Utilization of the heavy isotope of nitrogen as a tracer has found numerous applications in soil biology. It allows better definition of different stages of the nitrogen cycle, in particular the immobilization-mineralization cycle. In this work, the authors report the results of calculations of natural isotope ratios of nitrogen in samples of water, soil and vegetation prevailing in Dombes and discuss the possibilities of errors and coefficients of fractionation

Chalamet, A.

Domenach, A. M.

Pachiaudi, C.

English

Aquatic Commons

Freshwater Biological Association 
FBA Translation (New Series) No. 135 
Title: Ratio of natural isotopes of nitrogen. I. Primary results: 
Soils of Dombes. (Rapports isotopiques naturels de l'azote I. -. 
Premiers resultats : sols de Dombes.) 
Author(s) DOMENACH A.M., CHALAMET A. & PACHIAUDI C. 1977 
Reference: Revue Ecol. Biol. Sol 14, 279-287 
Original language: French 
Date of publication of original: 1977 
Translator: D. Powell 
Date of publication of translation: March 15, 1980 
Number of pages of translation: 10 
FBA Translations are to be considered as "provisional". As a rule 
they have not been prepared by expert translators, nor have, they 
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They are available from The Librarian, 
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England, 
at the current rate for xerox copying. 
FBA Translation (New Series) No. 135 
DOMENACH A.M., CHALAMET A. & PACHIAUDI C. 1977 
Ratio of natural isotopes of nitrogen. I. Primary results: Soils of Dombes 
Revue Ecol. Biol. Sol l4, 279-287 
Translated by: D. Powell. 
Utilization of the heavy isotope of nitrogen as a tracer has found 
numerous applications in soil biology. It allows better definition of 
different stages of the nitrogen cycle, in particular the immobilization-
mineralization cycle. (JANSSON, 1958). 
Recently some nitrogenous products have appeared on the market which 
are not enriched by 15N but impoverished: the nitrogen in the air naturally 
contains 0.3663% of nitrogen 15, while the impoverished products only 
contain 0.001% and thus can serve as tracers. Their gross advantage 
results from their cost, which is clearly lower than before, permitting 
a larger utilization. 
But use of heavy nitrogen as a tracer, if one wants to follow the 
added labelled substance for a long time, necessitates knowledge of the 
natural isotope ratio, which depends on the type of soil, the horizon of 
the same soil and/or of the nitrogen fraction considered (BREMNER, 1967 -
RIGA et col. 1971). 
Furthermore, the variations in the ratios of natural isotopes after 
the elimination of possible errors from dilution, may be able to be used 
themselves. Several articles have appeared in the last years which attempt 
to explain the significance of the natural variations but the conclusions 
which follow are different. 
According to KENNIE & PAUL (1974), the values for reflect the 
'cumulative effects that origin, biological processes and management 
have all had on the soil'. HAUCK (1973)affirms that the knowledge of 
variations of natural isotopes can give information on the changes which 
take place in the level of the soil-plant system. In KOHL, SHEARER, 
COMMONER (1971) and again LETOLLE & MARIOTTI (1974) the content of 
15 
N in nitrates from surface waters allows the detection of their origin: 
native organic nitrogen or nitrogenous fertilizers. In comparison EDWARS 
(1974) found that the isotopic variation does not permit identification 
of pollution sources of nitrates in the soil-fertilizer system. 
In this first work, we report the results of calculations of 
natural isotope ratios of nitrogen in samples of water, soil and vegetation 
prevailing in Dombes and discuss the possibilities of errors (BREMNER 1963) 
and coefficients of fractionation. 
Materials and techniques. 
The region of Dombes is part of the plateau of Bressan situated to 
the Northeast of Lyon. It is characterized by the number of ponds; the 
acid soil (pH 4 to 5), silt-clay, is constituted solely of fine elements, and 
has been the object of physico-chemical (CHALMET, 1970) and biological 
studies (BEAUPIED 1969). 
Different examples of water, soils and plants prevail in a transect 
following the water zonation pools; prairie with agrostis, plains (teppes) 
with sarothammes, coppice to forest of oak or birch. The levels of total 
nitrogen are calculated in the samples. 
To try to envisage at what level the isotopic fractionation took 
place, we measured the isotopic rates of nitrites produced by Nitrosomonas 
europaea in culture in the presence of sulfate of ammonium, as well as of 
the isotopic ratio in the remaining sulfate of ammonium. Nitrosomonas 
represents an important link in the nitrogen cycle in allowing the transformation 
of ammonium to nitrite. 
The bacteria are inoculated into a buffered medium at pH 7.9 to which 
has been added 100 ppm of N-NH4+. Measurements are taken when approximately 
half of the ammonium has been transformed to nitrite. 
Measurements of the isotopic ratio 29N2:28N2 by mass spectrometry 
are made as gaseous samples. It is thus necessary to collect the nitrogenous 
azo-compounds and transform them to N2. 
The preparation is made by the method used by BREMNER, 1965; BREMNER 
& EDWARDS, 1965) which necessitates converting the nitrogen compounds to 
ammonium. The ammonium is recovered after distillation of NH4+ with the apparatus 
from BREMNER. 
Nitrates and nitrites are reduced by the Dewarda reaction. The 
reaction can only be used on samples which have not undergone distillation 
because it will easily attack organic matter. Impurities such as amines 
can also be recovered at the same time as the sulfate of ammonium and can 
distort the final result. 
Transformation of sulfate of ammonium to gaseous nitrogen operates 
through a reaction with hypobromite of sodium in the Martin-Ross instrument 
(ROSS-MARTIN, 1970) modified by replacement of the air by helium. 
According to BREMNER (C.P.), during the reaction of sulfate of 
ammonium to sulfate hypobromite, nitrogen evolved at the beginning of the 
reaction contains less nitrogen 15 than that evolved at the end of the 
reaction. EDWARDS (C.P.) recommends heating and shaking the mixture of 
ammonium-sodium hypobromite to assure complete reaction and the expulsion 
of all the N2 gas in order to prevent fractionation. 
In addition to that transformation of nitrogen, a secondary reaction 
of oxidation to N2O can take place. But BREMNER concludes that this is not 
a difficulty in the spectrophotometry measurement, the gas being trapped 
by the liquid nitrogen. Indeed, we think, as does BREMNER, that there is 
no contamination of the nitrogen portion, but that variations observed in 
measurements of the same sample can be explained by the intervention of a 
lighter isotope fraction in the course of the reaction. Unhappily, we can 
do very little about it. 
The nitrogen thus formed is introduced directly into the mass 
spectrometer in order to isotopically analyze it. 
The mass spectrometer used is a AEI MS 20 with dual introduction: 
one for the sample to be analyzed, the other for a very pure (N 48) nitrogen 
gas standard. The gas standard allows comparison between measurements. 
The isotopic value in relation to air can be measured owing to a reaction 
of pyrogallol which retains the oxygen in the air. The percentage of 
nitrogen 15 in the standard is 0.3651%. The spectrometer is furnished with 
mechanisms allowing a vacuum extending into all the tubing connecting the 
2) Nitrosomonas E. 
We considered the values of and the percentage of Nitrogen 15 from: 
- the nitrite formed, 
- the sulfate of ammonium which remained, 
- the control sulfate of ammonium (half had not been reacted), 
The fraction f having reacted: 
Results and conclusions. 
1) Dombes. 
The number of samples varied from 3 - 6 . Values are given with a 
maximum variation of 2%. 
sample with the instrument. This assemblage follows the design given by 
VAN PRAAG (1971). Dead spaces are at a minimum and allow work on small 
quantities of sample (0.5 to 1 mgN). Results are given in where; 
The different steps of the method used are shown schematically in figure 1. 
FREYER and ALY (1974) are equally interested in the isotopic ratio 
The results presented in table 1 show that the forest soil has a 
equal to -5.2 while that of the prairie soil is +9.9. 
The soil of the forest has a smaller isotopic ratio than that of the 
air. This agrees with the results found by RIGA and colleagues (1971) 
as well as those of CHENG and colleagues (1964) who also found a negative 
for forest soils. The explanation of RIGA et al is that a large 
quantity of relatively transformed vegetable debris returns to the soil. 
This debris is subjected to impoverishment in living tissues. 
The soil under Sarothamme, and Sarothamme have similar (5.5 
and 5.2, respectively), lower than that of the prairie beside which they are 
found: . This seems to confirm the influence that vegetation, 
in this case Sarothamme, has on the supporting soil. 
The decrease in can be explained by preferential assimilation 
14 
of N from the air by the plants. HOERING (1955) found negative values 
of for leaves of clover. RIGA et al (1971) compared the isotope 
ratio of a field cultivated with vetch and that of the same field without 
vetch and established a decrease in the value of for the field with 
vetch. 
of water: water recovered from the soils of prairies, fields, forests. 
The values of of water reflect that of the terrain on which they are 
found. For example they are very low in a forest. 
In the Dombe, the isotope ration of the water of the pond: 
is very close to that of the prairie: . Is it possible then, as 
suggested by FREYER & ALY (1975) , to establish a relation between the soil 
nitrogen of the prairie and that of the pond water? 
SHEARER and colleagues (1974) tried to analyze the fractions that 
appeared with a mathematical model which schematically represents the 
transformation of nitrogen including ammonification, nitrification, 
denitrification. 
Given the knowledge of the constants of fractionation: 
nitrification constant for the immobilized ammonium, SHEARER 
et al have reached the following mathematical conclusions: if the immobilization 
increases, decreases and the nitrogen returning to an organic pool 
is more rich in than the nitrogen which disappears by ammonification. 
The organic nitrogen is enriched in while the nitrate nitrogen is impoverished. 
If one considers the ammonium - nitrite transformation which is effected 
by Nitrosomonas (Table II), one can see an important depletion of from 
nitrite while the ammonium remaining is enriched in . Nitrosomonas 
utilizes essentially the . A part of the isotopic fractionation takes 
place at the level of the microorganisms. Given the values found, it is 
possible to calculate the fractionation constant . 
If one allows that the ammonium-nitrite is a first order reaction 
as SHEARER et al found in accordance with other authors, (TONG & YANKWITCH, 
1957; DELWICHE & STEYN, 1970), the value of the factor of fractionation 
of nitrification is found by applying the formula of TONG & YANKWITCH. 
In the same way, DELWICHE & STEYN (1970) found a median value of: =1.026 
for a variation of f from 0.54 to 0.72. 
FREYER & ALY (1975) calculated the fractionation constant during the whole 
of nitrification (nitritation and nitratation). This constant has a slightly 
smaller value: 1.017. 
This method will only give a value approaching the constant of fractionation 
because the initial quantities are given by the control samples without 
Nitrosomonas europaea and can only be an approximation of the value from the 
where f is the proportion of transformation. 
percentage of from the accumulated product, in this case, nitrite, 
in time t. 
percentage of from the remaining product, in this case, ammonium, 
in time t. 
initial quantities of ammonium disseminated from the samples. 
Furthermore, one cannot measure the quantity of ammonium which is 
immobilized by the micro-organisms themselves or that which has volatilized 
in the course of the incubation. A raised pH (pH 7.9) favors this volatilization. 
The degree of fractionation seems to vary as a function of the quantity 
of transformation is larger. 
In the soil, it can be said that the quantity of ammonium stays constant 
(SHEARER, 1974) because it is produced in proportion to its utilization. In 
that case, the degree of fractionation is constant and can have a different 
value from that found in culture media. 
The constant of fractionation of nitritation which was used by 
SHEARER et al (1974) only corresponds to that of nitrification. It remains 
to be proved whether biological transformation of nitrites to nitrates is 
not accompanied by any isotopic fractionation. 
The problem remaining is to find if fractionation plays a role when 
using depleted or enriched nitrogen as a marker. 
For this we have inoculated a medium containing labelled sulfate 
of ammonium with Nitrosomonas europaea. 
We noticed a small difference between 
ammonium has 
a higher isotopic value (0.1 to 0.7%) than that of nitrite. 
In reality, these differences are of little significance because 
they are of the same order of magnitude as those found between different 
experiments. They will arise from the variations which one observes in 
all biological processes, and also from the precision of the measurements. 
Mass spectrometry gives less precise measurements on samples containing 
a large quantity of labelled product than on those containing nitrogen 
impoverished in N. One has to work within very sensitive limits and the 
influence of the standard of reference on the sample is of great importance. 
The values obtained seem less stable and less precise. 
If labelled nitrogen is used for the study of different stages of 
the nitrogen cycle, the value of the ratio of natural isotopes occurring in 
the dilution of the label becomes very important. On the other hand, it 
can be considered negligible compared to the values of of the products 
that we have utilized, which are of the order 10.000, soils themselves 
having a 
Conclusions. 
The content of measured in certain natural constituents is 
sufficiently small to be ignored during the use of substances labelled 
by enrichment or depletion of the heavy isotope; at the most, it will have 
to be taken into account at large dilutions of the labelled substances. 
The impoverished nitrogen used corresponds to a of about 10 000 
and the maximum difference between the of non-labelled samples is 70 
for Nitrosomonas europaea. When calculating the possibility of error (one 
must remember), the substance containing nitrogen impoverished in can 
be diluted about a hundred times in the course of the diverse transformation 
to which it is subject. This allows great possibilities of utilization 
in the field. 
By contrast, it allows emphasis on the natural variations of nitrogen 
15 in different soils: of the forest -4.5, of the teppes +5.2 and of 
the prairie +9.9 as well as the role of microorganisms in isotope fractionation; 
the coefficient fractionation by Nitrosomonas europaea is 1.04 
Without entering into a discussion per se, we would agree with the sentiments 
of RENNIE & PAUL (1975) (it is necessary] "to know and understand the processes, 
biological and other, which control the isotopic concentrations of nitrogen 
in the soil before being able eventually to use and interpret them". 
It appears from the model of SHEARER et al (1974), that laboratory 
studies allow us to better understand the diverse fractionation in the 
course of the nitrogen cycle. In addition, the prairie of the Dombes 
encompasses ponds fortified only by ground water and leached water seems 
to be an interesting field for following labelled . 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Notice 
 
 
Please note that these translations were produced to assist the scientific staff of the 
FBA (Freshwater Biological Association) in their research. These translations were 
done by scientific staff with relevant language skills and not by professional 
translators. 
 
 
 


Ratio of natural isotopes of nitrogen. I. Primary results: soils of Dombes [Translation from: Revue d'Ecologie et de Biologie du Sol 14, 279-287, 1977]

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Ratio of natural isotopes of nitrogen. I. Primary results: soils of Dombes [Translation from: Revue d'Ecologie et de Biologie du Sol 14, 279-287, 1977]

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