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Arch. Environ. Contam. Toxicol. 28, 508-512 (1995)

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Rapid Toxicity Assessment Using Ingestion Rate of Cladocerans and Ciliates C. M. Juchelka, T. W. Snell School of Biology, Georgia Institute of Technology, Atlanta, Georgia 30332-0230, USA Received: 15 July 1994/Revised:26 November 1994 Abstract. Ingestion rate of suspension feeding zooplankton is a useful endpoint for rapid toxicity assessment. Methods are presented for quantifying ingestion rate in single Ceriodaphnia dubia, Paramecium aurelia, and Brachionus plicatilis, using fluorescent beads and image analysis. Test organisms are exposed to toxicants for one hour, then allowed to feed on 2 Ixm fluorescent beads for 3 minutes. Toxicants reduced ingestion rate in a dose-dependent manner, permitting the calculation of NOECs. The NOECs for 7 toxicants are presented for the 3 species as well as comparative toxicity of pore water samples from 13 urban creeks. The sensitivity of the 1-h C. dubia ingestion rate test was compared to 48-h LC50 and 7-d reproductive tests for 2 and 4 compounds, respectively. The ingestion test was within a factor of 2 for 3 of the comparisons, but >8 times less sensitive for the other 3 comparisons. For B. plicatilis, 1-h ingestion rate NOEC was lower than the 24-h LC50s for 4 of the 5 compounds tested. The rotifer 48-h reproductive test was a more sensitive endpoint for 3 of 4 compounds. These methods are simple, rapid, and inexpensive, permitting several species to be incorporated into a test battery.

Previous studies have shown that zooplankton ingestion rate is a useful toxicological endpoint for daphnids (Capuzzo 1979; Cooley 1977; Day and Kaushik 1987; Flickinger et al. 1982; Geiger and Buikema 1981; Kersting and Van der Honing 1981; Lampert et al. 1989), rotifers (Fernandez-Casalderry et al. 1992, 1993, 1994; Juchelka and Snell 1994), and copepods (Reeve et al. 1977; Moraitou-Apostolopoulou and Verriopoulos 1979; Berman and Heinle 1980). Toxicant-induced reductions in ingestion rate eventually may lead to reduced survival and reproduction and adverse consequences at the population level (Halbach 1984; Kooijman and Metz 1984). Changes in feeding behavior could be used as rapid and sensitive indicators of toxic stress and may predict reductions in survival, growth, or reproduction (Geiger and Buikema 1981; Day et al. 1987). Most estimates of zooplankton ingestion rate in toxicological studies have relied on estimates of algal density before and after

Correspondence to: T. W. Snell

feeding. This method requires long feeding intervals, many test animals, and has large measurement errors. Fluorescent detection methods have been used by ecologists to measure ingestion rates in protozoan (Sherr et al. 1987) and rotifer (Rublee and Gallegos 1989) grazing studies. Fluorescent methods also have been developed for quantifying rotifer ingestion rate in ecotoxicological studies (Juchelka and Snell 1994). These authors used fluorescein labeled latex microspheres and image analysis to quantify ingestion rates in single rotifers. They showed that ingestion rate is a more sensitive endpoint than mortality in the rotifer 24-h acute test and approximates the sensitivity of the rotifer 48-h reproductive test for some compounds. Consensus is growing among aquatic toxicologists that single species tests are inadequate to characterize toxicity (Cairns 1983, 1986) and that a multispecies battery relevant to the receiving water is the preferred approach (Schimmel et al. 1989). Using a multispecies battery to assess toxicity requires that each test be rapid, simple, and inexpensive. Ingestion rate as determined with fluorescent beads has the potential to be useful in this application. The objective of this paper is to extend the methodology of estimating ingestion rate using fluorescent beads to other test animals. A test battery with representatives from cladocerans, rotifers, and ciliates will provide a better indication of how zooplankton assembleges might respond to toxicants than single species tests. The rotifer protocol has been adapted for the ciliate Paramecium aurelia, the cladoceran Ceriodaphnia dubia, and the marine rotifer Brachinousplicatilis. Ingestion rates decrease proportionally with increasing concentration of toxicants. Data are presented on pure toxicants and on pore water samples and the Sensitivity of ingestion rate to toxicant stress is compared to mortality and reproductive endpoints. Materials and Methods The B. plicatilis was the Russian strain (Snell et al. 1991). Test animals were obtained by placing about 2,000 cysts in a glass petri dish with 40 mL of ASPM synthetic seawater (Guillard 1983). At 15 ppt salinity, ASPM consists of 11.31 g NaC1, 0.36 g KC1, 0.54 g CaC12, 1.97 g MgC12 - 6H20, 2.39 g MgSO4 • 7H20, 0.17 g NaHCO3 in one liter of deionized water. The pH was adjusted to 8.0 with NaOH or HCI. The cysts were incubated at 25°C in continuous light of 20004000 lux. Hatching began after about 27 h and animals were used within five h of hatching.

Ingestion Rate of Cladocerans and Ciliates

A 24-well polystyrene plate was used to expose test animals to a control and several toxicant dilutions. Up to 2% of dimethyl sulfoxide was added to toxicants with low water solubility and this concentration also was added to the solvent control. The toxicants tested included the metals copper (CuC12 • 2H20), cadmium (CdCIa), and mercury (HgCI2); the pesticides pentachlorophenol and chlorpyrifos; and the organics phenol and naphthol. The synthetic beads consisted of fluorescein labeled, 2 p.m diameter latex microsopheres obtained from Molecular Probes, Inc. (Eugene, OR). The experimental design for estimating ingestion rate in B. plicatilis consisted of 15 replicates for each treatment, including the control. For each toxicant, there was a series of four concentrations. Thirty females were transferred into each exposure well and exposed for 1-h at 25°C. Microspheres then were added at a density of 106/mL and the rotifers were allowed to ingest them for 3 minutes. Tricaine methane sulfonate (TMS) was added to a final concentration of 24 p,M to anesthetize the animals. Test animals then were transferred to a microscope slide, a coverslip applied, and viewed at 50 × magnification. The illumination system had an excitation filter of 490 nm and an emission filter of 515 nm. The intensity of the localized fluorescence was quantified by an image analysis system with NIH Image software (National Institutes of Health, Bethesda, MD). A Javelin Newvichip CCD camera mounted on the microscope recorded the images of individual rotifers at 50 x magnification. These images were digitized with a Data Translation QuickCapture card on a Macintosh Ilci computer. The fluorescent intensity in each rotifer was quantified by centering a circle with a diameter of 41 p~m over the gut of the test animals. This size circle corresponded to the average gut diameter of the rotifer which was determined by gut measurements of 50 animals. This circle was used to measure the fluorescent intensity in the gut of each of the 15 replicate animals for each toxicant concentration. The fluorescent intensity of one microsphere was determined by taking the mean measurement of 25 individual spheres and was calculated to be 4.3 fluorescent units. The ingestion rate was computed by dividing the gut fluorescence intensity by 4.3. Treatment ingestion rates were compared to controls by a one-way analysis of variance and Dunnett's test to determine the highest toxicant concentration in which ingestion rate was not significantly different from that of the control animals (NOEC)..The normality of ingestion rate data was confirmed with a Shapiro-Wilks test and homogeneity of variances with a Bartlett's test. Similar tests were performed on the cladoceran C. dubia. Animals were continuously cultured in cladoceran medium (Keating 1985), consisting of 250 ml M-stock solution, 250 ml S-stock solution, 1.0 L deionized water, and 1.5 ml of vitamin stock. Twice weekly they were fed 5 × 105 cells/mL of Ankistrodesmus convolutus (UTEX#749) (Starr and Zeikus 1993). The C. dubia were tested in moderately hard synthetic freshwater (USEPA 1985). Animals used in a test were young females less than 24 h old. The same 7 toxicants were tested as for B. plicatilis in a similar experimental design with a few modifications. There were five replicates for each treatment, including the control. Ten animals were measured per treatment. The animals were exposed to test solutions for one hour at 25°C, then allowed to ingest microspheres at a density of 3 × 105/mL for 5 minutes. A concentration of 110 mM TMS was used to anesthetize the C. dubia at the end of the feeding period. The animals were then quickly transferred to a microscope slide because prolonged exposure to the TMS solution causes the carapace to fall off, rendering quantification of fluorescence impossible. The C. dubia images were recorded at 25 x magnification. The gut of C. dubia was traced freehand under white light, the tracing saved, and then restored for measuring gut fluorescence under epifluorescent illumination. Ingestion rates were calculated by dividing the average total gut fluorescence by the average intensity of one microsphere. A similar ingestion rate protocol was developed for the ciliate P. aurelia. These cells were maintained in serial batch culture in Sonnebore's Medium, which consisted of 2.5 g Cerophyll, 1.0 L deionized water, and 0.5 g Na2HPO4, diluted 1:10 (Sonnebom 1970). Dilution water was moderately hard synthetic freshwater. The paramecia used

509

in a test were selected from log-phase cultures. About 40 cells were added to each treatment well with a micropipet. After one h exposure, microspheres were introduced into the wells at a density of 2 x 106 /mL. The paramecia were permitted to ingest the spheres for 30 min, then quickly transferred to a microscope slide and a 1.2 mM TMS solution was added directly to the slide. Then replicate paramecia were measured per treatment at 50 x magnification. Fluorescent intensity of the ingested microspheres was measured by tracing the entire cell under white light, restoring the traced image under epifluorescent illumination, and quantifying fluorescence intensity. An ANOVA and Dunnett's test were performed to obtain an NOEC for each toxicant. The C. dubia, P. aurelia, and B. calyciflorus (Juchelka and Snell 1994) ingestion rate tests were conducted on pore water samples from 13 freshwater creeks that were part of an urban creeks study in the metro Atlanta area. The samples were obtained with a pore water extractor (Winger and Lasier 1991) from a depth of 1 cm in the sediments. The samples were stored at 4°C for no more than two weeks prior to their analysis. Before initiating a test, 1.2 ml of each sample was centrifuged for 5 minutes at 14,000 rpm to remove suspended particulates. The tests were then performed as previously described using undiluted pore water instead of the toxicant dilution series.

Results A comparison of ingestion rate N O E C s for C. dubia, P. aurelia, and B. plicatilis is presented in Table 1. Data for the ingestion test using the freshwater rotifer B. calyciflorus is included for comparison. The C. dubia ingestion test was most sensitive to cadmium, mercury, and chlorpyrifos, with NOECs of 0.063, 0.01, and 0.005 rag/L, respectively. The P. aurelia test was most sensitive to pentachlorophenol and copper, with NOECs of 0.1 and 0.0063 mg/L, respectively. The marine B. plicatilis ingestion rate test was most sensitive to mercury with an N O E C of 0.01 mg/L and to PCP with an N O E C of 0.5 mg/L. Examples of concentration-response curves for C. dubia are shown in Figure 1. Control ingestion rates measured over several weeks averaged 32.4 beads ingested per individual in 5 minutes with a coefficient of variation of 7.6%. The data show percent control ingestion rate as a function of log concentration. For example, ingestion rate at 0.005 chlorpyrifos/mL was 85% of control (0.85 x 33.4). Effect levels were examined for ingestion rate at the lowest observed effect concentration (LOEC) of the seven toxicants. Effect levels indicate how much ingestion rate must be reduced by toxicant exposure in order to detect a significant difference from control. On average, B. plicatilis, C. dubia, and P. aurelia ingestion rates were 4 1 - 4 9 % lower at the L O E C than in controls for PCP, copper, naphthol, and phenol. Similarly, ingestion rates were 25-26% lower for chlorpyrifos and cadmium, but 62% lower for mercury. The C. dubia 1-h ingestion rate test (i-NOECs) can be compared with the 7-d reproduction test (r-NOECs) (Winner 1989) and 48-h LC50s (Oris et al. 1991) for some of the toxicants tested. The i-NOECs were less sensitive endpoints than the rNOECs and the LC50s for all of the toxicants compared. The i-NOECs were about 8 and 125 times less sensitive than the r-NOECs for phenol and cadmium, respectively. However, the i-NOECs for PCP and copper were only 1.6 and 1.3 times less sensitive than r-NOECs, respectively. Data for 48-h LC50s was available only for phenol and copper and the i-NOECs were 10 and 1.8 times less sensitive, respectively. The 24-h LC50s were similar to the 1-h i-NOECs for P. aurelia. For cadmium and mercury, the LC50 test was about

510

C.M. Juchelka and T. W. Snell

Table 1. Comparison of ingestion rate NOECs (ing NOEC) with reproductive NOECs (rep NOEC). The ing NOECs are 1-h tests; the reproductive tests are 48-h for B. plicatilia and B. calyciflorus and 7-d for C. dubia. The Paramecium LC50 is for a 24-h test with P. caudatum. All values are in mg/L

B. plicatilis

C. dubia

P. aurelia

Compound

ing NOEC

rep NOEC

ing NOEC

rep NOECa

ing NOEC

Pentachlorophenol Phenol Cadmium Copper Mercury Naphthol Chlorpyrifos

0.5 250 30 0.1 0.01 8 1.5

0.5 125 30 0.1 0.01 --

0.5 31 0.06 0.03 0:01 2.5 0.005

0.3 4 0.0005 0.02 ----

31 0.25 0.006 0.05 7 3

-

-

0.1

B. calyciflorus LC50b -

-

-0.18 0.01 0.02 --

-

ing NOECc 0.13

250 0.25 0.05 0.1 0.63 0.25

rep NOECc 0.11 25 0.04 0.02 0.01 0.63 0.23

aWinner 1989 bMadoni et al. 1992 CJuchelka and Snell 1994

120

~ 100.

calyciflorus, C. dubia, and P. aurelia ingestion rate tests, along with the yeast Candida tropicalis and the bacterium Bacillis subtilis esterase enzyme inhibition tests (Burbank and Snell 1994), were performed on these samples. The sensitivity of the tests was determined by counting the number of sites found to be toxic and how many sites were uniquely toxic to one, two, or three species. The B. calyciflorus ingestion rate test detected toxicity at 9 of the 13 sites. The P. aurelia test showed 7 of the sites to be toxic, whereas both the C. dubia and B. subtilis tests detected toxicity at only 3 of the 13 sites. The C. tropicalis test failed to detect toxicity at any of the sites tested. The rotifer test alone found two of the sites to be toxic, and the P. aurelia test was unique in detecting toxicity at one site. Seven of the sites that were toxic with the rotifer ingestion rate test were also toxic with one other test. The C. dubia and P. aurelia tests both shared toxicity to one site, whereas the B. subtilis and P. aurelia tests shared toxicity to two sites. Only one of the 13 sites was not toxic by all five tests.

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Fig. 1. Concentration-response curves for C. dubia ingestion rates. Ingestion rates are beads ingested per animal in 5 minutes. Control ingestion rates are 30, 34, and 33 beads for cadmium, mercury, and chlorpyrifos, respectively

1.4 and 2.5 times more sensitive, respectively. In contrast, the ingestion test was about 1.7 times more sensitive for copper. The B. plicatilis ingestion test was generally more sensitive than the 24-h LC50s, but less sensitive than the 48-h reproductive test. The B. plicatilis i-NOECs were lower than the 24-h LC50s (Snell et al. 1991) for 4 of the 5 toxicants tested. The ingestion rate test ranged from 1.3 times more sensitive for cadmium to 6.1 times more sensitive for mercury. Only for copper was 24-h mortality a more sensitive endpoint than 1-h ingestion rate. Comparing B. plicatilis 48-h r-NOECs to the i-NOECs shows that 48-h reproduction was a more sensitive endpoint for three of the four toxicants tested. The i-NOEC and r-NOEC were equally sensitive to pentachlorophenol. The results of toxicity tests of the pore water samples from the 13 freshwater creeks are presented in Table 2. The B.

Discussion

Ingestion rate as a toxicological endpoint has been shown to be applicable to a variety of aquatic organisms, ranging from rotifers and cladocera to unicellular ciliates. Results further suggest that toxicity is more reliably characterized using a battery of test species since no single species is always the most sensitive. Quantification of ingestion rate in suspension feeders using fluorescently labeled microspheres and image analysis is rapid and sensitive. Using methods like these permit several test species to be included in a battery, making it more likely that toxicity will be detected. It is not known whether ingestion rates are an index of exposure rather than toxicity. If recovery to control ingestion rates after exposure occurs, then ingestion rate would be more appropriately viewed as an index of exposure. However, if no recovery is observed, then ingestion rate would be considered an index of toxicity. Exposure-recovery experiments are planned to clarify the type of endpoint ingestion rate represents. It should be noted, however, that Juchelka and Snell (1994) showed that there is a good correlation in the concentrationresponse curves for rotifer ingestion and reproductive rates for several toxicants.

Ingestion Rate of Cladocerans and Ciliates

511

Table 2. Comparison of B. calyciflorus, C. dubia, and P. aurelia ingestion rate tests of pore water samples from 13 urban creeks. Also compared are the Candida tropicalis and Bacillus subtilis esterase enzyme Inhibition tests

# # # # #

sites tested toxic uniquely toxic toxic to one other species toxic to two other species

Brachionus

Ceriodaphnia

Paramecium

Candida

Bacillus

13 9 2 7 0

13 3 0 3 0

13 7 1 6 0

13 0 0 0 0

13 3 0 3 0

A search of the literature revealed no work on the response of C. dubia ingestion rates to toxicants; several works on Daphnia were located. Flickinger et al. (1982) reported that D. magna filtration rate was significantly reduced after 24-h exposure to 10 Ixg/L copper. Fernando and Andreu-Moliner (1993) reported the D. magna 24-h ingestion rate NOEC in response to copper toxicity to be 20 Ixg/L, whereas the 24-h LC50 is 380 p,g/L. Bodar et al. (1988) reported that D. magna feeding rates were significantly reduced at cadmium concentrations as low as 1 p~g/L as compared to an 24-h LC50 of 970 Ixg/L. Gliwicz and Sieniawska (1986) found the filtering rate of D. pulex reduced by 25% at concentrations of 50 txg dichlorofenil/L, much lower than the 48-h LC50s. The 5-h ingestion rate of D. magna was significantly reduced at 31 Ixg methylparathion/L, whereas the 24-h LC50 was 0.3 Ixg/L (Fernandez-Casalderry et al. 1993). Fernandez-Casalderry et al. (1994) reported that the D. magna 5-h ingestion rate NOECs for endosulfan and diazinon were 0.15 and 0.23 Ixg/L, respectively, compared to 24-h LC50s of 0.62 and 0.90 Ixg/L. These data suggest that daphnid ingestion rate after a few hours of toxicant exposure is as sensitive an endpoint as 24--48h LC50s for many compounds. Some compounds, like methylparathion, might require longer exposures to detect toxicity. Much less work has been done on the ecotoxicology of ciliates than daphnids, but there are a few observations. When comparing the acute toxicity of some metals on seven species of ciliated protozoans, Paramecium caudatum proved to be one of the more sensitive test organisms (Madoni et al. 1992). Cadmium toxicity to P. aurelia has been studied by Sundararaman and Gupta (1990). Exposures to 100 txg/L cadmium for 24-h caused a decrease in the daily fission rate. At 100 Ixg/L, only about 60% of the cells divided normally, at 200 Ixg/L only a few cells underwent normal fission, and at 400 txg/L the surviving cells did not divide. The ultrastructure of P. aurelia was also altered after a 24-h exposure to 200 ~g/L cadmium. The observation that the P. aurelia 1-h ingestion rate NOEC for cadmium was 250 Ixg/L is consistent with the above results. Hoffman and Atlas (1987) have used a similar method to determine the effect of cadmium stress on bactivory by the ciliate Aspidisca costata in sewage slude. The sludge communities were exposed to 0 to 150 mg Cd/L for up to 6 h, and then bacterial grazing rates were measured by a double-staining technique and epifluorescence microscopy. They used a mixture of 0.91 and 0.53 Ixm diameter fluorescein-labeled latex beads at a concentration of 6.5 x 107/mL and a feeding time of 5 minutes. Grazing was significantly inhibited by exposures to cadmium concentrations > 2 5 mg/L. By comparison, P. aurelia ingestion rate NOEC for cadmium was 100 times lower. Ingestion rate ofB. plicatilis has been used to assess chlorine and chloramine toxicity by Capuzzo (1979). Rotifers were exposed to 1 mg/L free chlorine for 30 min, then transferred to

clean seawater with diatom food for 24-h. Filtration rates of rotifers surviving toxicant exposure over the 24-h period were about 50% lower than controls. This suggests that lower concentrations of free chlorine reduce filtration rate more than the concentrations causing mortality. The data presented also indicate that ingestion is reduced at lower toxicant concentrations than mortality and that ingestion rate responds more rapidly. Ingestion rate tests are simple, have wide species applicability, and provide toxicity results rapidly at low cost. Assessing toxicity with such techniques permits several test species to be included in a test battery. The results presented here suggest that using fluorescent beads to quantify ingestion rate could be a useful rapid screening tool for assessing toxicity in aquatic environments.

Acknowledgments. The manuscript was improved by comments from David Dusenbery.

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