Acknowledgment The authors thank John P. Bell for calculations of standard deviation by computer. Literature Cited Buck, M., Stratmann, H., Stauh 27, 11-15 (1967). Gill, W. E., Amer. Ind. Hyg. Ass. J . 21, 87-96 (1960). Kolthoff, I. M., Sandell, E. B., “Textbook of Quantitative Inorganic Analysis,” rev. ed., Macmillan, New York, 1948, p. 603. Kooiker, R. H., Schuman, L. M., Chan, Y. K., Arch. Environ. Health7,13-32, (1963). Lodge, J. P., Pate, J. B., Ammons, B. E., Swanson, G. A,, J . Air Poll. Control Ass. 16, 197-200 (1966). O’Keeffe, A. E., Ortman, G. C., Anal. Chem. 38,760-63 (1966). O’Keeffe, A. E., Ortman, G . C., ibid., 39, 1047 (1967). O’Keeffe, A. E., Ortman, G . C., ibid., 41, 1598 (1969). Saltzman, B. E., Anal. Chem. 26, 1949-55 (1954). Saltzman, B. E., ‘Selected Methods for the Measurement of
Air Pollutants,” Public Health Service Publication No. 999AP-11, pages C-4 and C-5, (1965). Saltzman, B. E., Wartburg, A. F., Anal. Chem. 37, 1961-64
(1969. ~S c a r i n g h , F. P., Frey, S. A., Saltzman, B. E., Amer. Znd. Hyg. ASS.J . 28,260-266 (1967). Scaringelli, F. P., Rosenberg, E., O’Keeffe, A. E., Bell, J. P., Anal. Chem. 42 (87). 1970. Shaw. J. T., Atmis. Environ. 1, 81-85 (1967). Stratmann, H., Buck, M., Air, Water Pollut. 10, 313 (1966). Thomas, M. D., MacLeod, J. A., Robbins, R . C., Goettelman, R. C., Eldridge, R . W., Rogers, L. H., Anal. Chem. 28, 1810-16 (1956). \-
Received for review February 24,1970. Accepted June 15, 1970. Presented in part at the Division of Water, Air, and Waste Chemirtry, 157th Meeting, ACS, Minneapolis, Minn., April 1969. Also, presented in part at the 11th Conferencen j Methods in Air Pollution and Industrial Hygiene Studies, Uniwr rity of California, Berkeley, Calif., March 30, 1970. Mention of any conimerrial product in this paper does not constitufe endorsement by the National Air Pollution Control Administration.
Nitrogen Isotope Fractionation in Soils and Microbial Reactions C. C. Delwiche and Pieter L. Steyn University of California, Davis, Calif. 95616
The isotopic composition of soil nitrogen in profile was examined and the extent of nitrogen isotope discrimination in various microbial reactions of the nitrogen cycle determined. The abundance of nitrogen-15 at various depths in profile differed among the soils examined. These differences appeared to be a function of soil texture or texture-dependent factors, and the possibility of isotope fractionation by chromatographic processes is likely. Because of the many factors contributing to variations in isotopic composition of soil nitrogen, the extent of enrichment with nitrogen-15 cannot be considered a direct measure of the degree to which nitrogen cycling has taken place in a given soil. In the fixation of nitrogen, the oxidation of ammonium ion to nitrite by Nitrosomonas and the assimilation of ammonium ion by the several species examined all showed some discrimination in favor of the lighter (nitrogen-14) isotope.
T
he rare stable isotope of nitrogen, N16, constitutes approximately 0.366 atom of atmospheric nitrogen (Nier, 1950). This value is relatively constant in the atmosphere (Dole, Lane, et al., 1954), but the isotope distribution can differ significantly in other nitrogenous forms, particularly in biological systems. Work in these laboratories and elsewhere (Cheng, Bremner, et al., 1964) indicated not only a variation in the abundance of the rare stable isotope of nitrogen from one soil to another, but also differences in distribution in profile. These minor variations in abundance become of concern because of their bearing on isotopic tracer studies of nitrogen fixation, and the potential errors they might introduce into any determination of fixation rates. The purpose of the present study was to determine in greater detail the nature of these variations in
isotopic composition, to examine some of the factors contributing to their existence, to evaluate the significance of such variations as they affect observations on nitrogen cycling, and to examine their potential contribution to our understanding of the biogeochemistry of nitrogen. Materials and Methods Nitrogen was determined by a modification of microKje1dah1 methods essentially as described by Bremner (1965) with use of a boric acid receiver with no internal indicator and titrating to the end point with a p H meter. The distillate was then evaporated to near dryness and converted to nitrogen gas by oxidation with alkaline hypobromite. Two successive liquid nitrogen traps were used to remove possible volatile contaminants, and the isotope ratio was determined o n a modified Consolidated-Nier Model 21-201 mass spectrometer with some alterations in circuitry and in mechanical features, including the substitution of an ion-getter pump for the diffusion pump with which the instrument was originally equipped. All samples were referred to a standard of ammonium chloride which in turn had been calibrated against atmospheric nitrogen. Since the purpose of the present study was to make comparative observations on isotope distribution, no effort was made to determine the absolute abundance of N15 in the atmosphere. The value of 0.366 atom “5, determined by Nier (1950) and established to be essentially constant by the work of Junk and Svec (1958), was used. Results Isotope Distribution in Profile. Studies were made of the isotopic composition of nitrogen in profile by using several soils of northern California which were part of a concomitant study of the rate of nonsymbiotic nitrogen fixation under field Volume 4, Number 11, November 1970 929
N ( p e e g . soil)
TOTAL
.ooo
,001
.002
.003
A T O M P E R C E N T E X C E S S NI5 ( R e f e r r e d t o a t m o s p h e r i c N,) Figure 1. Distribution of N1j and total nitrogen in two adjacent profiles of Yolo fine sandy loam
T O T A L N (,ue/g. s o i l ) 20 l
I
40 I
!
60 I
conditions. Figure 1 shows the distribution of nitrogen isotopes in two profiles of Yolo fine sandy loam taken to a depth of 140 cm. Total nitrogen content of soil samples taken a t the same level as those used for isotopic composition determination is also shown for a comparison. N15 content was greatest at a depth of approximately 20 crn., and all samples showed 0.001 or greater atom excess N15when compared with atmospheric nitrogen. This soil was a Yolo fine sandy loam from a n agricultural field near Davis, Calif., which had not been leveled and was subjected to only that tillage customary for a grain crop (barley). The second two profiles shown in Figures 2 and 3, respectively, were taken to a greater depth. These soils were also of the Yolo series, but both of them were in areas where some surface alteration probably had taken place during the past century by both leveling processes and alluvial activity. Total nitrogen values are also given for these soils. In each of the sites examined there appeared to be some correlation of isotopic composition with total nitrogen although this was not universally true. Isotope Effects in the Fixation of Nitrogen. Possible explanation for the difference in isotopic composition of the nitrogen of the atmosphere and that of the biosphere and soil may be discrimination in any of the reactions involved in the cycling of nitrogen. Bigeleisen (1949) made calculations o n the relative reaction velocities of isotopic molecules. Based upon these calculations, it could be assumed that discrimination between the isotopes of nitrogen could take place in any of the reactions involved in the cycling of nitrogen. The fixation reaction in Azotobacter was studied by Hoering and Ford (1959), who worked with four species of the organism Azotobacter. No consistent isotope effect was found
O
T O T A L N (,ue/g. s o i l )
I
0
d
I
20
40
100
80
60
I
r
d '
4'b
I -
---- --- -= k---
2-
h
P--
E
d
- 2
I F
a W a
ATOM % EXCESS N t 5
-E v
3-
I
P
TOTAL N
Il I
4-
4
I
3
F
a
I I I
5 -
I
W
n
I
I
6-
I I
I I
4
78-
-F
.ti000
.0010
.0020
ATOM PERCENT EXCESS ( R e f e r r e d t o atmospheric
.0030
Nt5 N2)
gt
P
n: ATOM % EXCESS N I 5
,I
; I
#
d
I
.0020 .004 .oooo A T O M P E R C E N T EXCESS NI5 ( R e f e r r e d t o a t m o s p h e r i c N,)
Figure 2. Distribution of N16 and total nitrogen in a deep profile
Figure 3. Distribution of N1j and total nitrogen in a deep profile
Yolo series near Davis, Calif.
Yolo series, second site, near Davis, Calif.
930 Envirppmental Science & Technology
from which the conclusion was drawn that the rate-determining step in the mechanism did not involve the bonding of nitrogen. Studies in our own laboratory with Azotobacter uinelandii are given in Table I. These show a discrimination in the reaction giving a value for P of 1.004 in which P is defined as fi = R , / R f , with R , = N15/N14in the atmosphere and R , = N15/NI4in nitrogen fixed. The ratio observed for A. cinelandii by Hoering and Ford was 1.0022. It is probable that differing results could be obtained with any species depending upon the conditions of observation, and it is difficult t o define what might be the ratelimiting factor under natural conditions. F o r this reason no definitive conclusion can be reached regarding the extent to which Azotobacter influences the isotopic composition of biologically fixed nitrogen until a more complete understanding of conditions is reached. The fixation of N14 appears to be favored slightly. Nitrification. The nitrification reaction was examined for possible isotope discrimination using Nitrosonionas europaea. Cultures of the organism were incubated in the presence of ammonium ion until approximately half of the substrate ammonium had been converted to nitrite. Cells were separated from the medium by centrifugation. Ammonium was then separated from the medium by distillation in the presence of 5 M sodium hydroxide and the residual nitrite was reduced with Devarda’s alloy and collected by distillation. Mass spectrometric determination of the isotopic composition of both fractions was then made. Results of this study are presented in Table 11. For the fixation reaction the supply of nitrogen available is essentially infinite, and /3 can be calculated directly from values of N15:N14ratios in atmospheric nitrogen and fixed nitrogen, respectively. F o r the nitrification reaction and other studies in which substrate (reactant) is limited and can be expected t o show a depletion in one or another of the isotopes, calculations are based upon the formulation developed by Tong and Yankwich (1957). For these applications, ratios were obtained of isotope distribution in nitrogen gas at nz./e. 29 and 28, respectively. m j e . 29 in unreacted fraction m.je. 28
Pa = -__-
m.je. 29 in product fraction nz.je. 28
Ph
= __-
f’
=
fraction reacted
The kinetic fractionation factor, P, was calculated from the relationship
P=
where and
R
-~ P b
*-2+Pb
Of the several reactions examined, this one showed the greatest isotope effect with a value for P of 1.026. As a result, for a reaction which does not go t o completion, N’j would tend to accumulate in the reduced nitrogen fraction and the nitrate formed would have depletion of the heavy isotope.
Table I. Isotope Fractionation During Nitrogen Fixation by Azotobacter Vinelandii Pf P 0.0073195 1 ,0054 0.0073278 1.0043 0.0073354 1 ,0032 0,0073364 1.0031 0.0073318 1.0037 0.0073328 1 ,0036 Av. 1.0039 m le. 29 .
where
m.je. 28 in atmospheric nitrogen = 0.0073590. m . / e . 29 PI = of fixed nitrogen. R R -Po ” p =2 (see text). a - 2 + P a ’ Rf = 2, R’ J pa = L -
;m8
Table 11. Values for Isotope Fractionation Obtained with Nitrosomonns Europaea Pfl pb f P 0.65 1 ,0251 0.007478 0,007186 0.65 1 ,0264 0.007490 0.007182 0.66 1.0183 0.007276 0.007494 0.59 1.0295 0.007472 0.007152 0.64 1 ,0360 0.006906 0.007304 0.68 1 ,0260 0.007220 0.006918 0.54 1.0176 0.007010 0.0071 88 0.64 1 ,0271 0.007200 0.006900 0.72 1,0284 0.007204 0.006858 Av. 1,0260 29 . where P a = - in unreacted ammonia. pb =
f p
= =
28 29
. nitrite formed. F~ in
fraction reacted. see text.
The other portion of the nitrification sequence, the oxidation of nitrite to nitrate, typified by Nitrobacter, also represents a possible source of fractionation of isotopes. To determine the magnitude of this fractionation requires a quantitative separation of nitrate from nitrite in the reaction products. Methods for this separation are presently under study. Denitrification. Similar studies were conducted with the denitrification reaction. I n denitrification there is a possibility of discrimination not only in the production of nitrogen gas, but also in the preceding reactions which include a reduction from nitrate to nitrite, from nitrite to a compound at the oxidation level of hyponitrous acid and in the production of nitrous oxide and its reutilization. For reasons which will be developed later, it was considered unlikely that the denitrification reaction would provide a complete explanation for the differences in isotopic composition observed between fixed nitrogen and that of the atmosphere. Although such a discrimination could be of considerable theoretical interest it was not pursued in detail as part of the present study. Wellman, Cook, et al. (1968), working with Pseudomonas denitrifcans, reported a n “instantaneous isotope fractionation factor” of approximately 1.02 or higher. Their studies also demonstrate that the extent of fractionation varies with conditions as would be expected because of the complex nature of the reaction. Particularly when nitrous oxide production was significant, it was difficult to achieve isotope balances. Moreover, for any given set of conditions the extent of isotope fractionation realized will be a function of the comVolume 4, Number 11, November 1970 931
Table 111. Isotope Effect in the Denitrification Reaction with Pseudomonas DenitriJcans Fraction Sample denitrified P" Pb P 1 0.63 0.007415 0.007261 1.0134 0.007410 0.007201 2 0.60 1.0189 3 0.75 0.007430 0.007210 1.0164 0.007418 0.007200 4 0.55 1.0208 0.007422 0,007232 5 0,60 1 ,0171 Av. 1.0173 where p a = pb =
p
=
29
y8i n unreacted nitrate. in nitrogen formed. see text.
pleteness of the reaction. The reaction undoubtedly influences terrestrial variations in abundance of N 1 j relative to N14, tending to favor the formation of N2140and N214and, therefore, the retention of NI5in the fixed form. Isotope distribution in nitrogen produced in the denitrification reaction was examined using P. denitrrFcans. Cells grown for 24 hr. in a n anaerobic environment with nitrate as hydrogen acceptor were separated from the medium by centrifugation and the resting cells were provided with a glucose substrate and nitrate. After a n incubation period of 4 hr. the product nitrogen and remaining nitrate were analyzed for their isotopic composition. Carbon dioxide was eliminated by passing the gases through a n ascarite column, nitrous oxide was separated from nitrogen by freezing in a liquid nitrogen trap and the nitrogen analyzed for isotope composition by mass spectrometry, determining the mass 29 :mass 28 ratio. Results of this study are given in Table 111. The value for p obtained in the conversion of nitrate to nitrogen was 1.017. The latter value is in close agreement with that of Wellman, Cook, et al. (1968). Discrimination of the nitrification reaction of Nitrosmonas would favor the production of NI4O2- and the further discrimination of the denitrification process in favor of the lighter isotope would be additive in its effect with the net retention of N l j in the soil system in excess of N14 when compared with atmospheric nitrogen. Nitrogen Assimilation. Isotope fractionation during ammonia assimilation was studied using Azotobacter rinelandii and three typical soil yeasts. A . cinelandii was grown on Burk's nitrogen free medium (Burk, Lineweaver, et a/., 1934) with 1 g. per liter of NH4C1 and incubated at 30" C. for 39 hr. Cells were separated from the medium by centrifugation. Hansenula californicu, Pichiu terricolu, and Schwanniomyces ullucius were grown o n Yeast Carbon Base (Difco) plus 5 g. N H K l per liter. Incubation was at 36" C. for 60 hr. Cells were harvested by filtering through a 0.45-p Millipore filter. Cell nitrogen and residual ammonia-nitrogen were quantitatively determined and analyzed mass spectrometrically as described. Since only a relatively small fraction of the supplied nitrogen = R,/R,, with pm = 29:28 was used, p was calculated: ratio of medium nitrogen and R, = pm/2 p m , while pc = 29 :28 ratio of cell nitrogen and R, = pc/2 pc. All organisms preferentially utilized NI4H4' (Table IV). Therefore, the unassimilated ammonia was enriched with respect to " 5 , provided assimilation was not complete. Further, the kinetic isotope effect was greater in the bacterium than in the yeasts, and also greater during assimilation than during fixation by A. cinelandii. Chromatographic Separations. The separation of isotopes of
+ +
932 Environmental Science & Technology
nitrogen in ammonia by means of ion-exchange resins was reported by Spedding, Powell, et al. (1955). These workers were able to accomplish an almost complete separation of nitrogen-14 and nitrogen-15 by cycling ammonium ion through a succession of ion-exchange resin columns. The soil colloid also could serve as a fractionating medium with the chromatographic fractionation of both cationic and anionic isotopic species. Comparison was made between the single plate fractionation factors obtained with a cation-exchange resin (Dowex 50), a n anion-exchange resin (Dowex l), and a kaolinitic clay soil (Ione series). Results of this study are given in Table V. The fractionation factors obtained were small in comparison with values for isotope fraction in the biological reactions studied. These represent only single plate fractionations, however, and the chromatographic process with the downward movement of ions in soil could be expected to introduce significant separation of isotopes. The cation-exchange resin and the kaolinitic clay showed similar values for /3, favoring the retention of the heavier isotope in the adsorbed fraction of NHa+.With the anion resin, by contrast, retention of the lighter isotope (N1403-) was favored. No soils have been examined for this property since the anion-exchange capacity of most of the soils examined was exceedingly low. As part of a future study it is intended t o examine these properties, including isotope distribution and znionic values for (3, in soils of significant anion-exchange capacity. Geoclzeniical Questions Because there is a demonstrable difference between NI5 content of the nitrogen of the atmosphere and that of the biosphere, the possibility arises that an examination of the fine structure of isotopic distribution between the atmosphere, the biosphere, and the soil might provide some geocheinical clue in the processes of weathering and atmosphere formation. Wlotzka (1961) reported that the nitrogen content of sedimentary rocks exceeds that of igneous rocks by a factor of 10 or more. H e did not report the isotopic composition of this nitrogen although studies in this laboratory suggests that the NI5 of nitrogen of sedimentary rocks is significantly higher than that of the atmosphere and is variable. Scalan (1959) reported a slight NlS excess in the nitrogen of igneous rocks compared with that of atmospheric nitrogen. It is probable that the nitrogen of sedimentary rocks is largely atmospheric in origin and that therefore concentration of has taken place due to biological or other processes. Gaebler, Choitz, et al. (1962) reported upon the N'" enrichment of some amino acids as did Hoering (1955) upon some animal and plant proteins. These differences in isotope abundance distribution are undoubtedly real and probably Table IV. Values for Isotope Fractionation during Assimilation of Ammonium-Nitrogen % "4-N P Organism assimilated pm P. 0,007252 1 ,0148 0,007359 2 . 5 A. uinelandii 0,007369 0.007346 1 ,0031 1 .07 H . Californica 0,007372 0.007359 1 ,0018 0.58 P. terricola 0,007367 0.007359 1.0011 1.16 Sch. allucius where
29
pm =
28 of ammonia-nitrogen in preincubation medium.
pc =
29 y8 of cell-nitrogen.
p
see text.
=
Table V. Isotope Fractionation with Ion-Exchange Systems System NH4Cl on Dowex 50
K N 0 3on Dowex 1
N H E 1 on Ione clay
P
Pa
Pb
f
0.007437 0.007436 0.007436 0.007437 0,007435 0.007413
0,007446 0.007442 0.007444 0.007445 0.007444 0.007418
0.4900 0,4900 0.4900 0,4900 0,4590 0.4590
0,99912 0.99941 0.99921 0,99922 0.99909 0.99949 Av. 0.99926
0.007454 0,007449 0,007447 0.007448 0.007449
0.007425 0.007431 0.007425 0.007431 0.007425
0.493 0.557 0.541 0.550 0.541
1,00283 1.00165 1.00205 1.00158 1,00225 Av. 1.0021
0,007441 0.007441 0.007438 0.007442 0.007440 0,007421 0.007421
0.007446 0.007452 0,007446 0,007447 0.007445 0.007427 0,007426
0.20 0.19 0.20 0.20 0.19 0.20 0.20
0.99939 0.99866 0.99903 0.99939 0,99939 0.99927 0.99939 Av. 0.99922
where
pa
29 .
= 28 I n
pb =
f
adsorbed fraction.
p
$ in solution fraction.
= =
fraction adsorbed. calculated as in nitrification reaction (see text).
~
have their explanation in countercurrent biological processes. The contribution of the biological system to minerals in the enrichment of nitrogen in N15appears to be variable, however. Smith and Hudson (1951) reported that the N1j content of the nitrogen of coal and petroleum is “essentially the same” as that of the atmosphere. Their nitrogen, obtained by the Dumas method, included nitrogen of organic sources and they emphasized the relative constancy of isotopic composition of nitrogen through recent geologic history. The extensive N1j enrichment of occluded nitrogen of pitchblende ores observed by White and Yagoda (1950) is probably not due t o chemical or biological discrimination, but more likely has its origin in differing cross-sections of the isotopes of nitrogen for the products of radioactive disintegration. They reported a n N 1 j content of the nitrogen occluded in uranites of l O Q years age, almost double that of present atmospheric nitrogen. If any single reaction in the cycling of nitrogen resulted in a concentration of the rare isotope, repeated cycling presumably would produce successive enrichment of the fixed portion of nitrogen. Table VI shows a comparison of the N‘j content of clover and grass collected from the same site on a n irrigated lawn in Davis, Calif. T h e clover shows a n average N 1 j content of 0.0008 atom excess N15 referred t o atmospheric nitrogen, significantly less than that of the grass or total soil nitrogen from t h e site. T h e conclusion is suggested that much of the nitrogen of the clover had its origin i n atmospheric nitrogen whereas that of the grass came primarily from the soil. There are other possible explanations, including fractionation in ion uptake or differences in isotopic composition of the nitrogen of soil at the levels from which the root system extracted nitrogen.
~~~
Table VI. Comparison of Isotopic Composition of a Legume and a Nonlegume Grown on the Same Site Nitrogen source Clover
Grass
Atom
122 92 105 17.4 10.3 14.4
Soil total nitrogen
a
Total N (mg./g. dwt.)
0.37 0.35 0.43
Nl5
content“ 0.0010 0.0007 0.0006 Av. 0.0008 0.0037 0.0037 0.0029 Av. 0.0035 0.0029 0.0033 0.0034 Av. 0.0032
excess relative to atmospheric nitrogen.
Table VII. Comparison of Isotopic Composition of Nitrogen in Soybean Plants as a Function of Fixation Nitrate Cobalt added added N15 Sample (pmoles/l.) (p.p.b.1 content“ 1 10 0.00 0.0023 =k 0.0002 2 10 0.01 0.0008 f 0.0002 3 10 0.10 0.00034 i. 0.00015 4 10 1.00 0.00037 f 0.00015 5 1000 0.10 0.0026 + 0.0003 6 K N 0 3 in culture solution 0.0028 i. 0.0003 a
Atom
excess relative to atmospheric nitrogen.
Volume 4, Number 11, November 1970 933
The validity of the conclusion that the difference in isotopic composition represents a distinction between atmospheric and soil nitrogen, respectively, is supported by the results of another study reported in Table VII, i n which isotopic composition of the nitrogen of soybean plants is a function of the level of cobalt in the nutrient medium provided. Cobalt is required for nitrogen fixation (Ahmend and Evans, 1959; Reisenauer, 1960) and in the absence of cobalt, essentially all of the plant nitrogen is from culture solution sources. Soybean plants were grown in the greenhouse in 4-liter culture solutions made up o f highly purified salts to which cobalt was added at varying levels. The medium was that of Delwiche, Johnson, et al. (1961), with cobalt chloride added at the level shown. Treatments 1-4 had nitrate added at the level of 10 kmoles per liter, and Treatment 5 contained 1mmole per liter of nitrogen as nitrate at the beginning of the experiment. When nitrogen in the culture solution was limiting, yield was roughly proportional to added cobalt at low concentrations. Cobalt at the level of 0.1 p.p.b. gave yields essentially the same as that at 1 p.p.b. When the primary source of nitrogen was through fixation, the isotopic composition was lowest, approaching that of atmospheric nitrogen. When nitrate was the primary source of nitrogen, isotopic composition approached that of the substrate nitrogen. From this it was also concluded that the isotopic composition of plant nitrogen was not significantly different from that of the source nitrogen and no demonstrable isotope fractionation took place. These results suggest that it might be possible to determine the source of nitrogen in plants under field conditions and to evaluate the extent to which the fixation of atmospheric nitrogen may be contributing to the total nitrogen of a n ecosystem. Moreover, if there are one or more discriminatory reactions in the biological cycling of nitrogen which contribute to the enrichment of N15 in the soil, it might be possible to determine the extent to which the nitrogen cycle has been operative during geologic time. A more detailed examination of the processes of nitrogen fractionation in soil, however, reveals that there are other more potent factors involved in the enrichment of N16 in soil nitrogen and insufficient information is presently available o n these processes to make possible their interpretation in terms of the geochemical history of nitrogen. What appears to be a chromatographic separation of isotopes in soil may be a n important factor and one which has been examined to only a limited extent. Reactions of nitrification and denitrification also contribute and their comparative effects are undetermined. Discussion Although there are significant differences in nitrogen isotope distribution from one soil t o another and in the soil profile, the various biological reactions examined are not the only contributors to these differences, Significant fractionation of’ nitrogen isotopes takes place in both nitrification and denitrification. I n addition, comparatively large fractionations of nitrogen isotopes were observed due to chromatographic phenomena with ion-exchange resins and with clay particles. These probably also play an important role in the separation of nitrogen isotopes in soil. As a result, it is unlikely that any regular biological process in the nitrogen cycle can be used t o predict the isotopic composition of soil nitrogen and also unlikely that the distribution of nitrogen isotopes in soil as contrasted with that in the atmosphere can be useful in evaluating the extent to which the nitrogen cycle has been operative through geologic time. 934 Environmental Science & Technology
No consistent relationship between N1j content of nitrogen and either soil particle size or total nitrogen content t o the soil has been demonstrated. Discontinuities in the isotope composition curves and total nitrogen curves in profile coincide in many cases, but the correlation may be positive or negative, depending upon the soil or the position in profile. These complex interrelationships appear to be a function of water (and therefore solute) movement and further study will be necessary to determine their significance. As a generalization, the abundance of N1”in soil nitrogen appears to be somewhat higher at a point 10 to 50 cm. from the soil surface than it is at either the soil surface or at greater depths. Moreover, at those points in the soil profile where there is a significant change in texture (e.g.,a point where sand content is unusually high), there is a significant enrichment in N1”. Further studies will be necessary to correlate isotopic composition with other soil properties, but there is little doubt that chromatographic phenomena as well as biological processes play a large role in the separations. Conclusions Various California soils were examined for their distribution of nitrogen isotopes in profile. These have N1j contents ranging from 0.004 atom excess N1j referred to atmospheric nitrogen to values in the neighborhood of 0.002 atom excess. No soils examined showed nitrogen with an N15 abundance less than that of atmospheric nitrogen. Examination of the nitrogen fixation reaction with A . uinelandii suggests a slight discrimination against fixation of the N1j molecule. It is pointed out, however, that any discrimination observed may be a function of conditions under which the organism was grown and may vary with species. Some discrimination takes place in the nitrification reaction using N . europciea with a value of /3 of 1.026 in which /3 is defined as
’
=
N15/N14in ammonia N15/N14in nitrite
The complexity of reactions in the denitrification process makes it difficult to establish any single figure for isotope discrimination in that process. Some discrimination against the heavier isotope takes place during assimilation of ammonia by A . cineiandii and three soil yeasts ( H . californica, P. terricola, and Sch. allucius). Leguminous plants fixing nitrogen have a nitrogen isotope composition similar to that of the atmosphere and nonleguminous plants or legumes which are not fixing N have a nitrogen isotope composition similar to that of the medium in which they are grown. Chromatographic phenomena on soil particles play a strong role in changing nitrogen isotope distribution in soil. Because of the comparative effect of chromatographic phenomena on nitrogen isotope distribution as related t o processes in the nitrogen cycle, it is unlikely that any quantitative conclusions can be drawn from isotope ratio data concerning the extent to which nitrogen has been cycled from the atmosphere to the soil and back. Acknowledgment The authors are indebted to Ellen Barker and Herman Phaff, Department of Food Science and Technology, University of California, Davis, who supplied yeast cultures.
Literature Cited Ahmend, S., Evans, H. J., Biochem. Biophys. Res. Cornmun. 1, 271 (1959). Bigeleisen, J., J. Chem. Phys. 17, 675-78 (1949). Bremner, J. M., “Methods of Soil Analysis,” Black, C. A., Evans, D. D., White, J. L., Ensminger, L. E., Clark, F. E., Eds., Agronomy No. 9, Part 2, Amer. SOC.Agron., Madison, Wis., 1965, pp. 1236-86. Burk, D., Lineweaver, H., Horner, K., J . Bacteriol. 27, 325-40 (1934). Chena. H. H.. Bremner. J. M.. Edwards. A. P.. Science 146. 1 5 G and 75’(1964). ’ Delwiche, C. C., Johnson, C. M., Reisenauer, H. M., Plant Phvsiol. 36. 73-78 (1961). Dole, M., Lane, G. A., Rudd, D. P., Zaukelies, D. A., Geochim. Cosmochim. Acta 6, 65-78 (1954). Gaebler, 0. H., Choitz, H. C., Vitti, T. G., Vukmirovich, R., Can. J. Biochem. Physiol. 41, 1089-97 (1962). Hoering, T. C., Science 122, 1233 and 34 (1955).
Hoering, T. C., Ford, H. T., J. Amer. Chem. SOC.82, 376-78 (1959). Junk, G. A., Svec, H. J., “Nitrogen Isotope Abundance Measurements,” U.S. Atomic Energy Commission, Office of Technical Information, ISC-1138, 1958. Nier, A. O., Phys. Rev. 77, 789 (1950). Reisenauer, H. M., Nature 186, 375 and 76 (1960). Scalan, R. S.. Ph.D. thesis, University of Arkansas,. Fayetteville; Ark., ‘1959. Smith. P. V.. Jr.. Hudson. B. E.. Science 113. 557 (1951). Spedding, F.’H.,’Powell, J: E., Svec, H. J., J. Amer. Chem: Soc. 77, 6125-32 (1955). Tong, J. Y . ,Yankwich, P. E., J . Phys. Chem. 61,540-43 (1957). White, W. C., Yagoda, H., Science 111, 307-9 (1950). Wellman, R. P., Cook, F. D., Krouse, H. R., Science 161, 269 and 70 (1968). Wlotzka, F., Geochim. Cosmochim. Acta 24, 106-54 (1561). Received for review January 5 , 1970. Accepted June 15, 1970.
Characterization of Bottom Sediments: Cation Exchange Capacity and Exchangeable Cation Status Stephen J. Toth and Arthur N. Ott Department of Soils and Crops, College of Agriculture and Environmental Sciences, Rutgers University, New Brunswick N.J. 08903
rn Two parameters, cation exchange capacity (CEC) and
exchangeable cation status (ECS), were used t o characterize bottom sediments collected from rivers, bays, and freshwater impoundments. It was necessary to investigate the effect of drying on CEC and exchangeable Fe and M n to arrive at satisfactory modifications of soil techniques used for these parameters. Wide variations were obtained in CEC and ECS values for the sediments. CEC and ECS values may be utilized for determining saltwater intrusions and pollution effects.
T
wo parameters that have been used to characterize soils are the cation exchange capacity (CEC), and exchangeable cation status (ECS). The cation exchange capacity is the ability of an exchanger, expressed in terms of me. per 100 g., to retain a specific cation at a certain p H value and salt concentration. The exchangeable cation status, also expressed in terms of me. per 100 g., refers to the amount of Na, K , Ca, Mg, and H held by the soil complex. The C E C values are used for estimating the total storage capacity of soils for a specific cation, and the ECS values are used for estimating the fertility levels of soils (Jackson, 1958). Values for these parameters are obtained by a variety of techniques (Bower and Truog, 1940; Chapman and Kelley, 1930; Jackson, 1958; Kelley, 1948). The factors that influence the magnitude of the CEC include: clay content, type of clay mineral, and organic matter content (Jackson, 1958; Kelley, 1948). Other factors include the p H of the displacing solution, the nature of the displacing cation, and the soil-to-solution ratio (Kelley, 1948). The purposes of the present study were to determine what modifications of existing soil methods were required for determining CEC and ECS values of bottom sediments and t o present data on sediments from some rivers, bays, and freshwater impoundments.
Methods Sample Preservation and Preparation. Bottom sediments collected either as core or grab samples require special handling to ensure minimal chemical changes. Grab samples must be transferred immediately to plastic bags and tightly sealed. Core samples will normally contain a water layer on the surface which must be retained by immediate sealing of the top and bottom of the core. Both types of samples must be frozen as quickly as possible after collection and stored. The frozen samples must be thawed rapidly prior to sample preparation. Some chemical changes occur on the outer surfaces of the samples, as indicated by an oxidized layer of Fe. This layer must be removed before the sample is mixed. Since stratification of both organic and inorganic materials commonly occurs, the sample must be mixed thoroughly until all signs of stratification disappear. If necessary, distilled water can be added until a thick paste forms. The paste should be passed through a stainless steel screen (opening 2 mm. in diameter) to remove coarse particles, wood, and shellfish fragments. Cation Exchange Capacity (CEC). Fifteen-gram samples of organic sediments or 25 g. of sandy sediments are rapidly weighed into a 250-ml. beaker, in duplicate, and immediately covered with 100 ml. of N neutral N H 4 0 A c solution (Schollenberger and Simons, 1945). At this time, additional samples are prepared for moisture determinations. After standing for 30 min., the solution plus the sample is transferred to a funnel fitted with a 12.5-cm. Whatman no, 40 filter paper. The sample is leached with small amounts of NHaOAc solution until 500 ml. of leachate are collected or until the pH of the solution, after passing through the sediment, is pH 7.0. The leachate obtained is used for the determination of exchangeable Fe, Mn, Zn, Cu, and Ni. The sediment remaining in the funnel, after leaching with N H 4 0 A c solution, is now washed five times with 25-ml. portions of 80% ethyl alcohol to remove excess NH,OAc. These Volume 4, Number 11, November 1970 935
