Ecotoxicological studies with the freshwater rotifer Brachionus calyciflorus. Resource competition between rotifers and daphnids under toxic stress

The Science of the Total Environment, Supplement 1993 Elsevier Science Publishers B.V., Amsterdam

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Ecotoxicological studies with the freshwater rotifer Brachionus calyciflorus. Resource competition between rotifers and daphnids under toxic stress M.D. Ferrando a, C. Janssen b, E. Andreu a and G. Persoone b aLaboratoryfor Ecotoxicology, Department of Animal Biology, Faculty of Biological Sciences, Universityof Valencia, Dr. Moliner 50, 46100 Burjasot, Valencia, Spain bLaboratoryfor Biological Research in Aquatic Pollution, Universityof Ghent, J. Plateaustraat 22, 9000 Ghent, Belgium

ABSTRACT This study examined the ability of the freshwater rotifer Brachionus calyciflorus to coexist with the large cladoceran Daphnia magna, and the effect of sublethal concentrations of copper on the competition between both species. Preliminary laboratory experiments showed that large Daphnia ( > 1.2 mm) can kill and rapidly exclude the rotifer B. calyciflorus in mixed-species cultures. Brachionus populations were suppressed by Daphnia both through exploitative competition for shared, limited food resources and through mechanical interference. At a food concentration of 1 × 105 cells/ml of Nannochloris oculata, competition caused high mortality rates and decreased fertility in the rotifer populations (1000 ind/1), even at low Daphnia densities (40 ind/1). Daphnia removed increasingly disproportionate shares of the daily algal food supply, leaving the rotifer populations to gradually starve to extinction. In other experiments the effect of sublethal copper concentrations (2.5, 5.0, 12.0 and 20 /zg/1) on the competition between both species was analysed. Short experiments (5 days) were carried out to study the effect of this toxicant on the rotifer populations with and without Daphnia. Changes in the competitive relationships between both organisms under toxic stress and the possible ecological consequences of these observations will be discussed.

Key words." Rotifer; Daphnids; Competition; Ecotoxicology; Copper INTRODUCTION

Much direct and circumstancial evidence from field and laboratory studies indicates that rotifers are strongly supressed by competition from large species of Daphnia (Gilbert, 1989). This supression is probably due to 1993 Elsevier Science Publishers B.V.

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a combination of exploitative competition for shared resources (Gilbert, 1985) and interference or encounter competition in which rotifers swept into Daphnia branchial chambers are eaten or rejected in damaged condition (Gilbert, 1988). Interrelationships between rotifers and cladocerans have drawn increasing attention in recent years (Gilbert, 1988, 1989; Matveeva, 1989; Kirk and Gilbert, 1990) but little has been done so far to study the effect of pollutants on competition behavior. Sublethal concentrations of pollutants may be expected to affect various behavioral capabilities of animals. For instance, Farr (1978) demonstrated that methyl parathion impairs the ability of grass shrimp, Palae~nonetes pugio, to escape predation by the gulf killifish, Fundulus grandis. We have been conducting laboratory competition experiments to test the ability of the rotifer Brachionus catyciflorus to coexist with Daphnia magna, and to examine changes in competition of both species as a result of sublethal concentrations of copper. MATERIAL AND METHODS

Test species Daphnia magna. Daphnia magna was provided by the Laboratory for Biological Research in Aquatic Pollution (Gent, Belgium). The stocks were reared in 4-L aquaria in a synthetic medium, according to the EEC prescriptions (EEC, 1984); the medium was renewed three times weekly. Culture densities were kept below 50 animals/l, and the daphnids were fed daily on Nannochloris oculata at a density of 5 x 105 cells/ml. The aquaria were placed in a temperature-controlled cabinet (22°C) with a 12:12 h light-dark cycle at 1000 lux light intensity at waterlevel. Offspring were separated three times per week.

Brachionus calyciflorus B. calyciflorus cysts were stored at 6°C in the dark. Cysts used in the experiments were produced in mass cultures maintained under rigorously controlled conditions (Dr. T.W. Snell, University of Tampa, USA). Cyst hatching was initiated by transferring to warmer temperatures and light. A standard synthetic freshwater (EPA) was used as hatching, culture and dilution medium (EPA, 1985). This medium is prepared from reagent grade chemicals and composed of 96 mg NaHCO 3, 60 mg CaSO4.2H20, 60 mg MgSO 4, and 4 mg KC1 per liter of deionized water. Standard environmental

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conditions for these bioassays were: temperature 22°C; pH 7.4-7.8; hardness 80-100 mg CaCO3/1 and alkalinity 60-70 m g / l .

Nannochloris oculata N. oculata, which was used as food in all experiments, was cultured in BBM medium (Bischoff and Bold, 1983). The log phase algae were harvested, centrifuged, washed, and resuspended in E P A water. Concentration of algae was measured by counting with a haemocytometer. The suspension was then diluted with E P A water to produce the desired concentration. Nannochloris density chosen in the experiments was 1, 5 and 15 x 105 cells/ml.

Competition experiments Three different competition experiments were conducted in 50 ml flasks ( T = 22°C). In each experiment, three replicate cultures were set up for each of three treatments: (1) rotifers alone, (2) Daphnia alone, and (3) rotifers and Daphnia. Details of experimental conditions are shown in Table 1. Each experiment was divided into three more experiments (A, B, C) according to the initial Daphnia number. Every day throughout the experiments, populations of both species in each culture were transferred to freshly prepared algal suspensions. The same days, population sizes were determined just before the organisms were transferred to new algal suspensions. The effect of D. magna on the rotifer population was determined by comparing the mean population sizes in the experimental TABLE 1 Details of competition experimentsbetween the cladoceran D. magna(2 days old) and the rotifer B. ca~ciflorus (< 24 h old) Experiment Number Rotifer: Daphnia: N. oculata number replicates Initialnumber Initialnumber density(c/ml)

Duration (days)

1A

3

50

2

1B

3

50

3

1C 2A 2B 2C 3A 3B 3C

3 3 3 3 3 3 3

50 50 50 50 50 50 50

5 2 3 5 2 3 5

10 10 10 10 10 10 10 10 10

15 × 105 15 × 105 15 x 105 5 × 105 5 × 105 5 × 105 1 × 105 1 × 105 1 × 105

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and control treatments and calculating the Daphnia-induced death rates of significantly suppressed rotifers from their population growth rates in the experimental and control treatments. The growth rate of a population (r) was calculated from (Gilbert, 1989): r = (In N t - I n No)/t, where N t and N O are final and initial population sizes and t is time in days. Three replicate r values were calculated for each population in each treatment. The mortality rate of a population caused by the presence of Daphnia (d o) was calculated from: d o =r c -re, where r c and r e are the population growth rates in the control and experimental beakers. Three replicate values of d D were obtained for B. calyciflorus from control and experimental r values.

Survivorship experiments An experiment was conducted to see if Daphnia had a direct inhibitory effect on Brachionus populations through eating or mechanically damaging the rotifers. The design of this experiment was to determine the survivorship of young (2 h old), non-ovigerous rotifers cultured in N. oculata (5 × l0 s cells/ml) with and without Daphnia for a 24-h period. The temperature of the experiment was 22°C. The initial number of rotifers was 50 individuals, the experimental vessels were 50 ml beakers containing 40 ml of algal suspension, and the Daphnia treatment consisted of 2 adults (7 days old). Treatments with and without Daphnia were replicated five times. At the end of the experiment, all rotifers were counted. Rotifers would not produce offspring during the experiment. Thus, since birth rates of the rotifers were zero, the survivorship results could be used to calculate instantaneous death rates (d) from the formula (Gilbert, 1985): d = (In N O- In Nt)/t , where Noand Art are initial and final population sizes, and t is time in days.

Effect of copper on competition experiments B. calyciflorus and D. magna were exposed to sublethal copper concentrations (0, 2.5, 5.0, 12.0 and 20 /~g/l) for 5 days (T = 22°C). Three replicate cultures were set up for each of three treatments: rotifers alone, Daphnia alone, and rotifers with Daphnia. The initial number of rotifers was 25 individuals, the experimental vessels were 50 ml beakers containing 40 ml of algal suspension (N. oculata: 5 x 105 cells/ml) plus the different copper concentrations, and the Daphnia treatment consisted of 2 individuals (4 days old). Every day population sizes of both species were determined by counting the organisms, and N. oculata density was also calculated. Then, the organisms were transferred to a new medium (food and toxicant).

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Rotifer survival under different conditions was c o m p a r e d and growth rates (r) were calculated.

Statistical analysis D a t a for control and experimental organisms were c o m p a r e d by analysis of variance (ANOVA); in case of significant differences ( P < 0.05), the m e a n values for exposed populations were c o m p a r e d to those of the control by Duncan's multiple range test. RESULTS AND DISCUSSION Three separate competition experiments showed that D. magna excluded B. calyciflorus from mixed-species cultures when algal food density (N. oculata) was low as well as when the n u m b e r of daphnids was higher (Table 2). In experiments 1-3, m e a n Daphnia-induced death rates of the affected species B. calyciflorus ranged from 0.22 d-1 (exp. 1B) to 0.34 d-1 (exp. 3C) (Table 2). Comparisons revealed that the m e a n d D values of B. calyciflorus in exp. 2 and 3 were significantly higher than the m e a n d D values of B.

calyciflorus in exp. 1, when N. oculata density was higher. The decline of the rotifer populations in mixed-species cultures a p p e a r e d to be due to starvation. F o r example, in experiment 3 (Tables 1 and 2), when algal density was 1 x 10 5 c e l l s / m l and Daphnia density high, Brachionus rarely p r o d u c e d eggs, they gradually died while exhibiting little TABLE 2 Population growth rates (r d -1) and Daphnia-induced death rates ( d D d - 1 ) of B. ca~yciflorus in control (without Daphnia) and experimental conditions (with Daphnia). All values are means _ S.D. Exp.

1A 1B 1C 2A 2B 2C 3A 3B 3C

r d -1

d D d -1

Control

Experimental

0.81 + 0.01 0.78 _+0.10 0.75 + 0.09 0.72 + 0.02 0.69 + 0.03 0.69 + 0.10 0.59 + 0.08 0.48 _ 0.01 0.52 + 0.05

0.57 + 0.09 0.56 _+0.08 0.51 + 0.06 0.47 __+0.09 0.39 ___0.04 0.40 + 0.01 0.30 + 0.02 0.21 _ 0.01 0.18 + 0.02

0.24 __0.07 0.22 + 0.05 0.24 + 0.04 0.25 + 0.07 0.30 + 0.02 0.29 + 0.08 0.29 + 0.06 0.27 + 0.01 0.34 _ 0.03

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or no reproduction. In contrast, Daphnia appeared to be unaffected by the rotifer. A second experiment indicated that the decline of Brachionus in cultures with Daphnia could be attributed not only to starvation but also to direct interference competition. The survivorship of young individuals of this rotifer was reduced by the presence of D. magna (7 days old), indicating that they were eaten or mechanically damaged by Daphnia (Table 3). Experiments with the rotifer Keratella cochlearis and Daphnia pulex have shown that such ingestion and damage consistently occurs, especially with larger Daphnia (Gilbert and Stemberger, 1985). The damage may occur when the rotifer is passed through and rejected from a Daphnia branchial chamber. On the other hand, experiments carried out by Gilbert (1985) showed that the rotifer B. calyciflorus was not mechanically damaged by D. pulex, maybe due to the smaller size of this cladocera. Although some of the mortality of B. calyciflorus in the cultures with D. magna from the competition experiments (Table 2) may have been due to direct competitive interactions, most of the mortality was probably due to starvation. Daphnia have very high clearance rates, so they were sequestering a disproportionate share of the food when it was provided each day. For example, at 22°C, the clearance rate of D. magna ( < 24 h old) was 1000/~l/animal/h (Fern~ndez-Casalderrey et al., 1992), while that of B. calyciflorus ( < 24 h old) was 5 . 6 / x l / a n i m a l / h (Ferrando et al., 1992). Daphnia population would remove enough food to sustain itself, leaving the rotifers to starve until the next pulse of food was added. The results of the present study suggest that when shared food is limiting, rotifers can be rapidly excluded by large cladocerans, such as Daphnia. Even if food is not limiting, rotifers can be killed by large cladocerans through interference competition (Gilbert and Stemberger, 1985; Gilbert, 1985).

TABLE 3 Effects of D. magna (7 days old) on the survival and instantaneous mortality rates (d) of young (0-2 h old) B. calyciflorus in 40 ml cultures with N. oculata (5 X 105 cells/ml) as food Experiment treatment

Number of replicates

Duration(h)

Number of Daphnia Initial Final

Rotifers Initial Final

Daphnia

5

24

2

2

50

No Daphnia

5

24

2

2

50

42.8 + 3.0 50.0

d 0.14 + 0.06 0

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TABLE 4 Density of N. oculata (cells/ml) remaining in cultures at the end of successive 24-h periods in a competition experiment with B. ca~yciflorus and D. magna fed on N. oculata (5 X 105 cells/ml) and copper treatment Exp.

Day

Culture

Brachionus

Daphnia

alone

alone

Brachionus and Daphnia

Control

1 2 3 4 5

2.90 × 2.90 x 2.70 x 2.60 × 2.00 x

105 105 105 105 105

1.10 x 1.20 x 1.20 x 1.20 x 1.10 x

105 105 105 105 105

1.10 × 1.10 × 0.90 x 0.80 x 0.70 x

105 105 105 105 105

2.5/xg/1

1 2 3 4 5

2.00 x 2.10 x 2.00 x 2.20 x 2.00 x

105 105 105 105 105

1.10 x 1.16 x 1.03 × 1.33 x 1.21 x

105 105 105 105 105

1.28 × 1.03 x 1.33 x 1.48 x 1.21 x

105 105 105 105 105

5.0/xg/1

1 2 3 4 5

1.88 x 1.77 x 1.95 x 1.72 x 1.68 x

105 105 105 105 105

0.94 x 0.84 x 0.87 x 0.93 × 0.90 x

105 105 105 105 105

0.87 x 0.85 x 0.76 x 0.78 x 0.71 x

105 105 105 105 105

12.0/xg/1

1 2 3 4 5

1.78 x 1.81 x 1.82 x 1.68 x 1.71 x

105 105 105 105 105

0.94 x 0.89 x 0.83 x 0.94 x 0.90 x

105 105 105 105 105

0.78 x 0.80 x 0.70 × 0.73 × 0.73 x

105 105 105 105 105

20/xg/1

1 2 3 4 5

1.22 x 1.62 x 1.38 x 1.37 x 1.48 x

105 105 105 105 105

0.70 x 0.71 x 0.73 × 0.62 x 0.68 x

105 105 105 105 105

0.59 × 0.61 x 0.59 × 0.59 x 0.61 x

105 105 105 105 105

I n o u r last e x p e r i m e n t , survival o f B. calyciflorus a f t e r 5 days e x p o s u r e to sublethal copper concentrations differed, depending on copper c o n c e n t r a t i o n a n d the p r e s e n c e o r n o t o f D. rnagna ( T a b l e 5). C o p p e r at a n o m i n a l c o n c e n t r a t i o n o f 2.5 / z g / 1 a l t e r e d t h e survival o f t h e r o t i f e r with a n d w i t h o u t Daphnia. W h e n c o p p e r c o n c e n t r a t i o n s i n c r e a s e d (5.0, 12.0, 20.0 /zg/1) r o t i f e r survival d e c r e a s e d ( T a b l e 5). N o e f f e c t o n Daphnia

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TABLE 5 Survival of B. calyciflorus after 5 days exposure to copper, with or without Daphnia as a competitor Copper concentrations (I-~g/l)

Rotifers only Rotifers and Daphnia Daphnia only

0

2.5

5.0

12.0

20.0

100% 80% 100%

29% 25% 100%

30% 21% 100%

7% 3% 100%

0 0 100%

survival was observed after copper treatment (Table 5). On the other hand, when rotifers were exposed to copper, in the presence of Daphnia, the survival of this species decreased more, indicating a combined effect between toxicant and competition behavior. Table 6 shows population growth rates (r) of B. catyciflorus with and without competitor, after 5 days exposure to the toxicant. As we can see in that table r mean values decreased as toxicant concentrations increased, especially in the presence of D. magna in the culture vessels. Statistical differences ( P < 0.05) were found among both cultures when animals where exposed to 5.0 and 12.0 /~g/1 copper. Thus, we can observe that rotifer survival is affected by the toxicant, mainly when there is also competition with D. magna. Table 4 shows N. oculata density during the experiment. As we can see, algal density was almost the same in the cultures with Brachionus and Daphnia as in those with Daphnia alone. So Brachionus contributed only slightly to

TABLE 6 Population growth rates (r d -1) of B. catyciflorus with and without D. magna after 5 days exposure to different copper concentrations. Copper concentrations (~g/1)

r d- a Rotifers only

0 2.5 5.0 12.0 20.0

0.50 + 0.02 0.20 + 0.05 0.20 ___0.01 - 0 . 1 0 + 0.09 --

*P < 0.05.

Rotifers and Daphnia 0.40 0.16 0.13 -0.3

+ + + + --

0.10 0.07 0.04* 0.01"

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the depletion of the food supply available to the Daphnia. After exposure to copper, algal density in all the cultures decreased as copper concentrations increased, probably due to the effect of the toxicant on algal growth; thus when rotifers were in mixed cultures with Daphnia, food supply was also lower when exposed to copper than without toxicant and then survival or reproduction decreased. Several researchers have suggested that increases in phytoplankton should occur in aquatic ecosystems after the removal of the filter-feeding zooplankton by a toxicant (Hurlbert, 1975; Papst and Boyer, 1980; Day et al., 1987); especially cladocerans which have a significant impact on the abundance of phytoplankton. The fact that cladocerans did not decrease after copper treatment in our experiment would also explain the reduction in algal density. From our results we can suggest that competition relationships can alter because of low concentrations of pollutants, since copper tends to be more toxic to rotifers than to crustaceans, and sublethal concentrations of this toxicant render B. calyciflorus more susceptible to competition. It can be hypothesized that low levels of copper, and maybe other toxicants, in the field could result in diminished numbers of rotifers, and subsequently increased numbers of crustaceans. Few studies have been done before to see the effect of pollutants on laboratory competition experiments. Farr (1978) studied the effect of the insecticide methylparathion on the predator choice of two estuarine prey species, the grass shrimp Palaemonetes pugio and the juvenile Cyprinidon variegatus. The predator, Fundulus grandis, consumed a greater proportion of grass shrimp when both prey species were exposed to methylparathion than when prey were not exposed. The predaceous rotifer Asplanchna sieboldi thus increased after Dursban treatments increased phytoplanktivorous rotifer populations, Asplanchna's preferred prey, by eliminating a cladocern (Moina) population that had competitively suppressed these populations (Hulbert, 1975). However, there are more studies that report the effect of pollutants on field competition experiments. Kaushik et al. (1985) and Solomon et al. (1985) reported that low concentrations of permethrin in situ lake enclosures resulted in reductions of populations of cladocerans; rotifers were not affected by this, and densities of these organisms increased when released from the pressures of competition and predation as other species were eliminated by the insecticide. Similar results were found by Day (1989), who studied the effect of fenvalerate on zooplankton. This compound reduced populations of cladocerans and allowed populations of rotifers and green algae (Chlorophyta) to increase because of removal of cladoceran competition and grazing, respectively.

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Thus, i n t r o d u c t i o n o f p o l l u t a n t s to the aquatic system m a y have a great effect on the interactions b e t w e e n species such as c o m p e t i t i o n or p r e d a t i o n , a n d this effect could have i m p o r t a n t ecological c o n s e q u e n c e s f r o m the point o f view of f o o d chain efficiency at lower trophic levels. ACKNOWLEDGEMENTS M . D . F e r r a n d o is recipient of a fellowship f r o m P l a n N a c i o n a l de P e r f e c c i o n a m i e n t o de D o c t o r e s y Tecn61ogos, M E C , Spain. C.R. J a n s s e n a n d G. P e r s o o n e a c k n o w l e d g e the s u p p o r t of the E u r o p e a n C o m m i s s i o n ( C E C contract No. EV4V-0110-UK(BA): D e v e l o p m e n t a n d validation of m e t h o d s for evaluating chronic toxicity to f r e s h w a t e r systems). REFERENCES Bischof, H.W. and H.C. Bold, 1983. Phycological studies. IV. Some algae from enchanted rock and related algae species. Univ. Texas Publ., 6318, pp. 95. Day, K.E., N.K. Kaushik and K.R. Solomon, 1987. Impact of fenvalerate on enclosed freshwater planktonic communities and on in situ rates of filtration of zooplankton. Can. J. Fish Aquat. Sci., 44: 1714-1728. Day, K.E., 1989. Acute, chronic and sublethal effects of synthetic pyrethroids on freshwater zooplankton. Environ. Toxicol. Chem., 8: 411-416. EEC., C.2. 1984. In: '84/449/EEC: Commission Directive of 25 April 1984 adapting to technical progress for the sixth time Council Directive 67/548/EEC on the approximation of the laws, regulations and administrative provisions relating to the classification packing and labelling of dangerous substances'. Off. J. Eur. Commun., L 251, 27: 155-159. Farr, J.A., 1978. The effect of methylparathion on predator choice of two estuarine prey species. Trans. Am. Fish. Soc., 107(1): 87-91. Fernfindez-Casalderrey, A., M.D. Ferrando and E. Andreu, 1993. Effect of the insecticide methylparathion on filtration and ingestion rates of Brachionus calyciflorus and Daphnia magna. Sci. Total. Environ., 134. Ferrando, M.D.C.R. Janssen, E. Andreu and G. Persoone, 1992. Ecotoxicological studies with the freshwater rotifer Brachionus calyciflorus. IlL The effect of chemicals on the feeding behavior. Ecotox. Environ. Safety., 26: 1-9. Gilbert, J.J., 1985. Competition between rotifers and Daphnia. Ecology, 66(6): 1943-1950. Gilbert, J.J. and R.S. Stemberger, 1985. Control of Keratella populations by interference competition from Daphnia. Limnol. Oceanogr., 30: 180-188. Gilbert, J.J., 1988. Suppression of rotifer populations by Daphnia: a review of the evidence, the mechanisms, and the effects on zooplancton community structure. Limnol. Oceanogr., 33(6): 1286-1303. Gilbert, J.J., 1989. The effect of Daphnia interference on a natural rotifer and ciliate community: short-term bottle experiments. Limnol. Oceanogr., 34(3): 606-617. Hurlbert, S.H., 1975. Secondary effects of pesticides on aquatic ecosystems. Residue Rev., 57: 81-148.

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Haushik, N.H.G.L. Stephenson, K.R. Solomon and K.E. Day, 1985. Impact of permethrin on zooplankton communities in limnocorrals. Can. J. Fish Aquat. Sci., 42" 77-85. Kirk, K.L. and J.J. Gilbert, 1990. Suspended clay and the population dynamics of planctonic rotifers and cladocerans. Ecology, 71(5): 1741-1755. Matveeva, L.K., 1989. Interrelations of rotifers with predatory and herbivorous cladocera: a review of Russian works. Hydrobiologia, 186/187: 69-73. Papst, M.H. and M.G. Boyer, 1980. Effects of two organophosphorus insecticides on the chlorophyll a and pheopigment concentrations of standing ponds. Hydrobiologia, 69: 245-250. Solomon, K.R., J.Y. Yoo, D. Lean, N.K. Kaushik, K.E. Day and G.L. Stephenson, 1985. Dissipation of permethrin in limnocorrals. Can. J. Fish Aquat. Sci., 42: 70-76. United States Environmental Protection Agency, 1985. EPA/600/4-85/013, 216p.