Aquaculture 215 (2003) 103 108 www.elsevier.

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Growth and survival of Rhamdia quelen (Siluriformes, Pimelodidae) larvae exposed to different levels of water hardness
C.R. Townsend, L.V.F. Silva, B. Baldisserotto *
Departamento de Fisiologia, Universidade Federal de Santa Maria, 97105.900 Santa Maria, RS, Brazil Received 16 July 2001; received in revised form 11 February 2002; accepted 4 April 2002

Abstract The response to water hardness increase varies from species to species. The purpose of this study was to verify the survival and growth of larvae of silver catfish, Rhamdia quelen, in water with different levels of water hardness. Larvae (2.75 mg and 5.00 F 0.05 mm) were randomly allotted to experimental units (three replicates per treatment) and kept in 44-l boxes (400 larvae/box). Larvae were exposed to five water hardness values (30, 70, 150, 300, and 600 mg l 1 CaCO3) at pH 8.25. Samples of larvae were collected on days 0, 7, 14, and 21, and the length, weight, and specific growth rate (SGR) were determined for each collection. Survival and biomass were calculated on day 21. Higher larvae growth, survival, and biomass were obtained at 30 and 70 mg l 1 CaCO3. Therefore, this is the recommended hardness range for growth and survival of silver catfish larvae. D 2003 Published by Elsevier Science B.V.
Keywords: Larvae; Survival; Growth; Calcium; Water quality; Rhamdia quelen; Silver catfish

1. Introduction Total hardness is the concentration of all divalent cations in water, and Ca2 + and Mg2 + are the most common cations in nearly all fresh water systems. The recommended value for water hardness for fish culture in ponds is above 20 mg l 1 CaCO3 (Boyd, 1998; Zweig et al., 1999), mainly because of its influence on phytoplankton growth and water

* Corresponding author. Tel.: +55-55-220-8342; fax: +55-55-220-8241. E-mail addresses: bernardo@ccs.ufsm.br, bbaldisserotto@hotmail.com (B. Baldisserotto). 0044-8486/03/$ - see front matter D 2003 Published by Elsevier Science B.V. PII: S 0 0 4 4 - 8 4 8 6 ( 0 2 ) 0 0 1 6 8 - 0

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pH control. However, Ca2 + is essential for fish for several biological processes such as bone construction, blood coagulation, and many other cellular functions (Flik et al., 1995). The internal sources of Ca2 + (bones) in fish are not easily accessible. Therefore, plasma Ca2 + is regulated by its ingestion with food or by branchial absorption (Flik and Verbost, 1995). Several teleost species exposed to acidic or alkaline water showed higher survival in hard than in soft water (Freda and McDonald, 1988; Yesaki and Iwama, 1992; Laitinen and Karttunen, 1994; Townsend and Baldisserotto, in press). The increase of water hardness from 40 to 440 mg l 1 CaCO3 also improved channel catfish (Ictalurus punctatus) survival in the presence of high nonionized ammonia levels (Tomasso et al., 1980), and levels of 300 500 mg l 1 CaCO3 are recommended for the successful hatching of eggs and viability of silver carp (Hypophthalmicthys molitrix) larvae (Gonzal et al., 1987). However, sunshine bass (Morone chrysops Morone saxatilis hybrids) and tilapia (Oreochromis mossambicus) growth was not affected by different Ca2 + concentrations (Seals et al., 1994; Hwang et al., 1996). Because the response to a water hardness increase seems to vary from species to species, the aim of this study was to analyze the survival and growth of silver catfish (Rhamdia quelen) larvae at different water hardnesses.

2. Materials and methods Silver catfish larvae (Pimelodidae) were obtained from one induced spawning (December) using carp pituitary extract in the fish culture sector of the Universidade Federal de Santa Maria. All eggs from the spawning were from the same cohort (female weighing 1.96 kg and male weighing 2.0 kg) and were incubated in Hungarian-model hatcheries (200 l) with running water. After absorption of yolk sac (3 days), larvae were randomly assigned to treatments at hardness of 30 (28 32), 70 (68 72), 150 (148 152), 300 (292 320), and 600 (590 620) mg l 1 CaCO3 and pH 8.1 8.3 (three replicates per treatment). These treatments were chosen because exposure of silver catfish fingerlings to this water hardness range did not induce any mortality or alteration in normal behavior (Townsend and Baldisserotto, in press). Larvae were maintained in continuously aerated 40-l freshwater polyethylene boxes for 21 days. Stocking density was 400 larvae/box (10 larvae l 1). These boxes contained a water reuse system (23 F 1 jC), and photoperiod was 10-h dark/14-h light, with luminosity of 0.6 lx (measured with a LI-COR photometer model LI-185B), since dark environments improve R. quelen growth (Piaia et al., 1999). Larvae were fed satiation six times a day (08:00, 10:00, 12:00, 14:00, 16:00, and 18:00 h) with fish food prepared according to Lopes et al. (2001). Fish food granule size was 100 200 Am (first week), 200 400 Am (second week), and 400 600 Am (third week). All feces and pellet residues were removed daily by suction, and consequently, 10% of the water in the boxes was replaced with water at previously adjusted pH and hardness. Samples of 20 larvae were collected from each replicate on days 0, 7, 14, and 21 after absorption of the yolk sac, mean length, and weight were measured. Specific growth rate (SGR) was calculated for each collection according to Jrgensen and Jobling (1993).

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Apparent accumulated mortality was determined by counting the dead larvae daily. This value could not be considered the real mortality because some larvae disappeared due to cannibalism, but could give an idea of larval mortality throughout the experiment. On day 21, all surviving larvae were collected to determine survival (%) and biomass (individual mean weight number of surviving larvae). Water was previously adjusted to the desired experimental pH by the addition of sodium hydroxide and water hardness was increased by the addition of calcium chloride (CaCl22H2O). Water pH was measured with a Hanna pH meter at 8:00, 12:00, 14:00, and 18:00 h and adjusted when necessary. Total water hardness was measured according to Adad (1982) and dissolved oxygen with a Digimed oxygen meter, model DM4. Both parameters were analyzed once a day. The following physicochemical parameters of the water were also analyzed according to Adad (1982), but at 3-day intervals: total alkalinity, total ammonia, and nitrite. All data are expressed as mean F S.E.M. Mean length, weight, G, and total biomass of treatment groups were compared by one-way ANOVA, and means were compared by Duncans multiple range test, using the SPSS statistical software (version 1986). Survival was analyzed by the chi-square test using the SAS statistical software (version 6.8, 1993). The minimum significance level was set at P < 0.05.

3. Results Water hardness and pH showed minor alterations in all treatments, remaining within the expected range throughout the experiments. Maximum total ammonia reached 0.8 mg l 1 and nonionized ammonia 0.10 mg l 1. Dissolved oxygen remained in the 5.0 5.5 mg l 1 range, while oxygen saturation was in the 60.0 76.6% range. Larvae had an initial individual mean weight of 2.7 mg and length of 5.0 F 0.05 mm. Throughout the period of 21 days, larvae maintained at water hardness of 30 and 70 mg l 1 CaCO3 showed greater length than the other treatments. At the first collection (day 7), higher weight was observed at a water hardness of 30 mg l 1 CaCO3, but at the end of the experiment, the highest value was obtained at a water hardness of 70 mg l 1 CaCO3. In the first week of the experiment, the SGR was lower in the treatments with water hardness of 150 and 300 mg l 1 CaCO3 than in the treatment with 30 mg l 1 CaCO3, but later, there was no significant effect of water hardness on this parameter (Table 1). Most larvae died in the first days of the experiment in the treatments with higher water hardness (300 and 600 mg l 1 CaCO3), but for larvae exposed to 150 mg l 1 CaCO3, higher mortality was spread throughout the first 2 weeks. Larvae submitted to 70 mg l 1 CaCO3 also showed higher mortality in the first week, while those maintained at 30 mg l 1 CaCO3 presented low mortality throughout the experiment. The highest survival rate at the end of the experiment was observed at the water hardness of 30 mg l 1 CaCO3 (80.4 F 4.1%), followed by 70 mg l 1 CaCO3 (62.0 F 5.9%). Larvae exposed to higher water hardness levels showed much lower survival rates: 150 mg l 1 CaCO38.7 F 0.9%, 300 mg l 1 CaCO31.0 F 0.8%, and 600 mg l 1 CaCO30.3 F 0.3% ( P < 0.05). Highest biomass values were observed in the treatments with water hardness of 30 and 70 mg l 1 CaCO3 (8.7 F 1.0 and 10.5 F 1.8 g, respectively). The increase of water hardness to 150, 300, and

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Table 1 Length, weight, and specific growth rate (SGR) of silver catfish larvae as a function of water hardness Hardness (mg l 1 CaCO3) Length (mm) 30 70 150 300 600 Weight (mg) 30 70 150 300 600 SGR (% day 1) 30 70 150 300 600 Days after absorption of yolk sac 7 7.02 F 0.06a 6.51 F 0.23a 5.91 F 0.14b 5.63 F 0.26b 5.02 F 0.10c 14 9.11 F 0.03a 8.32 F 0.41ab 7.88 F 0.62bc 7.20 F 0.55c 7.09 F 0.31c 21 11.82 F 0.41ab 13.26 F 0.24b 10.00 F 0.36c 09.72 F 1.28c 9.66ab,*

5.18 F 0.93a 3.50 F 0.48b 2.50 F 0.11b 2.49 F 0.25b 3.04 F 0.32b

14.25 F 2.57a 12.71 F 1.96a 11.58 F 0.95a 11.41 F 2.54a 13.71 F 3.06a

29.63 F 3.77a 45.41 F 4.58b 21.70 F 2.71a 17.16 F 2.28a 14.83a,*

8.86 F 2.53a 3.43 F 2.02ab 1.11 F 0.64b 1.28 F 1.41b 1.52 F 1.51ab

7.27 F 0.05a 9.16 F 2.16a 10.91 F 0.37a 10.54 F 1.87a 10.43 F 2.40a

3.55 F 1.01a 6.15 F 1.28a 2.96 F 0.63a 3.04 F 0.66a 2.97a,*

Means identified by different letters in the columns were significantly different ( P < 0.05) as determined by ANOVA and Duncans test for comparison of mean values. * Treatment with survivors in only one replicate on day 21.

600 mg l 1 CaCO3 reduced biomass values to significantly lower values (0.7 F 0.06, 0.06 F 0.04, and 0.01 F 0.01 g, respectively).

4. Discussion Dissolved oxygen levels were always above 5 mg l 1, the minimum level suggested by Boyd (1998). There are no studies about the minimum oxygen saturation level for this species, and as there were no differences among treatments, this parameter probably did not affect the results. The maximum nonionized ammonia levels observed in this experiment were within the maximum range suggested for growth in I. punctatus (Tomasso, 1994). Therefore, nonionized ammonia probably also did not interfere with the results. The apparent mortality of silver catfish larvae was higher in the first days of the experiment at higher water hardness. This mortality may be related to the osmotic stress induced by the increase in water hardness, which also reduced length, biomass, and survival of silver catfish larvae. Larvae were not adapted slowly to higher hardness levels because silver catfish fingerlings survived the increase of water hardness up to 600 mg l 1 CaCO3 (higher levels were not tested) without any problems (Townsend and Baldisserotto, in press). This difference in tolerance to high water hardness levels may be age-related, since the ability to maintain Ca2 + homeostasis improves with development (Grizzle and

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Mauldin, 1994). Recently hatched larvae have gills and renal complexes not fully developed, but maintain their internal concentration with chloride cells located on the skin (Alderdice, 1988; Rombough, 1999). Twenty-six days after hatching, rainbow trout larvae presented 75% of all chloride cells in the gills. As chloride cells in the gills are more efficient in terms of ion exchange than those on the skin (Rombough, 1999), the ionoregulatory capacity of fingerlings would be improved in relation to larvae, explaining their higher survival in water with high hardness levels. The postharvest survival of 5-week-old striped bass (M. saxatilis) fingerlings, a species extremely sensitive to handling stress, is improved by an increase in water hardness from 8 10 to 278 mg l 1 CaCO3 (with CaCl2). The addition of NaCl (5 g l 1) or MgCl2 instead of CaCl2 did not increase survival compared to control, showing the importance of the presence of Ca2 + in the water for this species (Grizzle et al., 1990). However, recently hatched larvae (9 days, still with the yolk sac) of this species showed higher survival after handling stress in water with high NaCl concentration (5 g l 1). In this stage, Ca2 + concentrations of 1.2 100 mg l 1 did not alter larval survival (Grizzle et al., 1992). However, sunshine bass (hybrids of M. chrysops M. saxatilis) submitted to waters with different Ca2 + concentrations (10, 20, 30, 40, or 80 mg l 1 CaCl2) did not show any significant difference in weight, food conversion, condition factor, or plasma Ca2 + concentrations under normal culture conditions (pH 6.9) (Seals et al., 1994). Also, neither the hatching rate nor the growth of tilapia larvae was affected by exposure to waters with 22 or 90 mg l 1 CaCO3) (Hwang et al., 1996). Therefore, it seems that the increase of water hardness up to 70 100 mg l 1 CaCO3 does not affect survival of teleost larvae at neutral pH. The effect of water hardness levels higher than 100 mg l 1 CaCO3 seems to vary according to the species studied, since 300 500 mg l 1 CaCO3 is recommended for efficient hatching and high viability of silver carp eggs (Gonzal et al., 1987), but reduces the survival of silver catfish larvae. Liming is recommended to stabilize low water pH, because calcium carbonate increases water buffering capacity (Boyd, 1998). For silver catfish, liming must not increase water hardness beyond 70 mg l 1 CaCO3, because this amount is enough to reduce the effect of acidic and alkaline pH on fingerlings (Townsend and Baldisserotto, in press), and allows good larval growth. Based on the present results, the best hardness range for survival and growth of silver catfish larvae is 30 70 mg l 1 CaCO3. Acknowledgements o Radu The authors wish to thank Dr. Joa nz Neto, from the Department of Animal Husbandry, for suggestions during the experiments, and CNPq for granting a fellowship to C.R. Townsend. References
mico de Qualidade. Guanabara Dois, Rio de Janeiro, 203 pp. Adad, J.M.T., 1982. Controle Qu Alderdice, D.F., 1988. Osmotic and ionic regulation in teleost eggs and larvae. In: Hoar, W.S., Randall, D.J. (Eds.), Fish Physiology: v. XIPart A. Eggs and Larvae. Academic Press, San Diego, pp. 163 251.

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Boyd, C.E., 1998. Water quality for pond aquaculture. Research and Development Series (Alabama) 43, Aug, 37 pp. Flik, G., Verbost, P.M., 1995. Celular mechanisms in calcium transport and homeostasis in fishes. Biochemistry and Molecular Biology of Fishes 5, 252 263. Flik, G., Verbost, P.M., Wendelaar Bonga, S.E., 1995. Calcium transport processes in fishes. Cellular and Molecular Aproaches to Fish Ionic Regulation. Academic Press, San Diego, USA, 317 341. Cap12. Freda, J., McDonald, D.G., 1988. Physiological correlates of interspecific variation in acid tolerance in fish. Journal Experimental Biology 136, 243 258. Gonzal, A.C., Aralar, E.V., Pavico, J.M., 1987. The effects of water hardness on the hatching and viability of silver carps (Hypophthalmichthys molitrix) eggs. Aquaculture 64, 111 118. Grizzle, J.M., Mauldin, A.C., 1994. Age-related changes in survival of larval and juvenile striped bass in different concentrations of calcium and sodium. Transactions of the American Fisheries Society 123, 1002 1005. Grizzle, I.M., Mauldin II, A.C., Young, D., Henderson, E., 1990. Effects of environmental calcium and sodium on postharvest survival of juvenile striped bass. Journal of Aquatic Animal Health 2, 104 108. Grizzle, I.M., Mauldin II, A.C., Ashfield, C.J., 1992. Effects of sodium chloride and calcium chloride on survival of larval striped bass. Journal of Aquatic Animal Health 4, 281 285. Hwang, P.P., Tung, Y.C., Chang, M.H., 1996. Effect of environmental calcium levels on calcium uptake in tilapia larvae (Oreochromis mossambicus). Fish Physiology and Biochemistry 15 (5), 363 370. Jrgensen, E.H., Jobling, M., 1993. Feeding in darkness eliminates density-dependent growth suppression in artic charr. Aquaculture International 1, 90 93. Laitinen, M., Karttunen, M., 1994. Effects of calcium and magnesium in acid water on the ion balance of eggs and alevins of rainbow trout (Oncorhynchus mykiss). In: Mu ller, R., Lloyd, R. (Eds.), Sublethal and Chronic Effects of Pollutants on Freshwater Fish. FAO-Fishing New Books, Cambridge, 371 pp. Lopes, J.M., Silva, L.V.F., Baldisserotto, B., 2001. Survival and growth of silver catfish larvae exposed to different water pH. Aquaculture International 9 (1), 73 80. Piaia, R., Baldisserotto, B., Townsend, C., 1999. Growth and survival of fingerlings of silver catfish exposed to different photoperiods. Aquaculture International 7, 201 205. Rombough, P.J., 1999. The gill of fish larvae. Is it primarily a respiratory or an ionoregulation structure? Journal of Fish Biology 55, 186 204. Seals, C., Kempton, C.J., Tomasso Jr., J.R., Smith, T.I.J., 1994. Environmental calcium does not affect production or selected blood characteristics of sunshine bass reared under normal culture conditions. The Progressive Fish-Culturist 56, 269 272. Tomasso, J.R., 1994. Toxicity of nitrogenous wastes to aquaculture animals. Review of Fisheries Science 2, 291 314. Tomasso Jr., J.R., Goudie, C.A., Simco, B.A., Davis, K.B., 1980. Effects of environmental pH and calcium on ammonia toxicity in channel catfish. Transactions of American Fisheries Society 109, 229 234. Townsend, C.R., Baldisserotto, B. Survival of silver catfish fingerlings exposed to acute changes of water pH and hardness. Aquaculture International (in press). Yesaki, T.Y., Iwama, G.K., 1992. Survival, acid base regulation, and ammonia excretion in rainbow trout in highly alkaline hard water. Physiological Zoology 65 (4), 763 787. Zweig, R.D., Morton, J.D., Stewart, M.M., 1999. Source Water Quality for Aquaculture. World Bank, Washington, 62 pp.