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Presenter

Leo Rivera operates as a research scientist and Hydrology Product Manager at METER Group, the world leader in soil moisture measurement. He earned his undergraduate degree in Agriculture Systems Management at Texas A&M University, where he also got his Master’s degree in Soil Science. There he helped develop an infiltration system for measuring hydraulic conductivity used by the NRCS in Texas. Currently, Leo is the force behind application development in METER’s hydrology instrumentation including HYPROP and WP4C. He also works in R&D to explore new instrumentation for water and nutrient movement in soil.

Transcript

LEO RIVERA 0:10
Okay, today we’re going to talk about methods of sampling and analyzing soil pore solution. And so just give you a brief outline of what’s going to be talked about in this presentation. First off, we’re gonna go into the importance of analyzing and sampling soil pore water. Then we’re gonna go into tools for sampling soil pore water, and we’re going to cover sampler types, material types and chemical suitability. We’re also going to go over vacuum systems and different extraction methods, and also important considerations for proper sampling. And then Chris Chambers will go into a section on analysis, going over common methods and sample preservation techniques.

LEO RIVERA 0:51
So soil water, why do we even care about soil water? Well, you know, one of the reasons we want to measure soil water is that it gives us the ability to measure solute concentrations in soil solution. And this can be important if you’re looking at plume assessment. It’s also important if you’re trying to understand leachate contamination of drinking water. An example of this is nitrates that could be leaching into the groundwater. Being able to sample a lot of different points can help us understand nitrate migration, and also other contaminants that move easily towards the groundwater that could potentially contaminate our drinking water. So sampling provides a strong benefit there. Also just helping to better understand the hydrologic cycle. And especially from a biology perspective, understanding what’s in the soil water helps us when trying to look at plant nutrient availability and how that’s going to affect plants, soil biology and microbiology and how things are changing over time, and just giving us a better understanding of the nutrient cycle.

LEO RIVERA 2:08
So first off, I want to go into — could talk about soil water, tension, suction, and potential and what the differences are. And so let’s go over some terminology in units. Aoil water tension and suction are used interchangeably. And essentially, they’re used to describe how tightly bound water is in soil. Soil water potential is essentially the negative of suction. So common units of that are kilopascals, or hectopascals and the way you can look at this is 100 kilopascals is equal to one bar of tension, if you’re used to using bars as your unit for suction or water potential. Another definition I want to go over is field capacity and just get that out of the way what field capacity is. Field capacity is, the definition for field capacity in the US is 33 kilopascals of suction. And essentially, this is the point that they have defined, where whether it’s wetter than field capacity, water will drain through the soil easily. And when you reach that field capacity point, it begins to retain the water and resist losing it due to gravity. You can still get water migration, even past fuel capacity when you get to gradients in water potential, but that’s a different story and not all that important for today’s topics.

LEO RIVERA 3:43
So when we’re looking at various approaches for soil water sampling, there’s a few different tools available, especially when using controlled suction or discontinuous suction. There are porous cups and plates available that you can use for sampling soil pore water. There’s some example pictures here of different pore water samplers and some different suction plates that can be used for sampling in situ. Suction is applied to the cup through a tube extending to the surface and that’s how we pull the sample through these materials. And accumulated soil water is pulled to the surface through the suction tube or through a companion tube — or sorry, there’s another technique where you can have soil solutions stored in a shaft and can be pushed or pulled through a secondary tube. And you can see some examples in the stainless steel pore water samplers. So some other things about suction cups and suction plates. With suction cups you have the option of shaft storage. And the nice thing about suction cups is installation is very simple. You can do just a simple augered hole and install this suction cup down the hole. And if you use the right size dogger, for example, we use an auger with a tapered head, that way, when you get the suction cup pushed down to the very bottom of the hole, it has better contact with the soil. And then with suction plates, the installation is going to be a little bit more labor intensive. You would have to likely either dig and auger a trench and come in through the side and install the suction plate that way. Or you could install the suction plate in the hole and then repack the soil over it, but then you would lose the intact soil. And that’s not always a good thing. And the nice thing about suction plates is unlike suction cups, suction plates can be used to measure drainage rates if they’re used with tensiometer controlled suction, and we’ll talk about that a little bit later.

LEO RIVERA 6:02
So when looking at suction cups or suction plates, one of the things that you have to consider is material of the cup. There are a few different material types available. And here’s some picture examples of them. There’s silicon carbide, aluminum oxide ceramic, there’s a polyethylene nylon membrane type sampler, borosilicate glass, and then there’s stainless steel. And each material type has a specific range of chemical suitability, and that is going to play a major role in your pore water sampler selection. So before making these selections, you really need to research what you’re trying to sample for and how the sampler is going to affect that. So here’s just a short list of some of the different suitabilities for the different types. So, with the aluminum oxide ceramic, you can typically sample for nitrates in common organic and inorganic substances. It does have a fairly limited range of chemical suitability, there are some compounds you could have issues with due to bonding with the ceramic. Another type is the polyethylene. And the polyethylene is especially suitable for heavy metals. So typically anytime I’m talking with somebody who’s interested in sampling for heavy metals, we typically lean towards the polyethylene nylon membrane. Now that one does have its limitations. Due to the nature of the material, it can only pull water samples in the zero to 20 kPa range. When the vacuum goes higher than that, it stops pulling water in although you haven’t reached actual air entry point of the material. Borosilicate glass is suitable for phosphate and dissolved organic carbon and a few various other compounds. Stainless steel is suitable for most organic and inorganic compounds. And then there’s the silicon carbide is probably one of the newest materials available on the market. And it is less absorbent or desorbent, than ceramic or borosilicate glass. And of all the materials, it probably has the broadest range of chemical suitability. And there’s still some more work that needs to go into its suitability with heavy metals and a few other compounds, but by far, the silicon carbide has the broadest range of chemical suitability.

LEO RIVERA 8:49
So again, when looking at porous cups or plates, remember the porous cup must not affect the solution chemistry or if it’s going to, you need to understand at what level and if there’s any treatments you can do to the cup to help reduce the effect it will have, got to think about sorption of ions and organics and metals. Remember, there is no universal material. Although the new silicon carbide cup from UMS is a big step forward, there’s still work that needs to be done to understand its full range of suitability. And it’s always good to look back into the literature to determine suitability. And there was a great paper that’s referenced here below by Weihermuller. And that’s a good place to start if you’re wanting to look at suitability of different compounds with different materials.

LEO RIVERA 9:45
Alright, so now let’s go ahead and move into vacuum systems and extraction methods. And so we’re going to focus on three different types of methods. We’re gonna focus on the discontinuous sampling method, where in this type you would usually use a hand operated vacuum floor pump or portable electric vacuum system. Then we’ll also focus on the continuous sampling method, where you would use a permanently installed vacuum station for continuous use. And then we’ll also focus on tensiometer controlled extraction, which goes along with the continuous sampling, but then using a tensiometer to vary the vacuum levels of soil water tension or soil water potential changes.

LEO RIVERA 10:33
So, with discontinuous sampling, it’s by far the simplest method. You essentially would just come out, maybe once a week, or maybe after an irrigation event, depending on your project, and apply a vacuum to the bottle, and essentially just walk away and come back maybe 12, 24 hours later, depending on how wet the soil is, to collect your sample. The applications of this are kind of just your typical qualitative analysis of soil water. And the benefits of the discontinuous sampling approach is it’s low cost and easy handling. And so definitely just the simplest technique to use, and you don’t have to worry about protecting electronics or anything when you’re out in the field — or about leaving electronics protected out in the field for long periods of time, because you’re just coming out periodically. The limits of discontinuous sampling, it is discontinuous. So because of that, you could miss events. And also, the sampling is undefined. You’re essentially, you’re guessing what level of vacuum to apply. So you’re gonna say, Okay, I’m going to set the vacuum to 50 kilopascals. And essentially, it’s going to just start pulling sample in, hopefully, unless the soil is too dry, and the vacuum will slowly begin to dissipate as it pulls sample in. And so it’s not very constant, and you really aren’t going to know how much solution you’re actually going to pull in.

LEO RIVERA 12:13
Now, the continuous sampling method, with this method, you have a constant vacuum applied or continuously maintained, using a vacuum system that’s meant to be left out in the field. And applications of the continuous sampling method are primarily for long term monitoring projects. If you have a site that you’re continuously having to monitor for any type of contaminants that could be moving, any studies on leachate, especially some compounds are very slow moving, and so it’s good to have continuous extraction, so you don’t miss when those compounds are coming by instead of having to guess when you want to come out for your discontinuous sampling approach. And depending on the vacuum that you apply, there is extraction from a certain pore size. And I think this primarily more so actually applies to the tensiometer controlled extraction, and we’ll talk about that here in just a minute. One of the benefits of continuous sampling is it’s defined sampling. You’re always sampling over the entire period of time, you’re not missing anything. And so you know that and it just, I think it helps make the projects a little more robust. Now, one of the limits of continuous sampling is the constant vacuum ignores changing soil water tensions. And if you have too high of a vacuum, you can have issues with carbon falling out, which can be an issue. So those are things that you kind of need to consider.

LEO RIVERA 14:09
So now, tension controlled extraction. When doing tension control extraction, you have a tensiometer that’s constantly measuring the soil water tension, and then the vacuum system is monitoring that and changing the level continuously as the soil water tension changes. The benefits of this, there’s constant sorption and filter effects. When you’re doing the, if you were to do the continuous extraction without the tensiometer control, you can get various filter effects depending on how the water potential is changing. And this is essentially what causes memory effects in your sampling. And so this can affect your actual solution chemistry. Some of the limits of tension controlled extraction is it samples from various pore sizes depending on the current vacuum. So, if the soil is really wet, and you’re only applying say 10 kilopascals of suction, you’re only going to be sampling from the very large pores and not the very small pores. And that will change as your vacuum level changes. But that is something to be understood. And here’s kind of an example setup of a tension controlled extraction unit. You have your vacuum system, your tensiometer. And then you have a buffer bottle in line between the vacuum system and the rest of your bottles and samplers, and you’re going to want to have a bottle for each sampler, because you don’t want to have, obviously you don’t want to mix solution from different samplers because you won’t know the chemistry at the different points.

LEO RIVERA 16:09
So we’ll go ahead and go into some temporal considerations. Spot measurements with the portable pumps. So if you’re doing the discontinuous sampling approach, only gives you a brief snapshot. And then again, if you’re only using a static vacuum, even with the continuous controlled extract extraction, the measurement is weighted heavily toward wet time periods.

LEO RIVERA 16:41
Using the controlled vacuum with the tensiometers. The pumping system is controlled by measurement of soil tension. So here’s an example graph, where you can see water potential measurements and the vacuum level changing, and then you see a drop, when we get a rainfall event, in the suction and — or in the water potential, and then the vacuum level also drops at the same time to maintain just a slightly higher vacuum than what the water is being held in the soil. And the nice thing about this is a representative sample is collected over time. So some considerations that you need to understand when looking at vacuum and how much vacuum you need to apply for soil water sampling. The vacuum that’s applied must be greater than soil water tension. So has to be at least slightly greater. An example is if the soil water tension is 10 kilopascals, your vacuum has to be at least one to two kilopascals higher to really be able to actually pull a sample in. So it always just needs to be slightly higher than how tightly bound the water is in the soil. You also want to think about the air entry potential of the porous material that you’re using, so the ceramics. So various ceramics do have different air entry points. And there are certain ceramics that are designed for different applications. So for example, if you’re doing more of a leachate or drainage application, then the ceramic you’re going to use is going to have a lower air entry point, closer to 10 kPa, because, one, they’re only designed to measure drainage. And in order to be able to handle the flow rates that you’ll see at the really wet end periods, they have to have a higher flow rate, and so in order to do that, that reduces the air entry point out ceramic. Other ceramics like aluminum oxide and silicon carbide, their air entry point ranges from 90 to 100 kPa. And with this higher air entry point, you can measure resident soil water, but it is slow. So the flow rate into these higher air entry point ceramics is going to be much slower.

LEO RIVERA 19:15
So you know, some other things to consider, really have the right tools for a successful sampling, setup, and experiment. There’s some things that you need to think about. One, creating a good seal is crucial. If you don’t have a good seal with your vacuum system, especially if you’re doing continuous or even discontinuous, you’re not going to be able to successfully pull in soil water samples. So making sure you have a good seal. Proper bottle selection is very crucial. I’ve run into people who oftentimes like to use older bottles that they have lying around the lab that might not be in very good shape. And they run into issues where they cannot create a seal, have a good seal and get a good vacuum. And so they wind up having issues pulling samples, and so it’s important that when you’re designing the system that you use proper bottles and also good connections. If one of your connecting point tests has a leak, again, you’re going to have issues maintaining vacuum and pulling samples. Other considerations, you will also want to protect the sample from the sun. You don’t want it to begin getting heated up, because you can get chemistry changes due to that. There are underground storage boxes available that can help protect the soil water samples, and it can also help maintain a more constant temperature for the sample, closer to that of what it is in the soil. There are also samplers with shaft storage, which are also a good option.

LEO RIVERA 20:59
Then some other considerations for the vacuum system, and primarily vacuum system protection, is you want to protect against overflow. So especially, one, you want to protect water from getting into the vacuum system. You also want to protect water from getting into different sampling bottles to prevent cross contamination. So here’s a picture right near the bottom of a Teflon stop that will actually seal up if it gets wet, and cut off the vacuum to the bottle. And then once it lets back down and is able to dry out, it will begin applying vacuum again. So these are good tools to use when setting up especially a continuous and tensiometer controlled system.

LEO RIVERA 21:49
Some other considerations. Over time, the pores for samplers can begin to clog. Fine particles, especially if you’re working in a clayey soil or a very silty soil, fine particles can clog their ceramics over an extended period of time. And I’m talking, this is usually over a couple of years. So if you’re really trying to set up a long term monitoring project, this is something that you’ll need to consider. What helps prevent this, one, you can prevent this with flushing the material out periodically. But also, maintaining the lowest possible vacuum will reduce the clogging rate and help prevent them from clogging over time. And so using tensiometer controlled extraction also helps in preventing pore clogging.

LEO RIVERA 22:40
Another thing you have to think about is level differences. If you are sampling from a certain depth, you need to make sure — you need to understand that you have to overcome that depth in order to actually be able to pull the sample up to the surface. Another thing is if you have a setup where your sampler is actually at a higher point than your bottle, then you will actually run into an issue where you’re getting a hanging water column inside of those tubes, actually, so you’ll have a higher tension being applied than should be, if you have this type of situation. So you’ll need to consider that when you’re deciding what vacuum levels you need to apply or when you’re setting up your vacuum systems and what level of over vacuum from the soil water potential that you want to apply. And just a good reference, one kilopascals approximately 10 centimeters — or approximately for 10 centimeters of level difference, you need to apply one kilopascal of suction. And another consideration if you’re trying to sample from very deep, water will begin to cavitate at around 85 kilopascals. So it’s going to be around, if you’re trying to sample deeper than eight meters, you could begin to run into issues pulling sample to the surface.

LEO RIVERA 24:17
Ready to go? Okay, good.

LEO RIVERA 24:21
So, some other considerations: flow rates. Remember the maximum flow rate that you can see from a sampler in free water with a vacuum of 50 kilopascals is approximately five milliliters per 10 minutes and this is in the higher air entry point ceramics, so around 90 to 100 kPa air entry point, and kind of a minimum in a sandy loam soil with 50 kilopascals of suction and so this is not going to be a completely saturated soil. You’re looking at around five milliliters per hour. So if you’re doing discontinuous sampling, you need to remember that when you’re making your decisions on when you’re going to apply your vacuum and when you’re going to come back and collect a sample, especially depending on how much sample you actually need to be able to collect for analysis. And if you’re doing analysis that requires high or a lot of sample, then you might want to consider the continuous or tension controlled extraction, because you’re always pulling a sample and you have a better capability of pulling in more sample and an opportunity to get more sample for your analysis. And sampling is only achievable if soil tension is lower than the vacuum applied. So if you’re working in too dry of conditions, then especially this can be an issue in sandy soils, because you go from a point where you can collect lots of samples to a point where you really can’t collect a sample very easily due to the soil moisture characteristic curve of sandy soils. So that is something to consider. But just remember, you’re not always going to be able to pull a sample if it’s too dry in your field. And so now, we’ll go ahead and move on to the analysis section. And Chris Chambers will take over from here.

CHRIS CHAMBERS 26:19
Thank you, Leo. Now that Leo has given us some methods for getting soil out of our pore spaces, I’m going to take over and talk about some ways to turn that water into data. This is by no means comprehensive, every single method out there that you could use to analyze your soil pore water. Mostly, we just want to give you some guidelines and put you on the path to finding out the things you need to get good data. One of the most important things is know your substrate. What kind of compound are you looking at? Most analyses are substrate specific. There aren’t very many places where you can just throw everything into some type of machine and then the data magically comes out. There’s usually more than one way to get the information that you want. And there’s usually some pros and cons with each method. There are some considerations that need to be made regarding sample preservation and collection. I’ll go over a few of those. And you also need to think about the difference between what you’re putting in — or what you’re pulling out of the soil and what you’re putting in your analyzer. So we’ll talk about some things to consider there as well.

CHRIS CHAMBERS 27:36
Okay, number one: know your substrate. What equipment do you need? How does that equipment analyze it? What precautions do you need to take with both your sample preparation and your sample storage? And what preparation do you actually need to do to turn that jar of water that you bring in from the field into actual numbers? There are some common analyses that are done. Are you looking at pore water chemical properties, inorganic compounds, their assays for organics, and also more complex pollutants or contaminants from say, landfills or waste sites? Pore water chemical properties, these are usually some of the most straightforward analyses. And here we’re talking about soil pH or electrical conductivity. And these are generally the most straightforward properties. You may have very little sample prep, say for pH it’s, take your sample out, insert your pH meter into your sample jar, and then you’ve got a number. And electrical conductivity is usually pretty straightforward like that as well.

CHRIS CHAMBERS 28:48
Inorganic compounds. In agricultural settings, these are some of the most common soil pore water assays that you’re going to be interested in. We’re talking about ammonium, nitrates and nitrites, are examples of inorganic compounds, particularly very important nitrogen containing compounds in ecosystems. There’s several ways to analyze these, you can use a colorimetric analysis. These are generally automated, you can generally run a lot of samples through at once. And basically you’re just measuring the absorbance of certain wavelengths of light. Ultraviolet spectrometry is becoming more and more common. You scan many wavelengths of UV light. And the nice thing about the ultraviolet spectrometry is there’s generally no reaction necessary. There’s not a whole lot of sample prep you can do, and these field portable devices like this are becoming more and more common.

CHRIS CHAMBERS 29:52
Organic compounds. You usually have some very specific analytical procedures when you’re getting organic compounds. Dissolved organic carbon is one common method. Basically, it’s small carbon molecules that are dissolved directly in the water. One way to get total DOC, you would filter the particulates out, you don’t want any of that, and then remove the inorganic carbon, your carbonates, through titration. Carbonate is pH specific. So by getting the soil extract to the right pH, you can have all of your inorganic dissolved carbon come out in gaseous form. Then you oxidize what’s remaining and measure the CO2 that is given off. Gas chromatography and HPLC are some other methods that let you get at carbon in your soil pore water, and I’ll go into those in a little bit more detail later. Dissolved organic nitrogen has a very similar method. Again, you filter out the particulate organic n, and then oxidize it to NOx, and then you detect the NOx. Note, methods like this don’t let you get at specific compounds. You’re looking at basically any organic nitrogen that’s in it, whether it’s an amino acid, or urea or any other compound, you just kind of take them off.

CHRIS CHAMBERS 31:37
So pollutants, say pollutants and contaminants. This is where we get into some very complex methods for analyzing what’s in your soil pore water. Gas chromatography, mass spectrometry, and HPLC are two common ways to get at these. Usually there’s a specific procedure available. For example, if you’re looking at hydrogen cyanamide, you can detect it through an HPLC procedure. And these are generally available — standard procedures like this are generally available on the web. And hexazinone you can find through a gas chromatography procedure.

CHRIS CHAMBERS 32:18
So just to go into a little bit more detail about this without running through all the technical details. So you can identify quantities of specific compounds or groups of compounds. For example, if you wanted to measure all of the lipids that are in some type of sample, you could use this to break out specific lipids. The name pretty much says the method, gas chromatography, so you need to have small compounds that you need to get into gaseous form. So you volatilized the compounds and then extract and concentrate them. And as I mentioned, it generally only works on small compounds like monomers or small polymers, so amino acids, sugars, and saccharides, other things of that nature. You can generally only measure certain suites of compounds at a time. So if you’re looking for, say, all of your sugars and all of your amino acids, you would probably have to run two analyses. So you may need to collect enough sample to have separate aliquots to run on your different analyses. And this is basically what gas chromatography gives you. Where the peaks are, each peak is a separate compound within the suite of compounds. Now this might be all of the sugars, and we’re looking at the abundance of individual sugars within a sample.

CHRIS CHAMBERS 34:01
Okay, and these analyzers are generally very high cost and usually have a lot of maintenance that goes along with them. But there are labs out there that can perform these procedures if you don’t want to invest in the equipment themselves. High performance liquid chromatography or HPLC is a method that’s much like gas chromatography. Only you can use bigger compounds. You still get same plot with peaks on it, where each peak is a separate compound. There’s frequently some very time intensive sample preparation. Again, you have to extract and concentrate your sample, and then you’re derivatizing it into a compound that is going to allow it to be separated in the instrument. And as I said, it works on much larger compounds, so if you’ve got some of the larger pollutants or contaminants in an ecosystem, you would look at a HPLC procedure rather than gas chromatography.

CHRIS CHAMBERS 35:18
So now, there’s this question, is what your — so your pore water analysis isn’t always going to match up with the most common measurement techniques. I’m going to use one example, for ammonium. It’s been very common that ammonium has been analyzed through KCL extractions in the soil. That creates a much different environment than just sampling the pore water sample. So be aware that using some of these methods, you won’t necessarily get the exact same data as the historical data from KCL extractions.

CHRIS CHAMBERS 36:00
Now sample preservation, recall in Leo’s talk, you’ve got a sample bottle out there collecting water. Some methods you’re collecting your sample over just a matter of hours or minutes; some methods, you may have your sample bottle out there for days. And you want to do your best to preserve that sample and be aware of what can happen to your sample while it’s out in the field or being transported to the laboratory. Volatile compounds are one thing you need to be aware of. Ammonium is in equilibrium with ammonia, which is gaseous. So you need to, if you’re interested in ammonium, you have to have some way of preserving your ammonium in that sample bottle. Labile compounds, carbon compounds that are very easily broken down, very mobile, basically just like candy for microbes that may or may not be in your sample bottle. And microbial contamination is something that you do need to take into consideration, depending on what substrate you’re looking at. Bacteria in the soil can be .2 to 2 microns in diameter. And fungal hyphae can be down to 1 micron. And some of these can penetrate the pores in the sample cups.

CHRIS CHAMBERS 37:29
Okay, so we’ll talk about ammonium a little bit. This is a very specific problem. Ammonium is a very common fertilizer that is frequently leached through the soil surface. If you’re wanting to measure ammonium in your soil pore spaces, you’ll have to be certain that you are actually preserving the ammonium that you pull out of the water. So it’s in equilibrium with ammonia, which is, as I mentioned, gaseous, and how much is in which fraction — ammonium versus ammonia — is pH dependent. It’s pretty easy to calculate how much — what that conversion rate will be at standard temperature and pressure, but you’ve got a sample bottle with a vacuum on and your substrate of interest is actually in equilibrium with the headspace. And this is what we’re talking about. At low pH, most of the ammonium is going to be in the form of — most of this is going to be in the form of ammonium. But as you get towards more alkaline conditions, then you start to get more and more ammonia, and then that is going to be in equilibrium with your headspace. So at a pH of 9.23, almost all of the ammonium or almost all of this is in the form of ammonia, and it’s essentially just going to evaporate. And if you’re maintaining a continuous vacuum, say sipping periodically to maintain a constant headspace or a constant vacuum, or if you’re using tensiometer control, where you’re constantly having to maintain that vacuum, then you could be losing all of your sample out through the vacuum if your pH is too high. One thing you can do, a few drops of strong acid will bring the pH down enough, less than four, that all of it will be in the form of ammonium, and it’s not going to volatilize away.

CHRIS CHAMBERS 39:47
And then labile compounds, things that are very easily broken down by exoenzymes that were pulled into your sample cups or by microbes that may have entered your sample cups. This is also a consideration for sample preservation, particularly if you’re going to have your samples out, you’re continuously sampling, and you’ll have your samples out for days or weeks before you collect them. Enzymes themselves can break down very quickly, and that can change your pore water chemistry as well. So the sample cups range from anywhere from about .2 to 2 microns, and they can actually let some microbes into your sample jars. It depends on which cup you use. One possibility to keep this activity at a minimum, if you’re going to have your samples out there for a long time, is to add a few drops of chloroform. And that will effectively shut down the microbes and your sample will be kind of at a steady state. Low temperatures are also a good idea. It’s why we bury our sample boxes. You don’t want to create a sun fuelled incubator out there for your sample to get eaten up by microbes.

CHRIS CHAMBERS 41:05
So some guidelines, questions that you should be able to answer when you’re deciding how you want to analyze your sample. Know how much sample you need for your analysis. Know the concentration required, how much, what’s the lower detection limit of the specific method that you’re using? Are your — if you want to sample multiple compounds, are your different analysis compatible? Can you do multiple compounds in one sample, in one analysis? Or do you have to have separate aliquots to send to different analyses? And do you need to take special sample preservation precautions? These are questions that you should be able to answer before you set up your experiment or choose the lab that you’re going to send your samples to.

CHRIS CHAMBERS 42:01
One example, I’m just gonna use nitrate as an as an example. It’s a common agricultural pollutant, a very common fertilizer, and it leeches through the soil very quickly. Environmental regulations are getting more and more strict, so you do, many places, have to monitor nitrate that’s leaching below their crop zone. And this is a very good method to do it, is to use these pore water samplers. So one way to analyze nitrates is through a process called cadmium reduction. You generally need about five milliliters a sample, and not all methods are going to require the same amounts. These are just some typical guidelines for this one type of analysis. Your minimum concentration can be up to one microgram — or can be as low as one microgram. If you need more sample, you may have to take some steps to concentrate it if you have very low sample concentrations. Is it compatible with another analysis? Many detectors can actually sample ammonium with this analysis. So not all methods, but some can. And is there any type of special sample preservation? For just nitrate analysis, generally not. Bear in mind that if you do want to measure the ammonium that goes along with this, then you’ll have to take some pH control methods to measure the ammonium. Consequences of this method. It can’t distinguish between nitrate and nitrite. So if that matters to you, you’ll need to find a different method. Otherwise, you’ve gone through all this work, you get your analyses back from the lab. And oh, all of our NO3 is in with our NO2-. So these are questions that you need to answer before you set up your experiment and choose a method. The UV spectrometry, I believe, can actually separate nitrate and nitrite. So you’ll have to tailor your analysis to what you hope to get from your data.

CHRIS CHAMBERS 44:12
I’m going to talk just briefly about stable isotope analysis. It does come up occasionally and there are some things to consider when using pore water samplers like this for stable isotope analysis. And we’re talking about, specifically in this case, deuterium, oxygen-18, nitrogen-15 and carbon-13. You should be aware of possible fractionating processes, and this is going to factor into your sample preservation. For deuterium and oxygen-18, evaporative enrichment is an important consideration. If you have a discontinuous sampling method, your headspace is closed, you probably don’t have to worry about this very much. The ratio of 18O to 16O isn’t going to change very much because you’re not losing any oxygen or hydrogen. So you probably don’t need to worry about it. However, if you have a continuous method that’s occasionally having to pull more vacuum, you may be losing some of your water vapor that is a different isotope ratio than that in your actual sample, and that can result in fractionation that isn’t the same as what you pulled out of the soil in the beginning. Again, this can have an enormous effect for volatilization of ammonium. If your pH isn’t right, and again, if you’re continuously sampling where you’ve got to maintain that vacuum, you could be losing ammonium that is much much generally lighter than the nitrogen dissolved in your sample. Enzymatic processes can also can also change the stable isotope ratio of a sample. So it is important to also control that. If you’re using a label, say if you’re extracting some label that you’ve added in an experiment, then you generally don’t have to worry about fractionating processes. So that’s not really a concern, but you should be aware of sample bottle contamination in these cases. And that’s all that I have for you. We thank you very much for listening and we will take some questions here.