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International Journal of Research in Fisheries and Aquaculture

Original Article Effect Of Water Hardness On Egg Hatchability And Larval Viability Of Angelfish (Pterophyllum scalare (Schultze, 1823)
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Milad Kasiri1, Mohammad Sudagar1 and Seyed Abbas Hosseini1 Department of Fishery, Gorgan University of Agricultural Sciences and Natural Resources Iran, Golestan, Gorgan, Shahid Beheshti Avenue, Postal code: 49138-15739 kasiri_milad@yahoo.com Received 28 August 2011; accepted 11 September 2011

Abstract The response to water hardness increase varies from species to species. The purpose of this study was to verify the effect of water hardness on angelfish (Pterophyllum scalare) egg hatchability and larval viability. The fertilized eggs and larvae were exposed to seven water hardness values (10, 50, 100, 150, 200, 250, and 300 mg/l CaCO3) at pH 7.25 and 28 C. Survival was calculated until day 21. The highest survival rate at the end of the experiment was (50.39 6.23%) observed at the water hardness of 100 mg/l CaCO3. The results imply that too soft water (10 mg/l CaCO3) and too hard water (300 mg/l CaCO3) are not suitable for Pterophyllum scalare incubation and larval rearing. A moderately hard water of 100200 mg/l CaCO3 is recommended for optimal normal hatching, high viability and maximum larval development of angel fish. 2011 Universal Research Publications. All rights reserved Keywords: hard water, soft water, incubation. 1 Introduction Ornamental fishes are often referred as living jewels due to their color, shape and behavior. They are quiet, generally small size, attractively colored and could be accommodated in confined spaces. Modern ornamental fish culture and breeding operations, have become vertically and horizontally intensified, necessitating a continuous supply of nutritionally balanced, cost-effective feed (1). The angelfish (Pterophyllum scalare (Schultze, 1823)), is a cichlid native to the Amazon Basin of Brazil and exported as an ornamental fish (2). This fish is one of most popular fish species among the freshwater aquarium trade industry (3). Angelfish are among the first fish purchased for beginner, As they are inexpensive and beautiful fish. They are ideal community tank fish and available in several color and forms (such as black zebra angelfish, Cobra angelfish, Half black angelfish and Koi angelfish) The past 50 years of commercial and hobby breeding of Pterophyllum scalare have produced a hardy fish tolerant of common tap water conditions, a great range of temperatures, and resistant to diseases (4). Total hardness is the concentration of all divalent cations in water, and Ca2+ and Mg2+ are the most common cations in almost all fresh water systems. Skeletal growth in vertebrates is partially controlled by the availability of minerals. The correlation between aqueous calcium content and fish skeletal growth (ossification) is especially important as calcium absorption from water is an essential route to acquire this mineral (5). The suggested value for water hardness for fish cultivation in ponds is above 20 mg/l CaCO3 (6, 7). One of the important processes during the early development of teleost egg is the swelling of newly fertilized eggs, which water hardness has been exhibited to have a direct effect on this stage (8). Abernathy (9) found that calcium and magnesium both contribute to water hardness, calcium, not magnesium, is necessary for the development and survival of rainbow sharkminnow eggs. Calcium, although necessary, was found to have a detrimental effect at higher concentrations due to its increasing the osmotic concentration of the incubation water which directly effected the swelling of the eggs, reducing larval survival. Different species of fish showed different percentage of survival at various different hardnesses. Channel catfish (Ictalurus punctatus (Rafinesque, 1818)) survival was improved when water hardness increased from 40 to 440 mg/l CaCO3 (10). Gonzal et al. (11) found that water hardness

International Journal of Research in Fisheries and Aquaculture 2011; 1(1): 6-10

Table 1: Nutriente composition of Prepared granulated feed diets (%) Diet Commercial extruder diet Protein 54 Lipid 18 Fiber 1.5 Ash 10 Vitamin 2 Table 2: Comparison mean ( SD) for Pterophyllum scalare egg hatchability and larval survival at different levels of total water hardness. Larval survival to end of yolk sac Total hardness (mg/l CaCO3) Hatchability (%) period (%) 10 64.71 5.58c 71.15 1.82c* 50 65.83 7.33bc 83.17 7.26a ab 100 73.26 4.66 85.31 5.58a abc 150 70.74 6.57 83.63 7.67a ab 200 73.57 1.61 80.73 3.48ab a 250 74.60 4.66 78.05 4.52abc bc 300 65.88 9.10 75.14 3.18bc Groups with different alphabetic superscripts differ significantly at P < 0.05. *Treatment with survivors in only two replicate in end of yolk sac period. Table 3: Survival rate (%) of Pterophyllum scalare during 21 days after yolk sac period as a function of water hardness Total hardness Day 7 Day 14 Day21 (mg/l CaCO3) ab 50 67.84 7.29 79.20 7.23 87.79 4.49 100 69.08 6.29a 81.87 1.94 88.78 4.33 150 68.82 7.09a 84.42 9.02 87.40 3.01 200 67.34 8.31ab 81.82 6.93 85.49 5.41 250 60.34 4.29bc 78.71 4.16 83.31 3.92 300 57.50 4.30c 76.59 8.18 84.21 3.96 Groups with different alphabetic superscripts differ significantly at P < 0.05. Table 4: larvae length of Pterophyllum scalare (mm) during 21 day. Total hardness Day 7 Day 14 (mg/l CaCO3) b 50 4.15 0.08 6.38 0.24ab a 100 4.38 0.17 6.45 0.06a a 150 4.30 0.16 6.43 0.13ab bc 200 4.04 0.06 6.35 0.25ab bc 250 4.02 0.14 6.33 0.17ab c 300 3.99 0.07 6.20 0.16b Groups with different alphabetic superscripts differ significantly at P < 0.05. from 100-600 mg/l CaCO3, in silver carp (Hypophthalmichthys molitrix (Valenciennes, 1844)) eggs had the greatest hatch and viability. However, another study showed higher larval growth and survival in Rhamdia quelen (Siluriformes Pimelodidae) were obtained at 30 and 70 mg/l CaCO3 and best hatching rate, high viability and maximum larval development of Clarias gariepinus (Burchell, 1822) were obtained in water with hardness of 3060 mg/l CaCO3 (12). The aim of this study, therefore, was to investigate the effect of water hardness for hatching and viability of

Day21 8.44 0.33a 8.46 0.15a 8.45 0.18a 8.40 0.12a 8.08 0.04b 7.90 0.18b

angelfish eggs. The results of this study will be useful in improving ornamental fish hatchery systems. 2 Materials and Methods Fourteen pairs of 15 months angelfish (average weight of 14.060.72 g and average length of 12.170.36 cm) were randomly stocked into each aquarium (4) with six replications per treatment. Angel fish were obtained from the Institute of Ornamental Fish Hatchery in Ghazvin, Iran and transferred to the place of experiment in Institute of Ornamental Fish Hatchery in Babol, Iran and were acclimatized for 2 weeks.

International Journal of Research in Fisheries and Aquaculture 2011; 1(1): 6-10

Figure 1: Survival from end of hatching period to day 21. Fish were fed with prepared granulated feed (table 1) until be satuated, twice per day (09:00 a.m and 17:00 p.m). Fish food granule size was 2.5 mm. The trials were conducted in 14 (803040 cm) glass aquaria. Gentle aeration was provided by air stones. After 30 days feeding, breeders were spawned during five month. After spawning the substratum containing eggs removed for mechanical hatching and fertilized eggs were incubated at 28 C at seven levels of water hardness: 10, 50, 100,150, 200, 250 and 300 mg/l CaCO3 using aerated 15 circular plastic tanks (20 cm in diameter). Methylene blue solution added to the water sufficiently to tint the water deep, dark blue (4). After hatching, the embryos were counted and the percentage determined for each water hardness. After yolk sac absorption (4 days), larvae of each treatment were maintained in continuously aerated 40 l polyethylene tanks for 21 days. Feces and other wastes were discharged every day and 50% of water was exchanged with fresh water has the desired hardness. During larval rearing, incubation remnants, dead larvae and waste were siphoned off every day to avoid any means of stress. Three developmental periods were defined: the hatching period, the yolk sac period and the growth period. The hatching period began when the first larvae was observed and ended when larvae had hatched. The yolk sac period extended from end of hatching period to the yolk-sac of the larvae was absorbed (yolk sac absorption was determined visually) (12). The growth period began from the end of yolk sac period to 21 days of the hatching. Larvae were fed with only Artemia nauplii to be satiated four times a day (08:00, 11:00 a.m and 14:00, 17:00 p.m) according to Goldestin (4). After yolk sac period, larvae were counted from all replicate on days 0, 7, 14 and 21 and samples of 20 larvae were collected from each replicate and the length were measured and noted. The percentage of eggs and larvae survival at the end of each developmental period

was determined. Data on hatchability and larval survival were included normality and variance homogeneity analysis analyzed with one-way analysis of variance (ANOVA) and Duncans Multiple Range Test (DMRT) (using SPSS 16.0 program) to test for significant differences in various water hardnesses. Significance was established at the 0.05 levels. 3 Results During the experiment, the water quality parameters were monitored during the trial and average value for temperature, dissolved oxygen, pH and salinity were 282 C, 5.8-7.5 mgl1 , 6.9-7.8 units and 0.1 mg l-1, respectively. Water hardness and pH showed minor alterations in all treatments remaining within the expected range throughout the experiments. The effects of water hardness on hatching rate and larvae survival were presented in table 2. The highest hatching rate observed at 250 mg/l CaCO3 and the lowest ones found at 10 and 50 mg/l CaCO3 (P < 0.05). There was a significant difference among experimental groups in percentage of larval survival to end of yolk sac period (P < 0.05). The best survival were observed at 50, 100 and 150 mg/l CaCO3. Furthermore, at 10 mg/l CaCO3 larvae just remained alive in two replicates to the end of yolk sac period, as well as to day 7 after this stage no larvae remained in this treatment (table 2). According to table 3 to day 7, higher larval survival (P < 0.05) were observed at 100 and 150 mg/l CaCO3 (69.08 6.29 and 68.82 7.09 (%), respectively) but in days 14 and 21, larvae survival didnt show significant difference ( P > 0.05) among experimental groups. The most larval mortality was observed in day 7 after yolk sac absorption and lower larval survival in this stage observed at water hardness of 300 mg/l CaCO3 (57.50 4.30%). The highest survival rate at the end of the experiment was recorded at the water hardness of 100 mg/l CaCO3 (50.39 6.23%) (Fig. 1). Throughout the period of 21 days, larvae maintained at water hardness of 100-200 mg/l CaCO3 showed better length than the other treatments (table 4). 4 Discussion The perivitelline space is located between the outer chorion of the egg and the vitelline membrane that surrounds the developing embryo. In a fertilized egg, the fluid filled perivitelline space provides room and protection for embryonic development (13). Ca2+ in incubation waters was ion. Thus, solute concentration (osmolality) was directly proportional to the ionic concentration (increased solute concentration = increased ionic concentration). Increasing the solute/ionic concentration of the incubation waters increased the osmotic concentration (increased solute/ionic concentration = increased osmotic concentration) (9). Hatchability in treatments, decreased in very hard and very soft water in effect of osmotic concentration. This can be explained by the fact that egg swelling is an osmoticaly driven process. When osmotic concentration is greater inside

International Journal of Research in Fisheries and Aquaculture 2011; 1(1): 6-10

the perivitelline space than the extracellular water surrounding the egg, extracellular water moves into the perivitelline space via osmosis causing the egg to swell. Typically, egg swelling increases when water hardness decreases because low water hardness usually means low osmolarity, and its swelling decrease when water hardness incrasees. The fact that, decreasing the hatchibility in very soft and very hard incubation waters mean that the CaCO3 in incubation waters were effective on angelfish eggs. In this study the highest larvae survival observed in soft water 50-100 mg/l CaCO3. It is agreed with studies results of Molokwu and Okpokwasili (12) examined the effects of increasing water hardness on hatch rates and larvae survival of Clarias gariepinus. They found that increasing in water hardness reduced larval survival. Also Abernathy (9) found that very soft waters (6.4-7.0 mg/l as CaCO3) produced the highest hatch and larval survival of rainbow sharkminnow (Epalzeorhynchos frenatum), In our study the best hatchability was found at 250 mg/l CaCO3 (74.60 4.64 %). Wild angelfish need to soft and acidic water but domestic angelfish dont need to this conditions. Many fish found under restrictive conditions in nature live better and easier when presented with entirely different conditions in captivity. The habitat conditions in nature may not indicate the fish's requirements, but rather its tolerance of conditions not tolerated by its competitors and predators (4). So, soft and acidic water is not a necessary condition for domestic angelfishes. The apparent survival of angel fish larvae was lower (57.50 4.30%) in the day 7 of the experiment at higher water hardness. This mortality may be related to the osmotic stress induced by water hardness, which also reduced length (3.99 0.07mm) of angel fish larvae. This difference in tolerance to high water hardness levels may be age-related, since the ability to maintain Ca 2+ homeostasis improves with development (14). Recently hatched larvae have gills and renal complexes not fully developed, but maintain their internal concentration with chloride cells located on the skin (15). 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 (15), the ionoregulatory capacity of larvae at next two week would be improved in relation to larvae, explaining their higher survival in water with high hardness levels. Townsend et al. (16) showed that the highest survival rate and growth of Rhamdia quelen (Siluriformes Pimelodidae) were obtained at the low water hardness (30 and 70 mg/l CaCO3), but in this study the highest survival rate was found at the 100 mg/l CaCO3 and growth rate obtained at 50 to 200 mg/l CaCO3 was higher. Morgan et al. (17) found that an increase in water hardness reduced or eliminated the effect of silver toxicity in early life

stages of rainbow trout (Oncorhynchus mykiss), while enhancing fish survival. However in our study the highest survival rate was found at moderately hard water (100 mg/l CaCO3). In this study we found the highest survival rate in 100 mg/l CaCO3 , but Blanksma et al. (2009) studied the effects of water hardness on skeletal development and growth in juvenile fathead minnows, and pointed that fathead minnows reared in calcium-abundant water (Ca = 65 1.5 mg/l as CaCO3; hardness 175 mg/l) had significantly increased survival but lower whole body mass when compared to their conspecifics in lowcalcium water. These results imply that hardness affects on water conditions for use in Pterophyllum scalare hatcheries and too soft water (010 mg/l) and too hard water (300 mg/l) are not suitable for Pterophyllum scalare egg incubation and larval rearing. References 1. B. Mandal, A. Mukherjee and S. Banerjee, Growth and pigmentation development efficiencies in fantail guppy, Poecilia reticulata fed with commercially available feeds. Agricult. Biol. J. North. America. (2010), 1(6): 1264-1267. 2. M.D.S. Cacho, S. Chellappa and M.E. Yamamoto, Reproductive success and female preference in the amazonian cichlid angel fish, Pterophyllum scalare. Neotropic. Ichth. (2006), 4:87-91. 3. M. Garcia-Ulloa, and H.J. Gomez-Romero, Growth of angel fish Pterophyllum scalare juveniles fed inert diets. Avanc. en Invest. Agrop. (2005), 9(3): 49-60. 4. R.J. Goldstein, Angelfish a complete pet owner's manual. Barron's Educational Series, New York (2001), 81 pp. 5. C. Blanksma, , B. Eguia, k. Lott., J.M. Lazorchak., M.E. Smith., M. Wratschko., T.D. Dawson., C. Elonen., M. Kahl and H.L. Schoenfuss, Effects of water hardness on skeletal development and growth in juvenile fathead minnows. Aquaculture (2009), 286: 226232. 6. C.E. Boyd,. Water quality for pond aquaculture. Research and Development Series (Alabama) 43 (1998), Auguest, 37 pp. 7. R.D. Zweig, J.D. Morton and M.M. Stewart, Source Water Quality for Aquaculture. World Bank, Washington (1999), 62 pp. 8. Spade, S. and B. Bristow, Effects of increasing water hardness on egg diameter and hatch rates of striped bass eggs. Nor. Americ. J. Aquacul. (1999), 61:263-265. 9. M.A. Abernathy, Effect of water hardness on the survival of rainbow sharkminnow (Epalzeorhynchos frenatum) eggs and larvae. MSc Thesis, University of Florida, Florida (2004), 63pp. 10. J.R. Tomasso, C.A. Goudie., B.A. Simco and K.B. Davis, Effects of environmental pH and calcium on

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concentrations of calcium and sodium. Trans. Americ. Fisher. Soc. (1994), 123: 10021005. 15. P.J. Rombough, The gill of fish larvae. Is it primarily a respiratory or an ionoregulation structure? J. Fish Biol. (1999), 55: 186204. 16. C.R. Townsend, L.V.F. Silva and B. Baldisserotto, Growth and survival of Rhamdia quelen (Siluriformes Pimelodidae) larvae exposed to different levels of water hardness. Aquacult. (2003), 215:103108. 17. T.P. Morgan, C.M. Guadagnolo, M. Grosell and M. Wood, Effects of water hardness on the physiological responses to chronic waterborne silver exposure in early life stages of rainbow trout (Oncorhynchus mykiss). Aqua. Toxic. (2005), 74 : 333350.

Source of support: Nil; Conflict of interest: None declared

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International Journal of Research in Fisheries and Aquaculture 2011; 1(1): 6-10