Dr. Stephen L Reinbold has had other classes do a laboratory study. Because of the difficulties with the field watershed study on the Longview campus, laboratory experiments were conducted with amphipods to simulate the field experiment. An increased amount of leaves did decrease oxygen, quite possibly reduced nitrates, and had little effect on mortality. The enzyme Oxyrase® decreased oxygen concentration and apparently caused denitrification in addition to assimilation. Both field and laboratory experiments will be repeated to further test the model of trees protecting watersheds, and it is hoped that more specific recommendations for protecting the watershed will be forthcoming.

 

Amphipod Laboratory Experiment

A controlled laboratory experiment was conducted in Fall 2007 to simulate the field experiment. Amphipods were used as experimental organisms because of their ready availability and importance in the field. Hackberry leaves were used as a food source because of their abundance in the field. Oxygen and nitrate concentrations were measured in the laboratory because they were the most significant abiotic data collected in the field.

Methods for laboratory experiment to determine the effect of amount of food given to amphipods on oxygen and nitrate concentrations.

You will be monitoring dissolved oxygen and nitrate concentrations using the Vernier program and equipment.  Be sure to follow the directions that come with the instruments.  The computers should be turned on and probes soaking in appropriate solutions and warming up before you set up the samples as follows. You will set up experimental and control populations of amphipods in jars with the variable being the quantity of food leaves.  Each group will set up two experimental jars and two control jars.  Half fill four jars with creek water. Place five amphipods in each jar using the large bore pipettes provided. Complete filling the jars with the water the amphipods came in. You will be provided with small discs of hackberry leaf.  Place two discs in each control jar.  Place four discs in each experimental jar. Leaf discs will be added in the same proportions each time after data are collected. Mark the initial water level on each jar with a wax marker. Distilled water will be added whenever the water level is observed to be below the mark, but water will not be added less than 24 hours before data collection.

Now return your attention to the Vernier equipment.  You will need to calibrate the nitrate probe in high concentration nitrate solution and then low concentration nitrate solution.  Use the factory setting for the oxygen probe for the calibration. After rinsing the oxygen probe with deionized water, place the probe in the first jar to be tested and click the Collect Data button on the computer screen. IMPORTANT the probe must be constantly stirred in the water. Let the program collect data long enough to get consistent results and then click the Stop button on the screen. Record the result and Clear Data. Lift the probe out of the jar and rinse with deionized water and continue in the same way until all jars have been tested. Now collect nitrate data.  Rinse the nitrate probe and insert it into the first jar.  (You do not need to stir it.) Collect data until a consistent result occurs and then stop it and record the data.  Clear the data, rinse the probe, and continue to the next jar in like manner until all jars are done. Data will be collected on the first day of the experiment and three subsequent dates, over a period of approximately two weeks.

In the experiment in 2008, denitrification will be enhanced by an enzyme named Oxyrase® that uses oxygen to convert lactate to pyruvate and water. Denitrification occurs when the oxygen level drops below 0.5 ppm (Ambler et al., 2001). To simulate natural conditions, the sediment with Oxyrase® will be partially separated from the clear water with the amphipods. Exchange of chemicals will be able to occur with the sediment below. The effect of the Oxyrase® will be compared to sediment without Oxyrase®. You will set up experimental and control populations of amphipods in jars with the variable being the presence or absence of Oxyrase® in the sediment.  Each group will set up two experimental jars and two control jars.  You will be supplied with sediment for experimental with Oxyrase® and sediment for control without Oxyrase®. Add 80 ml of experimental sediment to the two experimental jars and 80 ml of control sediment to the two control jars.  

Now insert the top jars in place in each sediment jar and insert the filter paper in each. Fill the four jars with creek water half way. Place five amphipods in each jar using the large bore pipettes provided. Finish filling the jars with the water the amphipods came in. You will be provided with small discs of hackberry leaf to add in each jar. Measure the oxygen in each top jar as explained above.  Also collect nitrate-N data from the top jars. Data will be collected again two days later. Collect oxygen and nitrate-N data for the top jars first. Remove the top jars and take the same measurements for the bottom jars. Count and record the number of amphipods surviving.

 

                                                                                                                                                                                                                          

                                                                                                                                                          

Results for Laboratory Study

The laboratory study was conducted in fall 2007. Data are located in Tables 1-3. As can be seen in Table 1, oxygen levels were significantly lower in experimentals than controls in groups 3 and 4 on October 18. Group 6 showed significantly lower oxygen level in experimental than control on October 23. No jars showed significantly higher oxygen in control than experimental. No significant differences were seen in nitrate concentrations between experimentals and controls (Table 2); however, 19 out of 24 comparisons showed lower nitrate concentrations in experimentals than controls, and only 7 showed lower or equal nitrate concentrations in control compared to experimental. Three of the 7 were on the first day of the experiment before any difference was expected. Table 3 shows mostly high positive correlations between oxygen concentraton and nitrate concentration. Survival rates were good throughout the experiment, and no significant differences were seen in survival between controls and experimentals when animals were counted on October 18 and 23.

Table 1. Effect of food quantity given to amphipods on oxygen concentration. Controls received two hackberry leaf discs four times during the experiment, and experimentals received four leaf discs on the same occasions.

oxygen oct. 9   oct. 11   oct. 18   oct. 23  
group control exper control exper control exper control exper
1#1 4.8 4.8 4.3 4.1 5.8 5.5 5.9 5.2
1#2 5.2 5 3.9 3.9 5.6 5 5.6 5.6
ave 5 4.9 4.1 4 5.7 5.25 5.75 5.4
t test con v exp 0.711723   0.711723   0.296585   0.305373
2#1 7.3 7.2 6.8 6.7 7.2 6.5 8.6 7.8
2#2 7.2 7.2 7.1 6.4 7.1 6.1 8.3 7.5
ave 7.25 7.2 6.95 6.55 7.15 6.3 8.45 7.65
t test con v exp 0.5   0.2   0.130019   0.063671
3#1 6 6 6.5 6.5 5.9 5.3 6.4 5.9
3#2 6 6.1 6.75 6.5 5.8 5.4 6.7 5.8
ave 6 6.05 6.625 6.5 5.85 5.35 6.55 5.85
t test con v exp 0.5   0.5   0.019419   0.107122
4#1 7.6 7.5 5.6 6.9 6.2 5.6 5.1 5.1
4#2 7.5 7.5 5.8 5.9 6.1 5.5 5.5 5.2
ave 7.55 7.5 5.7 6.4 6.15 5.55 5.3 5.15
t test con v exp 0.5   0.38824   0.013606   0.588295
5#1 6 6 5.8 5.5 5 4.7 5.8 5.4
5#2 6.1 6 5.7 5.3 5 4.8 5.1 5.7
ave 6.05 6 5.75 5.4 5 4.75 5.45 5.55
t test con v exp 0.5   0.129048   0.125666   0.82714
6#1 4.8 4.5 6.1 5.5 6 5.6 7.7 7.1
6#2 4.4 4.5 5.5 5.5 5.8 5.5 7.7 7
ave 4.6 4.5 5.8 5.5 5.9 5.55 7.7 7.05
t test con v exp 0.704833   0.5   0.129048   0.048875

 

Table 2. Effect of food quantity given to amphipods on nitrate concentration. Feeding schedule as in Table 17.

nitrate 9-Oct   11-Oct   18-Oct   23-Oct  
group control exper control exper control exper control exper
1#1 2 1.9 1.6 1.9 1.2 2.1 0.9 0.9
1#2 1.8 1.9 1.5 1.5 1.5 1.5 0.9 0.8
ave 1.9 1.9 1.55 1.7 1.35 1.8 0.9 0.85
t test con v exp 1   0.588295   0.349886   0.5
2#1 0.5 0.5 0.4 0.3 0.5 0.4 1.6 1
2#2 0.5 0.6 0.3 0.3 0.4 0.4 2 1.1
ave 0.5 0.55 0.35 0.3 0.45 0.4 1.8 1.05
t test con v exp 0.5   0.5   0.5   0.148663
3#1 3 2.9 3.4 3.2 0.5 0.6 1.1 0.8
3#2 2.9 2.9 3.4 3.2 0.5 0.5 1.2 1
ave 2.95 2.9 3.4 3.2 0.5 0.55 1.15 0.9
t test con v exp 0.5   #DIV/0!   0.5   0.198727
4#1 0.5 0.5 0.3 0.4 0.9 0.9 0.9 0.8
4#2 0.5 0.5 0.4 0.4 1 0.8 1.6 0.7
ave 0.5 0.5 0.35 0.4 0.95 0.85 1.25 0.75
t test con v exp #DIV/0!   0.5   0.292893   0.385136
5#1 5.3 5.1 1.6 1.5 0.6 0.6 0.9 1.1
5#2 5.1 4.9 1.7 1.7 0.7 0.5 0.9 0.6
ave 5.2 5 1.65 1.6 0.65 0.55 0.9 0.85
t test con v exp 0.292893   0.711723   0.292893   0.874334
6#1 2.3 2.4 1.4 1.3 1.1 0.8 1 0.7
6#2 2.3 2.1 1.4 1.2 0.9 0.9 0.8 0.8
ave 2.3 2.25 1.4 1.25 1 0.85 0.9 0.75
t test con v exp 0.795167   0.204833   0.349886   0.349886
ave. of ave.           1.15 0.858333

 

Table 3. Correlation between oxygen and nitrate concentrations in control and experimental jars over four sampling dates. Feeding schedule is given in Table 17.

    9-Oct 11-Oct 18-Oct 23-Oct correlation
  average average average average oxy/nitrate
control oxygen 5 4.1 5.7 5.75 -0.57827
group 1 nitrate 1.9 1.55 1.35 0.9  
exper oxygen 4.9 4 5.25 5.4 -0.43731
group 1 nitrate 1.9 1.7 1.8 0.85  
control oxygen 7.25 6.95 7.15 8.45 0.995591
group 2 nitrate 0.5 0.35 0.45 1.8  
exper oxygen 7.2 6.55 6.3 7.65 0.896288
group 2 nitrate 0.55 0.3 0.4 1.05  
control oxygen 6 6.625 5.85 6.55 0.389051
group 3 nitrate 2.95 3.4 0.5 1.15  
exper oxygen 6.05 6.5 5.35 5.85 0.889467
group 3 nitrate 2.9 3.2 0.55 0.9  
control oxygen 7.55 5.7 6.15 5.3 -0.49086
group 4 nitrate 0.5 0.35 0.95 1.25  
exper oxygen 7.5 6.4 5.55 5.15 -0.73965
group 4 nitrate 0.5 0.4 0.85 0.75  
control oxygen 6.05 5.75 5 5.75 0.711604
group 5 nitrate 5.2 1.65 0.65 0.9  
exper oxygen 6 5.4 4.75 5.55 0.808694
group 5 nitrate 5 1.6 0.55 0.85  
control oxygen 4.6 5.8 5.9 7.7 -0.84663
group 6 nitrate 2.3 1.4 1 0.9  
exper oxygen 4.5 5.5 5.5 7.1 -0.82127
group 6 nitrate 2.3 1.3 0.9 0.8  

Tables 4 and 5 record results for a follow-up experiment to the amphipod lab done on October 7 through October 9, 2008. Not surprisingly Oxyrase® lowered oxygen concentration in the bottom jars with sediment significantly over 48 hours compared to control jars, as seen in all groups. (The effect already occurred in groups 2 and 3 during the 30 minutes or so between adding the enzyme and measuring the oxygen.) Top jars in group 3 also had significantly less oxygen after 48 hours.. However, only one more amphipod died in experimental than control in group 3 (two vs. one). Group 1 lost one in experimental and four in control. Group 2 lost six in experimental and two in control. Nitrate-N concentration was significantly lower in experimental compared to control after 48 hours in group 1 bottom jars. In fact, only one of the groups had higher nitrate-N in experimental compared to control in top or bottom jars, and the difference was not significant. There was no significant difference in initial nitrate-N concentration in any top or bottom jars.  

Table 4. Effect of Oxyrase® on oxygen concentration in experimental jars compared to control jars without Oxyrase® over a period of 48 hours. The jars at the top of the table each contained clean water with five amphipods and two hackberry leaf discs (group 1) or four hackberry leaf discs (groups 2 and 3). The jars at the bottom of the table contained water with sediment and Oxyrase® (experimental) or no Oxyrase® (control).

water Ox Oct. 7   Oct. 9  
group exp control exp control
1#1 6.5 6.4 1.8 6.2
1#2 6.4 6.4 2.8 6.5
ave 6.45 6.4 2.3 6.35
t test exp v con 0.5   0.059052
2#1 7.2 7 4.3 6
2#2 7.1 7 1.7 6.4
ave 7.15 7 3 6.2
t test exp v con 0.204833   0.239124
3#1 8.2 8.6 2.3 6.8
3#2 8.4 8.6 1.9 7.1
ave 8.3 8.6 2.1 7
t test exp v con 0.204833   0.003719
sed. Ox Oct. 7   Oct. 9  
group exp control exp control
1#1 2.1 4.9 1.7 6.3
1#2 2.1 5 1.8 6.4
ave 2.1 4.95 1.75 6.35
t test exp v con 0.011168   0.000236
2#1 3.8 6 3.8 12.7
2#2 3.9 5.95 4 12.7
ave 3.85 5.975 3.9 12.7
t test exp v con 0.003531   0.007234
3#1 4.4 6.8 2.1 6.3
3#2 4.1 6.7 1.4 6.3
ave 4.3 6.8 1.8 6.3
t test exp v con 0.023219   0.048875

Table 5. Effect of Oxyrase® on nitrate-N concentration in experimental jars compared to control jars without Oxyrase® over a period of 48 hours. The jars at the top of the table each contained clean water with five amphipods and two hackberry leaf discs (group 1) or four hackberry leaf discs (groups 2 and 3). The jars at the bottom of the table contained water with sediment and Oxyrase® (experimental) or no Oxyrase® (control).

                                    

water N Oct. 7   Oct. 9  
group exp control exp control
1#1 1.4 1.2 0.4 0.7
1#2 1.5 1.2 0.3 0.7
ave 1.45 1.2 0.35 0.7
t test exp v con 0.125666   0.090334
2#1 1.65 1.7 0.4 0.8
2#2 1.7 1.6 0.4 0.7
ave 1.675 1.65 0.4 0.75
t test exp v con 0.711723   0.090334
3#1 1.4 1.2 0.1 0.2
3#2 1.3 1.2 0.1 0.2
ave 1.4 1.2 0.1 0.2
t test exp v con 0.204833   #DIV/0!
sed. N Oct. 7   Oct. 9  
group exp control exp control
1#1 1.1 1.2 0.5 0.9
1#2 1.1 1.3 0.4 0.8
ave 1.1 1.25 0.45 0.85
t test exp v con 0.204833   0.029857
2#1 1.2 1.1 0.6 0.8
2#2 1.7 1 0.5 0.7
ave 1.45 1.05 0.55 0.75
t test exp v con 0.347806   0.105573
3#1 0.8 1.1 0.1 0.2
3#2 0.7 1.1 0.1 0.1
ave 0.8 1.1 0.1 0.15
t test exp v con 0.090334   0.5

 

DISCUSSION

 

The laboratory experiment with amphipods suggested a similar effect to Bernhardt's field data. More food supplied as leaf discs apparently caused oxygen levels to drop, creating conditions favorable for denitrification, with consequent decrease in nitrate concentrations. Decreasing nitrate concentrations would be favorable for conditions downstream in the lake, where eutrophication from nitrates might otherwise occur. Oxygen concentrations were not dropped enough to increase mortality though, so as not to cause an undesirable side effect.

The laboratory experiment with amphipods done in 2008 using Oxyrase® showed that decreased oxygen levels caused decreased nitrate-N levels. Since experimental jars showed the greatest decrease in nitrate-N, then some of this decrease must be due to increased denitrification, as there is no apparent reason why decreased oxygen would lead to increased assimilation although Ambler et al.(2001) suggested lower levels of oxygen might be required for denitrication.

 

REFERENCES

Ambler, Pelovitz, Ladd, and Steucek. 2001. A demonstration of nitrogen dynamics in oxic & hypoxic soils & sediments. The American Biology Teacher 63(3): 199-206.