Stable Isotopes in Ecology
Gordon Holtgrieve ([email protected])
UW SAFS/AFS Workshop
May 23, 2012
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
Historic bear diets
Hilderbrand et al. 1996 Can. J. of Zoology
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
Cerling et al 1997 Nature
Modern Equus
• 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)
13C
of tooth enamel to reconstruct
plant distributions
Cerling et al 1997 Nature
Proton Number (Z)
16
15
Stable isotope
14
Long-lived radioisotope
13
Short-lived radioisotope
Al23
S29
S30
S31
S32
S33
S34
S35
S36
S37
S38
S39
S40
P27
P28
P29
P30
P31
P32
P33
P34
P35
P36
P37
P38
P39
Si25
Si26
Si27
Si28 Si29 Si30 Si31
Si32
Si33
Si34
Si35
Si36
Al24
Al25
Al26
Al27 Al28
Al31
Al32
Al33
Al34
23
24
Al29
Al30
12
Mg20 Mg21 Mg22 Mg23 Mg24 Mg25 Mg26 Mg27 Mg28 Mg29 Mg30 Mg31 Mg32
11
Na19 Na20 Na21 Na22 Na23 Na24 Na25 Na26 Na27 Na28 Na29 Na30 Na31 Na32 Na33
10
Ne17 Ne18 Ne19 Ne20 Ne21 Ne22 Ne23 Ne24 Ne25 Ne26 Ne27
9
F16
F17
F18
F19
F20
F21
F22
F23
F24
F25
O23
O24
8
O13
O14
O15
O16
O17
O18
O19
O20
O21
O22
N11
N12
N13
N14
N15
N16
N17
N18
N19
N20
N21
C9
C10
C11
C12
C13
C14
C15
C16
C17
C18
C19
B8
B9
B10
B11
B12
B13
B14
B15
Be10 Be11 Be12
7
6
C8
5
4
Be6
Be7
Be8
Be9
3
Li5
Li6
Li7
Li8
He3
He4
He5
He6
H
D
T
0
1
2
2
1
Li9
Isotones
B17
Be14
Isobars
Li11
He8
Isotopes
Isotopes
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
Neutron Number (N)
Neutron Number (N)
http://www2.bnl.gov/CoN/
Commonly used isotopes
•
– Climate, primary production, trophic, plant
13C/12C
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
Hydrogen 1H
2H
Carbon
12C
13C
Nitrogen 14N
15N
Oxygen 16O
17O
18O
Sulfur
32S
33S
34S
36S
Abundance
(%)
99.985
0.015
98.89
1.11
99.63
0.37
99.759
0.037
0.204
95
0.76
4.22
0.014
The “rare” isotope is generally heavier and really rare!
This can make analysis tricky.
Two approaches
Tracer studies
•Spike a pool with an enormous amount of the rare
isotope and watch where it goes.
Natural abundance
•Measure small differences among pools to infer
process or source.
RangesNatural
in naturalvariation
abundance for
isotopes
in three
isotopes
air
air
Delta notation – natural abundance
 R sample

δ  
 1 1000
 R standard 
  # atoms rare 




#
atoms


abundant  sample
δ
 1 1000
  # atoms rare 

  # atoms abundant 

standard


Delta notation
 R sample

δ  
 1 1000
 R standard 
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.
Example:
δ34S (‰ vs. CDT)
• “d” does not equal “∂” does not equal “δ”
Stable Isotope Standards (δ=0)
Primary
standard(s)
Standard Mean Ocean
Water (SMOW),
Vienna-SMOW
PeeDee Belemnite
(PDB)
Atmospheric nitrogen
(air)
Isotope(s)
2H/1H
18O/16O
17O/16O
Ratio
(mean ± 95% CI)
Reference
materials
0.00015576 ± 0.00000010 GISP, SLAP,
0.00200520 ± 0.00000043 NSB-1
0.0003799 ± 0.0000016
NSB-19, NSB-20,
NSB-21
17O/16O
0.0112372 ± 0.0000090
0.0020671 ± 0.0000021
0.0003859 ± 0.0000016
15N/14N
0.003663 ± 0.0000081
Air, NSB-14
0.0450045 ± 0.0000093
CDT
13C/12C
18O/16O
18O/16O
17O/16O
Cañon Diablo Troilite
meteorite
34S/32S
Per mil differences translate to very small
changes in the ratio of two isotopes.
25
δ15N (‰ vs. air)
20
15
10
5
0
0.3645 0.3665 0.3685 0.3705 0.3725 0.3745 0.3765
-5
Atom % 15N
Tracer studies are on a completely different scale
14000
12000
δ15N (‰)
10000
8000
6000
4000
2000
0
0
1
2
3
4
5
Atom % 15N
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
CO2
28
29
30
14N14N
14N15N
15N15N
SO2
44 12C16O16O
45 13C16O16O
46 12C18O16O
64
65
66
32S16O16O
33S16O16O
34S16O16O
14N16O
H2
1H1H
2
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
Nitrogen only
Carbon only
Carbon + Nitrogen
Analysis
15N
15N
& 13C
Material
plant
soil
animal
plant
soil
Range
10-100 µg N
100 – 800 µg C
10-50 µg N
Maximum
350 µg N
5000 µg C
<1500 µg C
Approx. weight of sample
~3-10 mg depending on %N content
~10-75 mg
~1 mg +/- 0.2 mg
~2-3 mg
~10-75 mg
Considerations when figuring out how much sample
to prepare (solid C and N)
• 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.
Additional considerations when analyzing samples
• 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.
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)
Universal standards
•
•
•
•
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
Reference gas for
unknown sample
Working
standard
Universal
standard
Working
Standards
δ15N (vs air)
60
USGS 41
(glutamic acid)
50
40
30
20
Bristol Bay
sockeye
10
Peach Leaves
(NIST 1547)
0
-30
-20
USGS 40
(glutamic acid)
-10
0
-10
10
20
30
40
δ13C
50(vs PDB)
Fractionations
Fractionations
Two types
Kinetic: difference in reaction rates among isotopes
A
RA
B
RB
R =Heavy/Light
Equilibrium: Distribution of isotopes is uneven at
chemical equilibrium.
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
R =Heavy/Light
RA
RB
Fractionations
• Both types of fractionations are usually mass
dependent (almost all fractionations are)
• Lighter isotope generally preferred to heavy
Examples….
Equilibrium fractionation of water between
phases
Relatively 18O depleted
H216Og
H216Oaq
+
+
H218Og
H218Oaq
Relatively 18O enriched
~9.8‰ difference at 20°C
~11.2‰ difference at 0°C
Lots of N
fractionations…
Soil-Plant
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.
A
RA
B
RB
 A B
RB

RA
 A B   A B  11000
Fractionations
Recommendation: Work through calculations
using isotopic ratios (R) rather than del values.
CO2
-8‰
ε = -20‰
CH2O
?
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.
product
5‰
ε=5‰
0‰
Rayleigh distillation
-5 ‰
Rt
( 1)
 ft
R0
reactant
-10 ‰
1
0.5
Residual fraction of reactant (f)
0
Isotopic Mixing
Example:
Marine-derived nutrients in
terrestrial plants using δ15N.
from Gende & Quinn
Scientific American 2006
Estimation of marine-derived nutrients
using stable isotopes of nitrogen.
Terrestrial end-member
~0‰
Salmon end-member
~11 – 14 ‰
3‰
δ15N
73%
27%
𝑃𝑡𝑒𝑟𝑟 =
𝑅𝑙𝑒𝑎𝑓 − 𝑅𝑠𝑎𝑙𝑚𝑜𝑛
𝑅𝑡𝑒𝑟𝑟 − 𝑅𝑠𝑎𝑙𝑚𝑜𝑛
Derive above equation from a simple mass balance on the board…
(some) Potential errors in mixing models
δ15N
2 sources, sample has
50% contribution from
each
2 sources, but
fractionation has
changed signal
0‰
50%
50%
obs
20‰
10‰
0‰
75%
true
3rd source, could be
100% from new source
or 50:50 from original
sources
20‰
10‰
25%
obs
ε = 5‰
20‰
10‰
0‰
100%
50%
obs
50%
Three source, two isotope mixing
↑
δ15N
Unconstrained
system
Fully constrained
system
Source
1
obs
obs
Source
3
Source
2
obs
δ13C →
Four source, two isotope mixing
↑
δ15N
Unconstrained system
Source
1
Source
4
obs
Source
3
Source
2
δ13C →
Prey
Trophic fractionations
15N
enriched
15N
depleted
What’s the reaction?
Deamination (removal of amino
group) favors 14N
You are what you eat + 3.4 ‰
is not universal.
Potential confounding factors
• Nutrient status
• Growth rate
• Resource partitioning/routing
Post 2002 Ecology
Some concluding thoughts…
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).
Slide glossed over in the
original presentation but
may be of interest
Mass Balance
Flux out 1
Flux in
Pool
Flux out 2
• Define system in terms of pools and fluxes
• Obey conservation of mass
• Common simplifying assumption of steady-state (d/dt = 0)
N2, N2O
assimilation
α ≈ 0.980
α ≈ 0.995
δ15N ≈ -2 – 0 ‰
NO3𝑑
= 𝐹𝑖𝑛 − 𝐹𝑎 − 𝐹𝑑 = 0
𝑑𝑡
𝑑
= 𝑅𝑖𝑛 𝐹𝑖𝑛 − 𝛼𝑎 𝐹𝑅𝑁𝑂3 𝐹𝑎 − 𝑅𝑁𝑂3 𝐹𝑑 = 0
𝑑𝑡
N2, N2O
assimilation
α ≈ 0.980
α ≈ 0.995
δ15N ≈ -2 – 0 ‰
NO3-
𝐹𝑖𝑛 = 𝐹𝑎 + 𝐹𝑑
𝑅𝑖𝑛 𝐹𝑖𝑛 = 𝛼𝑎 𝑅𝑁𝑂3 𝐹𝑎 + 𝑅𝑁𝑂3 𝐹𝑑
N2, N2O
assimilation
α ≈ 0.980
α ≈ 0.995
δ15NO3-
δ15N ≈ -2 – 0 ‰
𝑃𝑑 =
𝛼𝑎 𝑅𝑁𝑂3 − 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.
Trophic fractionations: a huge issue in food web
isotope mixing models
You are what you eat +3.4
Growth effects on trophic fractionations
Seabird chicks raised on a known diet.
Measured difference in δ15N between red
blood cells (RBC) and diet == trophic
fractionation
Growth rate is negatively related to trophic fractionation. More N
to assimilation, less to excretion.
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.
Sears et al. 2009, Oecologia
Lunch