COMMENT

Limnol. Oceanogr., 24(3), 1979, 593595

0 1979, by the American Society of Limnology and Oceanography, Inc.

A comment on the calculation of atom percent enrichment

for stable isotopes1

We would like to call attention to sev- The correct expression for atom % 13C
eral errors in the note by Slawyk et al. is:
(1977). In particular, two of the equations
they used to derive carbon uptake and atom % 13C = [ 12C1F13C] loo c2>
turnover rates based on changes in 13C
abundances in particulate material are in- where 13C and 12C are calculated from the
correct. mass signal intensities at 44 and 45 after
Equation 1 of Slawyk et al. (1977), appropriate corrections (e.g. 170, as
which they used to calculate atom % ex- above).
cess 13C (a,), is reproduced below: The atom % excess 13C reflects [13C]C02
incorporation into the particulate matter
R enriched sample during an experiment and is defined as:
- 1 x 100
a1 = R unenriched sample
atom % excess 13C
“where R is the ratio of mass signal in- = atom % 13C (enriched)
tensities 45 to 44 (R = 45/44).” This equa-
tion was adapted from the formula for - atom % 13C (unenriched)
613C in %0 (i.e. per mil): = 13C x 100 13C x 100
12~ + 13~ enriched - 12~ + 13~ unenriched
13C/12Csample
pc = - 1 1,000 (1) (3)
13CP2C standard I
where “unenriched” and “enriched” re-
which is routinely used to express differ- fer to the carbon isotope abundances of
ences in natural abundances of 13C and particulate matter before the addition of
12C (e.g. Craig 1953; Smith and Epstein [13C]C02 and at the end of the incubation
1971). As written, Eq. 1 of Slawyk et al. period. However, in practice a time-zero
cannot be used to calculate either P3C or atom % value is often used instead of an
atom % excess 13C. If the omitted brack- unenriched atom % value, as discussed
ets are replaced, then their value of “al” below.
approximates 613C/10. It is only an ap- Equation 2 of Slawyk et al. (1977),
proximation because of improper substi- which they used to calculate carbon turn-
tution of mass signal intensities of 45 and over rates (VJ, is also reproduced below:
44 for the abundances of 13C and 12C in
the 613C expression. There is a correction da,ldt
for the abundance of 170 in the CO, sam- [13c]co,
ple which must be applied for precise
calculation of 13C and lzC abundances
[‘3C]CO, +[W]CO,x loo - a1
(Craig 1957). Th is minor correction is im- where a, is obtained from their Eq. 1.
portant for P3C calculation, but should This equation is also incorrect because a,
not greatly affect calculations of atom %. appears in the denominator.
We note it because in a technique paper Carbon turnover rates (units = h-l) are
it should not be ignored. calculated from the rate of increase of 13C
atom % excess over time. Typically, this
1 Supported by NSF grant OCE 76-82084 to is obtained from a “time zero” and a final
T.R.F. value after some incubation time, t:
593
594 Comment

d(atom % excess 13C) = the above is not true, exponential equa-

dt tions must be used to compensate for a
declining atom % 13C in the ZCO, pool
(atom % excess 13C at time = t)
[ - (atom % excess 13C at time = 0)
t
1. (4) during incubation (see Sheppard 1962).
With appropriate substitutions and cor-
rections, the above equations also apply
The time-zero correction is necessary be- to the use of other stable isotopes such as
cause of chemical adsorption and ex- 15N:
change of CO, between particulate mat- 15N

ter and the aqueous inorganic carbon atom % 15N = 100

pool (X0,) in the samples, as well as
enriched particulate contamination in the
isotope stock solutions. One or more bot-
tles are filtered within minutes after iso-
tope addition. The use of time-zero bot-
tle(s) eliminates the necessity for the
unenriched sample referred to in Eq. 3
above. The “unenriched” atom % 13C is
a constant which appears twice on the
right side of Eq. 4 and is canceled out
during subtraction. Hence, atom % 13C
values can be substituted for atom % ex-
cess 13C values in calculating carbon
turnover rates:
d(atom % excess 13C)
dt
= d(atom % 13C)
dt
(atom % 13C at time = t)
= [ - (atom % 13C at time = 0)
t
1.
The complete expression for the turn-
over rate of particulate C is:
d(atom % 13C) [‘3c]co, + [12c]co,
X
dt lOO[ ‘3c]co,
(6)
where the expression on the right is
an inverted atom % l”C value for the
ZCO, pool. This compensates for the fact
that d(atom % 13C)/dt reflects only
[13C]C02 uptake whereas the turnover
rate reflects uptake of [12C]C02 as well.
Isotopic discrimination against the heavi-
er 13C during photosynthesis is l-3% (Pe-
ters et al. 1978), and this small correction
can be applied to Eq. 6 by multiplying
by 1.02. Note that our Eq. 6 holds only
where the XCO, pool turnover time is
long compared to the incubation time. If
15N + 14N
100
= (7)
2R + 1
where 15N and 14N are the number of 15N
and 14N atoms in N, gas generated from
the particulate sample, and R is the ratio
of peak heights 28 and 29, (m/e 28):(m/e
29), after correction for the residual spec-
trum and reagent blanks at those peaks.
Equation 7 assumes equilibrium be-
tween 14N2, 14N 15N, and 15N2, which can
be checked in a mass spectrometer with
highly enriched samples. Once it has
been established that equilibrium is ob-
tained by the sample preparation meth-
ods, it is only necessary to measure two
of the three peaks. Since in most ocean-
ographic and limnological applications,
enrichments are low (<lo-15%), the m/e
(5)
30 peak is frequently too small to be mea-
sured accurately. (For equations at higher
enrichments and discussion of equilibri-
um, see Fiedler and Proksch 1975.) Once
atom % 15N is obtained, Eq. 5 and 6 ap-
ply, with substitutions for atom % and
appropriate 15N substrate (e.g. [15N]NH3,
P5NlN0,, etc.). There is essentially no
isotopic discrimination between 15N and
14N during assimilation of inorganic nitro-
gen (Peters et al. 1978).
Since the equations of Slawyk et al.
(1977) for both atom % excess 13C and
particulate carbon turnover rate are in-
correct, we are at a loss to explain how
they produced reasonable comparative
data between [13C]C02 and [14C]C02 up-
take experiments. Although the values
are reasonable, we believe that the turn-
over rate data given in their table 2 are
probably erroneous.
Finally, we would like to comment on
the natural abundance of 13C of 1.28 atom
 Comment 595

% found by Slawyk et al. (1977) for batch Richard J. Volk

cultures of Fragilaria pinnata. This atom Department of soi1 Science
% abundance would correspond to a 613C North Carolina State University
much higher than any previously report- Raleigh 27607
ed value for natural material. In fact or-
ganic matter is generally depleted in 13C References
compared to the bicarbonate standard CRAIG, H. 1953. The geochemistry of stable carbon
which is about 1.1 atom % (e.g. Craig isotopes. Geochim. Cosmochim. Acta 3: 53-92.
1953). If correct, their value could only -. 1957. Isotopic standards for carbon and
result from prior exposure of the algae to oxygen and correction factors for mass spectro-
metric analysis of carbon dioxide. Geochim.
[‘“C]CO,.
Cosmochim. Acta 12: 133-149.
FIEDLER, R., AND G. PROKSCH. 1975. The deter-
mination of nitrogen-15 by emission and mass
Thomas R. Fisher, Jr. spectrometry in biochemical analysis: A re-
University of Maryland view. Anal. Chim. Acta 78: l-62.
Center for Environmental and PETERS,K. E.,R. E. SWEENEY, AND I. R. KAPLAN.
1978. Correlation of carbon and nitrogen stable
Estuarine Studies isotope ratios in sedimentary organic matter.
Horn Point Environmental Limnol. Oceanogr. 23: 598-604.
Laboratories SHEPPARD, C. W. 1962. Basic principles of the
Box 775 tracer method. Wiley.
Cambridge 21613 SLAWYK, G., Y. COLLOS, AND J. C. AUCLAIR.
1977. The use of the i3C and i5N isotopes for
Evelvn B. Haines the simultaneous measurement of carbon and
nitrogen turnover rates in marine phytoplank-
University of Georgia ton. Limnol. Oceanogr. 22: 925-932.
Marine Institute SMITH, B. N., AND S. EPSTEIN. 1971. Two cate-
Sapelo Island 31327 gories of i3C/‘*C ratios for higher plants. Plant
Physiol. 47: 380-384.

Limnol. Oceanogr., 24(3), 1979, 595-597

@ 1979, by the American Society of Limnology and Oceanography, Inc.

Reply to comment by Fisher et al.

As far as eq. 1 in Slawyk et al. (1977) Equation 2 is still not strictly correct be-
is concerned, we agree with the comment cause the ratio (m/e 45):(mle 44 + m/e 45)
of Fisher et al. (1979). If R = (m/e has to be used instead of R. However, in
45):(m/e 44), then the correct equation for our case, this does not significantly
calculating atom % excess 13C (a,) has to change uptake results since m/e 45 is al-
be written as follows: ways ~4% of m/e 44. This way of calcu-
lating leads to overestimates of uptake
r
R enriched R unenriched 1 values of about 5% on the average, de-
I
sample
a’ = R enriched
L sample + 1
sample
- R unenriched
sample + Ll
I
x 100. pending on the 13C enrichment (Table 1).
This is well within the experimental error
currently admitted for the 14C method
(1)
(Steemann Nielsen 1975) and the one
Equation 1 in Slawyk et al. (1977) should found for the 13C method in the field (un-
have read: publ. data).
a, = [R enriched sample - R unenriched sample] Regarding the 13C abundance value in
x 100
Fragilaria pinnata, we analyzed this se-
(2)
ries of replicates in order to determine
where R = (m/e45):(m/e44). what a significant enrichment is, instead