Written by Aida

**Introduction**

Metabolism has been a topic of interest for all living organisms. Many factors can affect the rate of a living being’s metabolism. Both ectotherms and endotherms differ in the way they control their temperature and so how they manipulate their metabolism. It is particularily interesting how poikilothermic organisms are affected by their surrounding environment. Indeed, Dr. Richard P. Brown investigated that body size has minimal direct influence on body-air temperature difference in ectoderms. Additionally, he discusses that a small directed dynamic action leads to a small increase in body temperature for these animals (2009). Metabolism requires a great amount of oxygen to produce energy in the form of ATP, therefore, oxygen is a good precursor to determine metabolism rates. Goldfish must consume oxygen to produce energy, but how do other factors like caffeine affect the metabolism of these animals? In this experiment, we will test the null hypothesis that the metabolic rates of goldfish as measured by oxygen consumption rate do not differ based on their exposure to caffeine.

**Materials and Methods**

This experiment focuses on the rate of oxygen consumption in goldfish to measure metabolism in the presence of caffeine as treatment. The goldfish used for this experiment is of the genus Carassius Species *Auratus Auratus*. To measure the rate of oxygen consumption to determine the activity of metabolism, an oxygen probe was used while being connected to *LoggerLite,* a program that records Oxygen consumption ( y axis) for a preset of time as the reaction advances ( x axis). For this experiment, a total of 4 fish were tested. Fish for trial 1 are weighted and fish for trial 2 also. In order to determine if caffeine has an effect on metabolism of goldfish, a control and experimental group was conducted on two fish for trial one and two different fish for trial 2. In trial one, place fish 1 and 2 on fish treated water ( 200ml) and add it to an oxygen probe chamber that was added 200 ml of fish treated water previously. No treatment (caffeine) was added during this step. Fish were left there 1.5 minutes for acclimation to the new environment. After, the *Loggerlite *program began to record data in segments of 20 seconds up to a total of 600 seconds. This is the control data of oxygen absorption for trial one. To test the effect of caffeine on these same fish for the experimental part, 50 ml of caffeine tablet was added to 200 ml of fish treated water on a separate cup. After, the caffeinated water was added to the oxygen probe with the first two fish. The same process was repeated as for the control group. For trial two, the remaining fish 3 and 4 (different fish) were tested the same as trial 1. **Figure 1** shows a flow chart with the experiment specifics. To finish the experiment, each data for both the control and experimental trials 1 and 2 was copied and pasted to excel to create data scatter plot and apply a linear regression line that determines the slope of the line (concentration of O2 consumed (mg/L)/time (sec)). If the slope is negative, the oxygen is depleted as the reaction goes forward. After recruiting data from a total of 5 other groups performing the same experiment in “ Caffeine 1” (**see figure 2**) two chi-square paired t tests were made ( on a computer based on slope values (ml/L/sec)for both experimental and control groups) to calculate a p-value to each corresponding group ( see **figure 2**). The null hypothesis was retained if the p-value is greater than .05 alpha value.

**Results**

The fish in the first group for trial 1 weight 13.477 grams. Trial two fish weight 11.407 grams. In the first trial control group, the fish moved very quickly and energetically. When added the caffeine, the fish moved slower, less grotesque and seemed to be more “relaxed”. In the second trial control group, the same observations were made as in the first trial control group, fish are observed to be more energetic and move quicker. At the 260 second, of this same control, oxygen consumption on the graph seems to slightly decrease and a fast hyper movement is observed on fish. After caffeine is added, fish are calmer throughout the 600 seconds. The regression line of the first trial control group has a slope of -.0005. The experimental group yielded a slope of -.0007 .The second trial control group gave a slope of -.0005 while the experimental group gave -.0006. See **figures 3a-3d **to see full linear regression equation.The mean and standard deviation for the “Caffeine group 1” group were used to determine a p value of 0.0569364 (see** figure 2 ** for complete mean and standard deviation of both control and experimental groups).

**Discussion**

Based on the resulting p- value being greater than an alpha value of .05 , we can retain the null hypothesis that caffeine has no effect in the rate of oxygen consumption in the metabolism of goldfish. The slopes of each of the linear regression graphs ( 3a-3d) have a negative slope, which means that oxygen concentration was always decreasing as the rate of the reaction advanced with time. However, the null is supported because the slopes are not too large to provide significance in the decrease of oxygen consumption. In other words, they did not vitiate much from each other (only a .0002 difference between the control and experimental group trial 1 and .0001 difference for the trial two experimental and control group. This means that despite the presence of caffeine or not, the fish metabolism was not really affected by it. For further study reference, the experiment can be conducted in a longer period of time beyond 600 seconds. This may be useful to test if the goldfish take longer to start depleting oxygen at faster rates. It may be that goldfish accelerate this depletion after a certain time that the slope becomes more negative. Also, it could be that goldfish are capable of storing certain oxygen molecules in the blood that they first must deplete before depleting surrounding oxygen at a fast rate.