Preparation of Aquaria
Glass aquaria of the size of 25x13x12 cm3 (24 tanks), simulating natural pond environment were used for the exposure. Being an aggressive fish, male B.splendens were kept individually in 20 separate cubic aquaria (12x12x12 cm3).
The experiment was composed of control (tap borne water 150 ppm CaCO3) and three treatment setups; 320, 540 and 900 ppm prepared by adding analytical grade CaCO3 to aged tap water. This series was selected to cover the hardness range, 100-1000 ppm in Vavuniya [8]. The hardness was determined by EDTA titration [12] and the treatment setups were screened weekly to maintain the relevant conditions. All the experiments were conducted as per the guidelines given by the research review panel of the Department of Bioscience, University of Jaffna.
Introduction of fishes to Aquaria & Maintenance
Healthy male and female fishes of P.reticulata and B.splendens were purchased from a nearby aquarium in Vavuniya and were transferred to the laboratory. Sexing was done by examining external morphology, where male fishes of both species possessed narrow and bright colored bodies and colorful caudal fins compared to round bellies and short caudal fins of the female fishes, displaying sexual dimorphism. Initial weight and standard length were measured, reporting 0.487 ± 0.008 g/ 2.984 ± 0.052 cm for P.reticulata and 1.266 ± 0.072 g/ 3.497 ± 0.012 cm for B.splendens. Then the fishes were acclimatized to aquarium condition (20 min. in each setup) and introduced to the experimental setups, 150 ppm (control), 320, 540 and 900 ppm CaCO3 and reared for 1½ month. In each experimental setup 15 females were introduced to 5 males of B.splendens, separately. For, P.reticulata, 25 females were introduced to 5 males. The exposure was conducted with three replicates.
Feeding (5% of the total body weight) were done twice a day at ad libitum with commercial fish pellets. The aquaria were maintained to keep the temperature 25-27 ºC and pH 6.5-7.5 by replacing the media with newly prepared hard water on weekly basis. Debris was siphoned out. Mild aeration level was maintained as the fishes are air breathers.
Determination of growth performance
Weight and length of adult fish were measured at the sexual maturation, and the length weight relationship was analyzed , using W= a TLb: (Log W = log a + b log TL) [13] to obtain the linear regression.
Determination of reproductive potential
Female fishes showing gravid spots (Oocyte stage III-IV) (N= 6per dose for each sp.) were randomly tested for fecundity (the number of ripening eggs found in the female just prior to spawning) and GSI (the ratio of fish gonad weight to body weight). Euthanizing was conducted with 0.02% MS222 solution.
Reproductive potential of B.splendens
To estimate the bubble nest size of B.splendens, three sets of clean breeding aquaria (size-60x30x30 cm3) were prepared without artificial bottom stones and aeration. A floating plant leaf was placed on the air water interface to facilitate the nest formation. After placing the male fish a clean glass cube containing a female fish was placed near the breeding aquarium to stimulate the nest building. Then the bubble nests built by male B.splendens (N=5) were measured (diameter) by a ruler [14].
Hatchability of B.splendens was estimated after allowing a successful courtship with a gravid female. Without disturbing the bubble nest, the number of eggs released was counted. After a successful mating female was removed, and male was allowed for 24 -48 hour pre hatched parental care. Next, the hatched larvae were counted, and the hatchability was determined, as the number of larvae hatched over the total number eggs [15].
Reproductive potential of P. reticulata
Fertility of P.reticulata was determined by counting the intra-follicular embryos inside the female by sacrificing few fishes (N=6) at the 21st day after mating [16]. The breeding tank for P.reticulata was formed with artificial aquarium stones and Vallisneria (a common water plant) to provide hiding place for the young ones. After the brooding parents were removed, and the hatchlings were counted.
Larval survival
Larval survival rate of both species (N= 20-30 per dose for each sp.) was determined after one week of exposure by counting the number of surviving one weeks-old larvae divided by the total number of hatched larvae / released young ones [15]. The larval growth performance (N=12 per dose for each sp.) was estimated in every 10 days interval by measuring their length gain.
Statistical analyses
Normality of the data set was tested before applying the statistics with SPSS 20.0 (IBM, USA).
Homogeneity and independence were tested for applying parametric analyses. One-way ANOVA and Tukey pairwise comparison were conducted to analyse the effects on weight, standard length, fecundity and Gonadosomatic Index, Laval growth (length) of the fishes. The linear regression analysis was used to find the length weight relationship (LWR). In LWR linear regression analysis, the slope of regression lines explicit the exponent coefficient value ‘b’. Variations in the estimates of ‘b’ for the fish species, examined from the expected value (ideal value ‘b’ = 3) were tested by t-test [17,18]. Students t-test was applied to analyze the variation i.e. derived by dividing the difference between ‘b’ and ‘3’ by standard error of ‘b’ [19].