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Chronic effect of NaCl salinity on a freshwater strain of Daphnia magna Straus (Crustacea: Cladocera): A demographic study
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Ecotoxicology and Environmental Safety 67 (2007) 411–416 www.elsevier.com/locate/ecoenv
Chronic effect of NaCl salinity on a freshwater strain of Daphnia magna Straus (Crustacea: Cladocera): A demographic study$ Fernando Martı´ nez-Jero´nimo, Laura Martı´ nez-Jero´nimo Laboratorio de Hidrobiologı´a Experimental, Escuela Nacional de Ciencias Biolo´gicas, IPN Prol. Carpio Esq. Plan de Ayala s/n, Col. Sto. Toma´s, Me´xico D.F. 11340, Mexico Received 24 November 2005; received in revised form 7 August 2006; accepted 27 August 2006 Available online 19 October 2006
Abstract Daphnia magna is mainly recognized as a freshwater cladoceran, but there are some strains that grow in brackish waters. The tolerance to salinity of a freshwater strain was assessed at NaCl concentrations of 0, 2, 4, 6, and 7 g L1. The green microalga Ankistrodesmus falcatus was fed at optimal concentration (4 105 cells mL1). Reproduction and survival were recorded in two experimental series: in the first one, 20 female neonates were individually studied for each treatment. In the second, cohorts of 10 female neonates were distributed in each of five replicates per treatment. In both cases, experiments were conducted over a full life-cycle. The determined 48-h LC50 for NaCl was 5.48 g L1, but we recorded reproduction at up to 7 g NaCl L1. The average clutch size, total progeny, number of clutches, and longevity were significantly reduced by the NaCl concentration (Po0:01); total progeny ranged from 467 to 25 neonates as edge values for NaCl concentrations of 0–7 g L1. Inter-brood time was significantly higher for females grown at 7 g NaCl L1 (3.9 days). The Life Table analysis demonstrates that average lifespan, life expectancy at birth, net reproductive rate and intrinsic rate of growth were also significantly reduced according to NaCl concentration. Based on the results for the two highest NaCl concentrations (6 and 7 g L1), we conclude that the used D. magna strain was acclimated to develop satisfactorily under concentrations of up to 6 g NaCl L1; however, the established salinity conditions reduced significantly reproduction and survival in this strain. r 2006 Elsevier Inc. All rights reserved. Keywords: Zooplankton; Salinity stress; NaCl toxicity; Asexual reproduction; Life table; Ankistrodesmus falcatus
1. Introduction Cladocerans are very important components of zooplankton, usually restricted to freshwater environments (Arne´r and Koivisto, 1993) with salinity values lower than 1 g L1 (Hart et al., 1991) or conductivity values less than 500 mS cm1 (Hebert et al., 2002). The genus Daphnia is freshwater in its origin and distribution (Peters, 1987; Teschner, 1995); for North America, there are 34 species in freshwater environments and only one for saline lakes (Daphnia salina) (Hebert et al., 2002). $ This study was supported by Coordinacio´n General de Posgrado e Investigacio´n-I.P.N. This study involved experimental animals, and it was conducted in accordance with national and institutional guidelines for the protection of human subjects and animal welfare. Corresponding author. Fax: +52 55 5396 3503. E-mail address: [email protected] (F. Martı´ nez-Jero´nimo).
0147-6513/$ - see front matter r 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.ecoenv.2006.08.009
Although more than 40 Cladocera species inhabit saline waters (five are marine and five dwell in highly mineralized continental water bodies), little information is available about the salinity tolerance and the osmoregulatory organs in these micro-crustaceans (Aladin, 1991), as well as on the biological response and adaptations in the freshwater species that invade brackish waters (Arne´r and Koivisto, 1993). Daphnia are hypertonic to the medium and the fluxes of water and solutes with the surrounding water could be considerable, but they have reduced their osmotic loads through the impermeability of their bodies and the low internal concentration of solutes, being sodium pumping from the epithelial cytoplasm to the hemolymph the major mechanism for osmoregulation in freshwater cladocerans (Peters, 1987). According to Arne´r and Koivisto (1993), although it is possible to find D. magna in rock pools with salinity values up to 12.5% in the Baltic Sea, they experimentally
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determined that the best development was achieved at 4%. Schuytema et al. (1997) also concluded that the best growth of D. magna occurs at salinity values lower than 4%. The freshwater Cladocera that successfully colonize brackish environments are smaller in size and have a reduced reproduction (Arne´r and Koivisto, 1993). Cowgill and Milazzo (1990, 1991) demonstrated that reproduction, population growth rate, and survival in D. magna decreased as NaCl concentration increased in the range of 0.08–6000 mg L1. Teschner (1995) experimented with freshwater and brackish water (2.5%) clones of D. magna exposed to media with 0% and 5% salinity and found that salinity tolerance is not restricted to brackish water clones because both showed the same negative effects on development and reproduction when grown at 5%; the author concluded that this salinity value was quite beyond the tolerance range for normal development of this species. On the other hand, Bailey et al. (2004) stated that, while there are some studies related to the consequences of salinity on growth and survival of freshwater cladocerans, the effects of salinity on ephippia (the resistance and dispersion structures produced during sexual reproduction in Cladocera) have not been sufficiently studied. It is possible that under some circumstances, a number of D. magna populations sporadically or seasonally cope with salinity conditions, hence it is necessary to study salinity tolerance in Cladocera and, specifically, to determine how biological responses, such as feeding and reproduction, are modified within the tolerance range of this environmental factor (He et al., 2001). The present study assessed the effect of different NaCl concentrations on reproduction, demographic parameters, and survival in a freshwater strain of D. magna and the responses to high salinity values in the tested ranges in previously acclimated organisms. 2. Materials and methods A freshwater D. magna strain that has been successfully grown in our laboratory for more than 15 years in reconstituted hard-water (Weber, 1993) was used as the test organism for this study, and as initial breeding population to measure survival of offsprings. The experiments were conducted in three phases:
2.1. Median lethal concentration (LC50) for NaCl and treatment description The acute toxicity (48 h) of sodium chloride (NaCl) was determined following the test protocol established in the Mexican Norm (NMX-AA087-1995-SCFI, 1996). Reconstituted hard water (192 mg L1 NaHCO3, 120 mg L1 CaSO4 2H2O, 120 mg L1 MgSO4, 8 mg L1 KCl; hardness ranged between 160 and 180 mg L1 as CaCO3) was used as dilution water and for controls. Temperature for tests was 2171 1C. Test neonates (age o24 h) were obtained from the original culture, maintained in reconstituted hard water. With the experimentally determined LC50 value, D. magna neonates were placed in reconstituted hard water plus 0, 2, 4, 6, 7, 8, and 9 g NaCl L1 to obtain brackish water acclimated lots. When reproduction occurred, neonates obtained from the third clutch were individually placed
in glass containers at the same salinity value, repeating this procedure for three generations; finally, neonates produced in the third generation in each salinity, were used as test organisms in all the experiments. At 8 and 9 g NaCl L1, specimens survived for up to 7 days, but they did not reproduce, so subsequent experiments were performed only for the following NaCl concentrations: 0, 2, 4, 6, and 7 g L1 (corresponding to salinity values of 0.3, 2.3, 4.3, 6.3, and 7.3 psu, and specific conductance values of 0.561, 4.316, 7.56, 10.92, and 12.72 mS cm1, respectively). The green microalga Ankistrodesmus falcatus was supplied as food at 400,000 cells mL1; it was previously determined that this cell concentration is an optimal food dosage for this strain (Martı´ nez-Jero´nimo et al., 1994). A. falcatus was cultured in autoclaved Bold’s Basal Medium, the biomass was harvested during the exponential growth phase and concentrated by centrifugation; microalgal concentrates were kept refrigerated (4 1C) and none was used for more than 1 week, when it was substituted with a fresh concentrate. These treatments were applied in the following two experimental series:
2.2. Reproduction and longevity individually determined From specimens previously acclimated to each experimental condition (as described), 20 D. magna neonates were individually distributed in twenty 150-mL Pyrexs borosilicate glass beakers (100 mL test volume) for each treatment. Once reproduction began, progeny per individual female was counted and discarded; this procedure was followed until all the reproducers had died, in this way recording individual longevity. Test media and food concentration were completely renewed daily. Age at first reproduction, inter-clutch time, number of clutches per reproducer, progeny per brood, total progeny, and longevity in each treatment were determined. A one-way analysis of variance (ANOVA I) was applied to all results to determine whether significant differences occurred among treatments; Tukey post-hoc comparison was also applied to identify treatments that were significantly different among them. Experiments were conducted at 2171 1C.
2.3. Life table analysis Cohorts of 10 neonates were randomly distributed in every treatment; five replicates for each were carried out in 600 mL Pyrexs borosilicate glass beakers, with 500 mL test volume. Test media and food concentration were completely renewed daily. Each day, the progeny and mortality of reproducers were recorded for all cohorts. With this information, the demographic parameters lx (proportion of survivals at each 24 h age group) and mx (average progeny per female in each 24 h age group) were determined. Both parameters were the basis for the calculation of the intrinsic population growth rate r (estimated by iteration with Euler’s P equation: nx¼0 erx l x mx ¼ 1), net reproductive rate R0, life expectancy at birth ex, generation time G, and the average lifespan. Survivorship curves, life expectancy curves, and fecundity tendencies were constructed. Results were compared with ANOVA I and Tukey’s post-hoc comparisons. Assays were conducted at 2171 1C. Actual salinity and specific conductance values corresponding to the different NaCl concentrations were determined with a YSI salineconductivity meter (Yellow Spring Instruments, mod. 30), duly calibrated; the equipment has a 70.5% precision.
3. Results 3.1. Median lethal concentration (LC50) for NaCl The Probit estimated LC50 (48 h) for NaCl was 5.48 g L1 (95% limits: 5.07–6.02 g L1); this value corresponds to a specific conductance of 9.79 mS cm1 (95% limits: 9.00–10.87 mS cm1). The tested concentrations ranged from 0 to 10 g NaCl L1, but at the highest salinity
ARTICLE IN PRESS F. Martı´nez-Jero´nimo, L. Martı´nez-Jero´nimo / Ecotoxicology and Environmental Safety 67 (2007) 411–416
in all the tests, all neonates died during the first 60 min. Some survivors at 48 h in concentrations above 5 g NaCl L1 were affected in their mobility and sometimes responded only to mechanical stimulation. 3.2. Reproduction and longevity individually determined
higher than those recorded with higher NaCl concentrations (Po0:001). The age at first reproduction ranged from 7.5 to 9.2 days in average (Fig. 1). According to ANOVA and Tukey’s post-hoc comparisons, females grown in 6 g NaCl L1 release their first clutch at a significantly lower age than the females from the other treatments (Po0:001). Inter-clutch time, ranging from 2.6 to 3.9 days, was affected by NaCl concentration (Po0:001), but only the value recorded at 7 g NaCl L1 was significantly higher (Po0:001). Average D. magna longevity ranged from 24.4 to 56.3 days and was affected by NaCl concentration (Po0:001), Tukey’s post-hoc comparisons demonstrated that the largest values were for females grown at 0 and 2 g NaCl L1, whereas for all the other treatments the longevity was significantly lower and different among them (Po0:001). 3.3. Life table analysis Fig. 2 shows the average values and the standard error values for the Life Table demographic parameters calculated for D. magna in the five treatments.
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Fig. 1 shows reproduction and longevity recorded for D. magna grown individually in all the treatments. The average clutch size ranged from 5.1 to 28.7 neonates (Fig. 1). ANOVA demonstrated that NaCl concentration had a significant effect on this parameter (Po0:001), and Tukey’s post-hoc test revealed that the clutch size was significantly different with all NaCl concentrations. Total progeny (equivalent to R0 in the Life Table analysis) ranged from 25.6 to 466.9 neonates (Fig. 1). The NaCl concentration had a significant effect (Po0:001), and the total progeny was lower as NaCl concentration increased (Tukey’s post-hoc test, Po0:001). Average number of clutches per female ranged from 5.0 to 16.4 (Fig. 1), and it was significantly affected by NaCl concentration (Po0:001). Tukey’s post-hoc test demonstrated that the number of broods was similar for females grown at 0 and 2 g NaCl L1, but both were significantly
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Fig. 1. Reproduction and longevity of Daphnia magna grown at different NaCl concentrations and fed Ankistrodesmus falcatus. Average values and standard error bars are shown.
ARTICLE IN PRESS F. Martı´nez-Jero´nimo, L. Martı´nez-Jero´nimo / Ecotoxicology and Environmental Safety 67 (2007) 411–416
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NaCl Concentration (g L-1) Fig. 2. Demographic Life Table parameters for Daphnia magna grown at different NaCl concentrations and fed Ankistrodesmus falcatus. Average values and standard error bars are shown.
ANOVA suggests that average lifespan was significantly affected by the NaCl concentration (Po0:001). The maximal average lifespan (57.7 days) was for D. magna grown in reconstituted hard water (0 g NaCl L1), whereas the shortest lifespan (25.2 days) was for females grown in 7 g NaCl L1. Tukey’s post-hoc test revealed that this parameter had a significantly different value for most treatments (Po0:001), except for 4 and 6 g NaCl L1, in which results were not statistically different (P40:05). With respect to the life expectancy at birth (e), results recorded were similar to the average lifespan (see Fig. 2), and ANOVA and post-hoc comparisons reflect similar conclusions. The highest life expectancy at birth (57.2 days) was for females grown in reconstituted hard water, whereas the lowest value was for D. magna females grown in 7 g NaCl L1. The Net Reproductive Rate (R0) was significantly reduced as the NaCl concentration increased (Po0:001) (Fig. 2), and the values recorded in all treatments were significantly different (Tukey’s post-hoc comparisons). The generation time (G) ranged from 17.8 to 30.5 days (Fig. 2), and it was affected by the NaCl concentration
(Po0:001), with the values for all the treatments being significantly different among them (P40:01). The intrinsic population growth rate (r), one of the most important demographic parameters, was also significantly affected by the concentration of NaCl (Po0:001). As can be seen in Fig. 2, r was reduced as NaCl concentration increased, but no significant differences were determined between reconstituted hard water (0 g NaCl L1) and the 2 g L1 concentration, nor between 4 and 6 g NaCl L1; r ¼ 0:2513 day1 was the lowest significant value recorded for the highest NaCl concentration tested. The survival curves for all treatments are shown in Fig. 3; as can be seen, the shape of the graph is very similar to the Type I survivorship curves, with mortality accentuated in the older organisms of the cohort. The highest mortality rates were for D. magna grown in 6 and 7 g NaCl L1. 4. Discussion For D. magna, Schuytema et al. (1997) determined a median lethal concentration (LC50) at salinity concentration
ARTICLE IN PRESS F. Martı´nez-Jero´nimo, L. Martı´nez-Jero´nimo / Ecotoxicology and Environmental Safety 67 (2007) 411–416
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Age (d) Fig. 3. Survivorship curves of Daphnia magna grown at different NaCl concentrations and fed Ankistrodesmus falcatus. Average values and standard error bars are shown.
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of 6.6 g L1 (measured conductivity, 10.0 mS cm1). Cowgill and Milazzo (1990) estimated an LC50 for NaCl at 6.034 g L1, and pointed out that the ‘‘non observable effects level’’ (NOEL) for NaCl was approximately 1.2 g L1. In the present study, LC50 was determined to occur at the NaCl concentration of 5.48 g L1 (measured conductivity, 9.79 mS cm1), and was lower than previously reported values. This situation could be related to a greater sensitivity to salinity by the experimental strain we used, a plausible situation since this strain has been grown with outstanding reproductive results in freshwater conditions for more than 15 years. Arne´r and Koivisto (1993) reported that they grew D. magna in salinities of 4% and 8%, but the conductivities reported (4.5 and 8.5 mS cm1) were lower than those determined here for the two highest NaCl concentrations (10.61 and 12.23 mS cm1), so we can suspect that the maximum salinity value they informed could in fact be lower. Intrinsic population growth rate (r) estimated in the present study for 6 g NaCl L1 (0.32 day1) was intermediate to the values obtained by Arne´r and Koivisto (1993) (0.52 and 0.299 day1, respectively, for 4% and 8%). They concluded that D. magna grew better at 4%, and, at this salinity, the cladoceran produced an average of 12.6 neonates per clutch, a value lower than that found here at a similar salinity (18.5 neonates). They also stated that D. magna can develop in salinities of up to 12.5%, but its presence declined abruptly above 4%, and no more information was provided for possible comparisons. As we said before, apparently in our study we tested higher salinity concentrations, taking into account the conductivity values for both studies and, should this be the case, the D. magna strain we used could be more tolerant to salinity. Cowgill and Milazzo (1991) observed that R0 and r values diminish as NaCl concentration increases, and they concluded that the reduction in R0 is manifested at values as low as 0.061 g L1, whereas the maximal reduction in r begins at 0.778 g NaCl L1. They stated that these demographic parameters are better indicators for assessing salinity stress than the traditional measures of toxicity (LC50, NOEL, NOEC, etc.). They calculated a R0 value of 10 neonates for D. magna grown at 6 g NaCl L1, lower than what we determined, even for a NaCl concentration of 7 g L1 (41 neonates, Fig. 2). Despite the reduction in growth rate recorded by us, reproduction in this strain was well above values reported in other studies at comparable salinity (or conductivity) conditions. Arne´r and Koivisto (1993) observed that r diminished at 8%, the specimens were smaller, and they moulted later than those grown at 4% and in freshwater conditions. They also concluded that the food concentration they supplied was high compared with other studies, but this could help maintain D. magna at 8%. As can be seen in Fig. 2, these two demographic parameters show a decline with respect to the increase in NaCl concentration, similar to that
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observed by Arne´r and Koivisto (1993), but better supported with experimental data. Other authors have shown that D. magna actively osmoregulates its body fluids up to a salinity of ca. 5%; higher salinities induce the organism to act as an osmo-conformer (Aladin, 1991; Arne´r and Koivisto, 1993). It can be argued that a reduction in the ability to osmo-regulate could be related with failure in salinity-tolerant clones to thrive in freshwater; this could explain the observations made by Teschner (1995), who reported that some brackish-water tolerant Daphnia clones lose their capacity to develop in freshwater. The change from a freshwater to a brackish medium, as was assayed here, suggests tolerance mechanisms that are inducible in the physiological tolerance range; the acclimation performed here enables this D. magna strain to endure salinity stress (provided as NaCl concentration). In this sense, Williams (1998) remarked that D. magna in slightly brackish waters has a narrow range of salt tolerance, whereas varieties found in highly saline lakes display a wide tolerance to this factor, as observed in Moina hutchinsoni, a cladoceran that flourishes in saline lakes in North America at up to 40 psu, but M. hutchinsoni can be grown under laboratory conditions at salinity values as low as 4 psu with similar results to those recorded at a higher salinity (Martı´ nez-Jero´nimo et al., 2004; Martı´ nezJero´nimo and Espinosa-Cha´vez, 2005). Considering its tolerance to salinity, Green et al. (2005) established that D. magna is a euryhaline species that is particularly tolerant to salinity conditions in brackish lakes; nevertheless, they concluded that the reproductive and/or survival rates of cladocerans are reduced at higher water conductivities. In our study, we demonstrated that the freshwater strain we used has in fact a relatively small range of tolerance to NaCl salinity; nevertheless it was acclimated to thrive at the upper salinity values within its tolerance range. Comparing results in the whole NaCl concentration range tested, we can conclude that the impairment effects on reproduction and survival under the current treatments were provoked by salinity stress, although it has been demonstrated that various freshwater invertebrate species (including cladocerans) are more sensitive to NaCl salinity than to the effect produced by the array of chemical compounds present in sea salt (Kefford et al., 2004). The present results might be useful for a better understanding of how salinity (as NaCl concentration) affected the demography, longevity, survival, and reproductive responses of a freshwater D. magna strain and which could be its maximum tolerance to this chemical compound. Results also indicate that the growth and survival of D. magna in NaCl stress conditions were suboptimal, yet for the strain here tested, they were clearly superior to those reported in other studies. These data provide support for the use of this strain, based on this capability to endure brackish waters, as test organism in toxicity assays performed in slightly saline conditions (up to 2.3 or 4.3 psu).
Acknowledgments Thanks are due to the Comisio´n de Operacio´n y Fomento de Actividades Acade´micas—I.P.N. and Estı´ mulo al Desempen˜o de los Investigadores—I.P.N. for the grants received. References Aladin, N.V., 1991. Salinity tolerance and morphology of the osmoregulation organs in Cladocera with special reference to Cladocera from the Aral sea. Hydrobiologia 225, 291–299. Arne´r, M., Koivisto, S., 1993. Effects of salinity on metabolism and life history characteristics of Daphnia magna. Hydrobiologia 259, 69–77. Bailey, S.A., Duggan, I.C., Van Overdijk, D.A.C., Johengen, T.H., Reid, D.F., Macisaac, H.J., 2004. Salinity tolerance of diapausing eggs of freshwater zooplankton. Freshwater Biol. 49, 286–295. Cowgill, U.M., Milazzo, D.P., 1990. The sensitivity of two cladocerans to water quality variables, salinity and hardness. Arch. Hydrobiol. 120, 185–196. Cowgill, U.M., Milazzo, D.P., 1991. Demographic effects of salinity, water hardness and carbonate alkalinity on Daphnia magna and Ceriodaphnia dubia. Arch. Hydrobiol. 122, 35–56. Green, A.J., Fuentes, C., Moreno-Ostos, E., Rodrigues da Silva, S.L., 2005. Factors influencing cladoceran abundance and species richness in brackish lakes in Eastern Spain. Ann. Limnol.—Int. J. Limnol. 4, 73–81. Hart, B.T., Bailey, P., Edwards, R., Hortle, K., James, K., McMahon, A., Meredith, C., Swadling, K., 1991. A review of the salt sensitivity of the Australian freshwater biota. Hydrobiologia 210, 105–144. He, Z.H., Qin, J.G., Wang, Y., Jiang, H., Wen, Z., 2001. Biology of Moina mongolica (Moinidae, Cladocera) and perspective as live food for marine fish larvae: review. Hydrobiologia 457, 25–37. Hebert, P.D.N., Remigio, E.A., Colbourne, J.K., Taylor, D.J., Wilson, C.C., 2002. Accelerated molecular evolution in halophilic crustaceans. Evolution 56, 909–926. Kefford, B.J., Palmer, C.G., Pakhomova, L., Nugegoda, D., 2004. Comparing test systems to measure the salinity tolerance of freshwater invertebrates. Water SA 30, 499–506. Martı´ nez-Jero´nimo, F., Espinosa-Cha´vez, F., 2005. Notes on the reproduction and survival of Moina hutchinsoni Brehm, 1937 (Moinidae: Anomopoda) grown in media of varying salinity. Aquat. Ecol. 39, 113–118. Martı´ nez-Jero´nimo, F., Villasen˜or, R., Rı´ os, G., Espinosa, F., 1994. Effect of food type and concentration on the survival, longevity, and reproduction of Daphnia magna. Hydrobiologia 287, 207–214. Martı´ nez-Jero´nimo, F., Elı´ as-Gutie´rrez, M., Sua´rez-Morales, E., 2004. A redescription of Moina hutchinsoni Brehm, a rare cladoceran (Branchiopoda: Anomopoda) found in remnants of a Mexican saline lake, with notes on its life history. J. Crustacean Biol. 24, 232–245. NMX-AA-087-1995-SCFI, 1996. Ana´lisis de agua. Evaluacio´n de toxicidad aguda con Daphnia magna Straus (Crustacea-Cladocera). Me´todo de prueba, Diario Oficial de la Federacio´n, Mexico. Peters, R.H., 1987. Metabolism in Daphnia. In: Peters, R.H., de Bernardi, R. (Eds.), Daphnia. Mem. Ist. Ital. Idrobiol. 45, 193–243. Schuytema, G.S., Nebeker, A.V., Stutzman, T.W., 1997. Salinity tolerance of Daphnia magna and potential use for estuarine sediment toxicity tests. Arch. Environ. Contam. Toxicol. 33, 194–198. Teschner, M., 1995. Effects of salinity on the life history and fitness of Daphnia magna: variability within and between populations. Hydrobiologia 307, 33–41. Weber, C.I., 1993. Methods for Measuring the Acute Toxicity of Effluents and Receiving Waters to Freshwater and Marine Organisms, fourth ed. United States Environmental Protection Agency, Cincinnati, Ohio, EPA/600/4-90/027F. Williams, W.D., 1998. Salinity as a determinant of the structure of biological communities in salt lakes. Hydrobiologia 381, 191–201.
Ecotoxicology and Environmental Safety 67 (2007) 411–416 www.elsevier.com/locate/ecoenv
Chronic effect of NaCl salinity on a freshwater strain of Daphnia magna Straus (Crustacea: Cladocera): A demographic study$ Fernando Martı´ nez-Jero´nimo, Laura Martı´ nez-Jero´nimo Laboratorio de Hidrobiologı´a Experimental, Escuela Nacional de Ciencias Biolo´gicas, IPN Prol. Carpio Esq. Plan de Ayala s/n, Col. Sto. Toma´s, Me´xico D.F. 11340, Mexico Received 24 November 2005; received in revised form 7 August 2006; accepted 27 August 2006 Available online 19 October 2006
Abstract Daphnia magna is mainly recognized as a freshwater cladoceran, but there are some strains that grow in brackish waters. The tolerance to salinity of a freshwater strain was assessed at NaCl concentrations of 0, 2, 4, 6, and 7 g L1. The green microalga Ankistrodesmus falcatus was fed at optimal concentration (4 105 cells mL1). Reproduction and survival were recorded in two experimental series: in the first one, 20 female neonates were individually studied for each treatment. In the second, cohorts of 10 female neonates were distributed in each of five replicates per treatment. In both cases, experiments were conducted over a full life-cycle. The determined 48-h LC50 for NaCl was 5.48 g L1, but we recorded reproduction at up to 7 g NaCl L1. The average clutch size, total progeny, number of clutches, and longevity were significantly reduced by the NaCl concentration (Po0:01); total progeny ranged from 467 to 25 neonates as edge values for NaCl concentrations of 0–7 g L1. Inter-brood time was significantly higher for females grown at 7 g NaCl L1 (3.9 days). The Life Table analysis demonstrates that average lifespan, life expectancy at birth, net reproductive rate and intrinsic rate of growth were also significantly reduced according to NaCl concentration. Based on the results for the two highest NaCl concentrations (6 and 7 g L1), we conclude that the used D. magna strain was acclimated to develop satisfactorily under concentrations of up to 6 g NaCl L1; however, the established salinity conditions reduced significantly reproduction and survival in this strain. r 2006 Elsevier Inc. All rights reserved. Keywords: Zooplankton; Salinity stress; NaCl toxicity; Asexual reproduction; Life table; Ankistrodesmus falcatus
1. Introduction Cladocerans are very important components of zooplankton, usually restricted to freshwater environments (Arne´r and Koivisto, 1993) with salinity values lower than 1 g L1 (Hart et al., 1991) or conductivity values less than 500 mS cm1 (Hebert et al., 2002). The genus Daphnia is freshwater in its origin and distribution (Peters, 1987; Teschner, 1995); for North America, there are 34 species in freshwater environments and only one for saline lakes (Daphnia salina) (Hebert et al., 2002). $ This study was supported by Coordinacio´n General de Posgrado e Investigacio´n-I.P.N. This study involved experimental animals, and it was conducted in accordance with national and institutional guidelines for the protection of human subjects and animal welfare. Corresponding author. Fax: +52 55 5396 3503. E-mail address: [email protected] (F. Martı´ nez-Jero´nimo).
0147-6513/$ - see front matter r 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.ecoenv.2006.08.009
Although more than 40 Cladocera species inhabit saline waters (five are marine and five dwell in highly mineralized continental water bodies), little information is available about the salinity tolerance and the osmoregulatory organs in these micro-crustaceans (Aladin, 1991), as well as on the biological response and adaptations in the freshwater species that invade brackish waters (Arne´r and Koivisto, 1993). Daphnia are hypertonic to the medium and the fluxes of water and solutes with the surrounding water could be considerable, but they have reduced their osmotic loads through the impermeability of their bodies and the low internal concentration of solutes, being sodium pumping from the epithelial cytoplasm to the hemolymph the major mechanism for osmoregulation in freshwater cladocerans (Peters, 1987). According to Arne´r and Koivisto (1993), although it is possible to find D. magna in rock pools with salinity values up to 12.5% in the Baltic Sea, they experimentally
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determined that the best development was achieved at 4%. Schuytema et al. (1997) also concluded that the best growth of D. magna occurs at salinity values lower than 4%. The freshwater Cladocera that successfully colonize brackish environments are smaller in size and have a reduced reproduction (Arne´r and Koivisto, 1993). Cowgill and Milazzo (1990, 1991) demonstrated that reproduction, population growth rate, and survival in D. magna decreased as NaCl concentration increased in the range of 0.08–6000 mg L1. Teschner (1995) experimented with freshwater and brackish water (2.5%) clones of D. magna exposed to media with 0% and 5% salinity and found that salinity tolerance is not restricted to brackish water clones because both showed the same negative effects on development and reproduction when grown at 5%; the author concluded that this salinity value was quite beyond the tolerance range for normal development of this species. On the other hand, Bailey et al. (2004) stated that, while there are some studies related to the consequences of salinity on growth and survival of freshwater cladocerans, the effects of salinity on ephippia (the resistance and dispersion structures produced during sexual reproduction in Cladocera) have not been sufficiently studied. It is possible that under some circumstances, a number of D. magna populations sporadically or seasonally cope with salinity conditions, hence it is necessary to study salinity tolerance in Cladocera and, specifically, to determine how biological responses, such as feeding and reproduction, are modified within the tolerance range of this environmental factor (He et al., 2001). The present study assessed the effect of different NaCl concentrations on reproduction, demographic parameters, and survival in a freshwater strain of D. magna and the responses to high salinity values in the tested ranges in previously acclimated organisms. 2. Materials and methods A freshwater D. magna strain that has been successfully grown in our laboratory for more than 15 years in reconstituted hard-water (Weber, 1993) was used as the test organism for this study, and as initial breeding population to measure survival of offsprings. The experiments were conducted in three phases:
2.1. Median lethal concentration (LC50) for NaCl and treatment description The acute toxicity (48 h) of sodium chloride (NaCl) was determined following the test protocol established in the Mexican Norm (NMX-AA087-1995-SCFI, 1996). Reconstituted hard water (192 mg L1 NaHCO3, 120 mg L1 CaSO4 2H2O, 120 mg L1 MgSO4, 8 mg L1 KCl; hardness ranged between 160 and 180 mg L1 as CaCO3) was used as dilution water and for controls. Temperature for tests was 2171 1C. Test neonates (age o24 h) were obtained from the original culture, maintained in reconstituted hard water. With the experimentally determined LC50 value, D. magna neonates were placed in reconstituted hard water plus 0, 2, 4, 6, 7, 8, and 9 g NaCl L1 to obtain brackish water acclimated lots. When reproduction occurred, neonates obtained from the third clutch were individually placed
in glass containers at the same salinity value, repeating this procedure for three generations; finally, neonates produced in the third generation in each salinity, were used as test organisms in all the experiments. At 8 and 9 g NaCl L1, specimens survived for up to 7 days, but they did not reproduce, so subsequent experiments were performed only for the following NaCl concentrations: 0, 2, 4, 6, and 7 g L1 (corresponding to salinity values of 0.3, 2.3, 4.3, 6.3, and 7.3 psu, and specific conductance values of 0.561, 4.316, 7.56, 10.92, and 12.72 mS cm1, respectively). The green microalga Ankistrodesmus falcatus was supplied as food at 400,000 cells mL1; it was previously determined that this cell concentration is an optimal food dosage for this strain (Martı´ nez-Jero´nimo et al., 1994). A. falcatus was cultured in autoclaved Bold’s Basal Medium, the biomass was harvested during the exponential growth phase and concentrated by centrifugation; microalgal concentrates were kept refrigerated (4 1C) and none was used for more than 1 week, when it was substituted with a fresh concentrate. These treatments were applied in the following two experimental series:
2.2. Reproduction and longevity individually determined From specimens previously acclimated to each experimental condition (as described), 20 D. magna neonates were individually distributed in twenty 150-mL Pyrexs borosilicate glass beakers (100 mL test volume) for each treatment. Once reproduction began, progeny per individual female was counted and discarded; this procedure was followed until all the reproducers had died, in this way recording individual longevity. Test media and food concentration were completely renewed daily. Age at first reproduction, inter-clutch time, number of clutches per reproducer, progeny per brood, total progeny, and longevity in each treatment were determined. A one-way analysis of variance (ANOVA I) was applied to all results to determine whether significant differences occurred among treatments; Tukey post-hoc comparison was also applied to identify treatments that were significantly different among them. Experiments were conducted at 2171 1C.
2.3. Life table analysis Cohorts of 10 neonates were randomly distributed in every treatment; five replicates for each were carried out in 600 mL Pyrexs borosilicate glass beakers, with 500 mL test volume. Test media and food concentration were completely renewed daily. Each day, the progeny and mortality of reproducers were recorded for all cohorts. With this information, the demographic parameters lx (proportion of survivals at each 24 h age group) and mx (average progeny per female in each 24 h age group) were determined. Both parameters were the basis for the calculation of the intrinsic population growth rate r (estimated by iteration with Euler’s P equation: nx¼0 erx l x mx ¼ 1), net reproductive rate R0, life expectancy at birth ex, generation time G, and the average lifespan. Survivorship curves, life expectancy curves, and fecundity tendencies were constructed. Results were compared with ANOVA I and Tukey’s post-hoc comparisons. Assays were conducted at 2171 1C. Actual salinity and specific conductance values corresponding to the different NaCl concentrations were determined with a YSI salineconductivity meter (Yellow Spring Instruments, mod. 30), duly calibrated; the equipment has a 70.5% precision.
3. Results 3.1. Median lethal concentration (LC50) for NaCl The Probit estimated LC50 (48 h) for NaCl was 5.48 g L1 (95% limits: 5.07–6.02 g L1); this value corresponds to a specific conductance of 9.79 mS cm1 (95% limits: 9.00–10.87 mS cm1). The tested concentrations ranged from 0 to 10 g NaCl L1, but at the highest salinity
ARTICLE IN PRESS F. Martı´nez-Jero´nimo, L. Martı´nez-Jero´nimo / Ecotoxicology and Environmental Safety 67 (2007) 411–416
in all the tests, all neonates died during the first 60 min. Some survivors at 48 h in concentrations above 5 g NaCl L1 were affected in their mobility and sometimes responded only to mechanical stimulation. 3.2. Reproduction and longevity individually determined
higher than those recorded with higher NaCl concentrations (Po0:001). The age at first reproduction ranged from 7.5 to 9.2 days in average (Fig. 1). According to ANOVA and Tukey’s post-hoc comparisons, females grown in 6 g NaCl L1 release their first clutch at a significantly lower age than the females from the other treatments (Po0:001). Inter-clutch time, ranging from 2.6 to 3.9 days, was affected by NaCl concentration (Po0:001), but only the value recorded at 7 g NaCl L1 was significantly higher (Po0:001). Average D. magna longevity ranged from 24.4 to 56.3 days and was affected by NaCl concentration (Po0:001), Tukey’s post-hoc comparisons demonstrated that the largest values were for females grown at 0 and 2 g NaCl L1, whereas for all the other treatments the longevity was significantly lower and different among them (Po0:001). 3.3. Life table analysis Fig. 2 shows the average values and the standard error values for the Life Table demographic parameters calculated for D. magna in the five treatments.
30
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Clutch Size
Fig. 1 shows reproduction and longevity recorded for D. magna grown individually in all the treatments. The average clutch size ranged from 5.1 to 28.7 neonates (Fig. 1). ANOVA demonstrated that NaCl concentration had a significant effect on this parameter (Po0:001), and Tukey’s post-hoc test revealed that the clutch size was significantly different with all NaCl concentrations. Total progeny (equivalent to R0 in the Life Table analysis) ranged from 25.6 to 466.9 neonates (Fig. 1). The NaCl concentration had a significant effect (Po0:001), and the total progeny was lower as NaCl concentration increased (Tukey’s post-hoc test, Po0:001). Average number of clutches per female ranged from 5.0 to 16.4 (Fig. 1), and it was significantly affected by NaCl concentration (Po0:001). Tukey’s post-hoc test demonstrated that the number of broods was similar for females grown at 0 and 2 g NaCl L1, but both were significantly
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Fig. 1. Reproduction and longevity of Daphnia magna grown at different NaCl concentrations and fed Ankistrodesmus falcatus. Average values and standard error bars are shown.
ARTICLE IN PRESS F. Martı´nez-Jero´nimo, L. Martı´nez-Jero´nimo / Ecotoxicology and Environmental Safety 67 (2007) 411–416
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NaCl Concentration (g L-1) Fig. 2. Demographic Life Table parameters for Daphnia magna grown at different NaCl concentrations and fed Ankistrodesmus falcatus. Average values and standard error bars are shown.
ANOVA suggests that average lifespan was significantly affected by the NaCl concentration (Po0:001). The maximal average lifespan (57.7 days) was for D. magna grown in reconstituted hard water (0 g NaCl L1), whereas the shortest lifespan (25.2 days) was for females grown in 7 g NaCl L1. Tukey’s post-hoc test revealed that this parameter had a significantly different value for most treatments (Po0:001), except for 4 and 6 g NaCl L1, in which results were not statistically different (P40:05). With respect to the life expectancy at birth (e), results recorded were similar to the average lifespan (see Fig. 2), and ANOVA and post-hoc comparisons reflect similar conclusions. The highest life expectancy at birth (57.2 days) was for females grown in reconstituted hard water, whereas the lowest value was for D. magna females grown in 7 g NaCl L1. The Net Reproductive Rate (R0) was significantly reduced as the NaCl concentration increased (Po0:001) (Fig. 2), and the values recorded in all treatments were significantly different (Tukey’s post-hoc comparisons). The generation time (G) ranged from 17.8 to 30.5 days (Fig. 2), and it was affected by the NaCl concentration
(Po0:001), with the values for all the treatments being significantly different among them (P40:01). The intrinsic population growth rate (r), one of the most important demographic parameters, was also significantly affected by the concentration of NaCl (Po0:001). As can be seen in Fig. 2, r was reduced as NaCl concentration increased, but no significant differences were determined between reconstituted hard water (0 g NaCl L1) and the 2 g L1 concentration, nor between 4 and 6 g NaCl L1; r ¼ 0:2513 day1 was the lowest significant value recorded for the highest NaCl concentration tested. The survival curves for all treatments are shown in Fig. 3; as can be seen, the shape of the graph is very similar to the Type I survivorship curves, with mortality accentuated in the older organisms of the cohort. The highest mortality rates were for D. magna grown in 6 and 7 g NaCl L1. 4. Discussion For D. magna, Schuytema et al. (1997) determined a median lethal concentration (LC50) at salinity concentration
ARTICLE IN PRESS F. Martı´nez-Jero´nimo, L. Martı´nez-Jero´nimo / Ecotoxicology and Environmental Safety 67 (2007) 411–416
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Age (d) Fig. 3. Survivorship curves of Daphnia magna grown at different NaCl concentrations and fed Ankistrodesmus falcatus. Average values and standard error bars are shown.
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of 6.6 g L1 (measured conductivity, 10.0 mS cm1). Cowgill and Milazzo (1990) estimated an LC50 for NaCl at 6.034 g L1, and pointed out that the ‘‘non observable effects level’’ (NOEL) for NaCl was approximately 1.2 g L1. In the present study, LC50 was determined to occur at the NaCl concentration of 5.48 g L1 (measured conductivity, 9.79 mS cm1), and was lower than previously reported values. This situation could be related to a greater sensitivity to salinity by the experimental strain we used, a plausible situation since this strain has been grown with outstanding reproductive results in freshwater conditions for more than 15 years. Arne´r and Koivisto (1993) reported that they grew D. magna in salinities of 4% and 8%, but the conductivities reported (4.5 and 8.5 mS cm1) were lower than those determined here for the two highest NaCl concentrations (10.61 and 12.23 mS cm1), so we can suspect that the maximum salinity value they informed could in fact be lower. Intrinsic population growth rate (r) estimated in the present study for 6 g NaCl L1 (0.32 day1) was intermediate to the values obtained by Arne´r and Koivisto (1993) (0.52 and 0.299 day1, respectively, for 4% and 8%). They concluded that D. magna grew better at 4%, and, at this salinity, the cladoceran produced an average of 12.6 neonates per clutch, a value lower than that found here at a similar salinity (18.5 neonates). They also stated that D. magna can develop in salinities of up to 12.5%, but its presence declined abruptly above 4%, and no more information was provided for possible comparisons. As we said before, apparently in our study we tested higher salinity concentrations, taking into account the conductivity values for both studies and, should this be the case, the D. magna strain we used could be more tolerant to salinity. Cowgill and Milazzo (1991) observed that R0 and r values diminish as NaCl concentration increases, and they concluded that the reduction in R0 is manifested at values as low as 0.061 g L1, whereas the maximal reduction in r begins at 0.778 g NaCl L1. They stated that these demographic parameters are better indicators for assessing salinity stress than the traditional measures of toxicity (LC50, NOEL, NOEC, etc.). They calculated a R0 value of 10 neonates for D. magna grown at 6 g NaCl L1, lower than what we determined, even for a NaCl concentration of 7 g L1 (41 neonates, Fig. 2). Despite the reduction in growth rate recorded by us, reproduction in this strain was well above values reported in other studies at comparable salinity (or conductivity) conditions. Arne´r and Koivisto (1993) observed that r diminished at 8%, the specimens were smaller, and they moulted later than those grown at 4% and in freshwater conditions. They also concluded that the food concentration they supplied was high compared with other studies, but this could help maintain D. magna at 8%. As can be seen in Fig. 2, these two demographic parameters show a decline with respect to the increase in NaCl concentration, similar to that
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observed by Arne´r and Koivisto (1993), but better supported with experimental data. Other authors have shown that D. magna actively osmoregulates its body fluids up to a salinity of ca. 5%; higher salinities induce the organism to act as an osmo-conformer (Aladin, 1991; Arne´r and Koivisto, 1993). It can be argued that a reduction in the ability to osmo-regulate could be related with failure in salinity-tolerant clones to thrive in freshwater; this could explain the observations made by Teschner (1995), who reported that some brackish-water tolerant Daphnia clones lose their capacity to develop in freshwater. The change from a freshwater to a brackish medium, as was assayed here, suggests tolerance mechanisms that are inducible in the physiological tolerance range; the acclimation performed here enables this D. magna strain to endure salinity stress (provided as NaCl concentration). In this sense, Williams (1998) remarked that D. magna in slightly brackish waters has a narrow range of salt tolerance, whereas varieties found in highly saline lakes display a wide tolerance to this factor, as observed in Moina hutchinsoni, a cladoceran that flourishes in saline lakes in North America at up to 40 psu, but M. hutchinsoni can be grown under laboratory conditions at salinity values as low as 4 psu with similar results to those recorded at a higher salinity (Martı´ nez-Jero´nimo et al., 2004; Martı´ nezJero´nimo and Espinosa-Cha´vez, 2005). Considering its tolerance to salinity, Green et al. (2005) established that D. magna is a euryhaline species that is particularly tolerant to salinity conditions in brackish lakes; nevertheless, they concluded that the reproductive and/or survival rates of cladocerans are reduced at higher water conductivities. In our study, we demonstrated that the freshwater strain we used has in fact a relatively small range of tolerance to NaCl salinity; nevertheless it was acclimated to thrive at the upper salinity values within its tolerance range. Comparing results in the whole NaCl concentration range tested, we can conclude that the impairment effects on reproduction and survival under the current treatments were provoked by salinity stress, although it has been demonstrated that various freshwater invertebrate species (including cladocerans) are more sensitive to NaCl salinity than to the effect produced by the array of chemical compounds present in sea salt (Kefford et al., 2004). The present results might be useful for a better understanding of how salinity (as NaCl concentration) affected the demography, longevity, survival, and reproductive responses of a freshwater D. magna strain and which could be its maximum tolerance to this chemical compound. Results also indicate that the growth and survival of D. magna in NaCl stress conditions were suboptimal, yet for the strain here tested, they were clearly superior to those reported in other studies. These data provide support for the use of this strain, based on this capability to endure brackish waters, as test organism in toxicity assays performed in slightly saline conditions (up to 2.3 or 4.3 psu).
Acknowledgments Thanks are due to the Comisio´n de Operacio´n y Fomento de Actividades Acade´micas—I.P.N. and Estı´ mulo al Desempen˜o de los Investigadores—I.P.N. for the grants received. References Aladin, N.V., 1991. Salinity tolerance and morphology of the osmoregulation organs in Cladocera with special reference to Cladocera from the Aral sea. Hydrobiologia 225, 291–299. Arne´r, M., Koivisto, S., 1993. Effects of salinity on metabolism and life history characteristics of Daphnia magna. Hydrobiologia 259, 69–77. Bailey, S.A., Duggan, I.C., Van Overdijk, D.A.C., Johengen, T.H., Reid, D.F., Macisaac, H.J., 2004. Salinity tolerance of diapausing eggs of freshwater zooplankton. Freshwater Biol. 49, 286–295. Cowgill, U.M., Milazzo, D.P., 1990. The sensitivity of two cladocerans to water quality variables, salinity and hardness. Arch. Hydrobiol. 120, 185–196. Cowgill, U.M., Milazzo, D.P., 1991. Demographic effects of salinity, water hardness and carbonate alkalinity on Daphnia magna and Ceriodaphnia dubia. Arch. Hydrobiol. 122, 35–56. Green, A.J., Fuentes, C., Moreno-Ostos, E., Rodrigues da Silva, S.L., 2005. Factors influencing cladoceran abundance and species richness in brackish lakes in Eastern Spain. Ann. Limnol.—Int. J. Limnol. 4, 73–81. Hart, B.T., Bailey, P., Edwards, R., Hortle, K., James, K., McMahon, A., Meredith, C., Swadling, K., 1991. A review of the salt sensitivity of the Australian freshwater biota. Hydrobiologia 210, 105–144. He, Z.H., Qin, J.G., Wang, Y., Jiang, H., Wen, Z., 2001. Biology of Moina mongolica (Moinidae, Cladocera) and perspective as live food for marine fish larvae: review. Hydrobiologia 457, 25–37. Hebert, P.D.N., Remigio, E.A., Colbourne, J.K., Taylor, D.J., Wilson, C.C., 2002. Accelerated molecular evolution in halophilic crustaceans. Evolution 56, 909–926. Kefford, B.J., Palmer, C.G., Pakhomova, L., Nugegoda, D., 2004. Comparing test systems to measure the salinity tolerance of freshwater invertebrates. Water SA 30, 499–506. Martı´ nez-Jero´nimo, F., Espinosa-Cha´vez, F., 2005. Notes on the reproduction and survival of Moina hutchinsoni Brehm, 1937 (Moinidae: Anomopoda) grown in media of varying salinity. Aquat. Ecol. 39, 113–118. Martı´ nez-Jero´nimo, F., Villasen˜or, R., Rı´ os, G., Espinosa, F., 1994. Effect of food type and concentration on the survival, longevity, and reproduction of Daphnia magna. Hydrobiologia 287, 207–214. Martı´ nez-Jero´nimo, F., Elı´ as-Gutie´rrez, M., Sua´rez-Morales, E., 2004. A redescription of Moina hutchinsoni Brehm, a rare cladoceran (Branchiopoda: Anomopoda) found in remnants of a Mexican saline lake, with notes on its life history. J. Crustacean Biol. 24, 232–245. NMX-AA-087-1995-SCFI, 1996. Ana´lisis de agua. Evaluacio´n de toxicidad aguda con Daphnia magna Straus (Crustacea-Cladocera). Me´todo de prueba, Diario Oficial de la Federacio´n, Mexico. Peters, R.H., 1987. Metabolism in Daphnia. In: Peters, R.H., de Bernardi, R. (Eds.), Daphnia. Mem. Ist. Ital. Idrobiol. 45, 193–243. Schuytema, G.S., Nebeker, A.V., Stutzman, T.W., 1997. Salinity tolerance of Daphnia magna and potential use for estuarine sediment toxicity tests. Arch. Environ. Contam. Toxicol. 33, 194–198. Teschner, M., 1995. Effects of salinity on the life history and fitness of Daphnia magna: variability within and between populations. Hydrobiologia 307, 33–41. Weber, C.I., 1993. Methods for Measuring the Acute Toxicity of Effluents and Receiving Waters to Freshwater and Marine Organisms, fourth ed. United States Environmental Protection Agency, Cincinnati, Ohio, EPA/600/4-90/027F. Williams, W.D., 1998. Salinity as a determinant of the structure of biological communities in salt lakes. Hydrobiologia 381, 191–201.