Collection of Pond water samples
Five days of test samples were taken from a fish pond with a diameter of 10 x 8 x 7 m, 500 liters of water, and 200 fingerlings. The sampling began the day the pond's old water was replaced with fresh water, and samples were taken every 24 hours. After collection, the pH of the samples was calculated and they were kept in the refrigerator.
This is an AlgalToxkit kit that includes all of the necessary ingredients, include test organisms, to conduct sensitive and repeatable toxicity experiments. The AlgalToxkit is designed to test the toxicity of fresh and waste water released from both aquatic and terrestrial habitats. Toxkits offer a significant advantage over traditional bioassays in that the test organisms are included in the kits in a "dormant" or "immobilized" state, ready to be activated before the test ISO 8692 is performed (2012).
Principle of Algaltoxkit Ftm
This bioassay kit is based on a 72-hour growth inhibition test using the green microalgae Selenastrum capricornutum (formerly Raphidocelis subcapitata and now Pseudokrichneriella subcapitata). After de-immobilization and transfer into an appropriate algal culture media, the green microalgae are immobilized in a specific matrix, where they persist for several months without losing viability; they resume normal growth ISO 8692 (2012).
The water samples were collected from the fish pond and were tested for their toxic effects on the microalgae as outlined below:
Sample 1: collected on day 1
Sample 2: collected on day 2
Sample 3: collected on day 3
Sample 4: collected on day 4
Samples 5 collected on day 5
Control – algal culturing medium for the algal test obtained from the Algaltoxkit; reference sediment.
The algal test was performed for 72 hours (with 3 replicates).
A battery of Algaltoxkit microalgae - Selenastrum capricornutum in speciation of the primary consumer was used for this study.
Testing with microalga
The growth inhibitory effects of pond water on the microalgae Selenastrum capricornutum were determined after a 72-hour test using the microalgae Selenastrum capricornutum. The tests were carried out in accordance with ISO (International Organization for Standardization) guideline 8692, with minor adjustments. This was attributable to the preference of cells with a path length of 9 cm over cells with a path length of 10 cm. The Algaltoxkit technology entailed using a spectrophotometer to measure the optical density of algal cells at 670 nm (ISO 8692). (2012).
Preparation of algal culture medium
A 1000 ml volumetric flask was filled with 800 ml deionized water. One of the two Nutrient Stock "A" vials was uncapped, and 10 ml of its content was placed into the flask. The caps of Nutrient Stocks B, C, and D were removed, and 1 mL of each was put to the flask. Deionized water was used to fill the flask to the 1 liter mark. To homogenize the algal culturing media, it was corked and shook. The solution was allowed to equilibrate in the air overnight. The pH of the solution was adjusted to 8.3 with 1 mol/l sodium hydroxide ISO 8692 before use (2012).
De-immobilization of the algae
One of the two tubes containing algal beads had its liquid content drained out, and a 5 ml of matrix dissolving solvent was placed into the tube, sealed, and rapidly shaken. Using a vortex shaker, the shaking was repeated every two minutes for 10 minutes until the algae were completely dissolved. The supernatant was decanted after centrifuging the tube containing the dissolved algal solution for 10 minutes at 3000 rpm. It was replaced with 10 mL deionized water, capped, and violently shaken to homogeneously re-suspend the algae. After a ten-minute centrifugation at 3000 rpm, the supernatant was decanted and the algae were re-suspended in 10 mL algal culturing media ISO 8692. (2012).
Preparation of concentrated algal inoculum
The algal suspension was poured into a 25 ml flask, which was then filled to the 25 ml mark with algal culture medium. To homogenize the algal suspension, the flask was corked and shook. Calibration and Algal Stock cells were labeled on two cuvette cells. The calibration cell was sealed with tape and filled with 2.5 mL algal growing medium. To zero-calibrate the spectrophotometer, the calibration cell was placed into the instrument. The algal suspension was then transferred to the Algal Stock cell in 2.5 mL increments and tightly taped. To properly disperse the algal suspension, the cell was violently agitated. After that, the Algal Stock was placed in the spectrophotometer, and the optical density (OD 1) was measured after ten seconds ISO 8692. (2012).
Preparation of the fish pond water samples
The bottles containing the five-day-old fish pond water samples were shaken, then 10 ml from each bottle was placed into five separate centrifuge bottles and centrifuged for five minutes. The supernatants were collected and poured into five separate flasks labeled C1, C2, C3, C4, and C5, with each flask containing 100 ml of the algal culturing medium. A control sample was made by pouring 110 ml of algal culturing media into a sixth flask. The algal suspension was taken and poured into each of the six flasks, 1 ml at a time. The flasks were corked and thoroughly shaken to equally disperse the algae ISO 8692 (2012).
Transfer of the algal-sample solutions into the test vials
The test vials were labeled in three sets: three (a, b, c) for each test sample collected over the course of five days: C0, C1, C2, C3, C4, and C5. The control was the sixth set marked - C6. 2.5 ml of the algal-sample solutions were put into the three test vials ISO 8692 after the flasks were opened (2012).
Incubation of the test vials
The cells were taped shut and placed in a holding tray at random. The tape was slightly punctured on one side of the cells to allow for gas exchange in the centre. The cells were incubated in a locker with continual sideways lighting of 10000 lux provided by cool white fluorescent lamps at a temperature of 23 oC according to ISO 8692. (2012).
Scoring the results
Every 24 hours, the amount of algal growth inhibition was measured in comparison to the control. They were measured as the optical density (at 670 nm) of algal suspensions in test cells after exposure to the toxicant for 24 hours, 48 hours, and 72 hours, respectively. After each measurement, the cells were returned to the holding tray and retained at random (ISO 8692). (2012).
Computation of Data
The mean values of the daily – optical density for the three replicates, the control cells, and the fish pond water (test samples) were calculated (ISO 8692, 2012).
Validating the test result
International Standard for Organization (ISO Standard 8692 – Section 8) stipulates that the average growth rate in the control shall be at least 1.4 per day, which corresponds to an increase in cell density by a factor - 67 in 72 hours ISO 8692, (2012).
Data are expressed as mean ± SD and, a test of statistical significance was carried out using a two-way analysis of variance (ANOVA). Mean values with p < 0.05 were considered significant.
Table 1 shows that the pH of the pond water samples increased from day to day for five days interval following the replacement of the old water with fresh water. The increments in pH levels from day-one to day-five were significant at p < 0.05.
Table 1 pH Values of the Fish Pond Water Samples
Data expressed as mean ± SD.
Mean values with p < 0.05 were considered significant.
Day 6: (Control – algal culturing medium for the algal test in Algaltoxkit)
Table 2 shows the extent to which pond water inhibited the growth of microalgae, S. capricornutum. Significant (p < 0.05) decreases in optical densities were observed when the pond water cultures were compared with the control culture.
Table 2 Algal Growth Inhibition Results
The initial nominal density = 0.002±0.001; Data were expressed as mean ± SD; Mean values with p < 0.05 were considered significant. Day 6: (Control – algal culturing medium for the algal test in the Algaltoxkit).