Hydrogen peroxide, iodine solution and methylene solution highly enhance the hatching rate of freshwater ornamental fish species

Abstract

The aim of this study was to evaluate the effect of hydrogen peroxide, iodine solution (PVP) and methylene blue on eggs disinfection of three ornamental fish species, Danio rerio, Pterophyllum scalare and Gymnocorymbus ternetzi. The main idea was to create conditions to enhance the hatching rates. Eggs of each species were exposed to different concentrations of hydrogen peroxide (5, 10, 15 and 25 mg/L), PVP (0.25, 0.5, 0.75 and 1 mg/L) and methylene blue (0.5 1, 2 and 3 mg/L). The optimal doses ranged between species and chemicals: for G. ternetzi, the concentrations that high enhanced the hatching rate were 1 mg/L for the PVP treatment, 25 mg/L for the hydrogen peroxide treatment and 3 mg/L for methylene blue treatment; for P. scalare, the best results were achieved with 25 mg/L for hydrogen peroxide treatment and 3 mg/L for methylene blue treatment. By contrast, for all the different chemical did not increased the D. rerio hatching rate. Results showed that hydrogen peroxide and methylene blue are the most versatile, effective and safe to use in these species. On the other hand, PVP can be used but with many precautions due to very low safety margin. Results clearly show that the optimal concentration of chemicals for eggs disinfection is fish species dependent and it is completely wrong to extrapolate concentrations between different chemicals and fish species. Our study suggests that P. scalare can be used as a model in study of effectiveness of new chemicals with potential to disinfect water and increase hatching rates.

Full text

Hydrogen peroxide, iodine solution and methylene
solution highly enhance the hatching rate of freshwater
ornamental fish species
Joa
˜o Chambel •Ricardo Costa •Mo
´nica Gomes •Susana Mendes •
Teresa Baptista •Rui Pedrosa
Received: 6 January 2014 / Accepted: 7 April 2014
ÓSpringer International Publishing Switzerland 2014
Abstract The aim of this study was to evaluate the effect of hydrogen peroxide, iodine
solution (PVP) and methylene blue on eggs disinfection of three ornamental fish species,
Danio rerio,Pterophyllum scalare and Gymnocorymbus ternetzi. The main idea was to
create conditions to enhance the hatching rates. Eggs of each species were exposed to
different concentrations of hydrogen peroxide (5, 10, 15 and 25 mg/L), PVP (0.25, 0.5,
0.75 and 1 mg/L) and methylene blue (0.5 1, 2 and 3 mg/L). The optimal doses ranged
between species and chemicals: for G. ternetzi, the concentrations that high enhanced the
hatching rate were 1 mg/L for the PVP treatment, 25 mg/L for the hydrogen peroxide
treatment and 3 mg/L for methylene blue treatment; for P. scalare, the best results were
achieved with 25 mg/L for hydrogen peroxide treatment and 3 mg/L for methylene blue
treatment. By contrast, for all the different chemical did not increased the D. rerio hatching
rate. Results showed that hydrogen peroxide and methylene blue are the most versatile,
effective and safe to use in these species. On the other hand, PVP can be used but with
many precautions due to very low safety margin. Results clearly show that the optimal
concentration of chemicals for eggs disinfection is fish species dependent and it is com-
pletely wrong to extrapolate concentrations between different chemicals and fish species.
Our study suggests that P. scalare can be used as a model in study of effectiveness of new
chemicals with potential to disinfect water and increase hatching rates.
Keywords Ornamental fish Disinfection fish eggs Hydrogen peroxide Iodine
solution (PVP) Methylene blue
J. Chambel (&)R. Costa M. Gomes S. Mendes T. Baptista R. Pedrosa
School of Tourism and Maritime Technology, Marine Resources Research Group, Polytechnic Institute
of Leiria, 2520-641 Peniche, Portugal
e-mail: joao.chambel@ipleiria.pt
123
Aquacult Int
DOI 10.1007/s10499-014-9779-1

Introduction
The ornamental fish sector is a widespread and global component of international trade,
with annual value of the world’s wholesale of one billion dollars, most of the aquaculture
production of ornamental fish focuses on freshwater species (Chambel et al. in press).
Currently, the development of an ornamental aquaculture protocol faces several critical
bottlenecks related to production processes and the competition of less expensive speci-
mens collected from the wild (Calado 2006).
The main constraint in the reliable production of most fish species is mortality during
the early developmental stages. The quality of eggs and larvae produced in hatcheries is
considered an important limiting factor in the larvae production and, consequently, in the
development of the aquaculture industry (Kjørsvik et al. 1990; Peck et al. 2004).
The damage by fungi in fish eggs results in an average annual lack of production of
about 20 %, with peaks higher than 40 % (Forneris et al. 2003). Due to heavily coloni-
zation by pathogens on the external surface of fish eggs, disinfection of eggs has been
widely used to reduce egg mortality and to improve rearing success during the yolk sac and
first feeding stages and also to reduce mortality of fish eggs incubated in hatchery tanks
(Morehead and Hart 2003; Madsen et al. 2005; Stuart et al. 2010).
There are some protocols for fish eggs disinfection, which include the use of malachite
green solutions, formalin, hydrogen peroxide, iodine solution (PVP), methylene blue,
ozone or sodium hypochlorite (Gaikowski et al. 1999; Arndt et al. 2001; Small and Wolters
2003; Rasowo et al. 2007). However, most published egg disinfection drug efficacy studies
were conducted on salmonids or on cool and warm water fish species and concentration of
the chemical to be used depends on fish species, the contact time of the chemical and the
water temperature (Rach et al. 1998,2004; Rasowo et al. 2007; Hirazawa et al. 1999; Eissa
et al. 2013), To the best of our knowledge, no studies have reported for the chemicals
antifungal efficacy on the ornamental fish eggs disinfection.
The goal of this study was to evaluate the effect of hydrogen peroxide, PVP and
methylene blue on eggs disinfection of three ornamental fish species, Danio rerio, verte-
brate fish model in research, Pterophyllum scalare and Gymnocorymbus ternetzi, two of
the most popular ornamental fish species, in order to promote high hatching rates and larval
survival to the first feeding.
Materials and methods
Eggs and experimental facilities
The study was conducted at the Ornamental Aquaculture Laboratory of Polytechnic
Institute of Leiria. All eggs were hatched in the laboratory. Males and females of D. rerio
and G. ternetzi were maintained separately in glass aquaria (60L) and P. scalare brood-
stocks were maintained in glass aquaria (100 l), both with external filtration, constant
aeration, photoperiod 14:10 light–dark cycle, temperature of 27 ±1°C, pH 6.5–7.5 and
fishes were fed ad libitum three times per day with commercial granulate food. Tanks were
cleaned daily, and water quality parameters were measured twice a week.
One day before reproduction males and females of each species D. rerio and G. ternetzi
were joined and in next morning fish spawned. After spawning, eggs were gently siphoned
and equally divided among experimental design. In the case of P. scalare 2–3 days before
spawning, the pair selects and begins cleaning the spawning site, using their mouths to bite
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and scrub the surface of the slate. After spawning, all eggs were gently siphoned from the
hatching slates and equally divided among experimental design.
Preliminary study
To determine the concentrations of each chemical used in this study, a preliminary test was
performed with the species G. ternetzi. To evaluate the effect of hydrogen peroxide, PVP
and methylene blue treatment dose on the hatching rate, 20 eggs were placed in aerated
400 mL beakers with the concentrations of 25, 500, 100 and 200 mg/L of hydrogen
peroxide (PANREAC QUIMICA, Spain), 1, 3, 5 and 10 mg/L of PVP (MEDA Pharma,
Portugal) and 1, 5, 15 and 25 mg/L of methylene blue (Merck SA, Germany). All con-
centrations were tested separately and defined on the active ingredient substance.
Each treatment was tested on three replicates, the disinfectants were applied at the
beginning of each experiment, and a complete water change was performed after 24 h
(time to eggs hatching).
Main study
The main study was realized testing the chemicals concentrations of 5, 10, 15 and 25 mg/L
of hydrogen peroxide, 0.25, 0.5, 0.75 and 1 mg/L of PVP and 0.5, 1, 2 and 3 mg/L of
methylene blue on D. rerio,G. ternetzi and P. scalare in same conditions as the pre-
liminary study. Complete water change was performed after 24 h to D. rerio and G.
ternetzi and 48 h for P. sclare (time to eggs hatching).
Determination of hatching rate
The hatching rate was expressed by the percentage of number of larvae hatched, 24 h (D.
rerio and G. ternetzi)or48h(P. sclare) after each treatment.
Statistical analysis
All data were checked for normality and homoscedasticity. One-way analysis of variance
(ANOVA) with Tukey HSD’s multiple comparison of group means was employed to
determine significant differences between the different treatments (Zar 2009). When
normality and homoscedasticity were violated, Kruskal–Wallis nonparametric test was
used with Games–Howell multiple comparison test (Zar 2009). Additionally, linear
regression analysis was used to measure the relationship between chemicals concentrations
and hatching rates.
Where applicable, results are presented as mean ±SEM. For all statistical tests, the
significance level was set at pB0.05. All calculations were performed with IBM SPSS
Statistics 20.
Results
During the experimental period, the water quality of broodstock was maintained in
appropriate values for maintaining these species, OD [8.0, pH between 7.1 ±0.4 tem-
perature 27 ±1°C, total ammonia and nitrite below 0.5 mg/L and nitrate \10 mg/L.
Aquacult Int
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Preliminary study showed that hatch rate obtained in control group and treatment groups
varied between 0 and 100 % (Fig. 1). The results showed that the average hatching rates in
all chemical treatments increased when compared with the control group (p\0.05). The
only exception was obtained for the treatment with PVP at concentration of 15 mg/L,
where no differences to control group were found (p[0.05). On the other hand, at 25 mg/
L, the hatch rate was lower compared with all treatments including the control group
(p\0.05). Furthermore, this study showed that the lowest concentration tested of PVP and
hydrogen peroxide promoted a hatching rate higher than the control group and no better
hatching rates were obtained for the higher concentrations.
Hatching rates observed in main study for each species and treatments are shown in
Figs. 2A–C, and Table 1. The control groups showed lowest hatching rates, namely,
53 ±7.7 % to D. rerio,50 ±2.8 % to G. ternetzi and especially 1.86 ±1.86 % to P. scalare.
The hatching rate for D. rerio (Fig. 2A) oscillated from 37.8 ±5.8 % (1 mg/L of PVP
treatment) to 66.7 ±10.1 % (25 mg/L of hydrogen peroxide treatment). The hatching rate
obtained for each chemical was 46.67 ±10.2–56.67 ±9.7 % with methylene blue treat-
ment, 37.8 ±5.8–66.63 ±6.7 % with PVP treatment and 42.2 ±5.9–66.6 ±10.1 %
with hydrogen peroxide treatment. However, no statistically significant differences were
found when compared between treatments (p[0.05).
The G. ternetzi hatching rate was significantly enhanced by all the chemical treatments
(p\0.05) when compared with the control, with the exception of 0.5 and 1 mg/L for the
methylene blue treatment, 5 mg/L for the hydrogen peroxide treatment and 0.25 mg/L for
the PVP treatment (Fig. 2B). Furthermore, the highest G. ternetzi hatching rate was
obtained for the highest concentration tested, which was 90 ±2.8, 86.6 ±1.6 and
1 3 5 10 1 5 15 25 25 50 100 200 Ct
0
20
40
60
80
100
M-B H-PPVP Ct
M-B > Methylene blue PVP > Iodine solution (PVP)
H-P > Hydrogen peroxide Ct >Control
Concentration (mg/L)
% of hatched eggs
abcd
abcd
abc abc
abc
abc
abc
abc abc abc
abc
bc
Fig. 1 Effect of methylene blue, PVP and hydrogen peroxide in percentage of hatched eggs of G. ternetzi
(preliminary study). Values are presented as mean ±SEM (n=3). Lowercase represents significant
statistical differences at level p\0.05: abetween treatments with control group; bbetween treatments with
15 mg/LPVP cbetween treatments with 25 mg/L PVP; dbetween 1 and 3 mg/L methylene blue
Aquacult Int
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0.5 1 2 3 0.25 0.5 0.75 1 5 10 15 25 Ct
0.5 1 2 3 0.25 0.5 0.75 1 5 10 15 25 Ct
0.5 1 2 3 0.25 0.5 0.75 1 5 10 15 25 Ct
0
20
40
60
80
100
A
CtM-B H-PPVP
Concentration (mg/L)
% of hatched eggs
0
20
40
60
80
100
Ct
B
M-B H-PPVP
Concentration (mg/L)
% of hatched eggs
bcde bcde
abce
bcde bce abce
ab
bcde
abce
ac
ad
ae
0
20
40
60
80
100
M-B > Methylene blue PVP > Iodine solution (PVP)
H-P > Hydrogen peroxide Ct >Control
C
ab
ac
ad
ae
acef
aa
af
CtM-B H-PPVP
Concentration (mg/L)
% of hatched eggs
abc
def abc
def
abc
def abc
def
Fig. 2 Effect of methylene blue,
PVP and hydrogen peroxide in
percentage of hatched eggs on
three species under study (main
study). Values are presented as
mean ±SEM (n=3). AD.
Rerio;BG. Ternetzi; lowercase
represents significant statistical
differences at level p\0.05:
(a) between treatments with
control group; (b) between
treatments with 3 mg/L
methylene blue (c) between
treatments with 25 mg/L
hydrogen peroxide; (d) between
treatments with 0.75 mg/L PVP;
(e) between treatments with
1 mg/L PVP; CP. scalare;
lowercase represents significant
statistical differences at level
p\0.05: (a) between treatments
with control group; (b) between
treatments with 0.5 mg/L
methylene blue (c) between
treatments with 1 mg/L
methylene blue; (d) between
treatments with 2 mg/L
methylene blue; (e) between
treatments with 3 mg/L
methylene blue; (f) between
treatments with 25 mg/L
hydrogen peroxide
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90 ±2.8 % for 1 mg/L of PVP treatment, 25 mg/L of hydrogen peroxide treatment and
3 mg/L of methylene blue treatment, respectively.
The results of the efficiency of eggs disinfection of P. saclare are showed in Fig. 2C.
All treatments and concentrations of methylene blue and hydrogen peroxide enhanced the
hatching rate comparatively to the group control (p\0.05). On the other hand, no dif-
ferences were found between hatching rate in PVP treatment and control group (p\0.05)
and with this chemical, hatching rates are lowest comparatively with other chemicals
independently of the concentrations (p\0,05). The hatching rate ranged from
1.86 ±1.86 % (control group) to 94.4 ±3.2 % (3 mg/L of L methylene blue). The higher
hatching rates obtained were 3.7 ±3.7 % at 0.5 and 0.75 mg/L of PVP, 88.87 ±3.2 % at
25 mg/L of hydrogen peroxide and 94.4 ±3.2 at 3 mg/L of methylene blue.
The results achieved by means of linear regression analysis (Table 1) showed statistical
significant dependences to hydrogen peroxide, iodine active and methylene blue for G.
ternetzi and hydrogen peroxide for P. scalare (p\0.05). The statical linear trends are
presented in Figs. 3a–d. The hatching rate increases when increases the chemical con-
centration used on the treatment, which means there is a direct positive correlation between
these two attributes. This relation was more prominent for PVP effect on G. ternetzi when
compared with other species. For this species, an increment of 1 mg/L of PVP leads to an
increase in hatching rate of 40 ±4.6 %.
Discussion
This study was intended to assess the effect of three of the most commonly used chemicals
on the hatching success of Danio rerio,Pterophyllum scalare and Gymnocorymbus ternetzi
eggs.
All of chemicals used in study showed capacity in increasing hatching rate at least in
one specie, and the optimal doses obtained varied between G. ternetzi and P. scalare
species and PVP, methylene blue and hydrogen peroxide. By contrast, for all the different
chemical did not increased the D. rerio hatching rate.
Table 1 Values of variation in hatching rate obtained as a function of the concentration of methylene blue,
hydrogen peroxide and PVP for the three species under study
Specie Chemical R
2
pvalue* Variation of hatching rate
(%)/mg L
-1
chemical
D. rerio Methylene blue 0.1041 0.3065 4.220 ±3.916
Hydrogen peroxide 0.2067 0.1375 1.042 ±0.6458
Iodine solution (PVP) 0.1431 0.2253 -24.83 ±19.21
G. ternetzi Methylene blue 0.7886 0.0001 13.28 ±2.174
Hydrogen peroxide 0.7857 0.0001 1.343 ±0.2218
Iodine solution (PVP) 0.9 \0.0001 44.00 ±4.638
P. scalare Methylene blue 0.04017 0.5322 3.702 ±5.722
Hydrogen peroxide 0.4035 0.0265 2.592 ±0.9966
Iodine solution (PVP) 0.01783 0.6791 -2.200 ±5.164
* Significance level was set at plevel B0.05. Values are presented as mean ±SEM (n=100 eggs/
chemical/species)
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The hatching rate obtained to P. scalare was very low in control group, however, in
the natural environment or in captivity without removing eggs from the parents, usually
hatching rate is higher (Farahi et al. 2011). This low percentage of hatching relates to
the fact that eggs are taken from the parents, to be subjected to treatment, since in
normal conditions, the breeders have parenting functions, removing most of unfertilized
and nonviable eggs by constantly agitating the water to hinder the attachment and
proliferation of fungi (Degani and Yehuda 1996), In Egypt, Saprolegniosis is consid-
ered one of the most important causes of mortality among angelfish (Ahmed et al.
1990). In the absence of parents, eggs are more vulnerable, leading to near zero
hatching rates.
The methylene blue showed capacity to increase hatching rate for G. ternetzi, and
hatching rate shows high dependence of concentration, by contrast for P. scalare all
concentrations increasing hatching rate but without dependency of concentration. The
safety of this chemical in disinfecting eggs is reported by Sanabria et al. (2009) who tested
01234
40
50
60
70
80
90
100
[Methylene blue] mg/L
A
% ofhatched eggs
0.0 0.5 1.0 1.5
50
60
70
80
90
100
B
[Iodine solution] mg/L
% of hatched eggs
0102030
50
60
70
80
90
100
C
[Hydrogen peroxide] mg/L
% of hatched eggs
0102030
0
20
40
60
80
100
D
[Hydrogen peroxide] mg/L
%hatched eggs
Fig. 3 Linear regression analysis for hatching rate (%) as function of methylene blue (a), PVP (b) and
hydrogen peroxide (c) concentration for G. ternetzi and hydrogen peroxide (d) concentration for P. scalare.
Values are presented as mean ±SEM (n=3)
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the effect of methylene blue in eggs and larvae of P. scalare showing that 24-h bath post
fertilization have no effects on survival and swim bladder.
Hydrogen peroxide shows high capacity in increase hatching rate in a concentration
dependent manner for P. scalare and G. ternetzi. In both species, the highest concentration
used (25 mg/L) can reach the maximum hatching rates. This chemical is currently used in
aquaculture, but some authors suggests use of hydrogen peroxide at a concentration of
1,000 mg/L in some species of fish and also that the use of 500 mg/L can be lethal for P.
scalare (Schreier et al. 1996; Barnes and Gaikowski 2004; Sanabria et al. 2009).
Iodine solution only proved to be effective for the G. ternetzi specie, although many authors
consider it a good chemical for fish eggs disinfection of several species. Stuart et al. (2010)
suggested the use of 50 mg/L PVP with a 5 min bath. On the other hand, Eissa et al. (2013)
obtained very efficientresults against the mold infection on egg stocksof P. scalare with the use
of 60 mg/L of PVP as immersion solution during 30 min. However, in our preliminary study,
the use of 1 or 5 mg/L PVP of and increased the G. ternetzi hatching rate. By contrast, the
treatment with 15 mg/L of PVP reduced the G. ternetzi hatching rate. Moreover, the treatment
with 25 mg/L of PVP was toxic for all the eggs. The toxicity was previously referred in baths of
fish eggs with 75 and 100 mg/L during 30 min for Chinook salmon and rainbow trout,
respectively (Alderman 1984; Fowler and Banks 1990). In line with the preliminary results
obtained in this study, Aydınetal.(2011) also found the hatching rates reductions and a
significantly increased abnormalities in turbot eggs after iodine treatments.
Our results showed that hydrogen peroxide and methylene blue are the most versatile,
effective and safe to use on the three tested species. PVP can be also used but with many
precautions due to the very low safety margin.
The results presented here confirm the importance of determining the optimal dose of
each chemical disinfectant for each species of fish in order to ensure the health and welfare
of the specimens used in aquaculture and laboratory work. One of the key points of our
study is related with the view that the optimal chemical doses used on each specie cannot
be extrapolated from species to species, independently of the similarity they have. Taken
together, both the low hatching rate observed on P. scalare control group and the high
increases of the P. scalare hatching rate induced by the disinfectants, this species could be
used as an experimental model in the study of effectiveness of new chemicals for water
disinfection and increase the hatching rates.
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