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Common uses of stable isotopes
• Identify a source
• Determine fate
• Estimate a rate
• Infer process/conditions (past and present)
Hobson et al. 1994 J of Animal Ecology
Hilderbrand et al. 1996 Can. J. of Zoology
Historic bear diets
The oceans as a source of plant nutrients
Chadwick et al. 1999 Nature
Water redistribution by plants (2H)
13C of tooth enamel to
reconstruct plant
distributions
• C3 and C4 plants differ in δ13C
(this because of different CO2
fixation pathways)
• Surveys of modern Equus teeth 13C reflect the global distribution
of C3 vs C4 grasses (horse teeth
are recording the dominant plant
type)
Modern Equus
Cerling et al 1997 Nature
Cerling et al 1997 Nature 13C of tooth enamel to reconstruct
plant distributions
Stable isotope
Long-l ived radioisotope
Short-l ived radioisotope
16
P2715
Si26Si2514
Al25Al24Al2313
Mg24Mg23Mg22Mg21Mg2012
Na23Na22Na21Na20Na1911
Ne22Ne21Ne20Ne19Ne18Ne1710
F21F20F19F18F17F169
O20O19O18O17O16O15O14O138
N19N18N17N16N15N14N13N12N117
C18C17C16C15C14C13C12C11C10C9C86
B17B15B14B13B12B11B10B9B85
Be14Be12Be11Be10Be9Be8Be7Be64
Li11Li9Li8Li7Li6Li53
He8He6He5He4He32
IsotopesTDH1
1211109876543210
Neutron Number (N)
S40S39S38S37S36S35S34S33S32S31S30S29
P39P38P37P36P35P34P33P32P31P30P29P28P27
Si36Si35Si34Si33Si32Si31Si30Si29Si28Si27Si26
Al34Al33Al32Al31Al30Al29Al28Al27Al26Al25
Mg32Mg31Mg30Mg29Mg28Mg27Mg26Mg25Mg24
Na33Na32Na31Na30Na29Na28Na27Na26Na25Na24Na23
Ne27Ne26Ne25Ne24Ne23Ne22
F25F24F23F22F21
O24O23O22O21O20
N21N20N19
C19C18
B17
24232221201918171615141312
Neutron Number (N)Neutron Number (N) http://www2.bnl.gov/CoN/
Pro
ton
Nu
mber
(Z
)
Isobars
Isotopes
Isotones
Commonly used isotopes
• 13C/12C – Climate, primary production, trophic, plant
physiology
• 2H/1H – climate, water cycle
• 18O/16O – climate, water cycle, primary production
• 15N/14N – nutrients, trophic
• 34S/32S – trophic (wetlands & estuaries)
Common isotopes in ecology
Element Isotope
Abundance
(%)
Hydrogen 1H 99.985
2H 0.015
Carbon 12C 98.89
13C 1.11
Nitrogen 14N 99.63
15N 0.37
Oxygen 16O 99.759
17O 0.037
18O 0.204
Sulfur 32S 95
33S 0.76
34S 4.2236S 0.014
The “rare” isotope is generally heavier and really rare!
This can make analysis tricky.
Two approaches
Natural abundance
•Measure small differences among pools to infer
process or source.
Tracer studies
•Spike a pool with an enormous amount of the rare
isotope and watch where it goes.
Natural variation in isotopes
air
air
Ranges in natural abundance for three isotopes
Delta notation – natural abundance
10001R
Rδ
standard
sample
10001
atoms #
atoms #
atoms #
atoms #
δ
standardabundant
rare
sampleabundant
rare
Delta notation
10001R
Rδ
standard
sample
Delta units (often shortened to “del”) are in units of per mil (‰)
•Smaller values are relatively “depleted”
•Higher values are relatively “enriched”
•Define your units what you used for Rstandard. Accepted
standards vary by discipline and application.
• “d” does not equal “∂” does not equal “δ”
Example: δ34S (‰ vs. CDT)
Stable Isotope Standards (δ=0)
Primary
standard(s)
Isotope(s) Ratio
(mean ± 95% CI)
Reference
materials
Standard Mean Ocean
Water (SMOW),
Vienna-SMOW
2H/1H
18O/16O
17O/16O
0.00015576 ± 0.00000010
0.00200520 ± 0.00000043
0.0003799 ± 0.0000016
GISP, SLAP,
NSB-1
PeeDee Belemnite
(PDB)
13C/12C
18O/16O
17O/16O
0.0112372 ± 0.0000090
0.0020671 ± 0.0000021
0.0003859 ± 0.0000016
NSB-19, NSB-20,
NSB-21
Atmospheric nitrogen
(air)
15N/14N
18O/16O 17O/16O
0.003663 ± 0.0000081 Air, NSB-14
Cañon Diablo Troilite
meteorite
34S/32S 0.0450045 ± 0.0000093 CDT
-5
0
5
10
15
20
25
0.3645 0.3665 0.3685 0.3705 0.3725 0.3745 0.3765
Per mil differences translate to very small
changes in the ratio of two isotopes.
Atom % 15N
δ1
5N
(‰
vs.
air
)
0
2000
4000
6000
8000
10000
12000
14000
0 1 2 3 4 5
Atom % 15N
δ1
5N
(‰
) Tracer studies are on a completely different scale
Significant potential for contamination if tracer and natural abundance are mixed.
Basics of measuring C and N
isotopes on organic samples
Mass Spectrometer
1. Separates compounds by mass (magnet)
2. Counts number of atoms of each mass (cups)
Commonly measured masses
N2 28 14N14N 29 14N15N 30 15N15N
14N16O
CO2 44 12C16O16O 45 13C16O16O 46 12C18O16O
SO2 64 32S16O16O 65 33S16O16O 66 34S16O16O
H2 2 1H1H 3 1H2H
H2O (bad trap)
18 1H1H16O
Ar (air leak)
40 40Ar
The Elemental Analyzer
Combusts solid organics into gases that can be
measured on an IRMS (CO2, N2, SO2, H2)
UC Davis requirements for C and N isotope analysis
Range Maximum
Nitrogen only 10-100 µg N 350 µg N
Carbon only 100 – 800 µg C 5000 µg C
Carbon + Nitrogen 10-50 µg N <1500 µg C
Analysis Material Approx. weight of sample 15N plant ~3-10 mg depending on %N content
soil ~10-75 mg 15N & 13C animal ~1 mg +/- 0.2 mg
plant ~2-3 mg
soil ~10-75 mg
• Generally target for the optimal amount of N in the sample because C
has a wider range and is more forgiving..
• To calculate the proper amount of sample you need an estimate of the
%N (by mass).
sample mass (mg) = target mass (μg) / %N / 10
• UC Davis calculator (good for Davis target of 80 μg N):
http://stableisotopefacility.ucdavis.edu/sample-weight-calculator.html
• BUT! If your material has a high C:N (~50 or more) there will be too
little N to get a good number and C will saturate.
Considerations when figuring out how much sample
to prepare (solid C and N)
• Use one lab consistently for all you samples. This is not only good
practice but there are often slight differences among labs because each
lab uses a different set of standards.
• Randomize your samples.
• Send duplicate samples to check repeatability.
• Try to send all your samples for a given project at the same time. If you
are sending samples in multiple batches, include a series of common
samples.
• If your samples don’t burn well (e.g., glass filters) you may want to add
an accelerant, usually VnO5 (adds O2).
• If you are interested in %C or %N data pay close attention to sample
weights.
Additional considerations when analyzing samples
Preparation
Determine target mass for your material
• Based on %N and C:N
• Labs vary in their target mass
- ~ 40 – 100 μg N
Stay consistent with weights
Pack tins tightly
• If a sample gets stuck the whole run can be off
• Excess air (N2) in tin can elevate background N
If material is hard to combust use an accelerant
• VnO5 at 1:1 by mass (also for working stds)
Match working standards to samples
• Span the expected range of del values
• Match C:N (if can)
Standards Universal standards
• Pee Dee Belmite (C, O)
• Standard Mean Ocean Water (O, H)
• air (N, O)
• Cañon Diablo Troilite meteorite (S)
Working standards
• Material of known isotopic composition (relative
to universal standards) included in every run
(n≈5-6)
• Used to calculate del values of reference gas
relative to universal standards
• Specific to each lab, although often shared
among labs
Reference gas
• Gas of unknown but consistent isotopic
composition injected with each sample
• Intermediary used to relate each unknown
sample to the working standard
Unknown
sample
Reference gas for
working standard
Working
standard
Reference gas for
unknown sample
Universal
standard
-10
0
10
20
30
40
50
60
-30 -20 -10 0 10 20 30 40 50
USGS 41
(glutamic acid)
δ13C
(vs PDB)
USGS 40
(glutamic acid)
Bristol Bay
sockeye
Peach Leaves
(NIST 1547)
δ15N (vs air) Working
Standards
Fractionations
Fractionations
Two types
Kinetic: difference in reaction rates among isotopes
Equilibrium: Distribution of isotopes is uneven at
chemical equilibrium.
A B
RA RB
R =Heavy/Light
A B
RA RB
Kinetic Fractionations
Difference in reaction rates among isotopes – It’s easier to make/break bonds with the lighter isotope
(extra neutron changes potential energy of bond)
– Molecular diffusion of a light molecule is faster than a
heavy molecule
A B
RA RB
R =Heavy/Light
Fractionations
• Both types of fractionations are usually mass
dependent (almost all fractionations are)
• Lighter isotope generally preferred to heavy
Examples….
H216Oaq + H2
18Oaq
H216Og + H2
18Og
Relatively 18O depleted
Relatively 18O enriched
~9.8‰ difference at 20°C
~11.2‰ difference at 0°C
Equilibrium fractionation of water between
phases
Lots of fractionations… Soil-Plant N cycle – ugly!
Fractionations
Notation and Terminology
– The amount one isotope is favored over the other
is called the fractionation factor (α). Equal to the
isotopic ratio of the products over the reactants.
RA RB
A
BBA
R
R
A B
10001 BABA
Fractionations
Recommendation: Work through calculations
using isotopic ratios (R) rather than del values.
-8‰ ?
CO2 CH2O
ε = -20‰
Answer in del units: -8 ‰ + (-20 ‰) = -28‰
Answer using R: 0.992 * 0.980 = 0.97216 = -27.84‰
Fractionations
Complete utilization
• Closed system = finite amount of reactant
• As the reactant pool declines the isotopic value of the product will return to
the starting condition.
ε = 5 ‰
0 ‰
-5 ‰
5 ‰
-10 ‰
1 0.5 0
Residual fraction of reactant (f)
reactant
product
)1(
0
t
t fR
R
Rayleigh distillation
Isotopic Mixing
from Gende & Quinn
Scientific American 2006
Example:
Marine-derived nutrients in
terrestrial plants using δ15N.
Estimation of marine-derived nutrients
using stable isotopes of nitrogen.
δ15N
Terrestrial end-member
~ 0 ‰ 3 ‰ Salmon end-member
~11 – 14 ‰
27% 73%
𝑃𝑡𝑒𝑟𝑟 = 𝑅𝑙𝑒𝑎𝑓 − 𝑅𝑠𝑎𝑙𝑚𝑜𝑛
𝑅𝑡𝑒𝑟𝑟 − 𝑅𝑠𝑎𝑙𝑚𝑜𝑛
Derive above equation from a simple mass balance on the board…
(some) Potential errors in mixing models
δ15N 20‰ 0‰
obs 50% 50%
2 sources, sample has
50% contribution from
each
2 sources, but
fractionation has
changed signal 25% 75%
10‰
20‰ 0‰ 10‰
true obs
20‰ 0‰
obs 50% 50%
10‰ 3rd source, could be
100% from new source
or 50:50 from original
sources
100%
ε = 5‰
obs
Source
1
Source
2
Source
3
↑
δ15N
δ13C →
Three source, two isotope mixing
obs
Fully constrained system Unconstrained system
obs
Source
1
Source
2
Source
3
↑
δ15N
δ13C →
Four source, two isotope mixing
obs
Unconstrained system Source
4
Trophic fractionations
15N depleted
15N enriched
What’s the reaction?
Prey
Deamination (removal of amino
group) favors 14N
You are what you eat + 3.4 ‰
is not universal.
Post 2002 Ecology
Potential confounding factors
• Nutrient status
• Growth rate
• Resource partitioning/routing
Stable isotopes are tracers of how elements move in nature. There is nothing
fundamentally special about 15N, 13C, etc. From a chemical perspective, N is N,
C is C, etc.
Most information on stable isotopes has been derived empirically. Our ability to
predict patterns in nature is generally based on observation and only minimally
based on first-principles. Much more to be learned….
As always, be aware of the assumptions and processes underlying analysis of
stable isotope data. For example, trophic level differences in δ15N are ultimately
based on physiology and bioaccumulation. How might changing physiology
affect your assumptions of εTL?
Stable isotope measurements of organics is common but the analysis is not
trivial. It is worthwhile to pay attention to QA/QC (both in the prep and at the
lab).
Some concluding thoughts…
Slide glossed over in the
original presentation but
may be of interest
Mass Balance
• Define system in terms of pools and fluxes
• Obey conservation of mass
• Common simplifying assumption of steady-state (d/dt = 0)
Pool Flux in Flux out 1
Flux out 2
NO3-
α ≈ 0.980
N2, N2O
α ≈ 0.995
assimilation
𝑑
𝑑𝑡= 𝐹𝑖𝑛 − 𝐹𝑎 − 𝐹𝑑 = 0
𝑑
𝑑𝑡= 𝑅𝑖𝑛𝐹𝑖𝑛 − 𝛼𝑎𝐹𝑅𝑁𝑂3𝐹𝑎 − 𝑅𝑁𝑂3𝐹𝑑 = 0
δ15N ≈ -2 – 0 ‰
NO3-
α ≈ 0.980
N2, N2O
α ≈ 0.995
assimilation
𝐹𝑖𝑛 = 𝐹𝑎+ 𝐹𝑑
𝑅𝑖𝑛𝐹𝑖𝑛 = 𝛼𝑎𝑅𝑁𝑂3𝐹𝑎 + 𝑅𝑁𝑂3𝐹𝑑
δ15N ≈ -2 – 0 ‰
δ15NO3- δ15N ≈ -2 – 0 ‰
α ≈ 0.980
N2, N2O
α ≈ 0.995
assimilation
𝑃𝑑 =𝛼𝑎𝑅𝑁𝑂3 − 𝑅𝑖𝑛
𝑅𝑁𝑂3 𝛼𝑎 − 𝑅𝑑
Note that the isotopic ratio of the pool is a function of whether N is
assimilated or denitrified. Thus, the δ15N of the pool can change through
time (with a shift in pathways) even though the fractionation factors remain
constant.
You are what you eat +3.4
Trophic fractionations: a huge issue in food web
isotope mixing models
Sears et al. 2009, Oecologia
Seabird chicks raised on a known diet.
Growth rate is negatively related to trophic fractionation. More N
to assimilation, less to excretion.
Measured difference in δ15N between red
blood cells (RBC) and diet == trophic
fractionation
Growth effects on trophic fractionations
Sears et al. 2009, Oecologia
Nutrient status effects on trophic fractionations
Again, seabird chicks raised on a known
diet.
Restricted diet for one group == poor
nutrient status.
Little effect of restricted diet on δ13C.
Significant effect on δ15N. Limited N
means a higher percentage is assimilated,
means a lower net trophic fractionation.
Lunch
