The Effect of Water Temperature, Water Acidity, and Animal Age and Body Size on the Opercular Respiratory Rate of Brown Bullhead Catfish

Introduction 

Brown Bullhead Catfish (Ameiurus nebulosus), one of the specimens used in my experiments.


My fascination with catfish started many years ago during my first natural science camp. When I first glimpsed a majestic catfish elegantly gliding under a big limestone rock in Rock Creek in Maryland, I decided I want to learn everything about these fascinating creatures. I started to go to and in the creek almost every day, and I learned how to recognize different catfish species, the habitats they can be found in, and their unique ability to adapt to environments with very poor water quality. Soon I noticed that on some days I was not able to see any catfish at all, while on other days I spotted multiple specimens. I was wondering whether the water quality might impact the life of catfish. I suspected that water temperature and acidity (pH) might affect the catfish a lot. Since the most obvious and least harmful measurement of catfish reactions to changes in their environment is to count their opercular respiratory rate, I designed my project to investigate how the opercular respiratory rate of catfish is affected by varied conditions in their natural range. I concentrated on the effect of water temperature and pH on the opercular respiratory rate of brown bullhead catfish.

I found a small branch of the creek with an abundant catfish population and gathered young specimens of brown bullhead catfish by very gently netting them, following Maryland Natural Resources regulations (Maryland Fishing Guide, 2014). The catfish were placed under appropriate conditions in my home aquarium tank, set up to model their natural habitat. The brown bullhead catfish (Ameiurus nebulosus) is very common species in North American waters (Pay & Burr, 1991). My catfish are fully acclimatized and suited for this study. None of the catfish has ever showed any sign of stress or discomfort.

 

pic 2 55 gallon tank

A brown bullhead catfish in a 55-gallon fully equipped home tank.


Background Research

My project is about how water temperature, water acidity (pH), and age and body size affect the opercular respiratory rate (ORR) of brown bullhead catfish. I investigated how much the opercular respiratory rate speeds up or slows down when different independent variables are changed within a natural range. Many catfish are tolerant of environmental changes. However, there is no data available about the ORR of these species.

Fish are poikilothermic organisms whose internal temperature varies considerably as a consequence of variation in the water temperature. Thus, their metabolic rate and respiratory rate depend on water temperature (Kapoor & Khanna, 2004).

Fish extract oxygen from water and release the carbon dioxide produced by tissue metabolism by passing water over their gills (Perry & Tuffs, 1998). There is a close coordination of pulsatile water flow and the counter-current pulsatile blood flow at the gills to optimize respiratory gas exchange (Taylor et al., 2009).


The operculum is a bony plate that covers the fish’s gills. It serves as a water pump; each time the fish respires, the operculum moves. Observation of the opercular movement allows one to evaluate the opercular respiratory rate (ORR). Effects of water temperature and acidity (pH) on respiratory rate have previously been studied in other fish species (Tantarpale et al., 2012; Heath & Hughes, 1973; Hargis, 1976; Stecyk & Farell, 2006; Reid et al., 2000; Borch et al., 1993; McKay, 2014; Zheng et al., 2014; Ramesh & David, 2009) but not in the brown bullhead catfish.

The brown bullhead catfish (Ameiurus nebulosus) belongs toIctaluridae family. The brown bullhead catfish lives in creeks, rivers, lakes, and ponds. It is common in most parts of North America (Pay & Burr, 1991). It is easy to tell it apart from other bullheads because of its distinctive light-brown checkered scales. The brown bullhead is a scavenger, searching for dead creatures. But it will also eat algae and different types of sea grass growing on the bottom of the lake or stream bed.

 

Testable Questions

To find out how basic water quality affects catfish, I asked three questions that I researched in three experiments.

Experiment 1: How does water temperature affect the ORR of brown bullhead catfish?

Experiment 2: How does water acidity (pH) affect the ORR of brown bullhead catfish?

Experiment 3: How does age and body size affect the ORR of brown bullhead catfish?

 

Hypothesis

Hypothesis 1:If the water temperature increases, then the ORRof brown bullhead catfish will also increase because of increased metabolic demand.

Hypothesis 2:If the water acidity increases, then the ORRof brown bullhead catfish will increase because of increased metabolic demand.

Hypothesis 3:If the age/size of the fish increases, then the ORR of brown bullhead catfish will decline because the metabolic rate of older, larger fish is lower compared to younger, smaller fish.

 

Materials List

Brown bullhead catfish in the experimental tank during experiments.


Materials used to conduct experiments.


Experimental organisms: Three brown bullhead catfish (Ameiurus nebulosus) caught in Rock Creek, Maryland, with a mean size of 5 centimeters, kept in a fully equipped 55-gallon tank for more than three years (water temperature 20–24oC, pH 7.0–7.2). This home tank is equipped with filtration and aeration, 12-hour-cycle lighting, gravel, sand, driftwood, and aquatic plants. About 30% of the water is regularly exchanged every week. Aquasana filter-purified water is used.

Experimental tank: One10-gallon tank with a canopy and medium-intensity lighting, constant aeration, a power head to keep the water temperature even in whole volume, a 100-watt heater, driftwood, two digital thermometers, and Aquasana filter-purified water.

Other Materials: Crushed ice made of Aquasana purified water, warm Aquasana purified water, bottle of vinegar (Heinz distilled white vinegar, 5%), pipettes, pH test kit for freshwater pH 6.0–7.0 (Aquarium Pharmaceuticals), ruler, cell phone for time measurement and taking pictures, two nets, paper towels, lab book, pencil to record data, computer.

Procedures

Experiment #1: Temperature change

pic 6 temp measurement

Temperature measurement.


Step 1: Water from the home tank was moved to the experimental tank.

Step 2: Heater thermostat was set to 20°C.

Step 3: Specimens A, B, C were transferred into experimental tank and kept for 30 minutes for acclimatization.

Step 4: ORR was measured by counting opercular movement for one minute for each fish at each temperature measurement point (20°C, 15°C, 12°C, 15°C, 20°C, 25°C and 30°C) three times (Trial 1, Trial 2 and Trial 3).

Step 5: Water temperature was gradually decreased to the measurement points 15°C and 12oC by adding crushed ice.

Step 6: Five-minute stabilization was reached before each ORR measurement.

Step 7: Water temperature was gradually increased back to the measurement points 15°C and 20°C by the water heater.

Step 8: Water temperature was increased to 25°C and 30°C measurement points by the water heater and by adding warm water.

Step 9: Catfish were returned to the home tank.

Step 10: Data was analyzed in Excel. Averages of three trials for each specimen were calculated.

Experiment #2: Change of pH/acidity

Before this experiment, I learned how to measure pH by using the pH test kit. I precisely followed the instructions included in the pH test kit. I filled a clean test tube with 5 mL of water to be tested for pH, added 3 drops of pH test solution, closed the test tube with a cap, and mixed the solution. I read the pH by comparing the color of the solution to the pH Color Card included in the kit. I rinsed the test tube with clean water after each use. A preliminary experiment was done without fish in the experimental tank to know how much vinegar to use to reach the desired pH.

Step 1: Water from the home tank was transferred to the experimental tank. The water temperature was set to 20oC.
Step 4: Acidity was changed to pH 7.0 by adding vinegar, 5 mL/minute over a five-minute period; pH testing until pH 7.0 was reached.
Step 5: ORR was measured for 1 minute for each fish three times.
Step 6: Acidity was changed to pH 6.5 by adding vinegar, 5 mL/minute over a five-minute period; pH testing until pH 6.5 was reached.

Step 7: ORR was measured for 1 minute for each fish three times.
Step 8: Gradual water exchange was performed over 15 minutes until pH 7.5 was reached.
Step 9: After 10 minutes, catfish were moved back to the home tank.
Step 10: Data was analyzed in Excel. Averages of three trials for each specimen were calculated.

Experiment #3: Change of animal age/body size

Step 1: Ruler was used to measure the length of catfish in centimeters.

Step 2: Evaluation of the effect of water temperature on the ORR according to the procedure described in Experiment 1 was performed on the same specimens—A, B, C—at approximate age 12 months. Exactly the same experiment was repeated 32 months later, at approximate age 44 months (2014).

Step 3: Data from Experiment 1, performed at ages 12 months and 44 months, was analyzed and compared in Excel. Averages of three trials for each specimen were calculated, and mean ORR values of the same three specimens at ages 12 months and 44 months were compared for each water temperature measurement point.

 

Variables, Controls and Sample Size

Jonathan counts the ORR using a cell phone to measure one-minute intervals.


Independent variables: Water temperatures, water pH and specimen age/size.

Specimens: Three brown bullhead catfish (Ameiurus nebulosus) of a total length (from nose to tip of tail filament) of (a) 10 cm, (b) 12 cm, and (c) 18 cm at approximately 12 months of age during the first measurement, and a total length of (a) 30 cm, (b) 30 cm, and (c) 33 cm at the time of second measurement at approximately 44 months of age.

Controls: To check the reliability of ORR measurement, I independently measured ORR three times for 1 minute under the same conditions. The measurements were not significantly different (62 ± 3ORRbeats/minute). 

Also, I measured ORR in the morning and in the evening to see if circadian rhythms affect the opercular movement. I did not observe any differences between morning and evening ORR measurements.

I measured ORR at least 12 hours after feeding and at least three days after water exchange in the home tank.

None of the catfish has ever showed any sign of stress or discomfort. 

 

Results, Discussion of Data, and Conclusions

In Experiment 1, on the effect of water temperature on opercular respiratory rate (ORR), the results show that the higher the water temperature, the higher the opercular respiratory rate observed. My data support my hypothesis. In warmer water, the metabolic rate of the catfish is higher, so the catfish produce more carbon dioxide and need more oxygen than in cold water. Therefore, the ORR was higher in warm water compared to cold water. I observed the same effect of water temperature on ORR on yearlings (Graph 1) as on the same fish 32 months later (Graph 2).

 

Graph 1: Effect of Water Temperature on the Opercular Respiratory Rate (in Beats/Minute) of Brown Bullhead Catfish, Measured on Yearlings (Approximately 12 Months Old)

graph_1

Graph 2: Effect of Water Temperature on the Opercular Respiratory Rate (in Beats/Minute) of Brown Bullhead Catfish, Measured on Specimens Approximately 44 Months Old

graph_2

In Experiment 2 on the effect of acidity on ORR, the results show that increasing the water acidity to pH 6.5 induced an increase in ORR. The data support my hypothesis. At higher water acidity, catfish are stressed, their body production of carbon dioxide is higher and their demand for oxygen is also higher. Therefore, catfish have higher ORR in water with higher acidity (lower pH).

Graph 3: Effect of Water Acidity/pH on the Opercular Respiratory Rate (in Beats/Minute) of Brown Bullhead Catfish; Temperature 20 ºC; Age 44 months

graph_3

In Experiment 3 on the effect of catfish age/size on ORR, the results show that the older, larger catfish, at an approximate age of 44 months, have markedly lower ORR compared to measurements taken under the same conditions on the same group of animals 32 months before, at the approximate age of 12 months and with a body length almost three times smaller. The data support my hypothesis. As yearlings, catfish are growing rapidly and their metabolic rate is higher; their body production of carbon dioxide is higher and their demand for oxygen is higher. Therefore, catfish have higher ORR at 12 months of age compared to an age of 44 months.

Graph 4: Effect of Animal Age/Body Size on the Opercular Respiratory Rate (in Beats/Minute) of Brown Bullhead Catfish at pH 7.5 and a Temperature of 20°C

graph_4

                      

Possible Errors and Further Research

My experimental setup worked well and the counting of ORR was reproducible and did not vary markedly between trials.

The results might be influenced by the stress caused by a bright light in the experimental fish tank (a different light than in the home tank). I would use a dimmer light to perform the experiment in future. I would also not put driftwood into the experimental tank. The catfish were hiding, which made ORR counting difficult.

Also, it would be helpful to video-record ORR to be able to count opercular movement more precisely from recordings, but my cell phone camera was not able to provide recordings clear enough for counting ORR. The best improvement of the ORR measurement method would be the application of the computer video technology to get a fully automated assay (Zheng et al., 2014).

During the experiment, the catfish had 30 minutes of acclimation in the experimental tank before specific tests were done. The 30 minutes might not have been long enough. If so, stress from being moved might have changed the results slightly. Also, the action of moving water between the tanks could cause it to be more highly oxygenated and thus affect ORR. This factor could change the measurements, since catfish need to respire more if there is not enough oxygen in the water. Therefore, they would obviously respire more slowly if more oxygen was available every time water was passed over their gills.

Also, ORR should have been measured at the regular acidity for Experiment 3. In future research, I would like to see how the water salinity affects the ORRof brown bullhead catfish, since this species is found also in tidal waters.I would also be interested in how the concentrations of agricultural pollutants, such as phosphates and nitrates, pesticides or insecticides, affect the ORR of brown bullhead catfish.

My ultimate goal is to study the respiratory rate of fish in the wild in their natural environment, using an underwater camera and other video techniques. This study revealed that the opercular respiratory rate is an easily and noninvasively assessed physiological parameter in brown bullhead catfish that showed sensitivity to changes in water temperature and pH and was dependent on the body size/age of the animals. I believe that the opercular respiratory rate measurement of various fish species is a simple, valuable, noninvasive method that should be more widely used for monitoring water quality and studying the toxicity of environmentally significant pollutants.

 

A brown bullhead catfish used in Jonthan's experiments.


Addendum

All the catfish specimens have been healthy and very active since I put them into my home aquarium more than three years ago. Since I kept all conditions during my observations within the natural range (temperature, pH) occurring in the creek, none of the catfish species has ever shown any signs of stress or discomfort during observation under different conditions in my aquarium. I plan to keep them in my aquarium tank and provide them with the same care I have to date.

Acknowledgements

I would like to thank my science teachers for their comments on my research project. I would like to thank my parents for getting me all the needed equipment for my research and helping me with the aquarium setup and occasional reminders to change the water and clean the filters in the aquarium.

 

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