BEHAVIOR RESPONSE OF ZEBRAFISH (DANIO RERIO) UNDER AMMONIA NITROGEN STRESS BASED ON ONLINE BIOMONITORING SYSTEM

Pollution by inorganic pollutants is a serious environmental problem worldwide. Among them, inorganic nitrogen is one of the persistent pollutants in the water ecosystem. Ammonia nitrogen is a common stress factor to cause several ecosystem issues, including toxicity, moreover, it is one of the key pollution indicators for water quality. Ammonia nitrogen could cause stress on organisms at trace level (environmentally relevant concentrations), but most of the available toxicity studies concentrations were higher than the environmental level. Studies on early detection of ammonia nitrogen stress on aquatic organisms are much warranted. Different bioindicators are used for toxicity studies, in which fish have widely used test specimens because they are highly sensitive to environmental stress (ammonia nitrogen toxicity) and an important member of the food web. Among different fish models, zebrafish are highly recommended for toxicity (neurotoxicity, behavioral toxicity and eco-toxicity) and water quality monitoring. Behavior change is a primary response of fish under environmental stress conditions. Moreover, behavior changes could reflect the internal and external stress of organisms. Thus, behavior changes could be a valid indicator for morphological or molecular or biochemical or physiological changes induced by chemical stress. Social behavior, locomotory behavior (swimming speed or swimming distance or average swimming speed), memory, mating behavior, feeding behavior and anxiety-associated behavior changes induced by chemical stress were studied using zebrafish. Different types of methods (traditional methods and video-tracking methods) were developed to assess these behavior changes. Noteworthy, we could quantify real-time behavior changes of fish using engineering technologies. In the present study, we used a homemade online biological behavior monitoring system-OBBMS (quadrupole impedance technology) and accessed the impact of ammonia nitrogen stress on zebrafish behavior strength towards early detection of external stress. The OBBMS consists of a fish chamber, water-circulation unit, automatic signal acquisition and processing unit and a computer. The fish chamber is cylindrical in shape (height 10 cm and width 5 cm) made-up of high-grade plastic materials. The fish chamber consists an inlet and outlet. Flow-through water system contains a water tank. Water from the tank flows to the fish chamber through the inlet and reaches back to the tank through the outlet. The water-flow was maintained automatically by a peristaltic pump (2 L/hour). The fish chamber has (in the inner wall) two pairs of metal sensors that harvest behavior signals regularly and automatically. The automatic signal acquisition unit receives and process (A/D) these signals. The processed data is displayed on the computer-monitor. Zebrafish were collected from our fish breeding laboratory, Institute of Environment and Ecology, Shandong Normal University, Jinan, China. Healthy zebrafish were used for toxicity assessments. We continuously exposed zebrafish (5 fish/fish chamber/group) to different concentrations (0.15 and 1.5 mg/L) of ammonia nitrogen for fifteen days and quantified the behavior strength. Simultaneously, four replicates were maintained for each concentration. A common control group was maintained with a similar setup. Test concentrations of this study were chosen based on the limit requirements of class I and class IV surface water ammonia nitrogen quality standards concentration, that in the National Surface Water Environmental Quality Standard, China (GB3838-2002). We processed the real-time data obtained from OBBMS using software (SPSS and MATLAB environment). Variations among the groups were validated through Self-organizing map and autocorrelation. The behavior strength of zebrafish was significantly (P<0.01) declined in ammonia nitrogen treatment groups throughout the study periods compared to the control group. However, the circadian rhythms (zebrafish behavior strength was higher during the daytime compared to the night) were not affected for the control group and the ammonia nitrogen treatment group (0.15 mg/L). In contrast, in the ammonia nitrogen treatment group (1.5 mg/L), the circadian rhythms were affected significantly (P<0.01). The variations in the circadian rhythms were visualized by Self-Organizing Map and autocorrelation analysis. We concluded that ammonia nitrogen has the potential to cause behavior toxicity on non-target organisms at environmentally relevant concentrations. The observed behavioral variations are concentration-dependent effects. Ammonia nitrogen could cause circadian rhythms abnormalities at higher concentrations. The real-time behavior response of zebrafish under chemical stress conditions was quantified efficiently in our online biological behavior monitoring system. We propose behavioral strength could be a useful non-invasive biomarker for early detection of chemical stress on organisms.