Org. Geochem. Vol. 17, No. 5, pp. 635-648, 1991 0146-6380/91 $3.00 + 0.

00
Printed in Great Britain. All fights reserved Copyright 1991 Pergamon Press plc
Statistical evaluation of the elemental composition of humic
substances
JAMES A. RIC~* and PATRICK MACCARTHY
Department of Chemistry and Geochemistry, Colorado School of Mines, Golden, CO 80401, U.S.A.
(Received 19 April 1990; accepted 7 December 1990)
Abstract--Elemental data (C, H, O, N, S, atomic H/C and O/C ratios) for humic acids (410 samples),
fulvic acids (214 samples) and humin (26 samples), isolated from environments all over the world, were
compiled from the literature. These data were analyzed statistically using the mean, median, mode, range,
standard deviation and t-test. The large data base allows statistically significant differences between
various humic substances to be established. Prior to some of the statistical evaluations, the humic
substances were grouped according to source---soil, freshwater, marine or peat. The average elemental
compositions for humic acid, fulvic acid and humin, in tow, and also when segregated by source, are
presented. Standard deviations for carbon contents are remarkably small suggesting that perhaps an
optimum composition exists for humic substances in nature. The evaluation shows that humic acids in
tow are significantly (P < 0.0005) different from fulvic acids in tote with respect to C, N, and O contents
and atomic H/C ratios. Fulvic acid in tote is significantly ( P < 0.0005) different from humin/n tote in
terms of C and O contents. When segregated by source, some significant ( P < 0.0005) differences between
humic acids isolated from freshwater, marine, and soil environments are evident; similarly, significant
differences are found between fulvic acids from freshwater and soil sources. Three van Krevelen diagrams
based on: (1) the 650 samples of humic acid, fulvic acid and humin; (2) humic acids segregated by source,
and; (3) fulvic acids segregated by source, are discussed.
Key words--humic acid, fulvic acid, humin, elemental composition, statistical analysis
I N T R O D U C T I O N
One of t he most f undament al charact eri st i cs of a
chemi cal subst ance is its el ement al composi t i on. De-
t ermi ni ng t he el ement al composi t i on (or empi ri cal
formul a) of a discrete chemi cal compound is a first
st ep t o obt ai ni ng its mol ecul ar formul a; this, in t urn,
is cri t i cal for det ermi ni ng its st ruct ural formul a.
However, in t he case of non- st oi chi omet r i c mat eri al s
such as coal , kerogen and humi c substances, the
concept of even a net mol ecul ar f or mul a is ext remel y
l i mi t ed, and a unique, st ruct ural f or mul a does not
exist for these mat eri al s.
Nevertheless, el ement al anal ysi s is a useful t ool for
charact eri zi ng non- st oi chi omet r i c mi xt ures such as
humi c substances. The appl i cat i ons of el ement al
anal ysi s in the st udy of humi c subst ances have re-
cent l y been reviewed by Steelink (1985). Because
humi c subst ances are non- st oi chi omet r i c mat eri al s
t hey must be char act er i zed in t erms of t hei r average
propert i es. Accor di ngl y, it is appr opr i at e t o conduct
a st at i st i cal eval uat i on of these pr oper t i es in or der t o
est abl i sh t he st at i st i cal l y val i d differences and t rends
in these mat eri al s. El ement al composi t i on is one such
average pr oper t y and it is st at i st i cal l y eval uat ed in
this paper .
*Present address: Department of Chemistry, South Dakota
State University, Brookings, SD 57007, U.S.A.
van Krevel en (1961) devel oped a graphi cal met hod
to st udy the coal i fi cat i on process in whi ch the at omi c
hydr ogen/ car bon ( H/ C) rat i o is pl ot t ed as a funct i on
of t he at omi c oxygen/ car bon (O/ C) rat i o. This t ype
of pl ot , now general l y known as a van Krevel en
di agram, is oft en used for the classification of coal s
and kerogens. A frequent appl i cat i on of the van
Krevel en di agr am is t o i l l ust rat e t he changes in
el ement al composi t i ons t hat occur dur i ng t he alter-
at i on of organi c geochemicals in a geologic envi ron-
ment; e.g. H/ C and O/ C r at i os have been used to
follow the effects of diagenesis on humi c substances
(Huc and Dur and, 1977; Reut er and Perdue, 1984).
van Krevel en di agr ams have also been used by vari -
ous workers t o i l l ust rat e composi t i onal differences
between humi c aci ds and fulvic acids, and also t o
show vari at i ons in humi c substances as a funct i on of
source. For exampl e, Kuwat suka et al. (1978) used a
van Krevel en di agr am t o compar e t he el ement al
composi t i ons of soil humi c and fulvic acids, coals,
pl ant tissues and vari ous classes of organi c com-
pounds. Visser (1983) empl oyed a van Krevel en
di agr am t o compar e fulvic and humi c aci ds from
aquat i c and t errest ri al sources.
The magni t ude of t he H/ C r at i o has al so been used
to i ndi cat e t he degree of ar omat i ci t y or unsat ur at i on
(a smal l value) or al i phat i ci t y (a l arge value) of a
subst ance (van Krevelen, 1961). Perdue (1984) has
poi nt ed out t hat the t ot al unsat ur at i on of a humi c
635
636 JAMES A. RJc'~ and P^rmCK M^CC~Th'V
material cannot be obt ai ned solely from the H/ C
ratio; in addition t o unsat urat ed forms of carbon the
H/C ratio is also a function of unsat urat i on present
in functional groups, primarily carboxyl and car-
bonyl groups, with lesser contributions from ot her
miscellaneous forms of unsaturation. I f H/C ratios
are calculated for the 21 humic material samples in
the study of Perdue (1984) and compared t o the
aromat i c car bon contents corrected for the various
forms of noncar bon unsat urat i on it is seen that,
t hough the actual numbers differ, samples which
exhibit a high aromat i c car bon cont ent also exhibit a
small H/ C ratio and vice versa. The lone exception is
a spodosol fulvic acid with high total acidity
(12mequiv/g), a low corrected aromat i c carbon
cont ent but a moderat e H/C ratio (0.85). The H/C
ratio thus appears t o be a qualitatively useful par-
ameter for compari ng the aromaticities of humic
materials.
To date, the number of humic samples plotted on
a single van Krevelen di agram has been relatively
small. The value of any such investigation would be
enhanced by enlarging the dat a base and by using
humic substances from a wider variety of source
environments. I n addition, when a large dat a set is
empl oyed one can justifiably apply statistical
met hods to quantify the relationships between the
various groups of humic substances by establishing
the statistically significant differences. For this study
we compiled from the published literature an exten-
sive dat a base consisting of the elemental contents of
650 samples of humic substances. The objectives of
this paper are:
(1) t o apply basic statistical methods to a very large
dat a base of elemental contents in order to quan-
tify the relationships between various classes of
humic substances; and,
(2) t o study van Krevelen diagrams for humic
substances prepared from this large dat a base.
DATA AND DATA ANALYSIS
Elements studied
Elemental contents (C, H, N, S, and O) of humic
acids, fulvic acids and humins were compiled from
the literature. Elemental contents are generally re-
port ed on a weight/weight percentage basis and t hat
is the form used in this paper. All elemental contents
are expressed on an ash-free basis, and, where necess-
ary, the ash-flee values were calculated by the present
aut hors using the published ash-contents. At omi c
H/C and at omi c O/ C ratios were comput ed for all
samples and were plotted on a van Krevelen diagram.
For this study, the samples were classified into humic
acid, fulvic acid and humin on the basis of the
conventional definitions of these fractions and based
on the descriptions of the samples presented in the
original publications. The samples were further
classified as soil, freshwater, marine, peat and coal
humic materials, depending on their sources. The
samples described as marine were obtained almost
exclusively from marine sediments; onl y two fulvic
acids and two humic acids in this dat a set represent
dissolved marine materials. Dopplerite, lignite and
leonardite humic substances were included with the
coal samples. The humic acid set consisted of 410
samples, the fulvic acid set had 214 samples, and
there were 26 samples of humin. These samples had
been isolated from locations all over the world.
Because of the large number of analyses compiled, no
single aut hor or publication dominates the contri-
butions to the overall dat a sets.
The elemental dat a for each sample consisted of
carbon, hydrogen, and oxygen contents in all cases,
nitrogen contents for all but two samples, and sulfur
contents for approx. 30% of the samples. Onl y in a
few cases had oxygen been determined directly;
generally it had been obtained by difference.
Statistical analysis
The mean, ($), was comput ed using the following
equation:
tl
~x~
=~=i (1)
n
where n is the total number of samples in a set and
x~ represents the individual values for a particular
parameter. The median (the value about which all
other values are equally distributed) and the mode
(the most frequently occurring range of values) were
also obtained from the dat a set. The mode was not
determined for dat a sets of <20 samples nor is i t
presented for small dat a sets which exhibited 2 or
more intervals of the same frequency. The mean,
median and mode are measures of the central ten-
dency for t hat paramet er and are the same for
normally distributed data.
The range, as presented, gives the low and high
values for a parameter in the dat a set. The st andard
deviation (s) was calculated using equation (2):
1-- n 2--11/ 2
s = L . - j . ( 2 )
The range is a measure of the dispersion of the data,
and st andard deviation is a measure of the dispersion
of the dat a ar ound the mean.
The t-test used here evaluates the probability t hat
the mean value of a particular paramet er exhibited by
two dat a sets (.~ and $2) could be observed within the
same population. The t-statistic is obtained using
equation (3):
t = [ n2 ( ~ l ) J I ' ` 2 (3)
Statistical evaluation of the elemental composition of humic substances 637
where Q is defined by:
r r T
Q = n , (x~, - x~,) nl
i L n 2 J J
[
" l rntll/ 212
--i~=l(Xl--XEi)L~J .] (4)
and nt and n 2 are t he number of measurement s in the
smal l er and l arger dat a sets, and x l, and x2, are the
i ndi vi dual values in t he smal l er and l arger dat a sets,
respectively (Crow et aL , 1960). Thi s st udy empl oyed
a one-si ded t-test.
Chi - squar ed (X 2) statistics are used to assess the
nor mal i t y of t he di st r i but i on of values in the dat a set
for a par t i cul ar paramet er. Thi s i nf or mat i on is
necessary because t he t -t est is par amet r i c, i.e. it
operat es on t he assumpt i on t hat t he dat a exhibit a
nor mal di st ri but i on. Consequent l y, t he dat a in an
unt r ansf or med st at e and l ogm-t ransformed st at e were
eval uat ed usi ng the fol l owi ng equat i on (Crow et al.,
1960):
( t / i - ei) 2
X2=
(5)
/ . d
i = t ei
where r is t he number of categories, r/~ are the
observed frequencies and ei are t he t heoret i cal fre-
quencies. The form of t he dat a which exhi bi t ed, or
most -cl osel y appr oxi mat ed, a nor mal di st r i but i on
was t hen used in t he t -t est . In most instances the dat a
in t he unt r ansf or med st at e exhi bi t ed, or most -cl osel y
appr oxi mat ed, a nor mal di st ri but i on. The use of dat a
t r ansf or mat i ons in st at i st i cal anal ysi s is discussed by
Bart l et t (1947). Fi gur e 1 gives a series of hi st ograms
whi ch i l l ust rat e t he di st r i but i on of the dat a which was
j udged t o exhibit, or most closely appr oxi mat e, a
nor mal di st r i but i on for t hree of the dat a sets used in
this study. A visual assessment of nor mal i t y can be
made for t he l arger dat a sets (e.g. all humi c acids) but
to do so becomes i ncreasi ngl y difficult as the number
of sampl es decreases. Chi - squar ed statistics are
usual l y a mor e sensitive i ndi cat or of the di st r i but i on
of t he dat a (Crow et aL , 1960) and for this reason
they were used t o eval uat e the nor mal i t y of t he dat a
when possible. Chi -squared statistics were not calcu-
l at ed for dat a sets cont ai ni ng < 50 samples. When an
assessment of nor mal i t y was needed for smal l dat a
sets it was made by i nspect i on of the hi st ograms.
The degrees of freedom (d.f.) were est i mat ed using
Sat t ert hwai t e' s (1946) appr oxi mat i on. Thi s met hod
est i mat es the degrees of freedom in si t uat i ons where
it is assumed or known t hat sl :/: s2. The degrees of
freedom are obt ai ned using:
r s;T
s ~+
d.f. = Lnl n2J (6)
nl m n2
Computer programs
The FORTRAN pr ogr am PHI ST (Kl usman, 1981)
was used to cal cul at e t he st at i st i cal quant i t i es de-
scribed in equat i ons (1), (2), and (5). The pr ogr am
orders the values in a dat a set from lowest t o highest
which facilitates t he det ermi nat i on, by inspection, of
the medi an and range. PHI ST al so pl ot s the distri-
but i on of the dat a as nor mal st andar d devi at e his-
t ogr ams from which the mode may be obt ai ned by
inspection.
Tests of significance [equation (3)] were per f or med
by TTEST ( Kl usman et al., 1980), a F ORTRAN
pr ogr am for cal cul at i ng the mean and t -st at i st i c for
two sampl e sets in which it is known, or assumed,
t h a t S 1 S 2 .
C H N
,(::,,,,II,,, ,,,,,dh,,,, ,,,,i,l,,,,,
S 0 O/C H/C
. . . . , , , . , , , , . . . . . , , , , , . . . . . . . , , t l , , , . . . . . . , , , , I t , , . . .
ALL humic acids
( b )
. . . . . . . . , , , , . . . . . . . , , , , h , l , , . . . . . . . , , 1 1 1 , 1 , . . . . , , , , L , , . . . . . , , l l l l , , , , . . . . . . .
Soil humic acids
, , , I I I I h, , . . . . . . . . . , , , , I I , , , . . . . . .
( c )
. . . . h i , , I . . . . . , t l l h . . . . . I , I I I l i . . . . . . . . . , , , . . , . . . . . . t i l l . . . . . . . , , t l . . . . . . h , , l m, . , . . , .
A
Mar i n e h umi c ac i ds
Fig. l. Elemental and atomic ratio histograms for (a) unsegregated humic acids, (b) soil humic acids and
(c) marine humic acids (based on data in Tables l and 3). All data except the S values are untransformed;
the S data are lOglo-transformed.
638 JAMES A. PaCE and P^TPdCK MACCARTHY
Limitations and assumptions in this study
This study is based on the analysis of experimental
dat a obt ai ned from the literature; as such, the quality
of the analytical met hodol ogy per se in the individual
publications from which the dat a were obtained
cannot be directly evaluated. However, even t hough
the elemental analysis of humic substances is not
wi t hout its problems (Huffman and Stuber, 1985),
carbon and hydrogen determinations can generally be
carried out with a high degree of accuracy and
precision. It is extremely rare for the results of
replicate determinations to be reported in the elemen-
tal analysis of humic substances and thus one cannot
evaluate the analytical precision pertaining to the
individual samples; Huffman and Stuber (1985) in a
unique study do, however, report on the precision
associated with the elemental analysis of humic sub-
stances. For the present study, it must be assumed
t hat the analytical dat a are accurate and t hat the
dispersion in the dat a results solely from genuine
differences between the samples.
Generally, oxygen cont ent is not determined
directly, but is obt ai ned by subtracting the sum of the
other elemental contents, plus the ash content, from
100%. As pointed out by Huffman and Stuber (1985)
this procedure is subject t o two serious limitations:
(1) the oxygen cont ent determined in this manner
includes the sum of all errors in the other elemental
determinations, and (2) the ash may contain elements
which have been previously determined, so that sub-
traction of the ash content, in effect, subtracts those
elements a second time. In some studies oxygen has
been determined directly (Huffman and Stuber, 1985;
Mal col m and MacCart hy, 1986) and generally if the
six elements C, H, N, O, S and P are determined, the
summat i on of elemental contents, on an ash-free
basis, comes close t o 100%, indicating that the
organic matter in humic substances is constituted
almost entirely from these six elements. However, in
the case of some high-ash samples, direct determi-
nation of oxygen can result in grossly erroneous
values (Malcolm and MacCart hy, 1986; MacCart hy
and Malcolm, 1989).
Anot her probl em in the determination of oxygen by
difference is that, generally, onl y C, H and N contents
are measured directly, and, consequently, the value
calculated for oxygen by subtraction includes other
elements such as sulfur and phosphorus. However, as
will become evident from the dat a compiled for this
study, sulfur cont ent can represent as much as 3% in
some samples of humic substances. I n addition, com-
paratively complete analyses of humic substances
(Thurman and Malcolm, 198 I; Huffman and Stuber,
1985) have shown t hat even phosphorus may consti-
tute a measurable fraction of humic substances. For
this study the published oxygen contents must be
accepted at face value for use in the statistical calcu-
lations, but the above limitations relating to the
oxygen values should be borne in mind.
The nontrivial problems associated with moisture,
and moisture determination, in humic substances are
discussed by Huffman and Stuber (1985), who point
out t hat even some commercial laboratories experi-
ence difficulty with this problem. The presence of
moisture can lead to two types of errors: (1) the
hydrogen and oxygen contents of the moisture may
be incorrectly attributed t o hydrogen and oxygen in
the organic matter, and (2) the presence of moisture
can lead to an incorrect value for the total organic
matter content unless properly corrected for. Again,
in this type of study, we cannot assess the extent of
these problems, but must assume t hat the moisture
contents were properly determined and corrected for
in the source publications, which most probabl y will
not be a correct assumption in some cases. As a result
of these considerations, the carbon dat a may be the
least susceptible t o error, and conclusions based
on the distribution of the element may be the most
reliable.
Overall, it is reasonable to assume, based on the
results which follow, t hat the majority of the elemen-
tal determinations were carefully conduct ed and t hat
grossly erroneous values represent only a small frac-
tion of the total sample popul at i on and will not
seriously influence the statistically-based conclusions
of this paper.
RESULTS AND DISCUSSION
Humic substances in general (humic and fulvic acids)
(1) General trends in elemental composition f or
humic substances. The mean, median, mode, range,
standard deviation and X2-statistics for elemental
compositions of the humic acids, fulvic acids and
humins are given in Table 1. The number of signifi-
cant figures for each entry in this and subsequent
tables is based on the values reported in the source
publications. It should be recognized t hat it is the
mean or average values t hat are being compared in
this paper, and t hat the ranges for elemental contents
of various humic fractions, as taken from the
literature, actually display considerable overlap
(cf. Table 1). Consequently, individual samples may
deviate from the general trends t hat are reported in
this paper.
In all dat a sets the range is defined by the outlier,
or extreme, values. Consequently, some of the values
specifying the range in Table 1 (and subsequent
tables) may appear highly anomalous; e.g. a soil
humic acid outlier has a H/ C ratio of 0.08, and a soil
fulvic acid outlier has a H/ C ratio of 2.13 (Table 1).
However, these are extreme, and possibly spurious,
values and their occurrence is t o be expected in a large
dat a set, particularly of the type under discussion
here. These extreme values are obviously not repre-
sentative of the dat a set as a whole, as evident from
compari ng them with the corresponding mean values
and st andard deviations. Table 1 incorporates humic
substances from all environments (soil, freshwater,
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640 JAMES A. RICE a n d PATRICK MACCARTHY
marine, peat, coal) and they are not segregated by
source. I n general, there is close agreement between
the mean, median and mode, consistent with the dat a
being normal l y distributed.
Even t hough the car bon contents of the humic acids
range from 37.18 to 75.76% the st andard deviation
for those samples is onl y 5.0% [relative st andard
deviation (RSD) = 9.1%]. Sixty-eight percent of the
humic acids have carbon contents in the interval
50.1-60.1% ( + 1 SD) and 96% of these samples have
car bon contents between 45.1 and 65.1% ( + 2 SD).
Considering t hat (1) humic acid is simply an opera-
tionally-defined product, (2) the samples involved in
this study were obt ai ned by numerous variations of
the traditional extractive technique, (3) t hat these
ht/mic acids were extracted by many different exper-
imentalists, and (4) t hat these humic acids represent
a br oad diversity of source environments (soil, fresh-
water, marine, peat and coal), geographical locations
and botanic precursors, the absolute st andard devi-
ation of onl y 5.0% (9.1% RSD) is noteworthy. In
fact, it was precisely this recognition of suprisingly
small st andard deviations, here and elsewhere in this
study, t hat prompt ed us t o embark on a more
rigorous statistical evaluation of the elemental com-
positions of humic substances. This observation may
suggest t hat an opt i mum composition exists for hu-
mic acids in nature. This, however, does not imply the
existence of molecular structural uniformity among
humic substances.
The range and relative st andard deviation for the
carbon contents of f ulvic acids are somewhat larger
t han those for humic acids, and the correspondi ng
values for humin are considerably smaller (Table 1).
However, one cannot speak as reliably of the humin
dat a because only 26 such samples were involved in
the study, and more t han hal f of these were from
peat; these limited dat a may not be representative of
humi n in general.
The mean values for the H/ C and O/ C atomic
ratios based on these large dat a sets are similar t o
those previously reported for humic acid and fulvic
acid (Visser, 1983; Steelink, 1985) and for humin
(Stuermer et aL, 1978) based on much smaller dat a
sets. The H/ C ratio (Table 1) indicates t hat fulvic
acids are, in general, more aliphatic t han humic acids.
It is seen from Table 1 t hat the ranges in reported
car bon contents for humic acids and fulvic acids are
similar. However, a single outlying dat um can have
an inordinate influence on the range, but the mean
carbon contents for humic and fulvic acids are
considerably different, displaying the net differences
between these materials.
The ranges for ot her elemental contents are also
rather large (Table 1). The relative st andard devi-
ations for hydrogen are moderately high, for nitrogen
are quite large and for sulfur are very large (88.8%
for humic acid and 83.3% for fulvic acid). The large
st andard deviations for nitrogen and sulfur may
simply be a result of inherent variability of these
Tabl e 2. t -St at i st i cs, a nd t hei r significance, used t o compar e unseg-
regnted humic acids (HA), fulvic aci ds ( FA) a nd humi ns ( HMN)
f r om Tabl e I
t -St at i st i c
t c C H N O O/ C H/ C
HA- F A 3.29 19.66" 2.08 6.47* 21. 41" 20.03* 7.67*
HA- HMN 3.58 1.64 2. I 1 1.48 I. 17 2.51 1.43
F A- HMN 3.48 17.68" 2.80 2.76 12.54" 8.75* 1.51
t c Represent s t he cri t i cal t - val ue at P = 0.0005 f or each compar i s on
and an ast eri sk (*) indicates a t -val ue significant at P < 0.0005.
elements in humic substances, and may also be due
to analytical errors in the determination of these
lesser constituents. On the basis of these dat a, and for
the reasons stated in the previous section, conclusions
based on observed trends in the means may be most
reliable in the case of carbon.
(2) Statistically significant differences in humic sub-
stances. Numerous trends can be discerned in the
dat a of Table 1. The importance of such relatively
large dat a sets is t hat they allow the statistically
significant trends t o be established by means of the
t-test. Table 2 presents the t-statistics, calculated
using equation (3), which compare humic acid with
fulvic acid, humic acid with humin, and fulvic acid
with humin for each of the elemental contents and
atomic ratios of the unsegregated samples described
in Table 1. Due to the comparatively small number
of sulfur analyses, the statistical significance of trends
observed in the sulfur contents are not evaluated. The
probability, P, at which to reject the null hypothesis
equating the means of a paramet er exhibited by two
dat a sets has been set at P < 0.0005. This means t hat
if the t-statistic for a particular parameter exceeds the
critical value (to) there is less t han one chance in 2000
t hat the means exhibited by those two dat a sets could
be observed in the same population; the values t hat
fall into this category are labeled with an asterisk in
Table 2. Many of the t-values in Table 2 are very
much larger than the critical values, to; in these cases
the null hypothesis is rejected at P-val ues t hat are
considerably less t han 0.0005.
Fulvic acid is statistically distinct from humic acid
on the basis of its C (lower), N (lower) and O (higher)
contents and its O/ C ratio (higher). Fulvic acid also
exhibits a larger and statistically different H/ C ratio
relative to humic acid. Because a larger H/ C ratio is
indicative of a more aliphatic character, this is con-
sistent with fulvic acid being, in general, more ali-
phatic t han humic acid. The only paramet er for
which fulvic acid is not significantly different from
humic acid in Table 2 is hydrogen content.
Fulvic acid differs statistically from humin with
respect to its C (lower) and O (higher) contents and
its higher O/ C ratio.
The statistical compari son of humic acid and
humin does not show these two materials to be
significantly different at P < 0.0005, and cannot dis-
count the possibility t hat the paramet er means exhib-
ited by the respective dat a sets in this study might
be observed in the same popul at i on at the chosen
T
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642 JAbl~ A. RICE and PATRICK MACCARTHY
probability level. This could be the result of real
similarities in the average elemental compositions of
humic acid and humin (on an ash-free basis) or it
could be due t o the small number of analyses from
which the humin dat a set was constructed. A small
dat a set tends t o diminish the ability of statistical
devices, such as the t-test, to discern subtle differ-
ences. Of course, humic acid and humin differ con-
siderably in other characteristics such as ash content,
solubility, and so on.
Humic acids segregated by source
(I ) General trends in elemental composition f or
humic acids. Table 3 contains the statistical dat a for
humic acids where the samples are segregated accord-
ing t o source: soil, freshwater, marine, and peat.
Coal-derived humic acids, which were included in
Table 1, are not included in this classification as there
were t oo few dat a t o make the presentation of
average elemental compositions meaningful for this
cat egory or t o perform tests of statistical significance.
The distribution of the data, on the basis of X2-stat -
istics, more closely approximates normal i t y when the
humic acids are segregated by source (Table 3) t han
when they are considered in toto (Table 1).
The most striking observation on compari ng the
dat a in Table 3 with those in Table 1 is t hat when the
humic acids are segregated by source, the range of
carbon values narrows dramatically and the st andard
deviations for carbon decrease remarkably except for
the marine humic acid, where only a small decrease
in the range occurs and an increase in the relative
st andard deviation is observed. For example, the
st andard deviations for carbon contents decrease by
about 25, 40 and 50% for soil, freshwater and peat
humic acids, respectively, compared t o the standard
deviation of humic acids considered in toto. This
observation, in conjunction with the noticeably
different averages for humic acids from different
sources (Table 3) suggests a "preferred" composition
for the humic acids from each environment.
The fact t hat the st andard deviations do not con-
tract in the case of the marine humic acids may result
from the variety of environments (near-shore, deep-
sea, etc.) in this study which were classified by the
original aut hors as being marine, some of which have
the likelihood of several sources of organic matter
input. For example, a near-shore marine environment
may receive organic mat t er from terrestrial as well as
algal sources due t o its proximity t o land masses.
Table 4. t -St at i st i cs, a nd t hei r significance, used t o compar e humi c
acids f r om soil, f r eshwat vr a nd mar i ne sources f r om Tabl e 2
t -St at i st i c
t~ C H N O O/ C H/ C
Soi l - f r eshwat er 3.39 8.98* 0.19 6.07* 8.43* 8.38* 2.68
Soi l - mar i ne 3.40 0. 79 7.37* 0.71 4.83* 3.70* 6. 01"
Fr e s hwa t e r -
mar i ne 3.29 6.56* 8.80* 5. 01" 10.82" 10.15" 4. 14"
tc Represents the critical t - val ue at P = 0.0005 f or each compar i s on
and a n ast eri sk (*) i ndi cat es a t -val ue si gni fi cant at P < 0.0005.
There is a similar pronounced cont ract i on
(except for the marine samples) of the st andard
deviation for oxygen contents when the humic
acids are separated by source. This is also generally
true for the st andard deviations of S, N and H
(with some exceptions) t hough the effect is not so
pronounced.
The larger H/ C ratio (Table 3), consistent with a
more aliphatic nature, for marine humic acid relative
to soil humic acid is in agreement with previous
reports based on considerably smaller dat a sets (Ishi-
watari, 1969; 1970; Rashid and King, 1970; Stuermer
and Payne, 1976; Steelink, 1985). The average el-
emental composition of soil humic acid in Table 3
falls within the range of values reported for an
"average" soil humic acid by Schnitzer (1977) based
on a substantially smaller dat a set.
(2) Statistically significant differences in humic
acids. Table 4 presents the statistical significance of
differences in the means of the elemental component s
of humic acid isolated from different sources. Peat
humic acid was not involved in this compari son
because of the comparatively small number of dat a in
this set. Table 4 shows t hat humic acid obtained from
freshwater sources possesses an elemental compo-
sition distinct from soil and marine humic acids.
Marine humic acid is statistically different from soil
humic acid with respect t o hydrogen and oxygen
contents and the H/ C ratio. The O/ C ratio suggests
t hat real differences exist between freshwater, soil and
marine humic acids.
A statistical distinction may be made between
marine humic acid and freshwater and soil humic
acids on the basis of the H/ C ratio. A similar
distinction cannot be made between soil and fresh-
water humic acids at the chosen probability level,
although they are statistically different at a lower
level, P < 0.005. This lower probability level still
allows only one chance in 200 t hat the means could
be observed in the same population, and suggests t hat
the lack of statistical distinction between the fresh-
water and soil humic acids at P < 0.0005 may be
due t o the small number of analyses of the fresh-
water humic acid relative t o the soil humic acid.
Thus, it is probabl y reasonable to infer t hat the
aliphaticity of humic acids from different sources
decreases in the order marine > freshwater > soil.
This would be consistent with the nature of the
organic input into soil and marine environments.
Marine organic detritus is believed t o be predomi-
nantly algal in origin and of a highly aliphatic nature
(Nissenbaum and Kaplan, 1972) while soil organic
input is believed to be more aromat i c in nature due
to the ubiquity of lignin in terrestrial plants (Flaig,
1972). A freshwater sample could have maj or inputs
of either algal or soil organic materials. In summary,
the dat a in Table 4 indicate t hat there are statistically
significant differences between the elemental contents
of humic acids from soil, freshwater and marine
sources.
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644 J ~ s A. RIcE and PATmCK MACCAItTm
Fulvic acids segregated by source
(1) General trends in elemental composition for fulvic
acids. Tabl e 5 cont ai ns the statistical dat a for fulvic
acids segregated by source. Gr oupi ng fulvic acids by
source causes a significant narrowi ng of the range and
st andard deviation of car bon values, except in the
case of the soil samples where the range is unaltered
and the relative st andard deviation is virtually un-
changed. The ranges and st andard deviations for
most other parameters also decrease when the fulvic
acids are segregated by source although there are
exceptions to this generalization.
Segregation of the fulvic acid samples by source
(Table 5) results in the dat a more closely approximat-
ing a nor mal di st ri but i on t han when those dat a are
evaluated in toto (Table 1). The most normal form of
the dat a in Tabl e 5, in cont rast to that in Table 1,
exhibits Z2-statistics very near or below the critical
values, confirming t hat these dat a exhibit or very
closely approximate a normal distribution.
Compared to soil, freshwater and mari ne fulvic
acids, all of which exhibit similar carbon and oxygen
contents, peat fulvic acid displays markedly different
values for C (higher), O (lower) and consequently, a
lower O/ C ratio. Hydrogen and H/ C values for
peat fulvic acid fall within the range of values
exhibited by other samples. However, caut i on must
be exercised in at t empt i ng to draw conclusions from
these observations in that only twelve samples of peat
fulvic acid are involved in this study and eleven of
these were collected by a single group from different
areas of one locale (Zelazny and Carlisle, 1974). This
may introduce some bias i nt o the estimate of this
sample popul at i on mean, and consequently peat ful-
vic acid will not be discussed any further in this paper.
Compari ng the soil, freshwater and mari ne "aver-
age" fulvic acids in Table 5 reveals that the C and O
cont ent s and the O/ C ratios are quite similar. Though
not as pronounced as when humic acids are segre-
gated by source, the H, N and S contents of the fulvic
acids do show different averages, when grouped
by source, compared to the average of all fulvic
acids combined. On the basis of the H/ C ratio the
"average" soil fulvic acid would appear to be more
aliphatic t han freshwater fulvic acid.
(2) Statistically significant differences in fulvic acids.
Table 6 reveals that the onl y statistically significant
difference between fulvic acids from soil and fresh-
water envi ronment s is in the hydrogen cont ent and in
the H/ C ratio. Because of the small number of mari ne
and peat fulvic acids for which dat a were available,
they were excluded from this type of comparison. By
Tabl e 6. t-Statistics, and their significance, used to compare fulvic
acids from soil and freshwater sources from Table 3
t -Statistic
t c c H N O O/C H/C
Soil-freshwater 3.29 2.56 6.88* 2.23 0.30 2.04 7.18"
t, Represents the critical t-value at P = 0.0005 and an asterisk (*)
indicates a t-value significant at P < 0.0005.
Table 7. t-Statistics, and their significance, used to compare humic
acids (HA) and fulvic acids (FA) from similar (soil and freshwater)
sources
t -Statistic
t C H N O O/C H/C
Soil HA-FA 3.29 16.75" 2.19 8.24* 17.85" 17.41' 8.36*
Freshwater
HA-FA 3.38 6.35* 4.35* 2.47 6.04* 6.42" 0.72
t c Represents the critical t-value at P = 0.0005 and an asterisk (*)
indicates a t-value significant at P < 0.0005.
inference the~, soil fulvic acid is more aliphatic t han
freshwater fulvic acid due to its larger H/ C ratio. This
result is interesting because it has often been stated
t hat soil humic materials are generally more aromatic
t han humic substances from other environments. For
example, Visser (1983) observed that humic materials
from freshwater sources generally exhibited H/ C
ratios greater t han that of soil humic substances. This
apparent difference is usually attributed to the ubiq-
uity of lignin, which some believe to be an i mport ant
humic precursor, in terrestrial plants. The H/ C ratio
for freshwater humic acid is greater t han that for
soil humic acid (Table 1) but the difference is not
statistically significant at P < 0.0005 (Table 5).
Intercomparison of humic and fulvic acids from similar
environmen ts
(1) General trends in elemental composition. When
segregated by source, fulvic acids (Table 5) consist-
ently exhibit a lower C content, higher O cont ent and
a higher O/C ratio t han do humic acids (Table 3)
from the same type of envi ronment . No clear distinc-
tions between humic acid and fulvic acid from similar
envi ronment s are evident when trends in the H, N
2.5
2 . 0
o
1. 5
"1-
i
~. o
0. . 5
HA
".%' ,%
%, fYiTI FA
HMN
o I
0. 5 1. 0 1. 5
A t o m i c O/ C r o t i o
Fig. 2, van Krevelen diagram based on humic acid (410
samples), fulvic acid (214 samples) and humin (26 samples).
Statistical evaluation of the elemental composition of humic substances 645
and S cont ent s are exami ned. The fulvic acids, wi t h
one except i on, exhi bi t a great er H/ C r at i o t han the
respective humi c acids.
(2) Statistically significant differences between humic
and f ulvic acids f rom similar environments. Visser
(1983) concl uded t hat t he maj or i t y of fulvic and humi c
aci d sampl es f r om freshwat er envi ronment s exhi bi t
si mi l ar H/ C rat i os. The results of t he t - t est in Tabl e 7
suppor t t hi s obser vat i on by i ndi cat i ng t hat t he differ-
ences in t he means of the H/ C r at i os in freshwat er
fulvic aci d and humi c aci d are not significant. How-
ever, freshwat er humi c aci d and fulvic aci d differ
significantly f r om each ot her wi t h respect to the ot her
par amet er s in Tabl e 7, except for the ni t rogen cont ent .
Tabl e 7 al so cor r obor at es st at ement s in the litera-
t ure ( Kononova, 1966; Schnitzer and Skinner, 1974)
whi ch poi nt out t hat soil fulvic aci d (Tabl e 5) is mor e
al i phat i c t han soil humi c aci d (Tabl e 3). Prest on and
Ri pmecst er (1982) r epor t a " pr edomi nant l y al i phat i c"
nat ur e for soil fulvic aci d based on ~3C-NMR spectra.
Soil fulvic aci d also exhi bi t s a st at i st i cal l y different
wei ght -percent , wi t h respect t o soil humi c acid, for
every el ement except hydr ogen (Tabl e 7) emphasi zi ng
t hat these two soil humi c subst ances differ in mor e
ways t han j ust t hei r degree of al i phat i ci t y.
Because of t he smal l t ot al number of humi n
sampl es in t he dat a set (Tabl e 1), humi n was not
segregat ed by source.
van Krevelen diagrams
Fi gur e 2 shows a van Krevel en di agr am based on
t he 650 sampl es in Tabl e 1, and i l l ust rat es t he compo-
si t i onal fields occupi ed by humi c acid, fulvic aci d and
humin. In this paper van Krevel en di agr ams are used,
as elsewhere ( Kuwat suka et al., 1978; Visser, 1983),
onl y t o summari ze composi t i onal differences bet ween
humi c mat eri al s. Humi c and fulvic aci ds occupy
br oad composi t i onal fields wi t h a consi derabl e regi on
of overl ap. The humi n samples occupy a compo-
si t i onal field t hat lies al most compl et el y wi t hi n t hat of
humi c acid.
The " t humb" ext endi ng from the humi c aci d field
i nt o the upper l eft -hand por t i on of Fig. 2 represent s
11 mar i ne humi c aci ds ext ract ed from Eocene chal ks
and cherts in t he i nvest i gat i on of Bein and Sandi er
(1983). These samples exhi bi t H/ C rat i os which are
not mar kedl y different from ot hers in t he humi c aci d
dat a set; the O/ C r at i os of these samples, however,
represent the l ower ext reme in the range of O/ C r at i os
listed in Tabl e 1 showing values of about 0.20 or less.
Even t hough these 11 humi c aci ds were ext ract ed by
a r at her convent i onal sodi um hydr oxi de- sodi um
pyr ophosphat e pr ocedur e (Bein and Sandier, 1983),
and consequent l y conf or m t o the oper at i onal defi-
ni t i on of humi c acid, some obvi ous differences exist
between t hem and t he bul k of t he humi c aci ds
represent ed in this di agram.
The van Krevel en di agr am present ed in Fig. 2
differs from t hat of Kuwat suka et al. (1978) in t hree
ways: (1) this di agr am includes dat a for humi n
samples, (2) appr oxi mat el y ten times as many humi c
aci d sampl es and t hi rt y times as many fulvic aci d
sampl es are utilized in the present di agr am and (3)
Kuwat suka et al. (1978) descri bed the di st r i but i on of
2. 5 2. 5
2.0
o . . . . . .
2 2 2 "~, ~:~i : * : ~ :
. . . . ~s*; t .s *. : ' o
._o : . ~ ~ . . .
1. 0 , ...,.~: . . . . . ..:::!:;::::.
o.5 ~ ii~ E ~ M
~ s
r r r n p
I I
0. 5 1. 0 1. 5
A t o m i c O/ C r o t i o
Fig. 3. van Krevelen diagram based on soil humic acids (215
samples), freshwater humic acids (56 samples), marine sedi-
ment humic acids (95 samples) and peat humic acids (23
samples).
2. 0
o
E 1.5
- r
E
0 1.0
0. 5
ffT~lM
~ s
FTmp
I I
0.5 1.0
A t omic O/C rot io
1.5
Fig. 4. van Krevelen diagram based on soil fulvic acids
(127 samples), freshwater fulvic acids (63 samples), marine
sediment fulvic acids (12 samples) and peat fulvic acids
(12 samples).
646 JAMES A. RICE and PATRICK MACCARTHY
the humic acids on their van Krevelen diagram as
being ' "J"- shaped' , but such a description is not
applicable to the larger data set employed in the
construction of Fig. 2 even though the data of
Kuwatsuka et al. are included in this figure. In
general, however, the regions occupied by humic and
fulvic acids are similar to those of Kuwatsuka et al.
(1978). The considerably larger data base used in the
present study enhances the reliability of conclusions
based on these diagrams.
Tissot and WeRe (1978) have described humic
substances as being similar to Type I I I kerogens. In
terms of Fig. 2 and the data in Table 1 (moderate H/ C
ratios, high O/C ratios), this appears reasonable; the
average humic material exhibits atomic ratios typical
of Type I I I kerogens.
Figures 3 and 4, which are van Krevden diagrams
for humic acids and fulvic acids, respectively, segre-
gated by source, emphasize the difference between
humic materials from different environments that
were described in earlier sections. Figure 3 shows that
freshwater, peat and marine humic acids exhibit
considerable overlap in their compositions. The soil
humic acid compositional field overlaps that of the
other three. The compositional fields occupied by
freshwater, marine and peat fulvic acids (Fig. 4)
appear to be mutually more distinct than their humic
acid counterparts (Fig. 3). Such apparent distinctions
may not be evident i f the marine and peat fulvic acid
data sets were larger. As with humic acids segregated
by source (Fig. 3), soil fulvic ac;d extensively overlaps
the compositional fields of the other three fulvic acids
in Fig. 4.
Average elemental compositions of artificial and
sewage sludge "humic " materials
Finally, in addition to the natural humic sub-
stances already discussed in this paper, the elemental
data for a very limited number of artificial and
sewage-sludge derived "humic materials" were also
briefly examined for comparative purposes. The
mean values for the dcmental contents and atomic
ratios of 17 microbially-produced "humic acids", 3
chemically synthesized "humic acids", and 3 sewage-
sludge "fulvic acids" are given in Table 8. The
microbially-produced "humic acids" have mean
values similar to those for unsegregated humic acids
(Table 1). For the chemically synthesized "humic
acids" the O/C ratio is similar to, and the H/ C ratio
is less than that of unsegregated humic acids. Only
one of these three "humic acids" contained nitrogen
(at a very high level). Chemically synthesized humic
Table 8. Mean elemental contents (weight pcrcant) and atomic ratios
for i 7 microbially produced "humic acids", 3 chemically synthesized
"humic acids" and 3 sewage sludge "fulvic acids". All values an: on
an ash-free basis
Sample C H N S O O/C H/C
Microbial 52.6 5.1 4.1 - - 38.1 0. 54 1.16
C h e m i c a l 58 . 1 3. 2 3. 5 - - 37 . 4 0 . 4 8 0 . 66
Se w ag e - sludg e 4 0 . 8 2 6. 57 2 . 8 3 8 . 1 5 4 2 . 2 7 0 . 7 8 1 . 9 3
acids do not contain nitrogen unless it is present in
one of the reagents. The O/C ratio for the sewage
sludge "fulvic acid" is similar to that of the unsegre-
gated fulvic acids (Table 1) while the H/C value is
considerably greater. The sewage sludge "fulvic
acids" have a very high sulfur content possibly due to
the presence of sulfonated detergents; all three of
these "fulvic acids" come from the same study
(Sposito et al., 1976). Differences between commer-
cial humic acids and soil and water humic substances
have also been reported (Malcolm and MacCarthy,
1986; MacCarthy and Malcolm, 1989).
SUMMARY
The statistical analysis presented in this paper
quantitatively identifies similarities and differences
between various humic substances, and in addition to
establishing new trends in the properties of these
materials, it provides a statistical basis for some
previously-reported trends.
The results of this study are summarized as follows:
(1) Humic acid, fulvic acid and humin exhibit
surprisingly small standard deviations in their el-
emental contents, particularly for carbon, even for
unsegregated samples. This may suggest that some
preferred composition, or a relatively narrow range
of compositions, exists for humic substances in
nature.
(2) The t-test indicates that the C, N, and O
contents and the O/C and H/ C ratios exhibited by
fulvic acid are significantly different from those of
humic acid at P < 0.0005. The carbon and oxygen
contents and the O/C ratio of fulvic acid are statisti-
cally different from those of humin. No statistically
significant differences in the elemental composition
exist, on the basis of these data sets, between humin
and humic acid.
(3) When segregated by source, humic acids from
different environments exhibit different means in the
elemental parameters; considerable decreases in stan-
dard deviations compared to humic acids in toto are
evident.
(4) Freshwater humic acid exhibits statistically
significant differences in its C, N and O contents and
O/C ratio compared to marine and soil humic acids.
Marine humic acids display statistically significant
differences in their H and O contents and in the O/C
and H/C ratios compared to soil humic acids. In
addition, two of the three humic acid source inter-
comparisons (marine-soil and marine-freshwater)
exhibit statistically different H/C ratios. By inference,
the degree of aliphaticity of humic acids decreases in
the order marine > freshwater > soil.
(5) Variations in means of the f ulvic acids, when
segregated by source, are not as pronounced as for
humic acids, but in most cases there is still a consider-
able decrease in the standard deviations compared to
unsegregated fulvic acids.
Statistical evaluation of the elemental composition of humic substances 647
The di muni t i on of the st andard deviation which
occurs when the samples are segregated by source,
coupled with the statistically significant differences in
the mean indicates, perhaps, t hat there is an opt i mum
composi t i onal range to be expected for each humic
material i n a particular envi ronment .
(6) Statistically distinct H cont ent s and H/ C ratios
exist i n fulvic acids from soil compared to freshwater
source environments. Because the H / C ratio exhib-
ited by the soil fulvic acids is larger, it is assumed to
be more aliphatic t han freshwater fulvic acid.
(7) Soil fulvic acid exhibits statistically significant
differences in the C, N and O contents and O/C and
H/ C ratios when compared to those of soil humic
acid.
(8) A van Krevelen di agram based on 650 samples
of humic acids, fulvic acids and humi ns shows that
humic and fulvic acids occupy distinct regions on the
di agram with an area of overlap. The humi n compo-
sitional field exists almost completely within the
humic acid field.
(9) A van Krevelen diagram based on humi c acids
segregated by source shows considerable overlap
between the compositional fields of soil, freshwater,
mari ne and peat humi c acids. A van Krevelen dia-
gram based on f ul vi c acids segregated by source
shows t hat soil fulvic acid overlaps the compositional
fields of freshwater, mari ne and peat fulvic acids.
Freshwater, mari ne and peat fulvic acids appear to
exhibit distinct compositional fields on the diagram.
(10) While chemically synthesized "humi c acids"
and sewage sludge "fulvic acids" conform to the
operat i onal definitions of these materials their mi nor
elemental component s indicate differences between
them and nat ural l y occurring humic substances.
Sewage sludge "fulvic acids" and chemically syn-
thesized "humi c acids" exhibit some pronounced
differences in their S and N contents, respectively,
compared to " nat ur al " humic materials. Microbially
produced humic acids are generally similar to the
humic acid data set in toto.
RE F E RE N C E S
Bartlett M. S. (1947) The use of transformations. Biom. Bull.
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