Ecotoxicology, 4, 206-218 (1995)

Life history characteristics of the freshwater ostracod Cyprinotusincongruensand their application to toxicity testing J O H N E. H A V E L * and B A R R I E L. T A L B O T T * * Department of Biology, SouthwestMissouriState University, Springfield, Missouri65804, USA Received 17 June 1993; accepted 30 September 1993

The biology of ostracod crustaceans suggests that this group could be a useful model for sediment toxicity testing. We examined life history characteristics of the freshwater ostracod Cyprinotus incongruens in the laboratory (25° C), then used length after 10 days as a response variable in a sediment toxicity test. Life history results indicated that both fecundity (0-64 total eggs per female), and egg development time (1->157 days), were highly variable. In contrast, juvenile development time showed less variation (7-16 days), and thus body length after 10 days was chosen as a sublethal bioassay character. A bioassay experiment using sediments isolated from mine-impacted cobble streams indicated that C. incongruens had a higher sensitivity to variation in sediment quality than the cladoceran Ceriodaphnia dubia. Surprisingly, the results indicated no correspondence between concentration of metals and toxicity in either C. incongruens or C. dubia. Overall our results indicate that ostracods are a good candidate taxon for sublethal toxicity tests of contaminated sediments. Keywords: development time; fecundity; life history; ostracods; sediment toxicology Introduction Environmental toxicologists have recently focused more attention on the study of sediments, which serve as an important sink for heavy metals and organic contaminants in aquatic systems (F6rstner and Wittmann, 1981). Attempts are presently in progress to identify appropriate test organisms from the benthic community and to establish standardized methods to quantify the bioavailability of sediment contaminants (Giesy and Hoke, 1989; Ingersoll and Nelson, 1990; Burton, 1991; Ingersoll, 1991; Landrum, 1991; Ross, 1991). A variety of benthic invertebrates have been employed, including nematodes, oligochaetes, amphipods, isopods, midges, and burrowing mayflies (Giesy and Hoke, 1989; Burton, 1991). Properties of effective bioassay organisms include a rapid response, ease of culture and handling, sensitivity to toxicants, low cost, broad geographical distribution, and ecological relevance (Giesy and Hoke, 1989). Each of the test systems currently in use has its advantages and disadvantages over the others (Giesy and Hoke, 1989). *To whom correspondence should be addressed. **Current address: Department of Zoology, Universityof Guelph, Guelph, Ontario N1G 2Wl CANADA 0963-9292 © 1995 Chapman & Hall

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207

Several features of ostracods suggest that this group may be especially useful for sediment bioassays. First, ostracods are common in many types of freshwater and marine habitats. Second, ostracods are primarily benthic in habit; various species live on or in the sediments as well as associated with littoral vegetation (Tressler, 1959). Third, ostracods are small and thus can be used in small-scale tests (Woodward et al., unpublished). Fourth, some ostracod species are easily reared in the laboratory (Kesling, 1951; Cohen, 1983; Havel and Hebert, 1989). An additional advantage of many freshwater ostracods, such as C. incongruens, is their parthenogenic mode of reproduction (Havel and Hebert, 1993). Such a breeding system allows isofemale lines (clones) to be established, thus excluding genetic variation from experiments (Havel and Hebert, 1989), and allowing life history studies with all-female populations. In the current study, we examined the feasibility of using freshwater ostracods for sublethal tests of sediments contaminated by heavy metals. We examined life history characteristics of the common epibenthic ostracod Cyprinotus incongruens Ramdohr (Crustacea, Cyprididae), focusing on juvenile development time and fecundity as possible sublethal traits. Using length after 10 days as a response variable, we carried out a bioassay with sediments from 36 Ozark stream locations showing variable levels of contamination by toxic metals. We hypothesized that C. incongruens would show a higher sensitivity to sediment-bound toxic metals than that of the planktonic cladoceran

Ceriodaphnia dubia. Methods

Life history studies Stock cultures of C. incongruens were obtained from a culture at the University of Windsor (Ontario), believed to have come from a Texas population (P.D.N. Hebert, personal communication). Stock cultures were started by placing 10-20 adult C. incongruens into glass jars filled with 500 ml 'artificial pond water' (recipe in Hebert and Crease, 1980) (pH = 7.4, conductivity = 150/~s, alkalinity = 32mg l -a, and total hardness = 54 mg 1-~ as CaCO3). Every other day, the ostracods were fed green algae (Selenastrum sp., final density about 10 6 cells ml-1), cultured by the methods of Goulden et al. (1982). Fresh lettuce leaf (about 5 cm 2) added once a week served as both a food and surface for grazing. All cultures were maintained at 25 + I°C with a 24 h photoperiod (cool fluorescent light, 50 lux). The life history study entailed observations recorded from three generations of ostracods (Go, G1, G2). Fecundity estimates were pooled from eggs laid by Go and Ga adults and body lengths pooled from measurements of G1 and G2 offspring. Pooling was valid, as statistical comparisons indicated no significant difference between the mean response for each of the generations. Egg development time was determined by observation of G1 eggs. Approximately 15 Go juveniles were removed from the stock culture jars and pipetted individually into 3 ml culture wells filled with artificial pond water and algae (Selenastrum sp.) at a final cell density of 8 x 10 6 cells per ml (standard algal diet). These ostracods were fed three times a week by adding the standard algal diet and observed daily until they reached reproductive maturity. When egg production began, fecundity was estimated by counting the number of eggs

208

Havel and Talbott

in each cluster using a Wild M5A dissecting microscope at 100x magnification. The eggs of C. incongruens are a bright orange colour and thus were readily detected in the well plates. Observations were made once a day on each of the Go adults for their G1 eggs, with all the eggs laid in one 24 h period considered a clutch. After each individual female had laid her first clutch, she was removed from the well and discarded to prevent her from laying additional eggs among the first clutch. To determine egg development time, the eggs were monitored daily for new hatchlings. As individual eggs hatched, the date was recorded and the G1 neonates were removed to a second well tray. The length of each G1 ostracod was measured daily at 50x magnification with an ocular micrometer. Careful observations of the wells allowed for the isolation of the exuvium of most newly-molted ostracods, thus confirming that a molt had occurred. G2 individuals (below) were similarly measured. After each G~ individual had matured and laid its first clutch, the adult ostracod was moved to a third tray, and its eggs (G2) were counted. Subsequently, after each clutch, the adult was moved to a new tray and eggs of each clutch counted until the adult died. All clutches of eggs were monitored daily and G2 neonates counted as they hatched.

Bioassay experiment C. incongruens were tested for their response to contaminated stream sediments in a 10 day bioassay and compared with results of the standard 7 day Ceriodaphnia bioassay (Weber et al., 1989). Since individual growth rates appeared to have a low variance in the life history experiment (see results below), body length after 10 days was used as a response variable for the bioassay. Survivorship over 10 days served as a second response variable. The experiment employed 36 different stream sediments as treatments, with nine replicate ostracods exposed to each sediment. The medium-sand-grain sediments (250-500ktm) were isolated by sieving samples from 12 Ozarks cobble stream sites. These sites showed high variation in heavy metal concentrations in the sediments (range for cadmium, not detectable (nd) to 125; copper, nd to 190; lead, 40 to 3700, nickel, 6 to 30; and zinc, 10 to 6960 ppm (Havel and Schultz, 1993). In place of artificial pond water (used above), 20/~m-filtered reservoir water was used for the bioassays with both Ceriodaphnia and Cyprinotus. The source of this water was Fellows Lake, Greene County, MO, USA (pH = 7.8, conductivity = 211/~s, alkalinity = 104mg 1-1 as CaCO3, and total hardness = 124 mg 1-1 as CaCO3). Ten to 15 days before starting the bioassay, well trays containing adult ostracods were established as a neonate source and fed the standard algal diet. Small blades of a local rescue, cut to lengths of about 5 mm and soaked in double distilled water, were added as a substrate to enhance egg laying. The neonates from each well were removed daily, providing a ready supply of known-age (<24 h) neonates for the bioassay. Since a pilot bioassay experiment using neonates resulted in unacceptably high mortality, we pooled hatchlings from each day and held them in a beaker for 2 days, during which time the ostracods molted once. These 2-day-olds were then randomly allocated to the 36 different treatments. To begin the bioassay, replicate 1.9 g samples of each of the 36 sediments were randomly allocated to tissue culture wells mixed with 2 ml of artificial pond water, and allowed to settle for 24 h. Individual 2-day-old juveniles were carefully pipetted into each well and fed the standard algal diet. Because of the juvenile's small size and delicate

Sublethal responses of a freshwater ostracod

209

carapace, pipetting was viewed through the dissecting microscope at 50 × to insure that the animal entered the well successfully and without obvious damage. The well trays were placed inside a GCA/Precision Scientific growth chamber at 25.6 _ 0.1°C and 16:8 light:dark photoperiod and kept under a translucent tupperware box in order to slow evaporation and keep light intensity at 1500 lux. Each day of the experiment 50% of the volume of each well was replaced with fresh renewal water (filtered reservoir water with Selenastrum sp. added to a final density of 105 cells per ml). The experiment was terminated on the tenth day, when each ostracod was removed and preserved. The number of survivors was counted and the body length of each survivor was estimated by measuring maximum carapace length at 50×. Survival and body length of the ostracods were compared to the results from the 7 day survival and reproduction test with Ceriodaphnia dubia (Weber et al., 1989) which had been exposed to the same sediments. Nine replicates were run for each of the 36 stream locations. For this test, C. dubia were isolated from stock cultures maintained at 25°C in filtered lake water and fed Selenastrum and YCT every day. Neonates (0-4 h old) were placed individually into 20 ml beakers containing 5 g of medium grain sand from each location and filled to 15 ml total volume with artificial pond water. Each day, the C. dubia were fed and the offspring were removed and counted until 7 days had elapsed (Weber et al., 1989). Since fecundities were lower than expected, a second fecundity endpoint was measured after 9 days. Body length of C. dubia was also measured after 9 days growth. In order to normalize the distributions and reduce heteroscedasticity, the proportion of survivors from each treatment was transformed by the arcsine square root function and fecundity by the square root function prior to statistical analyses (Sokal and Rohlf, 1981). Three-level nested A N O V A was used for each response variable to determine if significant differences could be attributed to region, stream site within region, and location within stream site (within region). The relationship between bioassay responses and the concentrations of single metals were examined by correlation analysis. The effects of concentration of all five measured metals on bioassay responses were examined by stepwise and multiple regression analyses (Sokal and Rohlf, 1981). All analyses were conducted using Minitab Statistical Software, release 8 (Minitab, 1991).

Results

Life history studies We observed the development of C. incongruens through eight instars, with a seven-fold increase in body length (Fig. 1). Growth stopped after the final molt (ninth instar) and egg production began. No further molts occurred although multiple clutches of eggs were laid. The duration of each juvenile instar ranged from 0.8 to 3.9 days (Table 1), with the time interval between hatching and laying the first clutch lasting a mean of 19.0 days (range 13-25). Adults lived for 14-37 days, with 1-4 days between individual clutches (Table 2). Fecundity was highly variable among individual females. Individuals laid 1-5 clutches of eggs (Table 2), with 1-36 eggs per clutch (Fig. 2). The females laid their eggs mainly in clusters, although eggs were occasionally laid singly. Lifetime fecundity ranged from

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211

Table 2. Time between clutches and fecundity for Cyprinotus incongruens. Time to 1st clutch was measured from the molt into the final (IX) instar Eggs per clutch

Interclutch period (days) Interval

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0 - 6 4 eggs (mean = 36.5, S E = 6.2, n = 15). Fecundity showed no apparent increase with age (Table 2). The egg d e v e l o p m e n t time was surprisingly variable. Only one-third of all eggs hatched within the 157 day period of observations. O f those that hatched, hatching times ranged from 1-157 days with peaks at 1, 4, and 21 weeks (Fig. 3). Clearly, egg d o r m a n c y was c o m m o n . Lifetime fecundity and egg development time were thus too variable under these control conditions to serve as response variables in the bioassay (coefficients of variation

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Bioassay experiment Bioassay experiments were run with both C. incongruens and C. dubia on medium-grain sands isolated from 36 locations within 12 stream sites (Table 3). Since some of the sites were downstream from mine tailings piles and had high levels of heavy metals (Havel and Schulz, 1993), we expected a correspondence between bioassay test responses and sediment source. Further, we expected that, as metal loadings increased, the mean responses from the bioassays would be depressed. Results from a pilot experiment in 1992 suggested that variation in C. incongruens body length and C. dubia fecundity were associated with sediment source and that a weak negative correspondence existed between these variables and the concentration of some metals (Havel and Schultz, 1993). However, this experiment had poor survival of C. incongruens (45% overall) and low statistical power, so the experiment was repeated. The 1993 experiment used higher replication (nine for each of the 36 treatments) and had good survival of C. incongruens (82% overall). Detailed comparisons among the 36 sediment locations are presented in Table 3 for several response variables from the C. incongruens and C. dubia bioassays. Because of the presence of old or active mines in the area, we expected the sites from the old and new lead belts to show the highest impacts, with two sites downstream from tailings piles (old-3 and new-3) to be most extreme.

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214 Table 4. Nested ANOVA results for Cyprinotus body length at 10 days Source

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0.644

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265

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Location (Site, region)

Survival of C. dubia over 7 days was high in sediments from all locations. In contrast, C. incongruens survival after 10 days showed large variation among sediment locations (range 11-100%, Table 3). The mean survival was lower for ostracods exposed to sediments from the old and new lead belt sites (mean 0.74) than to those from sites in Christian County and Springfield (0.90) (one-way ANOVA, F = 5.92, p = 0.02), indicating that at least some of the sediments contained toxic materials. Within the two lead belt regions, sediment quality showed higher variation among locations than those collected from Christian County and Springfield, as the variance in survival was higher in the former than the latter (F = 12.9, p < 0.01). Sublethal endpoints for both C. dubia and C. incongruens also detected variation in sediment quality. For C. dubia, fecundity at 7 and 9 days and body length at 9 days were all significantly correlated and showed substantial variation among the 36 locations (CV = 11%, Table 3). For each variable, three-level nested A N O V A revealed no significant variation among the four regions, but highly significant variation among sites (within regions). No significant variation was evident among locations (within sites). A comparison of means for each response variable indicated no significant difference between means from the locations in the two lead belt regions versus Christian County and Springfield locations. For C. incongruens, body length at 10 days showed greater variation among the 36 locations (CV = 12.5%, Table 3), than did body length for C. dubia (mean 0.95 ram, CV 3.5%; F = 12.8, p < 0.01). However, variances for C. incongruens body length and C. dubia fertility were not significantly different (Table 3). Nested A N O V A indicated that both region and site (within region) had highly significant effects on C. incongruens body length (Table 4), indicating that sediment quality varied at the scale of both regions and sites. Indeed, mean body length of C. incongruens exposed to sediments from locations in the new and old lead belts were significantly depressed relative to sediments from Springfield and Christian County (0.83 versus 0.93 ram, F = 6.34, p = 0.017). Surprisingly, there was no consistent reduction in body length at sites having the largest impacts of heavy metals (Old-3 and New-3). Indeed, all correlations between body length and concentrations of single metals were not significant. Stepwise and multiple regression indicated that concentration of all five metals together failed to explain any of the variation in body length (R 2 adjusted for degrees of freedom = 0%).

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Discussion

The current study revealed that body length of ostracods can be used as a sublethal endpoint for bioassays of contaminated sediments. Their small size, ease of culture, and ecological relevance make ostracods an attractive model for such studies. A 10 day test with metal-impacted stream sediments revealed that the ostracod Cyprinotus incongruens had a higher sensitivity than that of the 7 day survival and reproduction test using Ceriodaphnia dubia. Both survival and body length were effective measures for assaying the toxicity of sediments. Surprisingly, there appeared to be no significant impact of metal concentration, either singly or in combination, on either of these variables. Although heavy metals were highly concentrated in some of the sediments (Havel and Schulz, 1993), the lack of effect suggests that they were not in a bioavailable form and that unknown properties of the sediments were causing toxicity. Prior work employing ostracods in ecotoxicology has been limited. Acute bioassays to solutions of 16 different herbicides by a variety of species (cladoceran, Daphnia magna; ostracod, Cypridopsis vidua; amphipod, Gammarus fasciatus; isopod, Asellus brevicaudatus; glass shrimp, Palaemonetes kadiadensis; crayfish, Orconectes nails; and sunfish, Lepomis macrochirus) revealed that cladocerans and ostracods were both highly sensitive (low LCs0 values) to most herbicides and usually much more sensitive than the other species (Sanders, 1970). In a recent toxicity test, the sensitivities to heavy metals of three freshwater ostracods (Cyprinotus incongruens, Cypridopsis vidua, and Physocypria pustulosa) were compared to that of the cladoceran Ceriodaphnia dubia. Although C. dubia had a higher sensitivity than ostracods in all tests without sediment, addition of copper-spiked sediments to non-contaminated water led to a higher sensitivity in the ostracods, and this effect increased with longer exposures (Woodward, Havel and Stewart, unpublished data). Ostracods have also been included in several community-level microcosm experiments (Taub et al., 1981; Takamura and Yasuno,1986; Landis et al., 1992). Although densities of ostracods and algae were often depressed by pesticides, over the longer term their densities increased, presumably by indirect effects of depressing competitors and predators (Taub et al., 1981; Takamura and Yasuno, 1986). In a 2-month microcosm experiment investigating population and community responses to jet fuel pollution, Landis et al. (1992) found that population increases of both Daphnia and ostracods were delayed by up to 10 days and peak population densities were depressed. A depression of survival and/or individual growth rate, such as demonstrated in the current study, could account for these population-level responses. Our life history study with C. incongruens revealed that instar-specific body length had low variation under control conditions (Fig. 1), and thus body length after a brief period of growth could be useful as a sublethal character in sediment bioassays. In contrast, fecundity and egg development time were highly variable among individuals under control conditions and thus are currently not practical measures for bioassay experiments. However, the variation in these life history characters is intriguing from a biological perspective and merits further analysis. Individual C. incongruens females clearly laid both eggs which rapidly hatch (subitaneous) and eggs with delayed hatching. Eggs having a longer delay are also possible, as the experiment ended before all of the viable eggs had hatched. Indeed, only 33% of

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the eggs had hatched by the end of the experiment (157 days) and some remaining eggs could be recognized as still viable by their orange coloration. Such a pattern indicates that C. incongruens resting eggs can hatch after varying time intervals. Variable egg hatching times were also obtained in Angell and Hancock's (1989) experiments with Heterocypris incongruens (= C. incongruens; Delorme, 1970). In their study, 32% of the eggs hatched within 125 days, with half of these hatching within 10 days and half hatching at "indeterminant times in the future". Resting eggs appear to be a common characteristic among freshwater ostracods (Tetart, 1975; McLay 1978; Delorme, 1991) which enables many of these species to endure intermittent freezing and drying in temporary ponds (Tressler, 1959; McLay, 1978; Havel et al., 1990). Further work on the cues which trigger hatching of ostracod resting eggs should allow a better understanding of both the ecology of temporary ponds and the methods necessary to supply a reliable number neonates for bioassay experiments. Development of C. incongruens from hatching to laying the first clutch took about 19 days in the current study (at 25 ° C). In contrast, Marten's (1985) study of another cypridid ostracod (Mytilocypris henricae) showed that juvenile development in this species took about 25 days at 25 ° C and almost 60 days at 20 ° C. The marine myodocopid Skogsbergia lerneri required 65-93 days to reach maturity at 25 ° C (Cohen, 1983). It is clear that ostracods show a wide degree of variation in their rate of maturation, and thus species choice is critical for practical experiments. Because C. incongruens has a wide distribution (Tressler, 1959) and a rapid juvenile development time relative to other ostracods, this species should be a good choice for future studies of sediment toxicity. Sublethal bioassays examining impaired reproductive success are probably more practical for the cladocerans than ostracods. In contrast to the 19-days development time (hatching to egg laying) for C. incongruens, cladocerans typically pass through juvenile development in less than 7 days (Lynch, 1980). Also, the subitaneous eggs of cladocerans are segregated from resting eggs, are easily distinguished, and hatch quickly (about 1-2 days at 20° C; Lynch, 1980; Dodson and Frey, 1991). Thus, the 7-day bioassay with C. dubia can measure a combination of development time and fertility through three broods (Weber et al., 1989), and is now used routinely in aquatic toxicity testing (DeGraeve et al., 1992). However, the sediment-dwelling habit of ostracods makes them a useful surrogate for cladocerans in tests of sediments. The current study has shown that two characters, survivorship and body length after 10 days of growth, can be used in such a test with metal-contaminated sediments, and resulted in a higher sensitivity than C, dubia to toxic sediments. Future studies should determine which test conditions are optimum and compare the response of ostracods with that of other benthic invertebrates in standardized toxicity tests.

Acknowledgements We thank R. Farquhar, C. Gardiner and C. Hood for collecting sediments, K. Kimberley for metal analyses, and C. Hood for assistance with the Ceriodaphnia bioassay. Comments by C. Barnhart, P. Hebert, C. Ingersoll, and an anonymous reviewer improved the clarity of the manuscript. This study was supported by grants to JEH from the Coors 2000 Pure Water Fund and the Missouri Water Resources Research Center (USDI/GS 14-08-001-62029 01).

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