
60
.
I
70
I
80
,
90
(mg/l)
b
I I
I
0.01
,
C
,
,
,
10
20
30
, . , 40
50
Fig. 5. Effects of DCA on the swimming behaviour of B. calyciflorus b, sinuosity; and c, periods of swimming) as function of toxicant means with 1 s.e., **O.Ol 2 P > 0.001.
f
,
,
,
,
60
70
80
90
neonates. Swimming criteria (a, speed: concentration. C: control. Values are
rion as test endpoint. In this context, swimming activity of the rotifers (swimming of a grid) is defined as the number of 1 mm squares entered in a 30-s period of time (sq 30 s-l). This criterion was calculated only from the periods of displacements. The swimming activity criterion seems to be more sensitive than both the speed and sinuosity endpoints measured in the present study. However, except for lindane, the ratios
280
C.P. Charoy et al. IAquatic Toxicology 32 (1995) 271-282
of the speed and the sinuosity EC,,s to the swimming activity EC,,,s are small and varied from 1.08 to 1.76 depending on the chemical (3.19 for lindane). Comparing the 2-h EC&s obtained in the present study with the 24-h LC,,s reported by Janssen et al. (1994) and Ferrando et al. (1992) (Table 2), it can be concluded that the EC& resulting from behavioural tests were higher than the results from the conventional mortality tests. Even if the sensitivity of the behavioural toxicity test with copper was comparable to that of the acute toxicity test (Table 2), it is clear that the 2-h exposure period was too short to reach the same level of sensitivity as the 24-h LC,, tests. Despite this short exposure period, a clear concentration-response was observed. The mean coefficients of variation ranged from 24% to 34%; such values are satisfactory for behavioural criteria, and they suggest a good repeatability of the effects obtained. From the present study, it can be concluded that the locomotory behaviour of B. calycz~%rus is adversely affected by sublethal concentrations of chemicals. To date, very little information is available on the effects of toxicants on the locomotory behaviour of rotifers. Kleinow (1986) studied the qualitative effects of acrylamide on the movements of Brachionus plicatilis. Beauvais and Enesco (1985) examined the swimming activity of Asplanchna brightwelli exposed to curare by observing the rotifers’ behaviour as they swam over a grid. Using a similar method, Snell et al. (1987) showed that the swimming behaviour of B. plicatilis was adversely affected by sublethal levels of ammonia, and more recently Janssen et al. (1994) suggested that the swimming activity of B. calyczjkus could be used as a sensitive sublethal test criterion for ecotoxicological purposes. However, as mentioned, these three last workers used the same ‘open-field checkerboard grid’ method to record the rotifers’ movements. The swimming activity criterion obtained with this technique (counting the number of squares transversed by the rotifer) is, however, a very general parameter. With the method used in the present study the complex rotifer behaviour can be quantified and split up into simple and mathematically independent criteria. Indeed, the results of this study show that the four toxicants affect the various criteria in different ways (Table 2). Additionally, the sensitivity of these test endpoints depended on the compound used. Consequently, it can be concluded that as these various spatial and temporal behavioural endpoints seem to have quite different resolution powers in detecting sublethal effects, they may not only prove useful as ecotoxicological indices but also contribute to the understanding of some fundamental aspects of the effects of toxicants on the ecology of rotifers. Indeed, locomotory behaviour in general and rotifer swimming behaviour more specifically, can be considered as an integration of physiological, sensorial, nervous and muscular systems. It has been shown that rotifers’ swimming patterns are affected by the presence or absence of food (Charoy and Clement, 1993; Charoy, in press) and the oxygen content in the medium (Reale et al., 1993). Additionally, Gilbert (1963) reported that the presence of secretions from B. calyczjlorus females modified the swimming behaviour of conspecific males. Because of the integrative character of behavioural changes, it has been suggested that bioassays using behavioural test criteria could be very useful for rapid toxicity testing. Behavioural tests with fish have now become an accepted part of many mon-
C. P. Charoy et al. I Aquatic Toxicology 32 (1995) 271-282
281
itoring and testing programmes. However, the development of standardized toxicity tests with invertebrates using behavioural test endpoints is still in its infancy. The present study has shown that changes in the swimming characteristics of rotifers can certainly be considered as rapid and sensitive indicators of toxic stress.
Acknowledgments This work was supported by European Science Foundation grant RF/93/18/E. The authors would like to thank the anonymous reviewers for their constructive comments.
References Beauvais, J.E. and H.E. Enesco (1985) Life span and age-related changes in activity levels of the rotifer Asplanchna brightwelli: influence of curare. Exp. Gerontol. 20, 359-366. Borlakoglu, J.T. and R. Kickuth (1990) Behavioural changes in Gammaruspulex and its significance in the toxicity assessment of very low levels of environmental polluants. Bull. Environ. Contam. Toxicol. 45, 2588265. Bovet, P. and S. Benhamou (1988) Spatial analysis of animals’ movements using a correlated random walk model. J. Theor. Biol. 131,419433. Charoy, C. (in press) Modifications of the swimming behaviour of Brachionus calyct$orus (Pallas) according to both food environment and individual nutritive state. Hydrobiologia. Charoy, C. and P. Clement (1993) Foraging behavior of Brachionus CalyciJlorus: variations of the swimming path according to presence or absence of algal food (chlorella). Hydrobiologia 255/256, 95-100. Clement, P. (1977a) Introduction a la photobiologie des rotiferes dont le cycle reproducteur est controli. par la photoperiode. Approches ultrastructurale et experimentale. These Univ. Lyon I, 261 pp. Clement, P. (1977b) Ultrastructural research on rotifers. Arch. Hydrobiol. Beih. 8, 260-297. Clement, P. (1987) Movements in rotifers: correlations of ultrastructure and behavior. Hydrobiologia 147, 339-359. Clement, P. and E. Wurdak (1991) Rotifera. Microscopic Anatomy of Invertebrates 4: Aschelminthes. Wiley-Liss Inc., pp. 219-297. Clement, P., P. Mimouni, P. Fouillet and J.L. Chasst (1987) La discretisation spatiotemporelle de trajets obtenus en trajectometrie automatique. CR 112eme congrts des Societes Savantes. Clement, P.. P. Mimouni, P. Fouillet, P.Y. Coulon and M. Le Berre (1988) Recueil et analyse de deplacements de rongeurs par trajectometrie automatique. Sci. Tech. Anim. Lab. 13, 5-13. Cornillac, A., E. Wurdak and P. Clement (1983) Phototaxis in monochromatic light and microspectrophotometry of the cerebral eye of the rotifer Brachionus calyc$orus. Hydrobiologia 104. 191-196. Coulon, P.Y., J.P. Charras, J.L. Chasse, P. Clement, A.M. Cornillac, A. Luciani and E. Wurdak (1983) An experimental system for the automatic tracking and analysis of rotifer swimming behavior. Hydrobiologia 104, 1977202. Day, K. and N.K. Kaushik (1987) Short-term exposure of zooplankon to the synthetic pyredoid. fenvalerate, and its effects on rates of filtration and assimilation of the alga Chlamydomonas reinhardii. Arch. Environ. Contam. Toxicol. 16,423432. Fernandez-Casalderrey, A., M.D. Ferrando and E. Andreu-Moliner (1994) Effect of sublethal concentrations of pesticides on the feeding behaviour of Dapnia magna. Ecotoxicol. Environ. Saf. 27, 82-89. Ferrando, M.D., E. Andreu Moliner and A. Fernandez-Casalderrey (1992) Relative sensitivity of Daphnia magna and Brachionus calyciflorus to five pesticides. J. Environ. Sci. B 27, 51 l-522. Flickinger, A.L., R.J.F. Bruins, R.W. Winner and J.H. Skillings (1982) Filtration and phototactic behavior
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as indices of chronic copper stress in Daphnia magna Straus. Arch. Environ. Contam. Toxicol. 11, 451463. Geiger, J.G. and A.L. Buikema (1981) Oxygen consumption and filtering rate of Daphnia pulex after exposure to water-soluble fractions of naphthalene, phenanthrene, no 2 fuel oil, and coal-tar creosote. Bull. Environ. Contam. Toxicol. 27, 783-789. Gilbert, J.J. (1963) Contact chemoreception, matting behaviour and sexual isolation in the rotifer genus Brachionus. J. Exp. Biol. 40, 625-641. Goodrich, M.S. and J.J. Lech (1990) A behavioral screening assay for Daphnia magna: a method to assess the effects of xenobiotics on spatial orientation. Environ. Toxicol. Chem. 9, 21-30. Janssen, C., M.D. Ferrando and G. Persoone (1994) Ecotoxicological studies with the freshwater rotifer Brachionus calyc@orus: IV Rotifer behavior as a sensitive and rapid sublethal test criterion. Ecotoxicol. Environ. Saf. 28, 244255. Jones, M., C. Folt and S. Guarda (1991) Characterizing individual, population and community effects of sublethal levels of aquatic toxicants: an experimental case study using Daphnia. Freshwater Biol. 26, 3544. Kleinow, W. (1986) Effects of acrylamide on Brachionusplicatilis (Rotifera). Comp. Biochem. Physiol. 84C, 243-246. Luciani, A. (1982) Contribution a l’etude du vielhssement chez le rotifere Brachionus plicatilis: nage, cils et battements ciliaires, mttabohsme tnergetique. These Doct. Spec. Univ. Lyon I 1211, 86 pp. Reale, D., P. Clement and A. Esparcia-Collado (1993) Influence of the concentration of oxygen on the swimming path of Brachionus plicatilis (Rotifera). Hydrobiologia 255/256, 87-93. Snell, T.W. and CR. Janssen (in press) Rotifers in ecotoxicology: a review. Hydrobiologia. Snell, T.W. and G. Persoone (1989a) Acute toxicity bioassays using rotifers. 1. A test for brackish and marine environments with Brachionus plicatilis. Aquat. Toxicol. 14, 65580. Snell, T.W. and G. Persoone (1989b) Acute toxicity bioassays using rotifers. II. A freshwater test with Brachionus rubens. Aquat. Toxicol. 14, 81-92. Snell, T.W., M.J. Chidress, E.M. Boyer and F.H. Hoff (1987) Assessing the status of rotifer mass cultures. J. World Aquacult. Sot. 18, 270-277. Snell, T.W., B.D. Moffat, C.R. Janssen and G. Persoone (1991) Acute toxicity tests using rotifers. IV. Effects of cyst age, temperature and salinity on the sensibility of Brachionus calyciporus. Ecotoxicol. Environ. Saf. 21. 308-317.
Toxicology
32 (1995) 271-282
The swimming behaviour of Brachionus caZyczj2oru.s(rotifer) under toxic stress. I. The use of automated trajectometry for determining sublethal effects of chemicals C.P. Charoy”,*, C.R. Janssenb, G. Persooneb, P. Ckment” “Lahoratoire de TrajectomPtrie, Comportemrnts et Connai~ssances, Brit. 403 Universitk C. Berncrrd Leon I. 43. Boulevard du II Novembre 1918, 69 622 ViNeurbanne Cedex, France bLaborator_v,for Biological Resenrch in Aquatic Pollution, University of Ghent. J. Plateaustruat 22. 9000 Ghent. Belgium Received
14 September
1994; revised 28 November
1994; accepted
I I December
1994
Abstract Changes in the locomotory behaviour of the freshwater rotifer Brachionus culyctjkrus were used as sublethal indicators of toxic stress. To that end, the swimming behaviour of this rotifer was analysed using an automated tracking system. The swimming speed (temporal factor), the swimming sinuosity (spatial factor), and the periods of swimming were measured and the influence of four chemicals, each representing a distinct chemical class (copper, pentachlorophenol, lindane and 3,4-dichloroaniline), on the rotifer’s swimming characteristics were examined. The three test parameters exhibit different sensitivities depending on the chemical tested. The 2-h EC&s obtained with the behavioural test were of the same order of magnitude as the 24-h LC,,s resulting from conventional acute toxicity tests with the same test species. This potential use of behavioural test criteria for sublethal toxicity testing with rotifers is briefly discussed. Brachionus calycl$orus; Swimming Pentachlorophenol; Lindane; Dichloroaniline
Kqword.~:
behaviour;
Behavioural
toxicology;
Copper;
1. Introduction Although the use of invertebrates as biological models for testing the potential toxicity of wastewaters and chemicals is universally accepted, the test criteria used in such tests are usually restricted to survival, growth and/or reproduction. However, several authors have suggested that behavioural criteria may be more sensitive and rapid indicators of toxic stress than conventional criteria. Modifications of the feed*Corresponding
author.
Tel. (+33-72)
43 10 95; Fax (+33-72)
43 12 26.
0166-445X/95/$09.500 1995 Elsevier Science B.V. All rights reserved SSDI 0166-445X(94)00098-0
212
C. P. Charoy et al. I Aquatic Toxicology 32 (1995) 271-282
ing behaviour of various invertebrate species exposed to sublethal concentrations of chemicals have been reported by, amongst others, Geiger and Buikoma (1981), Flickinger et al. (1982) Day and Kaushik (1987) Jones et al. (1991) and FerandezCasalderrey et al. (1994). Changes in the locomotory characteristics of invertebrates were suggested as ecotoxicological criteria by Borlakoglu and Kickuth (1990) and Goodrich and Lech (1990). Nevertheless, to date, most ecotoxicological behavioural studies with invertebrates have been performed with respresentatives of the genus Duphniu. Other important components of freshwater communities such as rotifers have been rarely used in ecotoxicology in general, and in behavioural toxicology more specifically. A review of the use of rotifers as test organisms for toxicity testing purposes is given by Snell and Janssen (in press). Rotifers, however, especially those belonging to the genus Brachionus, are ideal biological test models because of their small size, short generation time, ease of culturing in the laboratory and the availability of resting eggs (test organisms available on demand) (Snell and Persoone, 1989a,b). In addition to these advantages, the selection of Brachionus caZyciJlorus for toxicological studies is ecologically well justified as this species is not only very abundant and plays a major role in several ecological processes in freshwater communities, but also has a cosmopolitan distribution. Furthermore, for pelagic rotifers like B. cuIyc@orus, the locomotory activity is essential to their survival as it enables the rotifers to stay in a favourable environment (Gilbert, 1963; Charoy and Clement, 1993). Swimming activity of this species is performed by the coordinated beat of the cingulum cilia, and is controlled (Clement, 1977a) by two innerved muscles inserted on the infraciliature (Clement, 1977b, 1987; Clement and Wurdak, 1991). Changes in the swimming characteristics can thus result from modifications of the beating ability of the cilia (Luciani, 1982) and/or from the independent contractions of the innerved muscles (Clement, 1987). In the present study we have examined the influence of four chemicals on different aspects of the locomotory behaviour of B. calyczjlorus using an automated video tracking system. The sensitivity of the different behavioural criteria was compared with the results of acute toxicity tests obtained with the same species.
2. Materials and methods The rotifer species Brachionus calyciJiorus used in the experiments was originally collected (1983) in the wild in Gainesville (Florida, USA) (Snell et al., 1991). Since then the rotifers have been cultured in the laboratory and were induced to produce resting eggs (cysts) at regular intervals. Details of the cyst characteristics are given in Snell and Persoone (1989b) and Snell et al. (1991). All test animals were obtained by hatching cysts. The hatching procedure consisted of transferring the dry cysts into a synthetic, moderately hard, freshwater medium (EPA, 1986) and incubating them in light (6000 lux, fluorescent tubes) at 25°C. Under these conditions, hatching was synchronous and the first neonates appeared after 16 to 18 h, at which time they were collected and used in the tests.
C.P. Charoy et al. IAquatic Toxico1og.v32 (1995) 271 282 INCUBATION MEDIUM
OF CYSTS IN THE SYNTHETIC
213
FRESHWATER
HATCHING, EXPOSURE OF NEONATES TO TOXICANTS
END OF EXPOSURE TIME, TRANSFER OF EACH INDIVIDUAL
TO THE RECORDING
CHAMBER
DATA PROCESSING Smoothing (mobile mean of 5 successiw
points)
Sampling (kept 1 point from every 5)
+
J Periods of swimming
Speed and sinuosity Fig.
FILE PROCESSING
I,
Experimental
design
The swimming behaviour of the neonates exposed to a range of toxicant concentrations was examined using an automatic tracking system (Coulon et al., 1983; Clement et al., 1988). Prior to the actual observations of the locomotory behaviour, the neonates were placed individually in small petridishes and exposed to the respective toxicant concentrations for a period of 2 h. The 2-h exposure period used in this study has been selected on the basis of preliminary experiments in which the swimming behaviour of B.calyc$orus exposed for 5 min, 2 and 6 h to the chemicals was examined. These results show that with the chemicals tested, the 2-h EC,,s obtained using effects on swimming behaviour as criteria are of the same order of magnitude as the 24-h LC,,s obtained in acute toxicity tests (manuscript in prep.). The experimental procedures involved in the observation and the analysis of the behaviour with this system are represented in Fig. 1. In the first data-aquisition phase, each animal is placed individually into a flat glass chamber (20 x 20 x 0.5 mm) containing the same
C. P. Charoy et al. I Aquatic
274
Toxicology 32 (1995) 271-282
test medium as that used during the 2-h exposure period. The behaviour of the rotifer was video-taped during 10 min, using a fixed camera placed above the test chamber. Recordings were performed at 23 ? 1°C red light was used in order to avoid possible phototactic effects (Cornillac et al., 1983). In the second data-processing phase, 25 XY coordinates of the barycenter of the animal were recorded per second, and stored in a computer. In the final phase of the procedure, the data files were analysed and the swimming parameters, speed (mm s-i) and sinuosity (rad mm-“‘) (Bovet and Benhamou, 1988), were calculated using the smoothed (mobile mean of 5 sucessive points) and the sampled (1 of every 5 points retained) rotifer trajectories. The last two procedures are aimed at removing computer-associated artifacts (non-biological phenomena) (Clement et al., 1987). The use of the sinuosity of the rotifer behaviour in addition to the speed makes it possible to describe the displacements in spatial and temporal terms separately. During the analysis, periods of rotifer immobility were timed and recorded. This latter factor was excluded from the files which were used to calculate the rotifer’s speed and sinuosity. The third test parameter, periods of swimming (s), was calculated from duration and frequency of rotifer immobility. Four chemical compounds were tested: copper (CuSO,.5H,O), sodium pentachlorophenol (PCP), lindane and 3,4-dichloroaniline (DCA). For lindane and DCA, acetone was used as solvent (6.32 g 1-l and 3.16 g 1-l for DCA and lindane respectively, according to the highest concentration used in the experiment). All chemical concentrations were nominal. For each toxicant, four toxicant concentrations were tested (Table 1). Per treatment, the swimming behaviour of 8 rotifers was recorded. Significant differences between the controls and the toxicant treatments were determined with the aid of a one-way ANOVA (P < 0.05). For each toxicant, a polynomial regression model was applied to determine the EC,,+ for the behavioural criteria. A polynomial regression model of the second order 0, = ax2 + bx + c) was used as it resulted in a better fit to the experimental data than commonly used linear models.
3. Results For both lindane and DCA, acetone was used as solvent, consequently
acetone-
Table I Test groups
Controls
(EPA ml)
Acetone
controls
Concentrations of test compound (mg I-‘)
CU
PCP
Lindane
3,4 DCA
1
1
1
1
Acetone 0.40% (3.16 g 1-l)
Acetone 0.80% (6.32 g 1-l)
5 10 20 40
20 40 60 80
2.5 6.2 12.5 25.
10-2 lO-3 1o-x lo-’
1 2.5 5 10
C. P. Charoy et al. I Aquatic Toxicology 32 (I 995) 2 71-282
Speed (mm/s)
1.0
275
a
0.8 0.6
I
2.0 Sinuosity (radlmm?
b
1.6
1.2
I
0.8
64xlPeriods of swimming (s)‘ 400-
Fig. 2. Effects of copper on the swimming behaviour of B. calyciJorus neonates. Swimming criteria (a, speed; b, sinuosity; and c, periods of swimming) as function of toxicant concentration. C: control. Values are means with 1 se., **O.Ol 2 P > 0.001, ***O.OOl 2 P.
control treatments were performed concurrently in each toxicity test. For all parameters measured in this study, no significant effect of the acetone was noted. Within the range of Cu concentrations tested, a gradual decrease of the linear speed with increasing concentrations was observed. Compared to the speed in the control treatment (0.472 mm s-‘) a decrease of 5.3, 16.3 and 41.5% was noted for 6.2, 12.5 and
216
C. P. Charoy et al. IAquatic Toxicology 32 (1995) 271-282
25 ,ug 1-l Cu, respectively (Fig. 2a). Only at this latter concentration, however, was the decrease in speed statistically significant. Moreover, copper induced a dose-dependent effect on the periods of swimming (Fig. 2~). Despite a small increase, the rotifers’ sinuosity was not significantly affected by Cu (Fig. 2b). An overview of the EC,& NOECs and LOECs for the different test endpoints is given in Table 2. Significant reductions in the swimming speed were noted at concentrations ~2.5 mg 1-l PCP (Fig. 3a). The mean swimming speed decreased from 0.532 mm s-l in the controls to 0.425, 0.379, 0.325 and 0.218 mm s-’ in the 1, 2.5, 5 and 10 mg 1-l PCP, respectively. The sinuosity of rotifers exposed to 2.5 mg 1-l PCP and higher was significantly affected. The gradual decrease of this parameter is presented in Fig. 3b in which a reduction of 25 to 66% within the PCP concentration range was noted. Furthermore, at 10 mg PCP l-l, the time of swimming decreased significantly. Compared to control treatment, the periods of swimming decreased by 47% at the highest concentration (Fig. 3~). Lindane did not, within the concentration range tested, significantly affect the rotifers’ speed and total periods of swimming (Fig. 4a,c). The sinuosity, however, was adversely affected by lindane and exhibited a gradual decrease with increasing toxicant concentrations. At 40 mg 1-l lindane the sinuosity of the B. calyczjlo~u~neonates was reduced by 41% compared to that of the control animals (Fig. 4b). Neither speed nor sinuosity was significantly affected by 3,4-DCA (concentration range: 20-80 mg 1-l DCA) (Fig. 5a,b). However, the periods of swimming of rotifers exposed to 80 mg 1-l DCA decreased drastically (Fig. 5~).
Table 2 Comparison between 2-h EC,,s obtained with behavioural toxicity tests and 24-h LC,, values based on acute toxicity tests using the same test species. a, from Janssen et al. (1994); B. from Ferrando et al. (1992) Acute test
Behavioural
tests
Swimming Speed activity’
Sinuosity
Period of swimming
2-h EC,,
2-h EC,, LOEC
NOEC
2-h EC& LOEC
16.0”
28.16
25.0
12.50
3.90”
6.52
2.50
1.o
Lindane 22.50” (mg 1-l) 22.5@
15.0”
61.50” DCA (mg I-‘) 61.508
63.50”
24-h LC,,
PCP
0.92”
NOEC
2-h EC,,, LOEC
NOEC
No significant effect
20.82
12.50
6.20
3.59
5.0
2.50
9.59
10.0
5.0
No significant effect
47.88
40.00
20.0
No significant effect
No significant effect
No significant effect
(mg I-‘) 2.168
102.64
80.0
60.0
C. P. Charoy et al. IAqualic Toxicology 32 (1995) 271-282
271
a Speed (mm/s)
0.6
*** I
I
C
I
2
4
6
PCP
2.0-
Sinuosity (rad/mmT
8
10
12
(mg/l)
b
_
1.6. 1.2 0.8 *** IA
0.4 -
o.o!
I
C
I
2
1
I
I
I
1
4
6
a
10
12
c
i‘/-200-
0.0:
I
C
I
I
2
4
.
I
I
I
6
8
10
Fig. 3. Effects of PCP on the swimming behaviour of B. caIyc$orus b, sinuosity; and c, periods of swimming) as function of toxicant means with 1 s.e., **O.Ol 2 P > 0.001. ***O.OOl 2 P.
1
12
neonates. Swimming criteria (a, speed; concentration. C: control. Values are
4. Discussion After a 2-h exposure, for all four chemicals, quantitative changes in the locomotory parameters of B. caZycz$orus neonates were noted; a gradual decrease of the values of
C.P. Charoy et al. I Aquatic Toxicology 32 (1995) 271-282
218
1
.o
a
Speed (mm/s) 0.8
0.6
I
Lindane
(mg/l)
b
2.0 Sinuosity (radlmm”l) 1.6 -
IIll
C
10
20
30
40
50
30
40
50
600 Periods of swimming
I (s
400
200
0.0
C
10
20
I
Fig. 4. Effects of lindane on the swimming behaviour of B. calycijlorus neonates. Swimming criteria (a, speed; b, sinuosity; and c, periods of swimming) as function of toxicant concentration. C: control. Values are means with 1 se.. *0.05 > P > 0.01.
the swimming criteria was observed. Rotifers swam less, and the characteristics of the locomotion were clearly affected. In Table 2, the results of the present study are compared with those reported by Janssen et al. (1994). These authors examined the effects of the same four chemicals on the swimming behaviour of B. cuZyczj7orus, but used the ‘swimming activity’ crite-
779
C. P. Charoy et al. IAquatic Toxicology 32 (1995) 271-282
1.0
Speed (mm/s)
0.8
1
0.6-
a
I
’
0.4
-
0.2
-
o.oI .
I
C
I
10
,
20
I
30
I.
40
DCA
I
50
I
60
.
I
70
I
80
,
90
(mg/l)
b
I I
I
0.01
,
C
,
,
,
10
20
30
, . , 40
50
Fig. 5. Effects of DCA on the swimming behaviour of B. calyciflorus b, sinuosity; and c, periods of swimming) as function of toxicant means with 1 s.e., **O.Ol 2 P > 0.001.
f
,
,
,
,
60
70
80
90
neonates. Swimming criteria (a, speed: concentration. C: control. Values are
rion as test endpoint. In this context, swimming activity of the rotifers (swimming of a grid) is defined as the number of 1 mm squares entered in a 30-s period of time (sq 30 s-l). This criterion was calculated only from the periods of displacements. The swimming activity criterion seems to be more sensitive than both the speed and sinuosity endpoints measured in the present study. However, except for lindane, the ratios
280
C.P. Charoy et al. IAquatic Toxicology 32 (1995) 271-282
of the speed and the sinuosity EC,,s to the swimming activity EC,,,s are small and varied from 1.08 to 1.76 depending on the chemical (3.19 for lindane). Comparing the 2-h EC&s obtained in the present study with the 24-h LC,,s reported by Janssen et al. (1994) and Ferrando et al. (1992) (Table 2), it can be concluded that the EC& resulting from behavioural tests were higher than the results from the conventional mortality tests. Even if the sensitivity of the behavioural toxicity test with copper was comparable to that of the acute toxicity test (Table 2), it is clear that the 2-h exposure period was too short to reach the same level of sensitivity as the 24-h LC,, tests. Despite this short exposure period, a clear concentration-response was observed. The mean coefficients of variation ranged from 24% to 34%; such values are satisfactory for behavioural criteria, and they suggest a good repeatability of the effects obtained. From the present study, it can be concluded that the locomotory behaviour of B. calycz~%rus is adversely affected by sublethal concentrations of chemicals. To date, very little information is available on the effects of toxicants on the locomotory behaviour of rotifers. Kleinow (1986) studied the qualitative effects of acrylamide on the movements of Brachionus plicatilis. Beauvais and Enesco (1985) examined the swimming activity of Asplanchna brightwelli exposed to curare by observing the rotifers’ behaviour as they swam over a grid. Using a similar method, Snell et al. (1987) showed that the swimming behaviour of B. plicatilis was adversely affected by sublethal levels of ammonia, and more recently Janssen et al. (1994) suggested that the swimming activity of B. calyczjkus could be used as a sensitive sublethal test criterion for ecotoxicological purposes. However, as mentioned, these three last workers used the same ‘open-field checkerboard grid’ method to record the rotifers’ movements. The swimming activity criterion obtained with this technique (counting the number of squares transversed by the rotifer) is, however, a very general parameter. With the method used in the present study the complex rotifer behaviour can be quantified and split up into simple and mathematically independent criteria. Indeed, the results of this study show that the four toxicants affect the various criteria in different ways (Table 2). Additionally, the sensitivity of these test endpoints depended on the compound used. Consequently, it can be concluded that as these various spatial and temporal behavioural endpoints seem to have quite different resolution powers in detecting sublethal effects, they may not only prove useful as ecotoxicological indices but also contribute to the understanding of some fundamental aspects of the effects of toxicants on the ecology of rotifers. Indeed, locomotory behaviour in general and rotifer swimming behaviour more specifically, can be considered as an integration of physiological, sensorial, nervous and muscular systems. It has been shown that rotifers’ swimming patterns are affected by the presence or absence of food (Charoy and Clement, 1993; Charoy, in press) and the oxygen content in the medium (Reale et al., 1993). Additionally, Gilbert (1963) reported that the presence of secretions from B. calyczjlorus females modified the swimming behaviour of conspecific males. Because of the integrative character of behavioural changes, it has been suggested that bioassays using behavioural test criteria could be very useful for rapid toxicity testing. Behavioural tests with fish have now become an accepted part of many mon-
C. P. Charoy et al. I Aquatic Toxicology 32 (1995) 271-282
281
itoring and testing programmes. However, the development of standardized toxicity tests with invertebrates using behavioural test endpoints is still in its infancy. The present study has shown that changes in the swimming characteristics of rotifers can certainly be considered as rapid and sensitive indicators of toxic stress.
Acknowledgments This work was supported by European Science Foundation grant RF/93/18/E. The authors would like to thank the anonymous reviewers for their constructive comments.
References Beauvais, J.E. and H.E. Enesco (1985) Life span and age-related changes in activity levels of the rotifer Asplanchna brightwelli: influence of curare. Exp. Gerontol. 20, 359-366. Borlakoglu, J.T. and R. Kickuth (1990) Behavioural changes in Gammaruspulex and its significance in the toxicity assessment of very low levels of environmental polluants. Bull. Environ. Contam. Toxicol. 45, 2588265. Bovet, P. and S. Benhamou (1988) Spatial analysis of animals’ movements using a correlated random walk model. J. Theor. Biol. 131,419433. Charoy, C. (in press) Modifications of the swimming behaviour of Brachionus calyct$orus (Pallas) according to both food environment and individual nutritive state. Hydrobiologia. Charoy, C. and P. Clement (1993) Foraging behavior of Brachionus CalyciJlorus: variations of the swimming path according to presence or absence of algal food (chlorella). Hydrobiologia 255/256, 95-100. Clement, P. (1977a) Introduction a la photobiologie des rotiferes dont le cycle reproducteur est controli. par la photoperiode. Approches ultrastructurale et experimentale. These Univ. Lyon I, 261 pp. Clement, P. (1977b) Ultrastructural research on rotifers. Arch. Hydrobiol. Beih. 8, 260-297. Clement, P. (1987) Movements in rotifers: correlations of ultrastructure and behavior. Hydrobiologia 147, 339-359. Clement, P. and E. Wurdak (1991) Rotifera. Microscopic Anatomy of Invertebrates 4: Aschelminthes. Wiley-Liss Inc., pp. 219-297. Clement, P., P. Mimouni, P. Fouillet and J.L. Chasst (1987) La discretisation spatiotemporelle de trajets obtenus en trajectometrie automatique. CR 112eme congrts des Societes Savantes. Clement, P.. P. Mimouni, P. Fouillet, P.Y. Coulon and M. Le Berre (1988) Recueil et analyse de deplacements de rongeurs par trajectometrie automatique. Sci. Tech. Anim. Lab. 13, 5-13. Cornillac, A., E. Wurdak and P. Clement (1983) Phototaxis in monochromatic light and microspectrophotometry of the cerebral eye of the rotifer Brachionus calyc$orus. Hydrobiologia 104. 191-196. Coulon, P.Y., J.P. Charras, J.L. Chasse, P. Clement, A.M. Cornillac, A. Luciani and E. Wurdak (1983) An experimental system for the automatic tracking and analysis of rotifer swimming behavior. Hydrobiologia 104, 1977202. Day, K. and N.K. Kaushik (1987) Short-term exposure of zooplankon to the synthetic pyredoid. fenvalerate, and its effects on rates of filtration and assimilation of the alga Chlamydomonas reinhardii. Arch. Environ. Contam. Toxicol. 16,423432. Fernandez-Casalderrey, A., M.D. Ferrando and E. Andreu-Moliner (1994) Effect of sublethal concentrations of pesticides on the feeding behaviour of Dapnia magna. Ecotoxicol. Environ. Saf. 27, 82-89. Ferrando, M.D., E. Andreu Moliner and A. Fernandez-Casalderrey (1992) Relative sensitivity of Daphnia magna and Brachionus calyciflorus to five pesticides. J. Environ. Sci. B 27, 51 l-522. Flickinger, A.L., R.J.F. Bruins, R.W. Winner and J.H. Skillings (1982) Filtration and phototactic behavior
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as indices of chronic copper stress in Daphnia magna Straus. Arch. Environ. Contam. Toxicol. 11, 451463. Geiger, J.G. and A.L. Buikema (1981) Oxygen consumption and filtering rate of Daphnia pulex after exposure to water-soluble fractions of naphthalene, phenanthrene, no 2 fuel oil, and coal-tar creosote. Bull. Environ. Contam. Toxicol. 27, 783-789. Gilbert, J.J. (1963) Contact chemoreception, matting behaviour and sexual isolation in the rotifer genus Brachionus. J. Exp. Biol. 40, 625-641. Goodrich, M.S. and J.J. Lech (1990) A behavioral screening assay for Daphnia magna: a method to assess the effects of xenobiotics on spatial orientation. Environ. Toxicol. Chem. 9, 21-30. Janssen, C., M.D. Ferrando and G. Persoone (1994) Ecotoxicological studies with the freshwater rotifer Brachionus calyc@orus: IV Rotifer behavior as a sensitive and rapid sublethal test criterion. Ecotoxicol. Environ. Saf. 28, 244255. Jones, M., C. Folt and S. Guarda (1991) Characterizing individual, population and community effects of sublethal levels of aquatic toxicants: an experimental case study using Daphnia. Freshwater Biol. 26, 3544. Kleinow, W. (1986) Effects of acrylamide on Brachionusplicatilis (Rotifera). Comp. Biochem. Physiol. 84C, 243-246. Luciani, A. (1982) Contribution a l’etude du vielhssement chez le rotifere Brachionus plicatilis: nage, cils et battements ciliaires, mttabohsme tnergetique. These Doct. Spec. Univ. Lyon I 1211, 86 pp. Reale, D., P. Clement and A. Esparcia-Collado (1993) Influence of the concentration of oxygen on the swimming path of Brachionus plicatilis (Rotifera). Hydrobiologia 255/256, 87-93. Snell, T.W. and CR. Janssen (in press) Rotifers in ecotoxicology: a review. Hydrobiologia. Snell, T.W. and G. Persoone (1989a) Acute toxicity bioassays using rotifers. 1. A test for brackish and marine environments with Brachionus plicatilis. Aquat. Toxicol. 14, 65580. Snell, T.W. and G. Persoone (1989b) Acute toxicity bioassays using rotifers. II. A freshwater test with Brachionus rubens. Aquat. Toxicol. 14, 81-92. Snell, T.W., M.J. Chidress, E.M. Boyer and F.H. Hoff (1987) Assessing the status of rotifer mass cultures. J. World Aquacult. Sot. 18, 270-277. Snell, T.W., B.D. Moffat, C.R. Janssen and G. Persoone (1991) Acute toxicity tests using rotifers. IV. Effects of cyst age, temperature and salinity on the sensibility of Brachionus calyciporus. Ecotoxicol. Environ. Saf. 21. 308-317.