Progress 04/01/24 to 03/31/25
Outputs
Target Audience:The target audience encompasses a diverse range of professionals and scholars, including academics, researchers, students, fish farmers, feed producers, and key stakeholders in the aquaculture industry. Additionally, it caters to water quality specialists, toxicologists, and food science professionals who play a critical role in ensuring the safety and sustainability of aquatic food production. While the primary focus may be on catfish, the research findings and methodologies have broader implications, benefiting other commercially and ecologically significant species such as largemouth bass, striped bass, baitfish, ornamental fish, and tilapia. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project has offered significant opportunities for training and professional development in aquatic toxicology, stress physiology, and experimental design. Key areas of skill enhancement include: (i) Training in range-finding tests and log-probit analysis for LC50 determination (ii) Proficiency in water quality monitoring, including ammonia quantification using the salicylate-hypochlorite method, dissolved oxygen regulation via the R362 Controller system, and iron measurement via flame atomic absorption spectrophotometry. (iii) Fish care and handling, including acclimation protocols, tank maintenance, and waste management. How have the results been disseminated to communities of interest?PI and students involved in the project actively disseminated their findings by presenting at prominent scientific conferences, including Aquaculture America in New Orleans and PAWC in Montgomery, Alabama. These conferences provided a valuable platform for sharing key research outcomes, engaging in methodological discussions with peers, and receiving constructive feedback from leading experts. Participation in such events not only enhanced the visibility of the research but also facilitated collaborations with academic and industry professionals, fostering opportunities for future interdisciplinary studies. The project team also participated in workshops and field days to directly engage stakeholders, including fish farmers, water quality specialists, and researchers. These interactive events featured poster presentations, in-depth discussions on stress physiology in aquaculture species, and hands-on demonstrations of key techniques used in the study--such as water quality monitoring, LC50 testing, and stress memory assessment. What do you plan to do during the next reporting period to accomplish the goals?In the next reporting period, we aim to accomplish other parts of sub-objectives of objective 1 which is, to examine stress memory phenomena in response to iron and hypoxia. Moreover, we also intend to accomplish objective 2. This focuses on assessing the potential effect of prior mild hyperammonia, iron exposure as well as hypoxia to protect catfish against future stress events.
Impacts
What was accomplished under these goals?
For specific objective 1 (Investigate the stress memory or imprints in catfish for the experimental conditions of elevated ammonia level (hyperammonia), reduced oxygen content (hypoxia) and high iron in the water), following sub-objectives were accomplished. Sub-objective 1:Determine the lethal dose response curve for ammonia, hypoxia and iron Establishing 10 day and 21 day -lethal concentration LC50 for ammonia After an initial acclimation period of two weeks in 2500 L flow-through holding tanks, catfish (15 -18 grams) were randomly distributed into ten 200 L glass aquaria. Each aquarium was equipped with an air-stone for aeration, and the water quality parameters were maintained at the same levels as those in the original holding tanks. 10-Day LC50 Determination To determine the lethal concentration (LC50) of ammonia over a 10-day period, a preliminary range-finding test was conducted. Based on the results, fish were exposed to five distinct concentrations of total ammonia: 5, 10, 15, 20, and 30 mg/L. 21-Day LC50 Determination For the 21-day LC50 assessment, another range-finding test was performed to refine the exposure concentrations. Fish were subjected to five different levels of total ammonia: 3, 6, 9, 12, and 15 mg/L. Each ammonia concentration was tested in triplicate to ensure reliability. The required levels of ammonia were achieved by spiking each exposure tank with a calculated amount of ammonium bicarbonate (NH?HCO?) stock solution. The 10-day and 21-day LC50 values were calculated using log-probit analysis, providing the lethal concentration values. Results The 10-day LC50 value for ammonia was calculated to be 21.70 mg/L. However, as the exposure duration increased, the fish exhibited a lower tolerance to ammonia. Over a 21-day period, the LC50 value decreased significantly to 9.44 mg/L, indicating that prolonged exposure exacerbated toxicity effects. Determining 10 day and 21 day -lethal concentration LC50 for hypoxia Acclimated catfish, as described previously, were stocked into 120 L glass aquaria (n = 8), each equipped with an air-stone to ensure adequate aeration. To determine the 10-day LC50 value under hypoxic conditions, a preliminary range-finding test was conducted, after which fish were exposed to five different hypoxic oxygen concentrations: 3.0, 2.5, 2.0, 1.5, and 1.0 mg/L. For the 21-day LC50 assessment, a separate range-finding test was performed, and fish were subjected to a different set of five hypoxic concentrations: 3.5, 3.0, 2.5, 2.0, and 1.5 mg/L. To ensure the reliability of the results, each hypoxic exposure group was conducted in triplicate. Dissolved oxygen levels in the hypoxic aquaria were continuously monitored and precisely regulated using the R362 Controller system, which is designed for oxygen control. The electrode of the R362 system was placed in the center of each aquarium to provide accurate readings. The system maintained the desired oxygen concentrations by automatically adjusting the supply of nitrogen or air through two connected valves, which were opened or closed automatically as needed. Additionally, oxygen levels recorded by the R362 electrodes were manually cross-checked at least twice daily using a separately calibrated oxygen electrode to ensure consistency and accuracy. To establish hypoxic conditions, the oxygen concentration in the aquaria was gradually lowered from normoxic levels (7.2-7.5 mg/L oxygen) to the designated hypoxic ranges. The experimental period commenced once the target hypoxic levels were reached and stabilized. To prevent unwanted oxygen diffusion from the surrounding air, each tank was covered with a plastic lid, ensuring that the intended hypoxic conditions remained undisturbed throughout the study. Fish mortality was recorded at 12 hours, followed by daily assessments on days 1, 2, and 3, and continued at regular intervals up to day 10 or day 21. Result The LC50 of hypoxia over a 10-day exposure period was determined to be 1.87 mg/L oxygen, with a 95% confidence interval (C.I.) ranging from 1.23 mg/L to 2.51 mg/L. This indicates that at this concentration, 50% of the catfish died of hypoxic conditions within 10 days. In contrast, the LC50 value for a prolonged exposure of 21 days was found to be slightly higher, at 2.21 mg/L (C.I. 1.47-2.95 mg/L). This suggests that organisms may exhibit some level of acclimation or physiological adaptation to hypoxic conditions over time. Determining 10 day and 21 day LC50 for iron Acclimated catfish (n = 10 per group) were stocked in 200 L glass aquaria equipped with air stones. To determine the 10-day LC50, fish were exposed to iron concentrations of 5, 10, 15, 20, and 25 mg/L, while for the 21-day LC50, concentrations of 4, 8, 12, 16, and 20 mg/L were tested. Each experimental group was conducted in triplicate. Iron exposure was maintained using a stock solution of FeCl?·6H?O (ACROS Organics, USA). Concentrations were monitored daily using the FerroZine method (Hach Method 8147) and flame atomic absorption spectrophotometry (iCE 3000 series, Thermo Scientific, USA), with adjustments made as needed. Mortality was recorded at 12 hours and daily until day 10 or 21. LC50 values with 95% confidence intervals (C.I.) were calculated using log Probit Analysis. Results The 10-day LC50 for iron was 13.97 mg/L (C.I. 10.62-17.27 mg/L), while the 21-day LC50 decreased to 8.41 mg/L (C.I. 5.29-11.60 mg/L). Sub-objective 2: Assessment of stress memory in response to the elevated ammonia conditions Under this sub-objective, we tested the hypothesis of whether pre-acclimation to a low concentration of ammonia could enable the catfish to develop an 'ammonia stress-avoidance' memory, enhancing their tolerance to subsequent lethal and sub-lethal ammonia threats. To test this, catfish were pre-exposed to 1.09 mg/L (total) ammonia (5% of determined 10-day LC50) and 2.17 mg/L (total) ammonia (10% of determined 10-day LC50), for 21 days. Each of these groups, including control (without pre-exposure 'naïve') was performed in triplicates in 300 L glass tanks, with 12 fish in each tank. To maintain the desired ammonia concentrations, NH4HCO3 stock solution was added to each exposure tank. Water quality was monitored at 12-hour intervals using the salicylate-hypochlorite method to ensure stable ammonia levels. A handheld HACH pH electrode (Colorado, USA) was used to monitor water pH continuously, ensuring it remained within the same range as the control group. Following 21 days of experimentation, each of these pre-exposed and respective parallel control (without pre-exposure 'naïve') groups were recovered for 7 days in clean (ammonia-free) water. Following this recovery phase, each group was subsequently exposed to a lethal (100% 10-day LC50) and sub-lethal (25% 10-day LC50 for 21 days) ammonia concentration. Results show that during the lethal ammonia challenge, the pre-exposed group with 2.17 mg/L (total) ammonia (10% of determined 10-day LC50) had a significantly longer survival time than the naïve group and 1.09 mg/L (total) ammonia (5% of determined 10-day LC50). Following 21 days of sub-lethal ammonia exposure, no significant difference was noted for 2.17 mg/L and 1.09 mg/L (total) ammonia pre-exposed group. Interestingly, 2.17 mg/L ammonia pre-exposed fish were able to excrete ammonia efficiently and retained ammonia load in the plasma within the basal level as compared to naïve group and 1.09 mg/L ammonia pre-exposed group. These findings suggest that catfish can develop an 'ammonia stress-avoidance memory' when pre-exposed to 2.17 mg/L (total) ammonia (10% of determined 10-day LC50) and it enables the catfish to resist a subsequent ammonia threat.
Publications
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