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
Sublethal responses of a freshwater ostracod
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 (
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
Sublethal responses of a freshwater ostracod
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 (
