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International Journal of Research in Fisheries and Aquaculture
Universal Research Publications. All rights reserved
Original Article
Effect Of Water Hardness On Egg Hatchability And Larval Viability Of Angelfish
(Pterophyllum scalare (Schultze, 1823)
1
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
[email protected]
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 100–200 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
6
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 30–60 mg/l CaCO3 (12).
The aim of this study, therefore, was to investigate
the effect of water hardness for hatching and viability of
7
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
14.06±0.72 g and average length of 12.17±0.36 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
(80×30×40 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
8
was determined. Data on hatchability and larval survival were
included normality and variance homogeneity analysis
analyzed with one-way analysis of variance (ANOVA) and
Duncan’s 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 28±2 °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 didn’t 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 don’t 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
9
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 (0–10 mg/l) and too hard water (300 mg/l) are not
suitable for Pterophyllum scalare egg incubation and larval
rearing.
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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