CHUCK NEWELL: Well, we've already looked at various attenuation processes that could be important for MNA. Now we want to transition to a discussion of several newer methods for gathering lines of evidence that natural attenuation is occurring. So Dave, what do we have today? DAVE ADAMSON: Well, the first is compound specific isotope analysis or CSIA. And the second one is molecular biological tools or MBTs. You see a lot of other terms used to describe MBTs, things like biomarkers or microbial or genetic analyzes. CHUCK NEWELL: Well, so that's right. And we'll go inot detail on these methods during the second half of this week's lectures. But it's the isotope analysis that are the focus of the first few lectures. So one benefit of CSIA is that to provide evidence a degradation is occurring. Now, this can be really important at sites where the normal lines of evidence, like decreasing concentration over time or production of daughter products, they either aren't enough or they can't be done. So think about aerobic sites with BTEX or chlorinated solvents. You really can't demonstrate daughter product formation at those sites. DAVE ADAMSON: Yeah. And there was a lot of great research, starting in the late 1990s, that showed the CSIA was a powerful forensic tool for doing this. Then in 2008, an EPA publication basically told the environmental remediation community as well as the regulatory folks that CSIA is important and valuable. Not only that, it went into great detail about how it should be done and how to interpret the data. So let's start out by showing you that document, here it is. A lot of you are probably familiar with it already. But let me highlight one important passage from it. Stable isotope analyzes can provide unequivocal documentation that biodegradation or abiotic transformation processes has actually destroyed the contaminant. CHUCK NEWELL: Wow, it's a pretty strong statement, but I think it really highlights that isotope data can be a really powerful tool. But I want to back up a minute and ask a question. So what do we mean when we say stable isotopes? DAVE ADAMSON: OK, sure. So let's take a look at this from a chemistries perspective on the next slide. So this graphic shows that the stable and most abundant isotope of carbon, we label it C12, because that's its atomic mass, which means it has six neutrons and six protons. It's by far the most abundant isotope of carbon at 98.9%, but there are other isotopes of carbon, right Chuck? CHUCK NEWELL: That's right. You know, the isotopes have a greater number of neutrons, and that's what makes them isotopes. So here's C13 on the right side, which has seven neutrons instead of six. DAVE ADAMSON: Yeah. And these two are considered the stable isotopes, because they're not subject to radioactive decay, unlike C14. That one's not stable, which is why it's so useful for age dating and that sort of thing. But it's not so abundant, and it's difficult to measure. And for CSIA, we want to eliminate the effect of radioactive decay in terms of tracking these isotope ratios. CHUCK NEWELL: So if that's a definition for stable isotopes, then it's easy to imagine that the elements and their isotopes might be relevant from an environmental standpoint. So that's what's shown on this next slide. In each case, you've got an isotope that's lighter, meaning it has fewer neutrons and a lower atomic mass, and an isotope that's heavier, it has more neutrons and a higher atomic mass. Now, this certainly isn't an all-inclusive list, but it does highlight that there are a lot of options, because these are prevalent in the types of contaminants that MNA is really concerned with. We'll take a look at this list later in this lecture. DAVE ADAMSON: OK. And so now let's take a look at this from the standpoint of one of those contaminants to show why we call these compound-specific stable isotopes. Here's PCE, which has two carbons. Most of the time, these are both C12, but sometimes one of these will be C13. And it's slightly heavier, and that's shown on the right. It turns out that these look different to microbes who are thinking about degrading a bunch of PCE molecules. CHUCK NEWELL: Right. And then this isn't physically correct, but I think of those microbes, they're small guys, right? And it's easier for them to chomp the smaller molecules, those smaller atoms, and so the ones without that extra neutron it. DAVE ADAMSON: Yeah. And that's basically what's called a kinetic isotope effect that's going on. There's a preference to degrade the lighter isotopes. So it's more rapidly degraded in this case to TCE. CHUCK NEWELL: And this preferential degradation causes the remaining PCE to become enriched and become heavier. Overall, we call this process fractionation, the change in the isotopic ratio due to degradation. DAVE ADAMSON: Yeah. And this is the process that we're really interested in. It's important to point out we're usually talking about bulk isotopic fractionation, even though fractionation is usually position-specific. Think about TCE being degraded to DCE. It's usually working on a specific bond, such that cis-1,2-DCE is the predominant product. CHUCK NEWELL: OK. Well, now maybe we'll talk about how we express this isotopic data. DAVE ADAMSON: Sure. Although I do have to admit it can get a little confusing for non-analytical chemists like myself. CHUCK NEWELL: And me. DAVE ADAMSON: Yeah. Fair enough. So anyway, the important thing is to remember that we're interested in this isotopic ratio or R between the heavy and the light isotopes in a compound. For carbon, that's C13 to C12. CHUCK NEWELL: Ah, but there's other convention that allows you to correct for that standard ratio of the isotopes, right? DAVE ADAMSON: Yeah. And the ratio that's usually reported accounts for this. So you're basically dealing with standard value for R or the standard ratio in terms of what you would expect for carbon-13 to carbon-12. There's this other convention that you report these on a per mL basis by multiplying the ratio by 1,000, so sort of like a percentage. But because the ratios can be really small, it's a little easier to work with. And finally, we use this del symbol to designate that the ratio has been corrected by both the standard, and it's using this per mL as the units. CHUCK NEWELL: OK. So if I see this del symbol out in front of the isotopic value, like carbon in TCE, I know we're using this convention to quantify the ratio of C13 to C12. DAVE ADAMSON: Exactly, exactly. Another important thing is that, for carbon, these ratios tend to be negative. And that's shown here on this next slide. Basically, you're showing the isotopic ratios associated with various products. CHUCK NEWELL: OK. So I see the isotopic ratio on the y-axis for all those different chlorinated solvents, and they're all negative. And I guess that's because these are manufactured products, and they would be expected to have a little less C13 than they would have naturally occurring. I know that these values, which in this case were compiled in that 2008 EPA document, can be very valuable in terms of source identification but also to determine if degradation is occurring. DAVE ADAMSON: Right. And that's because fractionation increases the isotopic ratios. So for these compounds, it means they become less negative as the relative amount of C13 increases. We'll talk a lot more about this in the next lecture when we talk about interpreting these data. And so what we need to remember is that the change in isotopic ratios are assumed to follow the Raleigh equation, which is shown here in the classic form. Chuck, you want to describe this equation for us? CHUCK NEWELL: OK. I'll do my best here. So if a compound is being degraded, it's undergoing this fractionation, the isotopic ratio R at any time can be calculated from the initial R value, the fraction of the compound remaining f, and this alpha term, which is called the fractionation factor. DAVE ADAMSON: And we rearrange this equation slightly when we perform CSIA to use the del convention that we showed earlier. So let's take a look at that here. This is what it looks like. We have the del value of the compound at any time being equal to the initial del value plus the product of the fraction of the compound remaining, that's that f term, times this epsilon value, which is termed the enrichment factor, sometimes fractionation factor. It's a re-expression of the fractionation factor that we showed on the previous slide. CHUCK NEWELL: OK. So some, maybe, unfamiliar terms here. How do we know what values to use? DAVE ADAMSON: Well, that's shown here. For the del at time t, that's what you're actually analyzing, so you're grabbing the samples, you're measuring what you see in those particular samples. That enrichment factor, usually that's something that's available in the literature. You're relying on somebody else to get that value for it. You could get that in your lab yourself if you had enough data, enough time to run those. The fraction of compound remaining, that's what you're trying to determine in a lot of these cases. And then you're at your initial del, you can either use that from a value from the field, from your field data or you can use estimates for what might be present in a manufactured product. CHUCK NEWELL: OK. So this is the Rosetta stone explaining how all this stuff works. But where you get the data to park in here? DAVE ADAMSON: Yeah. So it's actually not all that much different than what most people are used to in terms of collecting environmental data. In this case, you're mostly collecting groundwater samples. You're using conventional methods. You're filling up 40 mL VOA vials with a preservative and sending them off to a lab. Some labs will also do soil samples, assuming they're set up to do the extractions. And again, if you're interested in the methods, the 2008 EPA CSIA guidance lays out all of this stuff with a lot of additional details, of course. CHUCK NEWELL: OK. So that's pretty, that's good. How about the analysis part, is that easy or is that hard? DAVE ADAMSON: A little bit harder. So in this case, we're talking about pretty specialized methods. But this diagram over here lays out the basic method for carbon isotopes, which is using a GC method combined with an isotope ratio mass spectrometer. The setup's not really as common there's only a few labs, at least commercial labs, that have really specialized in doing this sort of thing. Then again, you've also got this idea, you've got initial analyzes, few hundred dollars usually per isotope at a minimum, plus you're going to need to analyze for the concentration as well. So there's additional cost with that. And some labs may ask for larger sample volumes for various isotopes, just due to detection limits and that sort of thing. CHUCK NEWELL: OK. But the good thing is that even though these methods have been adapted relatively recently, there's already lots of options in terms of what isotopes and what compounds you can analyze. That's shown on this list here, right? These chlorinated solvents are pretty common, but what else do you have? DAVE ADAMSON: Well, there's the petroleum hydrocarbons and fuel oxygenates, things like MTBE are pretty common to use. In all these cases, you're usually measuring carbon and hydrogen isotopes. And then you've got compounds 1,4-dioxane, which has pretty recently been added to this list. And there's a lot of interest in showing attenuation for this compound, because it's harder to use some of the more conventional lines of evidence for MNA. All right. Well, that's the principal. So let's wrap up with some of the key points. The measurements of the ratios of stable isotopes and contaminants can provide information about whether degradation has occurred. CHUCK NEWELL: OK. And these compounds become enriched in heavier isotopes as this degradation occurs, because the lighter isotopes are being preferentially used. This process is known as fractionation. DAVE ADAMSON: Yeah. And stable isotope analyzes are available from commercial labs and can be performed on groundwater and soil samples.