# Presentation: 429805

## Tracing sources of agricultural N using isotopic techniques:         the state of the science

- Carol Kendall
- USGS, WRD-National Research Program
- Menlo Park, CA
- (650-329-4576; ckendall@usgs.gov)

- December 2004

## My job description is “to develop new methods, new concepts, and new applications of environmental isotopes to solve problems of national importance.”

- I head a large project that uses our C-H-N-O-S-Cl-Br stable isotope capabilities, and our tritium and S-35 radio-isotopes capacilities, to study watersheds at scales ranging from small pristine watersheds in the Rockies, to large wetlands like the Everglades, to large basins like the Mississippi and San Joaquin River basins.

## This presentation will: 

**1)  provide a brief introduction to isotope fundamentals.**
**2)  review the basis of the original vivid debate about the use of nitrate isotopes to determine sources in agricultural basins. **
**3)  discuss how several new isotope tools can better provide critical information about sources of nutrients and associated organic matter, and**
**4)  show examples of the usefulness of these new multi-tracer approaches for quantifying nitrate and other agricultural products derived from different land uses.**

## Isotope hydrology/geochemistry textbook:  

**Isotope hydrology/geochemistry textbook:**
**Clark, I. and Fritz, P. 1997. Environmental Isotopes in Hydrogeology. Lewis Publisher (****http://www.science.uottawa.ca/~eih/****)**
**Streams/rivers/small watersheds:****  **
**Kendall, C. and McDonnell, J.J. (eds.), 1998. Isotope Tracers in Catchment Hydrology. Elsevier   (****http://wwwrcamnl.wr.usgs.gov/isoig/isopubs/itchinfo.html****)**
**Groundwater: **
**Cook, P. and Herczeg, A.L. (eds.), 2000. Environmental Tracers in Subsurface Hydrology, Kluwer Academic Publishers.**

## Slide 5

## What are isotopes?  

**Isotopes are part of an element that have different numbers of neutrons (but all the isotopes of an element have the same number of electrons and protons).**
**Example:****  ****The element N has 7 electrons and 7 protons.  Different isotopes of N can have various numbers of neutrons.  The two stable isotopes have 7 and 8 neutrons, respectively (****14****N, ****15****N).**
**What are stable isotopes?**
**Stable isotopes are ones that are NOT radioactive.**

## Basic principles behind the use of nutrient isotopes as tracers of sources and sinks:

**1)****	****Many different ****sources**** of nutrients have distinctive isotope ratios.**
**Many different ****processes**** (e.g., denitrification, nitrification) change the isotope ratios of the reactants and ****products**** such that the existence and extent of that specific process can be identified by measurement of the isotope ratios of the product and/or residual reactant.**
**In other words, different sources of nitrate and organic matter often have distinctive isotope “fingerprints” that can provide a better understanding of the system than just using ****mass balance “black box models”.**

**Basic principles behind the use of nutrient isotopes as tracers of sources and sinks:**

## Isotope Nomenclature

**A DELTA 15-N = +10 ‰**** ****means there is 10 parts per thousand (or permil) more 15-N in the sample than in the standard.  **
**A DELTA 15-N = -10 ‰**** ****means there is 10 parts per thousand (or permil) less 15-N in the sample than in the standard.  **

**Isotope Nomenclature**

**...****in other words......**

**Where ****Ratio**** is the ratio of 2 isotopes of an element, such as ****15****N/****14****N for nitrogen, for a sample or a standard.**

## Isotope Nomenclature

**Isotope Nomenclature**

**The standard for N isotopes is Air – which is defined as 0. **
**Other standards include:  the ocean (for water ****δ****18****O/ ****δ****2****H, and ocean carbonate (for ****δ****13****C).**

## People usually find the negative  values a bit confusing, but they are just a product of what material was chosen as the standard.

**Example:**
- For N, the ratio of 15N/14N in Air is about 1/250.
- If a sample has 1% more 15N than Air (=1/250), its ratio would be:  1.01/250.  This 1% corresponds to +10‰.
- If a sample has 1% less 15N than Air -- (=  0.99/250), this is -10‰.

## How isotopes are used to trace sources of nitrate:

**There are 2 stable nitrogen isotopes (****15****N,****14****N).  **
**	****Ratios of ****15****N/****14****N are reported as ********15****N. **
**	****(“******** values”, in units of permil = ppt = ****‰).**
**There are 3 stable oxygen isotopes (****18****O,****17****O,****16****O).  **
**	****Ratios of ****18****O/****16****O are reported as ********18****O.**
**	****Ratios of ****17****O/****16****O are reported as ********17****O.**
**Therefore, if different sources of nitrate have different ********15****N, ********18****O, or ********17****O values, we can sometimes determine how much nitrate comes from the different sources.**

## Fretwell’s Law

**To prevent upsetting reviewers’ stomachs and your own, take stable isotope data with a healthy dose of other hydrologic, geologic, and geochemical information. **
**Then, you will find stable isotope data very beneficial.”**
**	**
**	****(Marvin O. Fretwell, USGS, pers. comm. 1983)**

## Commonly Used Terms:

*heavy vs. light isotopes*
- 	the “heavy” isotope is the one with more neutrons; it is also generally the less abundant isotope.
*enriched vs. depleted*
- 	remember to state _what_ isotope is in short supply:
- 		does “enriched sulfate” mean that:
- 		the sulfate is enriched in heavy sulfur                                    _OR_                                                                                           	the sulfate is enriched in light sulfur?
*positive vs. negative*
*	*-10 ‰** **is more positive than -20 ‰** .**

## Isotopic Fractionation (the changes in the isotopic compositions of materials because of processes) can make different sources more distinctive or blur differences between sources.

-  During biological processes (e.g., assimilation, nitrification, denitrification), the lighter isotope (**14**N) ends up being concentrated in the products while the heavier isotope (**15**N) ends up being concentrated in the residual reactants.
- Hence, during the reaction NO**3**  N**2**, the resulting N**2** has a lower **15**N than the residual NO**3**.
-  Biological fractionations can make it very difficult to identify sources and quantify mixing proportions.

## Schematic showing how denitrification can increase the 15N value of residual NO3 derived from fertilizer, causing problems for interpretations of sources of N

**Schematic showing how denitrification can increase the ********15****N value of residual NO****3 ****derived from fertilizer, causing problems for interpretations of sources of N**

******15****N**

**NO****3**

**Cannot easily distinguish between: **
**(1) a manure source and **
**(2) a fertilizer source that has denitrified to have the same ********15****N as manure.**

**Solution:  use a multi-isotope approach**

## Another example of fractionation – but in this case, it is useful and provides a way of tracing sources of NH4 :

**Another example of fractionation – but in this case, it is useful and provides a way of tracing sources of NH****4 ****:**
**The ********15****N of NH****4 ****derived from volatilization of NH****3**** from animal waste lagoons is very low compared to the ********15****N of other N sources.**
**Nitrification of NH****4**** produces NO****3**** with a ********15****N that is only about 0 to 2‰  higher than the ********15****N of the NH****4  ****(if most of the NH****4**** is nitrified).**
**Contributions of atmospheric N from NH****3**** emissions, or leakage of lagoon waste into groundwater or streams. can often be readily quantified using ********15****N.  **

## Why is this?  

**Perhaps because one of the first attempts to use natural abundance 15N to understand the causes of the increases in nitrate concentrations in surface waters in many agricultural areas (Kohl et al., 1971, **_**Science**_**) elicited a hyper-critical response by 10 prominent soil scientists and agronomists (Hauck et al., 1972, **_**Science**_**), which concluded that use of 15N was a ****“questionable approach.”****  **

**While stable isotopes have become common tools for tracing sources of waters and solutes in small watersheds, they are currently under-utilized in larger basins, especially in agricultural and urban rivers.**

## In most instances, nitrate derived from fertilizer (~ 0‰) can be distinguished from nitrate derived from animal and human waste (10-20‰).

**In most instances, nitrate derived from fertilizer (~ 0****‰)** **can be distinguished from nitrate derived from animal and human waste (10-20****‰).**

**average**

## Kohl et al. (1971):  Revisited

**Kohl et al. (1971):  Revisited**

**Kohl et al. (1971)**
**Science, 174: 1331-34.**

**Hauck et al. (1972)**
**Science, 177: 453-54.**

## Trends attributed to mixing of NO3 from nitrification of soil N and fertilizer N

**Nitrate samples were collected from drain tile effluent, plus samples from a nearby lake, the Sangamon River, and a drainage ditch. **
**A linear regression through the data intersected the values measured for fertilizer and incubated soils.**

## The response by Hauck et al. (1972) made 5 main points:

**Analytical precision of natural abundance measurements insufficient for quantifying sources over the small range of differences in ****15****N**** ****between fertilizer and soil end-members (0.004 atom % or 10****‰)****. **
**Fertilizer NH4 mixes with soil N before it is oxidized to NO3, thus losing it’s identity. **
**Insufficient soil samples were analyzed to assess the true variability in the ********15****N of soils within the >900 square mile basin. **
**It is difficult to correct for the biological fractionation effects that cause great variability in ****15****N in soils. **
**The ****15****N of NO3 produced by nitrification of soil organic N is best determined by short-term incubations of soil, not the long-term incubations performed by Kohl et al.  **

**Response by Kohl et al. (1972) considered but ultimately dismissed most of the criticisms, saying that despite all the possible confounding complications, the surface water sample data themselves strongly supported their interpretation of mixing of soil and fertilizer NO3, and that their evaluation method probably under-estimated the true proportion of fertilizer-derived NO3.**

## Trends attributed to mixing of NO3 from nitrification of soil N and fertilizer N can also be explained by denitrification (or assimilatory fractionation) with an enrichment factor of ~5 ‰)

**Question:****  How do we better distinguish mixing from denitrification? ****	****Answer:****  Use a multi-tracer approach.**

## The response to this controversy was that the natural abundance approach for estimating the contributions of fertilizer-derived NO3 in surface waters was essentially abandoned in the USA for ~ 20 years, and efforts were concentrated on studying N transformations.  

**Interestingly, there were many studies in the 1970s and 1980s that estimated contributions of different N sources in ****groundwater**** that did not elicit the same kind of negative response. **
**In the late 1980s and 1990s, a number of new approaches for studying the impact of agricultural sources of N on groundwater and surface water were developed.  **
**Most are based on using a multi-isotope and/or multi-tracer approach to resolving N source vs cycling questions.**
**The result has been several dozen studies tracing sources of N and investigating N transformations in agricultural, urban, and forested watersheds, ranging from small to very large basins.**

## Ranges of δ18O and δ15N values of nitrate from different sources

**Ranges of ****δ****18****O and ****δ****15****N values of nitrate from different sources**

**A MULTI-ISOTOPE APPROACH**

## Analysis of the 15N of N2 and NO3, combined with water age-dating techniques, explains why:  

**different streams in an agricultural watershed in MD have ****different NO3 concentrations****, and **
**determines how long it will take for natural remediation processes to remove the fertilizer NO3 from groundwater.**

## Some even newer tools:  B, Li, Sr, U isotopes

**Current Isotopic Tools for Nutrient Studies**
** ****Nitrate:  analyzed for ********15****N and ********18****O ****(and sometimes for ********17****O).**
** ****POM (particulate organic matter):  filtered, acidified, and analyzed for ********15****N and ********13****C (and sometimes for ********34****S).**
** ****Water:  analyzed for ********18****O and ********2****H.**
** ****DIC (dissolved inorganic carbon):  analyzed for ********13****C. **
** ****DOC (dissolved organic carbon):  analyzed for ********13****C.**

**Some even newer tools:  B, Li, Sr, U isotopes**

** ****DON (dissolved organic nitrogen):  analyzed for ********15****N.**
** ****Ammonium: analyzed for ********15****N.**
** ****Phosphate:  analyzed for ********18****O. **
** ****Sulfate: analyzed for ********34****S ****(and sometimes for ********18****O and ********17****O).**
** ****O****2**** (DO = dissolved oxygen):  analyzed for ********18****O.**

## NEXT:  

- A brief review of several potential applications of isotopes to tracing agricultural pollution (and solving agricultural problems) – with my opinions about whether the applications are:
-  easily usable tools (i.e., “ready for prime time”), or that they still need more research;
-  under what conditions do the tools work best,
-  what additional isotope and chemical tracers appear to be beneficial for the application, and
-  selected examples.
-  **For more information, see my poster and the handout.**

## Fertilizer vs animal waste source of nitrate:  nitrate-15N 

**	****Tool usually works.**_** **_

## Fertilizer vs animal waste source of nitrate:  nitrate-15N 

- the effects of denitrification can be estimated using:
-  analysis of dissolved N2 for 15N (to correct for the N2 produced by denitrification),
-  nitrate-δ18O (this tracer usually is not as useful for this purpose as N2-15N); and
-  geochemical modeling using chemical data and perhaps DIC-13C, SO4-34S, 87Sr/86Sr, etc.
-  use of all 3 methods, along with age-dating of the water using CFCs or 3He/T, will improve quantification of source contributions.

**Fertilizer vs animal waste source of nitrate:  ****nitrate-********15****N **
**	****Tool often works. **
**But it is more costly if the soils are clayey or the groundwater is not well-oxygenated, so that significant denitrification is suspected. **

## What about use in rivers, streams, and wetlands?

## This can provide semi-quantitative to quantitative estimates of contributions in streams and rivers if:

-  denitrification in the riparian zone and/or hyporheic zone can be ruled out or quantified,
-  nitrate from soil organic matter is insignificant (or its 15N is similar to fertilizer),
-  uptake of nitrate by phytoplankton is minimal (or data are adjusted for this fractionation),
-  nitrification in the stream is minimal (or data are adjusted for this fractionation).

**Fertilizer vs animal waste source of nitrate:  ****nitrate-********15****N **
**	****Tool often works.  **
- But may require a fair amount of biogeochemical and hydrologic data.

## or ... 

**use other chemical and/or isotopic tracers (e.g., nitrate-δ****18****O or POM-********15****N/********13****C/********34****S) in addition to nitrate-********15****N. **

## The N isotope fractionation between NO3 and POM is dependent on NO3 concentration, and varies from ~ 0 ‰ in the Bay when NO3 is low, to ~ 4 ‰ in the SJR and Delta when NO3 is high. 

**The data are consistent with algae mainly growing in the SJR in contact with the observed nitrate**

**Transect in October 2002**

**San Francisco Ecosystem Restoration Program**

## Positive correlation of 15N and land use in major river basins in NE USA

-                from Mayer et al.  (2002)

**Positive correlation of ********15****N and land use in major river basins in NE USA**

## Soil vs animal waste source of nitrate:  nitrate-15N 

**	****Tool often works.**

**But...**

## Soil vs animal waste source of nitrate:  nitrate-15N 

**	****Tool often works...**
**But sometimes does not work because soil nitrates are often quite variable in ********15****N and sometimes overlap the ********15****N values of animal manure.****  ****More likely to work with pigs than other animals because pig waste from lagoons usually has a much higher nitrate-********15****N. **
**Quantification using **_**only**_** nitrate-********15****N can be successful if: **
-  the groundwater is well-oxygenated, the soils are sandy instead of clayey, so denitrification can be ruled out,
-  nitrate from soil organic matter is insignificant (or its **15**N is similar to fertilizer),
-  the fertilizer is nitrate, ammonium, or urea (not manure or green manure), and
-  nitrates from representative source areas (under the fields, feedlots, manure lagoons, septic fields, etc) are analyzed to insure that the nitrate-**15**N values of possible sources are a few ‰ different.

## Soil vs animal waste source of nitrate:  nitrate-15N 

- No ...
- but it probably would help improve the quantification if there is some difference in
-  the water-δ18O of soil water vs groundwater or lagoon water, or
-  in the O2-δ18O in the soil zone or groundwater;
-  these instances are not uncommon).
- Therefore, obtaining nitrate-δ18O is recommended.

**Soil vs animal waste source of nitrate:  ****nitrate-********15****N **
**	****Tool often works.**_** **_

## Surface water nitrate in the Mississippi River Basin has higher 18O and lower 15N values than nitrate in the San Joaquin Basin 

**atmospheric nitrate appears to be a significant source in urban and forested catchments, for small and large watersheds**

## Septic waste vs animal manure:  nitrate-15N 

**	****Tool usually does NOT work using only nitrate-********15****N.**
**(because the nitrate-********15****N values are usually overlapping).****  **

**But...**

## Septic waste vs animal manure:  nitrate-15N 

**	****Tool usually does NOT work using only nitrate-********15****N.**
**(because the nitrate-********15****N values are usually overlapping).****  **
- However, these types of waste can sometimes be distinguished if:
-  the average diets of the humans and animals are at different trophic levels,
-  nitrate-δ18O values of the wastes are different,
-  other isotope tracers that are specific for the different sources are used (B, Sr, S, C, Li, U),
-  other chemical tracers that are specific for the different sources are used (caffeine, pharmaceuticals, K, REEs, heavy metals, etc).

**See example of usefulness of K for distinguishing septic vs poultry waste...**

## Slide 41

## Isotopic compositions of fingernails as a proxy for diet

- Vegetarians (USA)

- Omnivores (Brazil)

- Omnivores (USA)

- Vegetarians (Brazil)

- Kendall and Martinelli, unpub. data

## Nitrate from waste from different kinds of farm animals: 

**	****Tool maybe will work.**
**There have been several very recent, very promising studies.**
- Therefore, semi-quantification of contributions of nitrate derived from different kinds of farm animals might be possible if:
-  nitrate-15N and nitrate-δ18O are not expected to provide clear separations without the combined use of other tracers,
-  other isotope tracers that are specific for the different sources are used (B, Sr, S, C, Li), **and/or**
-  other chemical tracers that are specific for the different sources are used (caffeine, pharmaceuticals, REEs, heavy metals, etc).

## Widory et al. (2004)

## Why do different kinds of animal waste-derived nitrate have different B-Li-S-Sr isotopic signatures?

**Mostly because of different sources of washing materials (for B, S, and maybe Sr) used for people vs AFOs (that result in differences in septic waste/WWTPs vs waste lagoons/spraying.**

## However, we need several more demonstration studies using a multi-isotope, multi-tracer, approach to see what kinds of tracers are most useful for what kinds of animals, under what kinds of conditions.

**	****Tool maybe will work.**
**There have been several very recent, very promising studies.**

## Ammonia from animal emissions vs other sources:

**	****Tool not ready for general use ****(needs further study).  **
**Quantification of ammonia from AFOs vs other sources (car exhaust, power plant exhaust, fertilizer volatilization) might be successful if:**
-  there is a lot of volatilization of NH3 from the waste (like hog lagoons), resulting in downwind ammonium-15N values that are very low.
-  the other sources of ammonium have 15N values that are significantly higher, and
-  the sources show relatively little temporal and spatial variability relative to the difference between the mean 15N values.

## Phosphate from fertilizer vs animal waste/septic waste vs soil organic matter:  phosphate-δ18O: 

**	****Tool maybe ready for use ****(needs further study).  **
**There have been a few recent and very promising studies of phosphate sources using various isotope tracers.**
_**U isotopes:**_  Uranium is a trace constituent of geologic sources of phosphate.  Hence, analysis of  234U and 238U can distinguish between “natural” and “geologic” sources.
_**Sr isotopes:**_  Strontium is a trace constituent of geologic sources of phosphate. Different sources of geologic phosphate (phosphorites vs carbonatites) appear to have different concentrations of trace metals and REEs; hence, these can be used as tracers of phosphate and/or fertilizer source.
- All of these isotope tracers will provide more quantitative information if they are combined with other isotope and chemical tracers.

## New isotope tool:  the 18O of phosphate (PO4)

**P is an essential nutrient influencing primary productivity, and is therefore an important factor in the cycling of C, N, and S in aquatic systems.**
**P has only 1 stable isotope so it cannot be used as a natural isotopic tracer.  However, the O atoms bound to the P are potentially useful tracers of P sources and sinks.**
**Main water management applications:****  **
** ****Quantify mixing of terrestrial vs marine sources.**
** ****Identification of point sources of P from waste-water treatment plants and fertilizer.**

## Organic matter from animal waste vs human waste:

**	****Tool often works.**
**Quantification of waste from human vs animals can be successful if:**
-  analyzed for 15N and/or 13C of the organic matter (both are best),
-  the average diets of the humans and animals are at different **trophic** levels (i.e., one group is herbivorous), **and/or**
-  one group eats a diet primarily composed of C3 plants (typical human diet in Asia, but not in Brazil; diets in the USA are intermediate), and the other eats a diet primarily composed of C4 plants (corn and sugar cane are the main C4 crops);
-  other isotope tracers that are specific for the different sources are used (B, Sr, S, C, Li, U),
-  other chemical tracers that are specific for the different sources are used (caffeine, pharmaceuticals, K, REEs, heavy metals, etc).

## Organic matter from animal/human waste vs natural organic matter

**	****Tool often works.**
**Quantification can be successful if:**
-  analyzed for 15N and/or 13C of the organic matter (both are best),
-  the average diets of the humans/animals is partially carnivorous),
-  the humans/animals eats a diet primarily composed of C3 plants when the natural organic matter is largely C4 (corn) or marine,
-  34S can also be useful for differentiating sources, especially when one source is marine (e.g., contamination of coastal waters with WWTP or AFO waste).
-  other isotope tracers that are specific for the different sources are used (B, Sr, S, C, Li, U),
-  other chemical tracers that are specific for the different sources are used (caffeine, pharmaceuticals, K, REEs, heavy metals, etc).

## Quantification of sewage contributions to coastal sediments

## Seasonal changes in the 15N13C, and C:N of POM

**Seasonal changes in the ********15****N********13****C, and C:N of POM**

- Most of the variability is caused by seasonal shifts in the isotopic composition of algae caused by seasonal changes in biogeochemical processes and the sources of dissolved C and N

## Other useful tools for agricultural studies:

**Sulfur isotopes:**  Analysis of sulfate for 34S and δ18O, or organic matter for 34S, can provide information about fertilizer and waste sources.
**Water isotopes:**  Analysis of the water for δ18O and δ2H (these are almost perfect tracers of the water itself) can provide extremely useful information about the sources of the nitrate and other solutes in the water.
**Age-dating nitrate contamination of groundwater:**  One powerful potential application of technological advances in the age-dating of young groundwater is to evaluate the impact of changes in agricultural management practices on water quality. Examples of environmental changes that can be assessed by this method include:  changes in the amount and types of fertilizers used, different fertilizer application times, relocation of potential pollutant sources, different remediation strategies, determination of timescales for remediation, and the installation of drainage systems.

## Bohlke (2002)

- Simplified representation of groundwater flow from an upland agricultural recharge area to a riparian wetland.
*Arrows *indicate directions of flow as determined from gradients in hydraulic heads and groundwater ages (ranging from about 0–50 years).

## Other useful tools for agricultural studies:

**Isotope biomonitoring:**  analysis of the 15N of algae, terrestrial plants, and animals as “proxies” for the isotopic compositions of nitrate and/or ammonium – and hence sources and land uses.
**Statistical, geochemical, and hydrologic modeling:**  We are past the time when simple bivariate plots and linear regressions are sufficient for interpreting data.  Other approaches:  EMMA (End Member Mixing Analysis), geochemical reaction path modeling, principal components analysis, and classification trees.
**Other chemical constituents:**  pharmaceuticals specific to different animal types, trace elements and REE for tracing fertilizers, and basic chemistry.  You can do a lot with just simple Cl concentrations for identifying animal waste contamination of rivers and groundwaters.

## Summary:  

- There has been a lot of progress with isotope characterization and tracing of agricultural contaminant sources in the last few years, particularly with regard to distinguishing between different kinds of animals (including humans) with “uncommonly” used isotopes.
- You should not rely on a single isotope (d15N) but instead use a multi-isotope, multi-tracer, approach if you want to answer questions about sources in complicated systems.
- Use a multiple lines of evidence (and multiple testable hypotheses).
- And don’t forget that you need to consider the hydrology and basic chemistry of the system – not just isotopes or organics.

## Value of isotopes for water resources management 

**1)  To tell us things about water resources that we didn’t know before.**
**2)  To tell us things about water resources that CONTRADICT what we thought we knew before.**

## Where to get isotope analyses?

-  EPA has several isotope labs.
-  USGS has 3 big stable isotope labs.
-  USDA has several labs.
-  Many universities have isotope labs in various departments.
- Some of these are mainly research labs, some are mainly service labs, and many do both.
- You will probably get better quality data if you send samples only to labs where they are using the same methods for their own research.

## Lab costs?  

- Isotopes are not very expensive – and they are a LOT cheaper than being wrong!
- How much do such isotope analyses cost?  $10-$200, depending on manpower requirements and market forces.  *(I could give some ranges of values). *
- Prices are dropping rapidly (or will!) due to increased automation and new microbial techniques.
- The best bargain is to interest someone who has an isotope lab – who might add in lots of extra analyses if he/she becomes intrigued or has spare mass spec and/or tech time.

## Some good advice:  

- Many of these stable isotope tools archive very well --  filtered, preserved, or frozen (depending on type). DIC and DO are the exceptions; they are good for months not years (unless you make special precautions).
- So collect many more samples that you can afford to analyze, and archive them.  Then used an “adaptive management practice” to decide what to analyze first.
- You might also want to archive samples for possible analysis for metal and semi-metal isotopes.  In general, these are filtered thru 0.2 micron and then acidified – using very pure reagents that don’t contain K or anything that might contribute radiogenic materials.

## Proposed sampling scheme for all future river nutrient studies

**Each time chemistry samples are collected at each site, collect and archive the following samples: **
**Nitrate isotope samples:****  Collect and archive ~20 ml, filtered (0.2 um), frozen, water samples.**
**POM isotope samples:****  Filter ~1L of water through a flat, pre-combusted, glass fiber filter (0.7 um), freeze the filters, and archive them. **
**Water isotope samples:****  Collect and archive 10-25 ml of water (unfrozen).**
**DOC/DON isotope samples:****  Collect and archive ~50 ml, filtered (0.2 um), frozen, water samples.**
**SO4 isotope samples:****  Collect, concentrate on resins, and archive.**
**Biota isotope samples:****  Archive ~1 cc frozen aliquots of biological samples (algae, riparian terrestrial plants, aquatic invertebrates, fish), especially ones collected along river reaches.**

**After chemical data are examined and modeled, these archived samples can be used to test hypotheses with new isotope data. **