Behavioral Responses in Zebrafish (Danio rerio) Exposed to Heavy Metal and Insecticide Induced Stress

Detecting contamination in real time is the best way to ensure an appropriate and timely emergency reaction. Biological indicators play an important role in the online monitoring of water quality in aquaculture and has gained great attraction in recent years. Alterations of fish behavioral responses are among the most sensitive indicators and earliest warning signals to identify accidental chemical pollution (Ayotunde et al., 2011; Xia et al., 2018). The lowest behaviorally effective toxicant concentrations that induced changes in fish behavior within 96 h of exposure ranged from 0.7 to 5% of their LC₅₀  (Huang et al., 2014). Fish behaviors, such as avoidance, cough rate, predator avoidance, feeding behavior and learning or social interactions were demonstrated by some studies on exposure to single or multiple toxicants (Afifi et al., 2016; Jingchun et al., 2019; Zhang et al., 2020).
       
Heavy metals and pesticides are two of the most commonly pollutants in water pollution accidents, which are always the target studied in fish behavioral alterations (Iryna et al., 2013). Although, the patterns of effect on behavioral reaction differed among the test species and test system, similar pollutants had the same effects on locomotion activity, resting metabolic rates and levels of metabolic substrates. Atchison and Cope (2010) summarized the effects of six metals on fish behavior and found fish can move out rapidly from the most heavily contaminated areas with Cd, Cu, Zn, Fe and Ni exposure. Huang and Zhou (2011) proved that zebrafish showed similar behavior patterns when exposed to Zn and Cr. When exposed to pesticides, fish had the same abnormal behaviors, including rapid transfer and staying just below the water surface layer of the aquarium (Jie et al., 2018). Numerous researches have been conducted to understand the effects of pollutants on fish behavior, but less is known about the differences in the stress behavioral responses of fish exposed to pesticides and heavy metal contamination.
       
Quantifiable fish behavioral changes can be observed from short-term and sublethal exposure effects, mechanisms of effects and interaction with environmental variables (Gerhardt, 2007). With the recent development of computer-imaging techniques, trajectory modeling has become one of the most effective tools for studying complex fish behavior. Video images of fish behavior are taken using a digital video camera set above or in front of the tank to record the position of every fish (Kang et al., 2009; Banna et al., 2014). Some studies have been developed for capturing trajectories with a camera fixed above a fish tank monitoring a school of fish instead of individuals. Based on this, the quantitative analysis of fish activity is useful to explore the effects of their exposure to environmental stressors.
       
The aim of this study was to investigate the differences in the behavioral responses of zebrafish exposed to two different pollutants i.e., heavy metal and pesticide. An experiment was performed using cadmium (CdCl₂; 3.5 mg/L) as the model for heavy metals, deltamethrin (C₂₂H₁₉Br₂NO₃; 15×10^-3 mg/L) as the model toxicant for pesticides and zebrafish (Danio rerio) as the test species.

Test species and chemicals
 
Adult zebrafish (6-8 months) were obtained from the Institute of Hydrobiology and cultured in a glass chamber for more than three weeks before the experiments. Dechlorinated water with DO of 6.8±0.2 mg/L, kept at 28.5±1°C and 10:14 (L:D) photoperiod was used. Cadmium chloride was obtained from Sigma (St. Louis, MO, USA) and Deltamethrin was purchased from Shanghai Pesticides Research Institute, China. All compounds were technical grade (>95% purity).
 
Experimental procedure
 
The experiment was conducted session of 2020-11 and 2021-03 at Water Supply and Drainage Science and Engineering Laboratory, Xi’an. Swimming trajectories of zebrafish were record by a real time CCD camera, which was placed in the front of the test chamber (400 × 75 × 300 mm) (Fig 1). In Table 1, swimming speed, turning frequency, depth and distance between fish every 10 s are converted from two-dimensional trajectory data. The study lasted 180 minutes, during which unexposed behavior of fish was observed with dechlorinated water as a reference and deltamethrin (C₂₂H₁₉Br₂NO₃) and cadmium (CdCl₂) were then introduced to the respective treatment groups for 120 minutes (4 replicates each). The control test was conducted for 180 minutes with dechlorinated water flowing through the chamber.
 

 

 
Data analysis
 
To reduce tracking error, the data was first preprocessed by equation (1):
 
Where,
M_d is the median of each parameter during 1-min; x_n/2 and x_(n/2+1) are the third and fourth ranked values for the parameter during the 1-min., respectively and n=6. The endpoints were described by boxplot graphs at every 10-min. Control test values were compared with those during each of the other intervals and exposure test values were compared with those during the first 60 min. before exposure. Differences between the means were considered significant when p<0.01 and marked as the asterisk.
       
Statistical tests were performed using GraphPad PRISM software (San Diego, CA). Assumptions of homogeneity of variance across treatments were cached by Levene’s test and one-way analysis of variance (ANOVA) analyzed by Dunnett’s test. If homogeneity was not observed, differences between unexposed and exposed conditions were detected by nonparametric statistical comparisons (Wilcoxon test).

Effects on zebrafish swimming speed and turning frequency
 
In the control group, swimming speed were stable at the range of 25~45mm/s. Speed increased significantly after deltamethrin and cadmium exposure, which continued for 10 to 50 minutes, even up to 110 mm/s and then abruptly returned to unexposed levels (p<0.01). As shown in Fig 2, turning frequency varied with the changes in swimming speed. It generally fluctuated within the range of 2-10n/10s (Fig 3) in the control period but increased significantly after exposure (p<0.01), up to 15n/10s. Swimming speed and turning frequency were considered the best endpoints to describe behavioral changes in fish activity (Yang et al., 2018), where sudden increase in activity of fish was recorded on exposure to listed stressors, followed by a period of high activity and then a period of low or normal activity. It’s worth noting that the duration of hyperactivity differed between cadmium and deltamethrin exposure, lasted nearly 15 mins and 50 mins, respectively.


 

       
The swimming activity of zebrafish exposed to both the toxicants changed within 20 min. Pollution induced changes in zebrafish have been shown to be consistent with the stepwise stress model (SSM), which postulates that if the exposure level was higher than the thresholds any aquatic organism can resist, a time-dependent sequence regulatory or compensatory behavioral stress responses will be triggered (Ren et al., 2015). The theoretical behavior responses of zebrafish to pollution may include no effect, stimulation, adjustment, or a toxic effect (Jie et al., 2018).
 
Effects on zebrafish swimming depth and swimming distance
 
The zebrafish were distributed uniformly across the test chamber and the average swimming depth was mostly within the range of 100 to 200 mm in the control group (Fig 4). Swimming depth did not change immediately after exposure, but it suffered a significant change from the 80^th min (p<0.01). The average swimming depth showed totally different trends with increased from 140 mm to 226 mm during deltamethrin exposure but decreased from 161 to 84 mm during cadmium exposure. As shown in Fig 5, zebrafish aggregated after cadmium exposure. The distance between fish decreased significantly during the period from the 60^th to 80^th min (p<0.01) but recovered to a normal range in the subsequent exposure period. In contrast, the distance between fish after deltamethrin exposure increased significantly after 60^th min of exposure (p<0.01).
 

 

       
As escape from the closed test chamber was not possible, immobile or hypoactive zebrafish remained at the top or bottom of test chamber, which could be designated as secondary response to the said exposure stress (Kang et al., 2009). Swimming depth changed lagged other responses by at least 20 mins. Through extensive research, Gerhardt (2007) showed that ventilation responses need a higher threshold of both response intensity and contaminant concentration. It is proved that avoidance with increased swimming activity is the first response, followed by the changes in the swimming depth and the distance between fish.
 
Different behavioral responses of zebrafish
 
There were some obviously different responses of zebrafish under sublethal cadmium or deltamethrin exposure, such as the completely different trends in swimming depth and the duration of increased speed and turning frequency, which could be attributed to the different pathways and mechanisms of toxic biological effects (Chakraborty et al., 2016; Reddy et al., 2021). Necessary for survival, the adaptive  response of aquatic organisms involves many physiological changes at neurological, endocrine, olfactory and metabolic levels (Rama-Krishnan et al., 2020).
       
The duration of the increased speed and turning frequency after deltamethrin exposure lasted 35 minutes larger than cadmium exposure. It was deduced that hyperactivity is often linked to disruptions in metabolic and physiological function, including altered oxygen consumption and ventilation rates, altered levels of metabolic substrates and effects on the abundance or activity of metabolic enzyme (Drummond and Russom 2010; Gandar et al., 2016). Brain AChE activity may cause hyperactivity, loss of coordination, muscle twitching, convulsions, paralysis and other kinds of behavioral changes (Zhang et al., 2016). Deltamethrin is a direct-acting inhibitor of AChE in fish bodies and which leading to accumulation of acetylcholine in synapses with a potentially lethal disruption of the nerve functions (Assis et al., 2010). Sandahl et al., (2005) further demonstrated that brain AChE inhibition was significantly correlated with the reductions in spontaneous swimming activity. Beauvais et al., (2001) also corelated decreased brain AChE activity with decreased swimming behavior of rainbow trout exposed to carbaryl. Behavioral alteration caused by cadmium exposure does not always coincide with brain AChE inhibition but more associated with the impaired olfactory ability (Korkmaz et al., 2018; Beyers and Farmer, 2010). Cadmium often moved along olfactory system neurons by axonal transport mechanisms. After exposed to 2 μg/L cadmium for seven days, juvenile rainbow trout behavior changed with significant accumulation of cadmium in the olfactory system (Scott and Sloman, 2004).
       
Mainly through the gills and body surface absorption, deltamethrin leads to damage to fish respiratory system leading to hypoxia and driving fish to engulf atmospheric air at the water surface, with increased ventilation rate to allow more water to flow over the gills as an attempt to compensate low body oxygen levels and remove the toxins from the body (Xing et al., 2017). Fish is reported to swim near the water surface with a tilted body because of respiratory damage from 1 h of exposure to sublethal concentrations of potassium chloride, phenol and benthiocarb (Kang et al., 2009). Unlike deltamethrin, cadmium is distributed throughout the body with the greatest burdens in the kidneys and liver after gills absorption. Damage to kidneys and liver might be catalyze reactions, which generate reactive oxygen species (ROS) and lead to environmental oxidative stress (Boughammoura et al., 2013). Farombi et al., (2007) studied that the activity of metabolic enzymes in the liver and kidneys is disturbed earlier and more strongly than gills following exposure to Cd. Although, zebrafish were also observed to have breathing difficulties, which may be associated with the response of fish to reducing the intake of metal ions that adsorb to the gill membrane that covers the metal binding site of Ca (Patra et al., 2010).

Zebrafish are sensitive to sudden exposure to cadmium and deltamethrin, adjusting to toxicity stress through behavioral and physiological adaptations. Behavioral responses are time dependent and accordant with the stepwise stress model (SSM). Avoidance with increased swimming activity is the first response, followed by changes in the swimming depth. Due to different pathways and mechanisms of toxic biological effects, fish behavior differs under different acute and chronic stresses. The duration of hyperactivity during deltamethrin exposure was lasted 35 minutes larger than Cd exposure. The average swimming depth showed totally different ways with increased during deltamethrin exposure but decreased during cadmium exposure. It is proved that these different responses of zebrafish do exist and this provide another tool that may be used to help separate chemicals according to mode of action. It will increase the significance and usefulness of fish behavior as indicators for water monitoring.

This work was supported by the National Natural Science Foundation of China (51909203), Special Scientific Research Program of Shaanxi Provincial Department of Education (19JK0392) the Project of Shaanxi Province Key Laboratory of Environmental Pollution Control and Reservoir Protection Technology of Oilfields and Natural Science Basic Research Program of Shaanxi (Program N0.2021JM-424).