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Environmental Significace of the Groundwater-Surface Water Interaction Zone

WHONDRS Surface Water Sampling for Metabolite Biogeography

Columbia river shoreline

Protocol

Watch the Surface Water Protocol video protocol for more information.

In addition to the sampling kit provided by WHONDRS, you will need the following:

  1. Cooler with wet ice or blue ice for keeping samples cold in the field
  2. Celsius thermometer for taking water temperature
  3. A method for collecting latitude and longitude in decimal degrees in the field (a smart phone is sufficient)
  4. A method for taking pictures of the field site (a smart phone is sufficient)
  5. A -20 C freezer for freezing the ice pack
  6. A 4 C refrigerator for storing the samples until shipping
  7. Access to a FedEx office for shipping samples back overnight
  8. Access to stream gauge data (that can be made public) for at least the 3 months prior to collecting the samples. Data must be recorded at a minimum frequency of every two hours.

The sampling kit should contain the following items:

sampling kit

  1. Hard copy data sheet
  2. Shipping label that is pre-filled for sending samples back via FedEx overnight
  3. Freezer pack (place this in a -20C freezer for at least 48 hours prior to shipping)
  4. 20mL syringe (should be new, and sealed)
  5. 3mL syringe (should be new, sealed)
  6. One pair of nitrile gloves to minimize contamination
  7. Two filters. One has a needle attached, this is one that will be used for sampling. The other is a spare and needs to remain sealed unless it is needed
  8. One needle to fill the 5mL syringe with RNALater from the small epi tube after sampling
  9. A spare needle (keep it sealed unless it is needed)
  10. Four 40 ml vials, each with a unique label. Do not unscrew the cap from any vial. A sample will be collected by piercing the septum with the needle attached to the filter.
    • One vial will be clear. Fill this vial first.
    • Three vials will be amber. The amber vials each contain 10 microliters of 85% phosphoric acid to aid in sample preservation. Fill these vials after the clear vial to minimize acid transfer to the clear vial.
  11. Small measuring tape (you may want to bring your own)
  12. Pencil for recording field metadata
  13. pH strip and color matching chart
  14. One small plastic epi tube filled with RNALater, a preservative to use on the filter after sampling
  15. Two luer lock caps to seal the ends of the used filter after it has been filled with RNALater
  16. One small whirlpak bag to store the used filter in

After confirming that you have all of the needed gear, choose the sampling location and collect field metadata as follows:

water sample

  1. Choose your location to be as close as possible to a stream gauge. Also try to sample in a location with natural sediments as opposed to locations with artificial substrates.
  2. Attempt to sample ~1m from the shoreline, and record the distance from the shoreline where samples are collected.
  3. Confirm there is downstream flow at the sampling location (e.g., drop in a leaf and make sure it moves downstream)
  4. Record the date, local time, time zone, and your name on the data sheet
  5. Measure the water depth at the sampling location and record it on the datasheet in cm
  6. Measure the temperature of the water at 50% of the water column depth; record it on the datasheet in degrees C
  7. Record latitude and longitude in decimal degrees. For this, feel free to use a GPS or a smart phone app such as 'My GPS Coordinates' or Google maps.
  8. Remove the pH strip from its protective plastic bag, making sure to keep the paper dry.
  9. Dip the strip into the river water upstream of where you are standing.
  10. Wait 15 seconds after wetting the pH strip and then compare the color to the color chart provided with the pH strip. Record the pH value on the metadata sheet.
  11. Place the pH strip on a white background (the back of the metadata sheet) and photograph the pH strip. This picture will be uploaded with the metadata.

After collecting field metadata, collect samples as follows:

  1. Put on the provided nitrile gloves (do your best if they don't fit well)
  2. Open the filter package that has a needle attached to the filter but leave the needle/filter assembly in the package
  3. Open the syringe package and remove the syringe. Please don't touch the outlet of the syringe and do not fully remove the plunger from the syringe body. This is important to avoid contamination.
  4. While sampling, please stand downstream of the sampling location. This is important to avoid a non-representative sample.
  5. Fill the syringe with river water, collecting water from 50% of the water column depth. Expel the syringe contents into the river and repeat this two more times.
  6. After flushing the syringe 3 times, fill the syringe again from 50% of the water column depth; this is the sample water for the first replicate.
  7. Screw the syringe onto the filter that has a needle connected to it. Remove the plastic cover that is protecting the needle while the needle is still in the filter package. Please don't touch the outlet of the syringe, the inlet of the filter, or any part of the needle. This is important to avoid contamination. Also, retain the plastic needle cover. Note that it is okay to touch the cylindrical filter housing.
  8. Push 2-3 mL of water through the filter/needle assembly. This water is not collected.
  9. Beginning with the clear glass vial that has '-1' at the end of its sample identification number, remove the flip top cap on the glass vial. Please don't touch the septum that is exposed after removing the flip top cap. This is important to minimize contamination. Do not unscrew the top of the vial. Only remove the white disc that rests on top of the cap and conceals the septum.
  10. Pierce the septum with the needle and fill the vial to the pre-marked fill line (approximately half way full). Please don't fill more than the pre-marked fill line. If the vial is more than half full, it will burst when frozen.
  11. After injecting sample water into the vial, withdraw the needle from the septum and carefully place the filter/needle/syringe assembly back into the plastic needle cover that is still in the filter package (see the video protocol for demonstration of this method).
  12. Place the vial containing the sample into the cooler with wet or blue ice. Note there is no need to record any information on the sample label. There is no need to replace the flip top cap that previously covered the septum.
  13. Unscrew the syringe from the filter/needle assembly, expel any remaining liquid into the river, and then refill the syringe from the same sampling location, again at 50% depth of the water column.
  14. Screw the syringe back onto the filter/needle assembly, expel a small volume of sample water through the filter/needle assembly, and repeat the above sampling procedure for the vials with '-2', '-3' and '-4' at the end of their sample identification numbers. These are the second, third, and fourth replicates. Note that one filter and one needle should be used to collect all four replicates. The second filter and second needle sent with the kit should only be used if there is an issue with the first filter or needle. If an extra filter or needle is needed, please do not touch the inlet or outlet of the filter, and please don't touch the inlet of the needle or any part of the metal needle (it is okay to touch the plastic needle cover). If the extra filter or needle are needed, please record the reason on the paper metadata sheet. If the extra needle or filter are not needed, they should be shipped unopened when the samples are shipped.
  15. After filling the amber vials, shake each one gently to incorporate acid into the sample. Then place back into cooler.
  16. After filling the vials, you will need to preserve the filter.
    • To do this, first detach the 20mL syringe from the filter, expel any remaining water onto the ground, and fill the syringe with air.
    • Then attach it to the filter again, and push the air through the filter. The goal is to expel as much water from the filter as possible. Repeat 2 or 3 times if needed.
    • Discard the needle attached to the filter.
    • Take one of the small luer lock caps provided and attach it to the open end of the filter. Note that the filter has a "male" side and a "female" side and therefore there are two types of caps provided.
    • From the provided supplies, take out the small plastic epi tube filled with RNALater. This is the preservative for the filter. Also take out a 3mL syringe and a new needle.
    • Connect the new needle to the 5mL syringe, carefully open the small epi tube, and fill the syringe with the RNALater by simply putting the needle down into the liquid.
    • After filling the syringe with RNALater, invert the syringe so that the needle is facing up. Discard the needle.
    • Take the used filter off the 20mL syringe, taking care not to touch the inlet of the filter.
    • Attach the 5mL syringe to the filter and rotate so that the syringe is facing down and the filter is below it. Push the plunger to slowly fill the filter with RNALater. Fill until you feel some resistance or until you have used all the RNALater.
    • Detach the syringe and put into a waste container. Cap the filter using one of the small luer lock caps provided and shake the sealed filter gently.
    • Put the capped filter into a small whirlpak bag, tie up to seal, and place in the cooler. Please ensure no water is entering the whirlpak bag.

Prior to leaving the field site:

river photos

  1. Take the following photos with a smart phone or camera (note these will be uploaded along with metadata). For the last three pictures (b, c, d), lay out a measuring tape to 30cm on the shoreline as a reference for scale and make sure it can be seen in the photos. The first figure (looking across the river) doesn't need to contain the measuring tape.
    1. Looking across the river to give a sense of how broad the river is
    2. Looking upstream, showing the river surface, shoreline sediments, and vegetation
    3. Looking downstream, showing the river surface, shoreline sediments, and vegetation
    4. Looking straight down onto a location with representative shoreline sediments
  2. Please confirm that all fields on the paper metadata sheet are filled out prior to leaving the field, and take a picture of the data sheet. This picture will also be uploaded.

After samples are collected, transport them to a refrigerator, keeping them on wet or blue ice during transit:

  1. Place the ice pack in a freezer
  2. Place all samples (all vials and the filter preserved in RNAlater) in a refrigerator. Please don't use a freezer for these as they may burst.

Metadata:

  1. After the samples are stored in the refrigerator, please enter the metadata on the Metabolite Biogeography Metadata form If there is difficultly in accessing the form, please email for a copy of the form.
  2. Please enter as much information as possible. The form indicates what information is required and what is optional.
  3. The form also provides instructions on how to submit the field photos and the stream gauge data. Please name all files with the Sample ID. For the photos, please follow the naming convention described in the metadata form (e.g., S000001-across, S000001-up, S000001-down, S000001-sed, S000001-data, S000001-pH).
  4. For the stream gauge data, it is critical to follow the instructions carefully to ensure consistent data format and consistent units across sampled locations. If the closest stream gauge provides downloadable real-time data, it is acceptable to provide the url and the PNNL WHONDRS team will download and format the data.

Shipping:

  1. All shipments should be stored in the refrigerator until they are shipped on a Monday or Tuesday. It is critical that overnight shipments be made on Monday or Tuesday. Please don't ship later in the week. This is important in case there are shipping delays. If it is impossible to wait until a Monday or Tuesday, please contact prior to sending.
  2. It can be useful to remove materials from the refrigerator and pack the cooler as close as is reasonable to the time FedEx will ship the package. This minimizes time spent out of the freezer.
  3. When you're ready to ship, place the glass vials back in the vial holder located in the cooler and place the vials and the vial holders into a ziplock bag. Unless a vial broke, please include all four vials. This is important to make sure the vials don't break due to rolling around during shipment. If there is a broken vial, please do not send it back; instead find some material to place in the vial holder to take the place of the missing vial.
  4. Inside the cooler, also include the unused filter, unused needle, measuring tape, and absorbent padding that came with the supplies. Pack those materials into the cooler and then place the frozen ice pack on top.
  5. Put the lid of the cooler on and then place the paper metadata sheet on top of the lid. This will keep it dry.
  6. Please don't return gloves, the syringe, or the used needles. Please dispose of the used needle in an appropriate sharps container.
  7. Tape the outer box closed. Please use enough packing tape to make sure it won't come open during shipping.
  8. Adhere the pre-printed shipping label to the outside of the box by removing the backing from the plastic sleeve that contains the shipping label.
  9. Drop the package off at FedEx, or have it picked up by FedEx.
  10. On the day you ship the package, notify that you shipped the package, and include the FedEx tracking number in your email. This is critical to guarantee sample integrity and timely delivery. The subject line of the email should be "SHIPPED SAMPLES - [unique sample ID #]". If you wait until the day after you have shipped the samples, the samples may have already arrived prior to our notification, which will increase the likelihood of delivery issues and potential warming.

WHONDRS