Introduction

Aquaculture refers to the practice of cultivating populations of aquatic plants and animals in a variety of water conditions, including freshwater, brackish, and marine water^1,. Fish is believed to offer around one-sixth of the world’s protein, making aquatic foods a major source of protein and other nutrients³. The feeding habits, development rate, illnesses, and survival value of pond fish can all be influenced by the water quality^{4, 5}. Several elements, including dissolved oxygen (DO), temperature, pH, organic load, and ammonia, as well as meteorological conditions, influence pond quality⁶. Phytoplankton production and respiration are linked to dissolved oxygen⁷; pH and ammonia are linked to temperature and the number of organic matter inputs, as well as ammonia excretion by fish⁸. Nutrients such as nitrogen and phosphorus are supplied by the fish diet, with roughly 35% of these nutrients partitioned in fish biomass⁹. The excretion of metabolic waste is caused by the uptake and metabolism of nutrients⁹. Sediment can hold 100–1000 times more nutrients than water and accumulates unused nitrogen and phosphorus¹⁰. The concentration of stored nutrients, organic matter, and bacterial density in the sediments may rise and then gradually be released for uptake¹¹. Under aerobic conditions, mineralization of stored organic debris results in the development of harmful compounds, which degrades pond water and health⁷. The quality of the fish pond is determined by the amounts of dissolved oxygen, ammonia, and nitrite, which are regulated by the feeding rate. Microorganisms such as microalgae are employed to monitor aquatic and terrestrial environments as indicators. It is simple to test them and they are readily available. When microorganisms are exposed to toxins in the environment, such as heavy metals like cadmium and polycyclic aromatic hydrocarbons, they produce new proteins called stress proteins, which are employed as early warning indicators¹². Microorganisms make up a large portion of marine biomass and are responsible for the nutrient cycle in the ocean¹³. Environmental contaminants in water can be detected using microbial indicators¹⁴. The presence of toxins in water can easily be detected by observing changes in bacteria’ digestive systems, which are rejected or affected by toxins that may restrict their growth¹⁵. This test is speedy in comparison to other procedures. This test is quick to monitor when compared to other test procedures. However, the ability to only detect changes in organisms caused by poisons is a significant shortcoming¹². The bacterium Vogesella Indigofera, for example, reacts quantitatively with heavy metals^16,14. Since various studies have shown that baseline toxicity often demands chemical activities of 0.01-0.1,²² whereas chemical activity measures the energy level of an organic molecule relative to its liquid purity of (0–1)²². This baseline toxicity test was intended to monitor the build-up of toxicity in pond water for five days. The microalgae Selenastrum capricornutum were used as quick test organisms in this study. Therefore, This work was designed to monitor the onset of pond water toxicity using Selanastrum capricornutum.   

Materials and methods 

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 stored in the refrigerator.

Toxkit

The kit used for the study was AlgalToxkit which was having all of the necessary ingredients, including 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^17.

Principle of algaltoxkit F^tm

This bioassay kit is based on a 72-hour growth inhibition test using the green microalgae Selenastrum capricornutum (formerly Raphidocelis subcapitata and Pseudokrichneriella subcapitata). After de-immobilization and transfer into an appropriate algal culture media, the green microalgae were immobilized in a specific matrix, where they persist for several months without losing viability and resume normal growth¹⁷.

Experimental design

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).

Toxicity tests

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. The inhibitory effects of poorly soluble organic and inorganic materials, volatile chemicals, heavy metals, and waste water can be tested using this method as described in ISO 14442 and ISO 5667-16. 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^17.

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 into 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 shaken. 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 ISO¹⁷.

De-immobilization of the algae

One of the two tubes containing algal beads had its liquid content drained out, and 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 of deionized water, capped, and violently shaken to homogeneously re-suspend the algae. After 10^th minute centrifugation at 3000 rpm, the supernatant was decanted and the algae were re-suspended in 10 mL algal culturing media¹⁷

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 an algal culture medium. To homogenize the algal suspension, the flask was corked and shaken. 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. For zero-calibration, the 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 10 seconds ISO 8692¹⁷.

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 5 separate centrifuge bottles and centrifuged for 5 minutes. 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 8692¹⁷. 

Transfer of the algal-sample solutions into the test vials

The test vials were labeled in 3 sets: 3 (a, b, c) for each test sample collected for 5 days: C0, C1, C2, C3, C4, and C5. The control was the 6^th  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.

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 center. 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 ⁰C according to ISO¹⁷.

Scoring the results

Every 24 hours, the amount of algal growth inhibition was measured in comparison to the control. They were measured as the OD (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 (OD) for the three replicates, the control cells, and the fish pond water (test samples) were calculated¹⁷.

 Validating the test result

The International Standard for Organization (ISO Standard 8692 – Section 8) requires that the average growth rate in the control to be at least 1.4 per day, which corresponds to an increase in cell density by a factor – 67 in 72 hours ISO¹⁷.

Statistical Analysis

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.

Results

Table 1 shows that the pH of the pond water samples increased from day to day for 5^th days interval following the replacement of the old water with fresh water.

Table 1: The pH values of the fish pond water samples.

Table 2 shows the extent to which pond water inhibited the growth of microalgae, S. capricornutum. Significant (p < 0.05) decreases in OD were observed when the pond water cultures were compared with the control culture.

The initial nominal density = 0.002±0.001;

Table 2: algal growth inhibition results

Discussion

Fish consume nitrogen and phosphorus from their food and produce ammonia as a waste product of metabolism^18,19. The elevation in pH observed from day one to 5^th day was most likely due to the release of ammonia into the pond water by fish and the decomposition of organic materials as shown in Table 1. Fish feed and excrement provide nutrients to the fish pond². Absorption and metabolism of nutrients result in the production of metabolic products, which cause pond water to deteriorate and discolor. The oxidation process, as well as the degradation of organic particles by bacteria, definitely contributed to the decline of pond water quality over time^7,21  The explanation for this can be traced back to pond water inhibiting the growth of the microalgae S. carpricornutum. Due to their presence in the first level of the trophic chain, algae are sensitive and crucial in elutriate toxicity testing to check the impacts of dangerous compounds in water⁷. They created oxygen, which is required in the marine ecosystem’s food chain.

Algal growth in the cultures caused significant increases in algal density in both the tests (days 1, 2, 3, 4, and 5) and the control group (day 6) after 24 hours. Table 2 summarizes the outcome. The maximum algal growth was seen on 6^th day (control), compared to the original nominal algal density at zero hours. This was expected because it didn’t include any toxicants that may have stopped microalgae from growing¹⁰. The rate of inhibition rose from day 1 to days 2, 3, 4, and 5, according to the measured algal densities. This may be as a result of  degradation of organic waste such as feeds, toxicant production such as ammonia, and pond water deterioration over time ^9,8,.

The accumulation of pollutants may have contributed to the decrease in algal growth in the fish cultures after 48 hours, compared to an increase in algal growth in the control culture after 24 hours. The toxicological effects of pond water on microalgae development. S. capricornutum had been severely harmed. Considering that microalgae are a type of algae, because microalgae are photoautotrophs, their viability is dependent on nitrogen and phosphorus availability^19,20. The pollutants in the test cultures interfered with the algae’s normal photosynthetic activity, causing growth suppression. The reductions in the recorded algal densities of the test samples – days (1, 2, 3, 4, and 5) were linked to the growth inhibition of the microalgae, S. capricornutum, in the fish pond water cultures, and therefore implicated the pond water in the light of toxicity. At the end of 72 hours, growth inhibition of microalgae was found in both the control and test cultures. In the test cultures, this was due to the accumulation of a toxicant from excretion and decomposition, but in the control cultures, it was due to a shortage of nutrients, gas depletion, insufficient irradiation, and a natural fall in algae growth, among other things. The maximum average microalgae growth in the control was 1.1 percent per day (measured after 24 hours), which fell short of the validity criteria of at least 1.4 percent per day¹⁷. The inconsistencies and contradictions in this study’s elutriate toxicity test could have been caused by minor changes to ISO guideline 8692. Using the results of this investigation, one may estimate the pond’s poisonous level and possibly anticipate the fish’s perceived risk and survival value.

Conclusion

The use of microalgae toxicity as a bioindicator was used to examine the in-situ assessment of pond water. The microalgae growth rate was used as an indicator of pond water toxicity without measuring or defining the nature of the contaminants in the pond. This experiment was beneficial simple, and repeatable. This technique revealed information about how well aquatic life was doing in respect to its surroundings. As a result of this finding, it’s reasonable to conclude that the toxicity of pond water was significantly increased on 5th day.

Acknowledgment

The MicroBiotests firm, located at Kleiner 15 9030 Mariakerke (Gent), Belgium, www.microbiotests.be, has generously donated these Toxikits. I am grateful to Professor Ikechukwu NE Onwurah, who made the Toxikits available.

Conflict of Interest 

No conflict of interest was reported as all the  authors contributed equally.

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