Preparation of Aquaria
Glass aquaria of the size of 25x13x12 cm3 (24 tanks) were used for the exposure. Being an aggressive fish, male Betta splendens were planned to keep in 20 separate cubic aquaria (12x12x12 cm3) at a rate of each male per cube. The interior of aquaria was prepared as a simulation of natural pond using stones, aquatic plants and aeration.
The experiment 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. The hardness of the medium was determined by EDTA titrimetric method [9] and the treatment setups were monitored weekly basis to maintain the water hardness. Experiment setups were maintained at the Environmental Biology Laboratory (EBL) at the Vavuniya Campus in Sri Lanka. 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
Ovo-viviparous fish as Poecilia reticulata and oviparous fishes as Betta splendens were chosen for the experiment. The virgin fishes of both species (males and females) were purchased from a nearby aquarium in Vavuniya and were transferred to the EBL. Sexing was done by examining external morphology as both species showed sexual dimorphism. Initial weight and standard length were measured. Then the fishes were acclimatized to aquarium condition 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. To avoid aggression each males of B. splendens were kept separately in the smaller aquaria. For, P. reticulata, 25 females were introduced to 5 males. The exposure was conducted with three replicates.
Determination of growth performance
Weight and length of adult fish were measured, and the length weight relationship analysis was conducted, using W= a TLb: (Log W = log a + b log TL) [10] to obtain the linear regression.
Determination of reproductive potential
Two gravid females from each aquarium were randomly tested for fecundity (the number of ripening eggs found in the female just prior to spawning) and Gonadosomatic Index (GSI- the ratio of fish gonad weight to body weight). Euthanizing was conducted with 0.02% MS222 solution.
To estimate the bubble nest size of B. splendens, clean breeding aquarium (size-60x30x30 cm3) were prepared without artificial bottom stones and aeration. A floating plant leaf was put on the air water interface to facilitate the nest formation. After adding the male in to the breeding aquarium, a clean glass cube containing female fish was placed near it to stimulate the nest building. Then the bubble nests built by male B. splendens were measured (diameter) by a ruler [11].
Hatchability of B. splendens was estimated after allowing successful courtship with a gravid female inside the breeding aquarium. Repeated introductions of gravid females were required to obtain a successful breeding. Without disturbing the bubble nest, the number of eggs released was counted. At the end of the matting, female was removed, and male was allowed for 24 -48 hour for pre hatched parental care. Next, the hatched eggs were counted, and the hatchability was determined, as the number of larvae hatched over the total number eggs [12].
Fertility of P.reticulata was determined by counting the intra-follicular embryos inside the female fish by sacrificing few fishes at the 21st day after mating [13]. Breeding tank of P. reticulata was formed with the artificial aquarium stones and Vallisneria to provide hiding place for young ones. After female broods release young ones, they were separated from their parents and counted.
Larval survival rate was considered after one week of exposure to the water hardness treatments by counting the number of surviving one weeks-old larvae divided by the total number of hatched eggs / released young ones [12]. The larval growth performance was estimated in every 10 days interval by measuring the length gain.
Statistical analyses
One-way ANOVA and Tukey pairwise comparison were used to analysis the significant differences of 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) of the fish to know the growth pattern. In LWR linear regression analysis, the slope of regression lines explicit the exponent coefficient value ‘b’. Significant variation in the estimates of ‘b’ for the fish species was examined from the expected value (ideal value ‘b’ = 3) was tested by t-test [14,15]. Students t-test was employed by dividing the difference between ‘b’ and ‘3’ by standard error of ‘b’ [16].