DFO eDNA field sampling guide

Alex: Hi, my name is Alex Price and this is Trisiah Tugade, and we are biologists with Fisheries and Oceans Canada. We perform on the water sampling for aquatic invasive species. Today we will show you how to perform environmental DNA sampling. It might look like a lot of steps, but don't worry: you'll quickly get the hang of it. You'll be using an eDNA sampler.

The eDNA sampler pumps water out of the water body that you're testing. That water then goes through a specialized filter that collects environmental DNA. These filters will be sent back to our lab for analysis to determine whether an aquatic invasive species is present in your water body. By taking part in this program, you are helping protect the Great Lakes for the benefit of all of Canada.

Before you set up your eDNA sampler, it's important to make sure you have all the other necessary equipment ready to go. You'll need:

• a sterile bucket that contains 5 sterilized aluminum filter housings for every location you plan to sample;
• multiple pairs of new disposable gloves;
• plastic bags with labels; paper envelopes;
• a container of silica beads; and
• sterile tweezers.

Two buckets of clean water for each sampling location.

The water should be deionized, distilled, or even bottled spring water. Municipal water can be used only if other options are not available. A dirty bucket or container to collect all of the used and contaminated supplies. These include all of the filter housings, gloves, or tweezers that were used during sample collection, and any other items that had been removed from the sterile container.

The sterile bucket we only want to take things out of. We never want to put things back in, as that can be a significant cause of contamination in our samples. A separate container for your samples. Pencils, pens, or markers and paper for recording information. A GPS if one is available. If a day of sampling will include visiting more than one site, additional items will include:

• a bucket of household bleach solution diluted to 25% to decontaminate field equipment prior to sampling a new site;
• clean water to rinse away bleach from equipment after decontamination;
• clean paper towels for cleaning and drying equipment;
• bags to collect garbage.

It is important that the buckets containing the sterile supplies and buckets containing clean water are kept as far away as possible from the used and contaminated equipment, as well as the cleaning supplies.

This is to minimize the risk of cross-contamination. For example, if the sterile supplies are kept inside your vehicle, the contaminated equipment and cleaning supplies should be kept in the trunk.

Here we have our eDNA sampler. And the first thing that I want to show you is the LCD screen. When you turn the unit on, the LCD screen lights up and takes you to the settings menu. The keypad has different arrow buttons and the enter button, which help you maneuver through your settings before you run your sample. We will go through all of the settings later in this video. On the top of the backpack is an inlet socket, and this is where the staff is attached to the unit. And where the water will be pumping into the unit.

Beside the inlet socket is a Y-trap, which has a screen in it that prevents large particles from going into the water pump. Throughout the season, you will need to clean out this Y-trap to ensure that water can flow freely through the system. We also have the status light, which flashes on and off when a sample is being taken. And this is to ensure that someone doesn't turn off the backpack or take the filter housing out of the water while the backpack is pumping water. On the side of the unit, we have a speaker, which has three sound indicators built into it. The first sound is a constant tone that you can hear when the pump is in use. The second indicator is a higher beep, that indicates that the sample has been completed and the filter housing can be taken out of the water.

The third indicator tells you that the pump is working beyond its capacity and that a filter might have to be cleaned or changed. When this alarm goes off, the pump will automatically shut off, and this is the time where you need to check your filters to ensure they're not clogged. Now we're going to look inside the unit. This is where the battery is housed. The battery goes inside and is locked in place. And this is where you connect the battery via a small cable.

You always want to make sure that when you're moving between sites, you disconnect the battery to prevent damage to the unit. If you're traveling long distances, you should remove the battery from the unit altogether.

This is the on and off switch. Beside the on/off switch is the pump fuse. This fuse protects the pump from overheating or short circuiting if it is using too much energy. And this is a USB connection, which allows you to connect a computer to the unit. This is a feature that we don't normally use for our sampling procedures.

The serial number is located in the bottom righthand corner. This is unique to each backpack unit and is used when you're calling to have the unit serviced. Now we're going to look at the back of the backpack. The backpack's equipped with a sturdy frame, which makes it easier to carry the backpack from site to site. It also allows users to walk through shallow water and collect samples without having to put the backpack down. On the bottom of the backpack, there is a connection where you can add a block heater, which prevents your sample from freezing in colder temperatures. Our sampling takes place during the warmer seasons of the year, so a block heater is not necessary for our sampling. There is also an outlet socket here where you connect a hose and allows your wastewater to drain away from the backpack.

This is the staff that's included with the backpack. It's comprised of a hard housing with a hose that fits in the middle of it. On one end of the staff, there is a filter housing connection, and this is where the filter housing is connected and goes into the water to take your sample. Beside the filter housing connection is a check valve, which allows water to be pulled up through the hose and past the filter, but stops water and eDNA from leaking back out when the pump is turned off.

Located on the end of the staff are several extension latches, which allow the staff to be extended. This allows the filter housing to reach the water without the user having to go into the water to collect the sample. The staff can be extended up to 15 feet in length. On the other end of the hose, there is a quick connect, also known as the inlet coupler. It's used to attach the staff to the inlet socket on the top of the backpack, and this is where the water will be pumping into the unit.

This is the drain hose, which connects to the bottom of the backpack via the outlet socket. This is the tripod that's included with the backpack. It allows your staff to be extended into the water and stay balanced while you're taking your sample. On top of the tripod is a swivel bracket that helps position the staff when taking samples from the water and when draining the unit after a sample is taken. This is the filter housing that comes with your backpack unit, and it's comprised of two sections. The lower section is where the water will come into the housing. On the top part of the lower housing is an O-ring. Before you use the filter housing, please make sure this O-ring is not visibly worn, bent, or discoloured.

If it is damaged in any way, the bottom part of the housing may fall into the water during sampling. On the top part of the housing unit, there is a quick connect that attaches to your backpack staff. Underneath the housing is a backing screen and a rubber gasket, and this is where you will be placing your filter.

So, Trisiah and I are going to be putting a filter into the filter housing. When installing a filter, you must first make sure you're using a fresh pair of sterile gloves. I'm going to open the filter housing, and remove the rubber gasket. Trisiah is going to install a filter membrane into the filter housing using sterile tweezers. Once the filter membrane is installed, we're going to place the rubber gasket onto the filter, and we are going to close the filter housing. We're going to do that by pushing firmly on both pieces, not twisting, as this can rip and tear the filter membrane. And now we are ready to attach this to our staff and take a sample.

So, when you're choosing your site, some things that you want to look for is accessibility, so how easy can you get to the water's edge and back. We're looking for sites where water might be pooling, or stagnant water that might hold environmental DNA longer. We're also looking for flowing areas and rivers, where we think fish might be migrating up towards a suitable spawning location.

At each location you have chosen, you will be running the eDNA sampler 6 times. You're going to start with the pre-priming process, to prime the system with sterile water. This will be done without a filter. Then you're going to do a control sample, using sterile water and a fresh filter. After that, we're going to run it three times in order to get three replicate samples in the water body.

In total, the unit will be run 5 times with water, twice with sterile water, and three times using water from the water body. Finally, you will run the unit for a sixth time in order to drain the system of any remaining water from that site before you move to a new sampling site. At the next location or water body, the unit will be primed with sterile water again, flushing out any residue from the previous site. So we're going to get a total of 4 samples for each site. We're going to have one control and we're going to have three replicates. Please remember that you must use a new sterile filter head every time you take a sample. The more careful we are about avoiding cross-contamination, the more confident we can be in the results that we receive after the samples have been analyzed in the lab.

Trisiah and I are at a sampling site right now, and we're going to show you how to set up the eDNA backpack and take all of our samples. And we'll also show you how to tear down the sampling equipment. We're keeping the sterile equipment out of the mud and away from the water to avoid any contamination risk. So now we're looking for a suitable spot to set up the tripod where the backpack and the equipment's not going to get too muddy, where we can extend our staff so that it reaches the water. Next step is to set up the backpack, and it has a red hanger that goes onto the tripod and balances the tripod out, so when the staff is fully extended, it has a counterweight to hold it in.

Next, I'm going to insert the staff hose into the swivel bracket, and I'm going to pull the hose through. And I'm going to secure the staff onto the tripod. When handling the staff, it is important not to touch the brass connections at the end of the staff with your bare hands. We want to do everything we can to avoid contaminating the water during sample collection. If you need to touch the brass connections for any reason, you should always wear a fresh pair of gloves. Next, I'm going to insert the staff quick connect into the inlet socket on the top of the backpack and install the drain hose at the discharge outlet underneath the backpack.

I'm going to turn the staff around, and I'm going to extend the staff while Trish prepares the filter housing for the pre-prime. Trisiah will put on fresh gloves and take a sterile filter housing out of the sterile bucket. Keep in mind that there should not be a filter in the filter housing during the pre-priming process. Now that the filter housing is attached, I'm going to open the unit, and I'm going to connect the battery.

And I'm going to turn the unit on. [unit beeps] And the unit is going to go through its opening sequences. So now that I'm back on the main screen, I'm going to check the settings before we run the samples. So with the LCD screen showing settings, you click the enter button to continue. And the first option we have is to enable the depth mode. To change, you press the up arrow, and the options are yes or no. I'm not going to enable depth mode because our samples are taken at the surface of the water. And so we keep the depth mode as no. We press enter to continue to the next setting, which is the hose length. The hose length that we use for our sampling is 15 feet, but other programs use longer hoses if they're trying to get deeper in the water column. In which case, you would use the up and the down arrows to change the hose length in one foot increments.

Our hose length is going to remain at 15 feet. We're going to click the next button, which goes to the volume limit. This is the volume of water that is going to be brought up through your filter housing. We use 2 liters of water for each sample, but you can use the up and the down arrows to change the volume of water in .25 litre increments.

Once we have our volume limit set to two litres, we're going to click enter to hit the next setting, which is the pump speed. Using the up and down arrows, you can change the pump speed from automatic to half speed and to three quarter speed. For our purposes, we keep the pump speed on automatic, but you would use the half and three quarter speed if you're working in areas where there's lots of particulate matter in the water that might clog your filter faster.

We're going to click enter and go to the next setting, which is the pressure limit. The unit has a maximum pressure limit of 80 Kilopascals. If the water pump goes above 80 for an extended period of time, that's where you will hear the warning buzzer. And then the pump will automatically shut off to protect itself from damage. Our water pump pressure is going to be 80 Kilopascals, because we are typically working in shallow, turbid waters, and we are going to have filters that are going to become clogged with particulate fairly quickly.

But you can change the pressure in the unit by using the up and down arrows, and it goes in 5 Kilopascal increments. We're going to click enter to go to the next setting, which is enable heater. This is the block heater that prevents their sample from freezing when you're sampling in colder conditions. We use the up arrow to switch between enabling the heater on or off.

We sample in the warmer seasons of the year, and so our block heater is going to be turned off. The next setting allows a remote switch to be used. When you have the backpack on your back and you're traveling through a water system, instead of taking the backpack off to start your sample, you have a remote switch that the backpack user can press to start a sample.

Again, you use the up arrow to enable it or disable it. We are not going to use the remote switch for our purposes, so we keep that as no. The next setting that we can change is do we want the pump to shut off automatically once the sample has been completed. And we use the up arrow to switch if we want to do manual. For our purposes, we're going to have the shut off set to automatic. Next, this is the setting that enables the pre-prime of the unit. Use the up arrow and you can change between off and on. We want to keep enable pre-prime on, and what that does is it allows all the air to get out of the system before we take a sample.

It helps us have more accurate volume measurements and it also clears out the backpack of any contaminants before we are at a new site. And now we can start the pre-priming process. It is telling us that the pre-prime process is ready. So we're going to use clean water only. So this is the clean deionized or distilled water that we bring separately in a bucket.

We're not going to have a filter inside the housing. So Trish is going to move the deionized clean water bucket underneath the filter housing. And I'm going to lower the housing into the water. So I want to make sure that I'm only submerging the filter housing because no parts of the staff itself should come into contact with the water.

You want the drain hose to be away from the unit to avoid the unit getting wet and contaminated while making sure that it doesn't empty back into the water that is being sampled. Once the filter is submerged, I'm going to press enter to start the pre-prime process. And what the pre-prime process is doing is getting any air out of the system.

And what that means is we have a steady flow of water coming through the system which is going to translate to a more accurate volume measurement when we are taking our sample. The status light is flashing, indicating that the water pump is currently on and pulling water in.

So this can take up to a minute for the water to get into the entire system.

That buzzer has indicated to us that the pre-priming process has been completed. Once the pre-prime process is complete, I'm going to lift the filter housing out of the clean water, and Trisiah is going to remove the filter housing. The housing we just used for the pre-prime is now considered dirty and can be placed into the dirty bucket. So now we will run our control sample. For the control, we will use our other, second bucket of clean water that we brought with us. Trisiah is going to put a fresh filter into our new sterile filter housing.

Remember, use a new pair of sterile gloves when you are accessing sterilized equipment inside the clean container. If in doubt, use new gloves.

Trisiah: I'm going to prepare the filter housing. And it's important to note that whenever I'm touching anything within the sterile bucket, that it doesn't touch anything else outside of the bucket. As soon as it does, it becomes contaminated. So first I'm going to grab a pair of sterile tweezers, always doing everything with gloves when I'm working within this sterile bucket.

I'm going to open up the filter housing, and I'm going to remove the rubber gasket. I'm just going to place it down too. Take a piece of filter. Reinsert the rubber gasket. And then once that rubber gasket is reinserted, I'm going to reassemble the filter housing. Push firmly, don't twist. And now the filter housing is ready to be placed.

Alex: Trisiah is now going to attach the filter housing onto the staff and we are going to put it into our second bucket of clean water to run our control sample. It's asking if we want to push enter to start a new sample or if we want to push down to drain the system. Because we're doing our control sample, I'm going to press enter to start. The water pump is engaged and we are now taking a sample. So right now the screen is showing us the pressure that's inside the water pump.

It's showing us the temperature of the water. It's showing us the flow rate that the water is moving through the filter membrane, which is currently between 1 and 1.6 litres per minute. And lastly, we have the quantity of water that's gone through the filter membrane. Once the quantity reaches 2 litres, the sample is going to automatically stop and we're going to be able to take that filter out and put it into our coin envelope.

It's important to do a control sample at each site because it tells us whether or not our equipment has been contaminated or not. If a control comes back as a positive reading, we can't be confident that the replicate samples that we're taking in the water body is accurate. So this is a very important step, to do a control at each site.

Okay, so our control sample has been completed. The pump is now shut off. We collected 2 litres of water through the filter membrane. Our average flow rate was 1.6 litres per minute, and it took approximately 79 seconds. Now we'll lift our filter housing out of the clean water.

We will remove the filter membrane out of the housing without contaminating it and put in our coin envelope. I put on fresh gloves, so I'm going to be able to go into the sterile bucket and I'm going to grab tweezers and a new baggie with a coin envelope. I'm going to take the coin envelope out of the bag and I'm going to fold it open. And with the tweezers, I'm going to take out the rubber gasket. You can use the same tweezers for the filter paper, but make sure they don't touch anything else. When you're taking the filter out of the housing, you want to fold it in half once. This keeps the DNA on the filter. And we're going to put the filter membrane inside the coin envelope.

We're going to press it down. This helps to get the moisture out of the membrane. We are going to fill out the label on the coin envelope. Because this is our control sample, we're going to put an X beside control. And we're going to put the coin envelope in a bag. We are going to fill out all the information on the bag.

The last thing we're going to do is fill the bag about halfway with absorbent beads. Those beads pull the moisture out of the sample and preserve it until it gets back into the lab where it can be analyzed. Do not put this bag into the sterile bucket, as it can cause contamination. We are going to put this bag into the separate container we brought for our samples.

The filter housing is now considered dirty, so we want to put that into the dirty bucket along with our gloves and with our tweezers. So now that we've completed our control sample, Trisiah is going to prepare another filter housing with a filter membrane, with new sterile gloves and tweezers. And now we are ready to do our first sample in the water body.

We are going to move the filter housing towards the water. And now I'm going to extend the staff until the filter housing is able to reach the water's edge. I'm going to lower the housing into the water. I'm going to press enter to start. And we're going to repeat what we just did for the control sample.

Once the backpack has completed its sample and shut off, we are going to pull the sample out of the water. We are going to pull the staff closer to us so that the center of gravity is closer to the backpack.

I'm going to move the staff over to Trisiah, who's going to once again remove the filter membrane, fold it in half, place it in a coin envelope, fill out the label with all of the information. She's going to place the coin envelope back into the bag, fill out the information on the bag, fill the bag about halfway with absorbent beads, and then place the bag into the separate container we brought for our samples. All while using a fresh pair of gloves.

We're going to repeat this another 2 times for each sample.

Okay, so now that we have completed our 4 samples, our control and our 3 replicates at this site, we want to drain all the water out of the system to avoid contamination from this site to the next site, as well as to get all the water out so that the users aren't getting wet. We're going to press continue. And we're going to push the down arrow to drain the unit of all the water. Before it starts draining the system,

it's going to ask us to invert the staff pole so that the filter housing connection is facing up. This is to allow the water to fall gravity down and out of the system easily and not overwork the pump. Next we're going to go to the settings. And I'm going to press enter. The water pump is turned on and it is now pumping all of the water out of the system.

This can take up to a minute or two. We know that the system has completed its draining process when the water pump is shut off and the flow rate has gone to zero. After that, it's going to ask us if we want to run the last settings, or if we want to go back into the settings menu and change any variables for the next site. In most cases, you will choose continue.

Now that we are moving sites, we want to ensure that we open up the backpack, we switch off the backpack, and we disconnect the battery for transport. I'm going to disconnect the staff and I'm going to take it out of the swivel mount. I'm going to be careful not to get any mud or any debris inside of the connection, as this directly links to the water pump inside the backpack unit.

Before we go to another site, we want to decontaminate the tip of the staff with a bleach solution. We have to consider this part of the staff contaminated because it goes into the water. And because we have our bleach solution back in our vehicle, we are going to place the contaminated part of the staff inside a bag to ensure that we're not transferring any contamination from the head of the staff to the vehicle or to any other parts of the equipment that we're using. Supplies used on this site should not be placed near other sterile supplies that may have been brought into the field for a day of sampling. Everything that was on this site should be considered dirty until it is properly decontaminated. And then it will be sterile and ready to use at our next sampling location.

The eDNA backpack, staff, and buckets should also be cleaned between sites and prior to putting them into a vehicle. But it's enough to wipe them down with bleach solution instead of soaking them. See the cleaning protocol for more information. Remember, we want to try to minimize contamination wherever we can.

Thank you for participating in the eDNA sampling program. We hope this video was helpful. For troubleshooting, please refer to the included manual.