The overall goal of this nutrient pulse method is to indirectly manipulate the ambient pool of dissolved organic matter to understand how this pool of organic matter responds to changes in nutrient concentration. This method can help answer key questions in the stream biogeochemistry field, such as how organic matter and nutrient cycling are linked. The main advantage of this method is that it represents a whole ecosystem manipulation and can be easily replicated.
The implications of this technique extend to watershed export of carbon and nitrogen as the interaction of these elements determines how they are retained within the stream. We first had the idea for this technique when we realized that the nutrient slug approach created a dynamic range of nutrient concentrations and that the concentrations of dissolved organic matter were sensitive to these changes. To begin, deploy the field conductivity meter, selecting a location approximately 0.5 to 1.0 meters upstream of the sample collection site.
The meter will remain in place throughout the experiment. Next rinse acid-washed, high-density polyethylene bottles three times and use them to collect 125 milliliters of background grab samples in triplicate at the addition site and then at the collection site of the experimental reach. These are used to determine ambient concentrations prior to the addition of the solution.
Record the instream background conductivity prior to the addition of solutes. Then record the time of collection and the conductivity of the collected background samples. First collect stream water with a large container.
Then add all of the reagents and ensure that the solutes are completely dissolved. Keep track of the amount of water added. Rinse the reagent vessels three times with additional stream water and pour the rinse into the solution container.
Stir the solution with a clean stir stick until all the reagents have dissolved. Label a 60 milliliter high-density, polyethylene bottle appropriately and into it, collect the 60 milliliter aliquot of the addition solution. Store it separately from other samples to avoid contamination.
Next, pour the sample into the addition site in a smooth and quick motion to minimize travel lag time and to avoid splashing. Immediately rinse the container and stir the stick in the stream three times to release all reagents. Record the time in hours, minutes, and seconds.
Record the masses of tracers added, alongside the time of release and take care not to disturb the stream benthos or solution until the conclusion of the experiment. At the collection site, first order labeled sampling bottles in ascending order while waiting for the solution. Use the field conductivity meter to take readings at the collection site.
Arrival of the solution will be detected by an increase in conductivity. Once solution arrival has been detected, take a clean 125 milliliter bottle and rinse it in the stream water. Discard the rinse downstream of the sampling site, and then collect the sample by holding the bottle into the main flow of the water at the sampling point.
Repeat this collection process, using one new bottle after another throughout the passage of the solution, and record the collection time and conductivity of each sample. Continue sampling until conductivity returns to within five microsiemens per centimeter of background level. Cap the samples and store them in a cooler.
First arrange the samples for filtration so that the rising lim is filtered in ascending specific conductivity until the peak. Next arrange the falling lim samples to be filtered in ascending order of specific conductivity. To filter samples, take a 60 milliliter syringe, remove the plunger and then close the stopcock.
Pour around 10 milliliters of sample into the syringe and replace the plunger. Shake the syringe so the sample rinses the internal walls. Next, attach a filter holder to the syringe and open the stopcock.
Push the sample through the filter by depressing the plunger and discard the rinse. Remove the plunger and close the stopcock. This time, pour 30 milliliters of the same sample into the syringe and replace the plunger.
With the syringe positioned to expel into a clean 60 milliliter sample bottle, open the stopcock and push around 10 milliliters through the filter. Cap the bottle, swirl it, and discard the filtrate. Repeat three times to ensure impurities are removed from the bottle and the walls are coated with sample.
Remove the plunger again and close the stopcock. Pour around a 60 milliliter sample into the syringe and replace the plunger. Push the sample through the filter holder into the 60 milliliter sample bottle.
Filling bottles no higher than the shoulder, cap the bottles and place the samples into a cooler. Finally, analyze samples for dissolved organic carbon and nitrogen and any other parameters of interest in order of low to high conductivity. These plots are linear regressions of measurements of dissolved organic nitrogen concentrations, or DON, after addition of nitrate via the pulse method.
Results show that dissolved organic nitrogen concentrations change in response to nitrate addition. Variation in the response of dissolved organic nitrogen was also observed within sites at different sampling times, which suggests that aquatic microbial communities may be using different components of the dissolved organic matter at different times of the growing season. Positive correlations are interpreted to reflect dissolved organic nitrogen's role as a nutrient source, while negative correlations are interpreted to reflect dissolved organic nitrogen acting as an energy source.
Experiments that showed non-significant relationships reflect a non-responsive DON pool or one in which the nutrient-based processes and energy-based processes are off-setting. Here, the plots show linear regressions of dissolved organic carbon, or DOC, after nitrate addition. Contrary to the DON plots, these show few instances of significant changes in the DOC concentration due to nitrate addition.
This may indicate that the nitrogen-rich and carbon-rich fractions of the dissolved organic matter pool have their own particular set of ecological and biogeochemical controls and that the ambient pool of DOC is highly recalcitrant. Once mastered, this technique can take between three to six hours depending on the system. While performing this method, it is important to avoid contamination of low conductivity samples by high conductivity samples.
Following this procedure, other methods, such as nutrient uptake connectics can be calculated to determine how the rate and flux of nutrient uptake determines the concentrations of dissolved organic carbon and dissolved organic nitrogen. After its development, this technique paved the way for researchers in the field of aquatic biogeochemistry to explore the controls on nutrient retention within stream ecosystems. Although this method can provide insights into stream nutrients cycling, it can potentially be applied to other environments, such as groundwater and estuarine systems.
After watching this video, you should have a good understanding of how to perform nutrient addition experiments in headwater stream ecosystems.
