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

1
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_at_usgs.gov)

December 2004
2
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.
3

• 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.

4
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.go
v/isoig/isopubs/itchinfo.html) Groundwater
Cook, P. and Herczeg, A.L. (eds.), 2000.
Environmental Tracers in Subsurface Hydrology,
Kluwer Academic Publishers.
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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 (14N, 15N). What are
stable isotopes? Stable isotopes are ones that
are NOT radioactive.
7
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.

8
Isotope Nomenclature
Where Ratio is the ratio of 2 isotopes of an
element, such as 15N/14N for nitrogen, for a
sample or a standard.
in other words
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.
9
Isotope Nomenclature
The standard for N isotopes is Air which is
defined as 0. Other standards include the
ocean (for water d18O/ d2H, and ocean carbonate
(for d13C).
10
People usually find the negative d 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.
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How isotopes are used to trace sources of
nitrate There are 2 stable nitrogen isotopes
(15N,14N). Ratios of 15N/14N are reported as
?15N. (? values, in units of permil ppt
). There are 3 stable oxygen isotopes
(18O,17O,16O). Ratios of 18O/16O are reported
as ?18O. Ratios of 17O/16O are reported as
?17O. Therefore, if different sources of nitrate
have different ?15N, ?18O, or ?17O values, we can
sometimes determine how much nitrate comes from
the different sources.
12
Fretwells Law
Warning! Stable isotope data may cause severe
and contagious stomach upset if taken alone. 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)
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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 .

14

• 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 (14N) ends up being concentrated in the
  products while the heavier isotope (15N) ends up
  being concentrated in the residual reactants.
• Hence, during the reaction NO3 ? N2, the
  resulting N2 has a lower d15N than the residual
  NO3.
• Biological fractionations can make it very
  difficult to identify sources and quantify mixing
  proportions.

15
Schematic showing how denitrification can
increase the ?15N value of residual NO3 derived
from fertilizer, causing problems for
interpretations of sources of N
Cannot easily distinguish between (1) a manure
source and (2) a fertilizer source that has
denitrified to have the same ?15N as manure.
NO3
Solution use a multi-isotope approach
?15N
16

• Another example of fractionation but in this
  case, it is useful and provides a way of tracing
  sources of NH4
• The ?15N of NH4 derived from volatilization of
  NH3 from animal waste lagoons is very low
  compared to the ?15N of other N sources.
• Nitrification of NH4 produces NO3 with a ?15N
  that is only about 0 to 2 higher than the ?15N
  of the NH4 (if most of the NH4 is nitrified).
• Contributions of atmospheric N from NH3
  emissions, or leakage of lagoon waste into
  groundwater or streams. can often be readily
  quantified using ?15N.

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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.
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.
18
Ranges of soil water d15N values for different
land uses
average
In most instances, nitrate derived from
fertilizer ( 0) can be distinguished from
nitrate derived from animal and human waste
(10-20).
19
Kohl et al. (1971) Revisited
Kohl et al. (1971) Science, 174 1331-34.
Hauck et al. (1972) Science, 177 453-54.
20
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.
21

• 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 15N
  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 its identity.
• Insufficient soil samples were analyzed to assess
  the true variability in the d15N of soils within
  the gt900 square mile basin.
• It is difficult to correct for the biological
  fractionation effects that cause great
  variability in 15N in soils.
• The 15N 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.
22
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.
23
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.
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A MULTI-ISOTOPE APPROACH
Ranges of d18O and d15N values of nitrate from
different sources
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• 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.

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• Current Isotopic Tools for Nutrient Studies
• Nitrate analyzed for d15N and d18O (and
  sometimes for d17O).
• POM (particulate organic matter) filtered,
  acidified, and analyzed for d15N and d13C (and
  sometimes for d34S).
• Water analyzed for d18O and d2H.
• DIC (dissolved inorganic carbon) analyzed for
  d13C.
• DOC (dissolved organic carbon) analyzed for
  d13C.

• DON (dissolved organic nitrogen) analyzed for
  d15N.
• Ammonium analyzed for d15N.
• Phosphate analyzed for d18O.
• Sulfate analyzed for d34S (and sometimes for
  d18O and d17O).
• O2 (DO dissolved oxygen) analyzed for d18O.

Some even newer tools B, Li, Sr, U isotopes
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• 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.

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Fertilizer vs animal waste source of nitrate
nitrate-?15N Tool usually works.
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Fertilizer vs animal waste source of nitrate
nitrate-?15N 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.

• In this case, quantification can be successful if
  
• the effects of denitrification can be estimated
  using
• analysis of dissolved N2 for ?15N (to correct
  for the N2 produced by denitrification),
• nitrate-d18O (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.

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What about use in rivers, streams, and wetlands?
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Fertilizer vs animal waste source of nitrate
nitrate-?15N Tool often works. But may
require a fair amount of biogeochemical and
hydrologic data.

• 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).

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or use other chemical and/or isotopic tracers
(e.g., nitrate-d18O or POM-?15N/?13C/?34S) in
addition to nitrate-?15N.
33
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.
Transect in October 2002
San Francisco Ecosystem Restoration Program
The data are consistent with algae mainly growing
in the SJR in contact with the observed nitrate
34
Positive correlation of ?15N and land use in
major river basins in NE USA
from Mayer et al. (2002)
35
Soil vs animal waste source of nitrate
nitrate-?15N Tool often works.
But
36

• 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 ?15N and sometimes
  overlap the ?15N values of animal manure. More
  likely to work with pigs than other animals
  because pig waste from lagoons usually has a much
  higher nitrate-?15N.
• Quantification using only nitrate-?15N 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 ?15N 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-?15N values of possible sources are a few
  different.

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Soil vs animal waste source of nitrate
nitrate-?15N Tool often works.

• Do you need nitrate-d18O?
• No
• but it probably would help improve the
  quantification if there is some difference in
• the water-d18O of soil water vs groundwater or
  lagoon water, or
• in the O2-d18O in the soil zone or groundwater
• these instances are not uncommon).
• Therefore, obtaining nitrate-d18O is recommended.

38
Surface water nitrate in the Mississippi River
Basin has higher d18O and lower d15N 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
39
Septic waste vs animal manure nitrate-?15N
Tool usually does NOT work using only
nitrate-?15N. (because the nitrate-?15N values
are usually overlapping).
But
40

• Septic waste vs animal manure nitrate-?15N
• Tool usually does NOT work using only
  nitrate-?15N.
• (because the nitrate-?15N 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-d18O 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
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Isotopic compositions of fingernails as a proxy
for diet
Omnivores (Brazil)
Vegetarians (Brazil)
Omnivores (USA)
Vegetarians (USA)
Kendall and Martinelli, unpub. data
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• 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-d18O 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).

44
Widory et al. (2004)
45
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.
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Nitrate from waste from different kinds of farm
animals Tool maybe will work. There have been
several very recent, very promising
studies. 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.
47

• 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.

48
Phosphate from fertilizer vs animal waste/septic
waste vs soil organic matter phosphate-d18O
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.
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New isotope tool the d18O 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.

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• 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).

51

• 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).

52
Quantification of sewage contributions to coastal
sediments
53
Seasonal changes in the d15N, d13C, and CN 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
54
Other useful tools for agricultural
studies Sulfur isotopes Analysis of sulfate
for ?34S and d18O, or organic matter for ?34S,
can provide information about fertilizer and
waste sources. Water isotopes Analysis of the
water for d18O and d2H (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.
55
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
050 years).
Bohlke (2002)
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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.
57

• 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 dont forget that you need to consider the
  hydrology and basic chemistry of the system not
  just isotopes or organics.

58
Value of isotopes for water resources management
1) To tell us things about water resources
that we didnt know before. 2) To tell us
things about water resources that CONTRADICT what
we thought we knew before.
59

• 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.

60
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.
61
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 dont contain K or
anything that might contribute radiogenic
materials.
62
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.