Environ. Sci. Technol. 1903, 17, 412-417
Application of Van Krevelen's Graphical-Statistical Method for the Study of Aquatic Humic Material Simon A. Visser Department of Soil Science, Lava1 University, CitQ Universitaire, QuQbec, Canada G1K 7P4
By means of H:C and 0:C atomic ratios information was obtained on the basic structure, and on structural changes with varying molecular weight and origin, of fulvic and humic acids sampled at various locations of a watershed within the Laurentide provincial park, Quebec. In order to investigate also the effect of humification, humic matter produced by a streptomycete and sampled at regular intervals during a 6-month incubation period was included in the investigation. Also studied was the effect of ultraviolet irradiation on the structure of either recently formed or more humified humic substances. It was shown that fulvic and humic acids are more alike in aquatic than in terrestrial environments. They are also less aromatic in natural waters than in soils and, at least in the case of fulvic acids, are richer in oxygen. Contrary to what is normally observed for humus from natural environments, it would appear that for certain types of humus such as fulvic acids from a microbial culture, increases in molecular weight or in the degree of humification would result in material with a less aromatic character. Humus obtained from natural waters was found to be in a more advanced state of humification at the end of summer than at the beginning. Recently formed fulvic acids appeared to be very sensitive to ultraviolet irradiation.
Introduction Graphical-statistical methods have often proved to provide valuable information concerning structure and reaction processus involving products of natural origin. In order to investigate and to describe changes taking place in the structure and elementary composition of plant substances, in particular during processes such as humification and coal formation, Van Krevelen (1) made use of a diagram in which the atomic hydrogen to carbon ratio was plotted vs. the atomic oxygen to carbon ratio. Principal reactions such as decarboxylation, dehydration, dehydrogenation, hydrogenation, and oxidation could be represented in this diagram by straight lines. Other workers have later used this method for the comparison of the elementary compositions of coals, soil fulvic acids (FA'S), and humic acids (HA's) as well as plant constituents (2). Soil HA's normally occupy a broad J-shaped area in the diagram with H:C ratios ranging from 0.5 to 1.5 and with 0:C atomic ratios of between 0.35 and 0.55 (Figure 1). Their C, H, and 0 composition are very close to those of lignins. Soil FA'S occupy a wide area to the right side of HA's, with their elementary composition being similar to those of fresh plant constituents such as stems, barks, and celluloses. As Van Krevelen has shown, from the value of the H:C ratio, obtained after substracting the C and H contents of the functional groups (as partly indicated by the O C ratio) from the total C and H content, information can be obtained on the structure of the carbon skeleton of polymers: whereas a low H:C value of around 0.3 would indicate a substance with a very highly condensed aromatic ring system, a ratio of approximately 0.7 would be obtained from a noncondensed aromatic structure; values of between 0.7 and 1.5 would correspond to material of which the basic unit consists of an aromatic nucleus with an 412
Envlron. Scl. Technol., Voi. 17, No. 7, 1983
aliphatic side chain of up to 10 carbon atoms. Furthermore, a H:C ratio of between 1.5 and 1.7 would be indicative of a cyclic aliphatic compound, whereas values of approximately 2 would be typical of a paraffin. In the case of an aromatic ring system it can generally be stated that the lower the H:C ratio, the higher its degree of condensation (3). During the process of humification of organic matter, changes have been reported involving (4-8) increases in the aromatic character of the material, condensation of unsaturated aliphatic chains with each other, increases i n carbon and decreases in hydrogen contents, increases in carboxyl and methoxyl contents (and to a lesser extent, in carbonyl groups), and also decreases in alcoholic hydroxyl groups. It would appear, therefore, that by studying H:C and 0:C atomic ratios of humic matter, information can be obtained on its structure and on changes occurring in it during the process of humification. In the present study, by the use of the Van Krevelen diagram, information will be presented on differences in the structure and composition of humic fractions obtained from various natural aquatic environments and from microbial cultures, structural differences between humic compounds of different molecular weight, changes taking place in the structure of the humic compounds during the process of humification, and structural changes occurring in humic matter exposed to ultraviolet irradiation.
Experimental Section Origin of Material. Humic matter of natural aquatic origin was investigated, as well as humic material of microbial origin in various stages of humification. The aquatic humic material was obtained from water samples collected during the months of May, July, and Sept 1974 from a watershed comprising a lake (Lake Pikauba), a river (Chicoutimi River), a forest stream and a swamp. The watershed is situated in the Laurentide Provincial Park in a cryoboreal region of the Precambrian Shield in the province of Quebec (Canada). The dominant geological formations in the area are made up of granites. Vegetation consists mainly of conifers dominated by firs. Average summer temperatures are between 13 and 17 "C with average winter temperatures of around -25 to -30 "C. Spring floods occur in the months of April and May. Lake Pikauba is subject to overturn in autum and usually also in spring; its hypolimnion is situated below a depth of approximately 7.5 m. The lake was sampled at its point of outfall into the Chicoutimi River. In order to investigate the effect of humification on the structure of humic compounds, material of microbial origin was included in the investigation. It was obtained from a simple glucose-yeast extract medium (glucose, 0.1%; Difco yeast extract, 0.19'0) inoculated with an active humus-producing actinomycete isolated from a Quebec forest soil and incubated under constant conditions of aeration, temperature (25 "C), and pH (7.0). Samples were collected for recovery of FA'S after incubation periods of 4, 7, 14, 60, and 180 days. For samples of both natural and microbial origins, fractionation procedures were started within 1 day of collection.
0013-936X/83/0917-0412$01.50/0
0 1983 American Chemical Society
1j1I1 0500b0
050
040
I
I
0J0
oko
Fractionation Procedure. After the pH of the samples was adjusted to 8.4, they were centrifuged at 28000g in a Sorval ultracentrifuge fitted with a continuous-flow head in order to remove any particulate organic and inorganic matter. The supernatant was next filtered consecutively over Diapor (Amicon Corp., Lexington, MA) filters DP06, DP045, and DP02 with respective pore sizes of 0.6,0.45, and 0.2 pm. Very large volumes, such as was the case of samples collected from natural environments (150 L), were reduced by concentrating the organic matter by means of a Diafiber (Amicon Corp., Lexington, MA) hollow fiber filter HlOPlO until the eluate became colored. Although the nominal molecular weight cutoff of this filter is quoted by its manufacturer as being 10000 when used with proteins, it was found that as long as the ionic strength of the oligotrophic natural waters remained low, humic materials with a molecular weight of around 500 did not normally pass. The HA fraction was precipitated by adjusting the pH to 1.5 after which the above-mentioned centrifugation and Diafiber filtration procedures were repeated. The final solution (containing the fulvic fraction adjusted to pH 6.0) was then reduced in volume by ultrafiltration using a Diaflo UM05 ultrafilter. A t this pH no colored material passed into the filtrate. The HA's were purified by repeating the cycle of precipitation at pH 1.5 followed by dissolution at pH 8.0 until the supernatant a t pH 1.5 remained virtually colorless. In the case of the FA'S,the concentrate, after adjustment to pH 1.5, was passed over Amberlite resin (BDH Chemicals, Toronto) XAD-2, previously purified and activated by refluxing in a Soxhlet apparatus with methanol for 2 days. The adsorbed fulvic matter was partially eluted (up to 90%) by using a Tris buffer consisting of 0.03 M tris(hydroxymethy1)aminomethaneand 0.15 M KCl adjusted to pH 8.4 and 10.15, after which the eluate was concentrated over a D i d o UM05 ultrafiltration membrane. The cutoff under these experimental conditions was at approximately 500 daltons. Some residual brown material obtained from the XAD-2 resin by washing it subsequently with methanol appeared to contain a high percentage of lypophilic material. This fraction, which amounted to approximately 10% of the
0 k
0:0
swarm
090
FA-sw HA-sw J
160
I ;0
120
total FA content of the original sample, was not further investigated in this study. Both fulvic and humic acids were next suspended in a Tris buffer (pH 8.4 10.15) and, by using Diaflo ultrafiltration membranes fitted into continuously stirred ultrafiltration cells, were fractionated into fractions with molecular weight ranges of -500-1000,1000-10000,1000020 000, 20 000-30 000, 30 000-50 000, 50 000-100 000, 100000-300000, and >300 000 daltons. In order to allow calculations on these fractions, their average molecular weights were arbitrarily set at respectively 750, 5000, 15000,25 OOO, 40 000,75 OOO, 200 OOO, and 500 000 daltons. By always applying during the ultrafiltration procedure the pressures as prescribed by the manufacturer and by maintaining the pH and ionic strength of the solutions at the levels previously indicated, the average particle size of these humic matter fractions was found to match reasonably well with the average molecular sizes of the humic compounds as obtained by equilibrium ultracentrifugation (9). After fractionation, the concentrates were thoroughly washed with Tris buffer, until the ultrafiltrate remained completely colorless. The buffer was finally removed by washing the humic fractions (present in the form of their K salts) with distilled water, until the filtrate had a pH of 7.0 and was completely chloride free. The concentration of the humic fractions was then adjusted to 0.5%, after which the dry material was obtained by freeze drying. It was stored in the dark at -40 "C until further analysis. Ultimate Analysis. The C, H, and N contents of the FA and HA molecular weight fractions were determined on P205-driedsamples by using a Hewlett-Packard HP 185 carbon hydrogen nitrogen analyzer. Besides these elements the samples contained only less than 0.2% sulfur, determined according to Safford and Stragand (10). Accordingly, the oxygen content of the samples was calculated as the difference between 100% and the sum of the carbon, hydrogen, and nitrogen content of the ash-free material.
Results and Discussion The results of the elementary analyses obtained on the fractionated fulvic and humic acids from the various loEnviron. Scl. Technol., Vol. 17, No. 7, 1983
413
Table I. Elementary Composition (%) of Molecular Weight Fractions of Humic Substances from Various Aquatic Environmentsa fulvic acids mw
H
C
750 5 500 1 5 000 2 5 000 40 000 7 5 000 200 000
49.6 46.5 46.7 45.2 45.5 43.7 42.4
7 50 5 500 1 5 000 25 000 40 000 75 000 200 000
46.3 45.1 44.3 41.4 42.7 40.8 39.2
f f f f f
7 50 5 500 1 5 000 25 000 40 000 7 5 000 200 000
47.5 46.1 45.9 44.7 44.8 40.9 40.7
f
7 50 5 500 15000 25 000 40 000 75 000 200 000
49.9 48.7 47.9 45.7 46.8 45.1 44.8
f f f f
f i:
f f
f f
f f f f
f f f f f f
i
humic acids
N
0
H
C
N
0
1.0 0.8 0.3 0.3 0.8 0.8 0.4
4.8 f 4.8 f 4.7 f 4.2 ?: 4.3 f 3.8 f 3.6 t
0.1 0.1 0.2 0.1 0.2 0.2 0.1
1.9 f 1.8 f 1.8 f 2.0 f 2.0 f 1.8 f 2.0 f
0.8 0.6 0.3 0.1 0.1 0.4 0.4
Lake 43.7 f 1.8 46.8 f 1 . 4 46.8 f 0.6 48.7 f 0.2 48.2 f 0.8 50.6 f 0.4 52.0 f 0.7
48.9 f 51.0 f 50.2 f 52.6 i: 53.5 t 53.0 f 52.5 f:
0.7 0.4 1.0 0.5 0.3 1.4 0.5
5.3 f 5.3 f 5.2 f 4.9 f 4.8 f 4.8 f 4.7 f
0.1 0.1 0.1 0.2 0.1 0.1 0.1
2.7 f 0.2 2.7 i: 0.1 2.5 f 0.3 2.0 f 0.2 2.0 f 0.2 2.1 f 0.4 1.7 f 0.1
43.0 41.0 42.1 40.5 39.7 40.1 41.1
f f f f
0.8 0.4 1.3 0.4 0.5 1.7 0.5
0.8 0.3 0.6 1.3 0.4 1.1 1.4
4.8 4.5 4.3 3.9 3.9 3.6 3.3
0.1 0.2 0.3 0.2 0.3 0.2 0.3
2.3 f 0.2 2.2 f 0.2 2.1 f 0.3 1.9 f 0.4 2.1 f 0.3 1.8 i: 0 . 5 1.7 f 0.6
River 46.7 f 0.9 48.2 f 0.5 49.3 0.3 52.8 f 0.9 51.3 ?: 0.7 53.8 f 0.5 55.8 f 0.7
54.4 f 51.7 f 51.1 f 48.3 f 49.1 f 48.7 f 48.1 f
0.6 1.7 2.2 0.7 2.0 0.3 2.0
5.1 f 5.0 f 4.8 i 4.8 f 4.6 f 4.4 f 4.4 f
0.3 0.3 0.1 0.1 0.0 0.0 0.1
2.7 F 2.0 f 2.0 f 1.9 f 2.0 f 1.7 f 1.6 f
0.3 0.4 0.3 0.1 0.1 0.1 0.0
37.8 f 41.3 f 42.1 f 45.1 f 44.4 f 45.4 f 45.9 f
0.6 1.0 2.5 0.6 2.0 0.4 2.0
0.7 1.3 1.6 1.3 1.6 0.7 0.4
4.6 i: 0.2 4.3 f 0.3 4.3 i: 0.4 3.7 f 0.4 3.8 f 0.4 3.4 f 0.2 3.4 f 0.2
1.6 f 1.5 f 1.5 f 1.5 f 1.6 f 1.5 f 1.6 f
0.4 0.2 0.2 0.0 0.1 0.2 0.3
Stream 46.3 ?: 1 . 3 48.1 f 1.8 48.3 f 1 . 9 50.1 f 1.7 49.9 f 2.0 54.2 f 0.8 54.3 f 0.4
51.6 f 49.5 f 49.0 f 48.3 f 48.6 i 46.1 f 45.3 i:
0.9 0.3 1.1 0.2 0.3 0.6 1.4
5.2 f 5.0 f 5.0 f 4.6 f 4.5 t 4.5 f 4.4 f
0.2 0.2 0.3 0.2 0.4 0.6 0.2
2.9 2.7 2.6 2.5 2.5 2.2 2.1
i:
0.2 0.2 0.2 0.3 0.3 0.4 0.3
40.2 f 1.0 42.8 f 0.2 43.4 f 1.4 44.7 f 0.5 44.4 f 0.6 47.2 i: 0.8 48.2 f 1.7
0.4 1.1 0.9 1.0 0.1 0.2 0.4
4.5 f 4.5 f 4.4 f 4.0 f 4.1 f 3.8 f 3.4 f
2.4 f 2.2 f 1.9 f 1.3 f 1.7 f 1.3 f 1.7 f
0.0 0.1 0.1 0.3 0.1 0.1 0.6
Swamp 43.2 f 0.3 44.7 f 0.8 45.9 f 0.5 48.9 f 0.8 47.4 f 0.6 49.8 f 0.4 50.1 f 0.3
50.8 i 52.2 f 52.7 ?r 53.4 f 54.0 f 54.6 f 55.9 f
0.5 0.3 0.8
5.1 f 4.9 f 4.3 ?: 4.4 f 4.0 f 3.8 t 3.7 f
0.1 0.2 0.3 0.4 0.1 0.1 0.1
3.0 t 2.8 f 2.2 f 1.7 f 1.8 f 1.7 f 1.6 f
0.1 0.0 0.1 0.1 0.2 0.1 0.1
40.9 40.3 41.4 40.5 40.8 40.1 38.8
f f
f f f
f f
0.4 0.5 0.4 0.5 0.5 0.5 0.1
+_
0.1 0.8 0.3 0.4
f f f f f
*
f f
0.5 0.1 ~t:0.6 f 0.2 ?r 0.1 f 0.2 f 0.4 f f
a Average of three samples obtained in May, July, and Sept 1974.
cations of the watershed are indicated in Table I, whereas the results obtained on the fractionated FA'Sof microbial origin have been entered in Table 11. It appears that the aquatic FA'S had a lower carbon and normally a lower hydrogen and higher oxygen content than their HA counterparts. This is generally also the case for soil humic substances. I t is interesting to note that the carbon, hydrogen, and nitrogen contents of the aquatic humic matter normally decreased with increasing molecular weight of the material, whereas for oxygen the reverse was often true (Table 111). For the more mature humic matter of microbial origin the same type of relations existed between the molecular weight of the humic fractions and their carbon, nitrogen, and oxygen contents as observed in the case of the aquatic humic substances (Table IV). In order to allow comparison of H:C and 0:C atomic ratios of FA'S and HA's of aquatic and microbial origin with those obtained from soils and with similar ratios of other types of natural substances (2), the position of the areas occupied by these products in the Van Krevelen diagram have been indicated in Figure 1. Observations on the Humic Matter in General. Comparison of the H:C ratios obtained on soil, aquatic, and microbial fulvic and humic acids shows; in this order, gradually higher values. A narrow H:C ratio is normally considered to be indicative of a high degree of condensation (3, 11). Whereas soil FA'S, as a result of their higher content of side radicals, normally show a higher H:C ratio than comparable HA's, it appears that this is less the case with the aquatic humic matter from the Laurentides, where the H C ratios of the fulvic and humic acids are very similar. The closer likeness of fulvic and humic acids from 414
Environ. Sci. Technol., Vol. 17, No. 7, 1983
natural waters as compared to those from other types of environments has also been observed in another context (12).
It is shown that the lower molecular weight fractions of the FA'Sand HA's of any of the origins being investigated occupy a relatively small area in the diagram. It can be assumed, therefore, that these low molecular weight FA'S and HA's have a similar basic carbon structure. The low molecular weight aquatic FA'S are positioned to the right of their HA homologues, their higher 0:C ratio indicating a greater richness in oxygenated groups such as COOH, OH, and OCH,. The higher molecular weight humic compounds occupy positions that appear to be radiating out from the above-mentioned rather restricted area, indicating that as the molecular weight increases, more profound differences are observed between the various types of humic matter. The higher molecular weight fractions normally show a lower H:C ratio (i.e., a more condensed aromatic system) than material with a lower molecular weight. This is illustrated in more detail in Figure 2 for fulvic and humic acids from the lake and river waters. This observation is an agreement with findings by Rashid and King (13)that the C:H ratio of humic substances from marine sediments was generally directly correlated with their molecular weight. On the other hand, none of these results, except one, can support the proposal by Swift et al. (14) that the degree of aromaticity of humic matter increases toward the lower molecular weight range, inferring, as did also Inoko and Tamai (15),that the process of humification leads to lower rather than to higher molecular weight material (3). Only in FA'S of microbial origin did the
I
I
I
I
I 0
Figure 2. Position in the Van Krevelen diagram, relatlve to their molecular welght, of fulvlc and humic acids Isolated from lake and river waters.
Table 11. Elementary Composition (%) o f Molecular Weight Fractions of Fulvic Acids Obtained from a Microbial Substrate in the Course of a 6-MonthIncubation Period
C
H N 0 C
H N 0 C
H N 0 C
H N 0
C H N 0 C
H N 0
750
5500
50.3 6.6 9.1 34 .O
50.0 6.4 9.8 33.8
4 Days 50.9 47.1 6.4 6.2 7.1 8.2 38.5 35.6
49.4 6.3 7.7 36.6
48.9 6.3 6.9 37.9
50.9 6.4 9.6 33.1
49.4 6.4 9.7 34.5
7 Days 45.9 49.3 6.2 6.2 8.7 7.1 39.2 37.4
48.1 6.2 7.8 37.9
48.1 6.5 6.8 38.6
50.9 6.5 9.8 32.8
49.1 6.4 9.5 35.0
1 4 Days 47.6 48.9 6.4 6.4 7.0 9.1 36.9 37.7
47.3 6.4 7.9 38.4
45.6 6.3 6.7 41.4
50.6 6.4 9.9 33.1
49.6 6.2 9.5 34.7
28 Days 48.9 49.3 6.2 6.5 9.3 7.0 35.6 37.2
47.7 6.3 7.9 38.1
46.8 6.4 6.6 40.2
50.7 6.0 9.9 33.4
49.9 6.2 6.5 34.4
60 Days 48.1 48.6 6.3 6.0 9.3 6.9 36.3 38.5
47.0 6.3 7.9 38.8
46.3 6.1 6.5 41.1
49.4 6.2 9.5 34.9
1 8 0 Days 48.8 50.1 5.9 5.8 9.3 6.9 36.1 37.1
50.7 5.2 9.9 34.2
15000 25000 40000 75000
0 50
0 54
0 58
0 62
0 66
0 70
O K I 70
47.5 6.3 7.9 38.3
45.4 5.6 6.4 42.6
degree of aromaticity increase toward the lower molecular range. Observations on Humic Matter of Microbial Origin. The unique position in the upper middle section of the diagram of the microbial FA'S is, in particular, the result of their wide H C ratio. This is indicative of a poorly developed aromatic structure, in particular where the higher molecular weight fractions are concerned. It is interesting to note the almost diametrically opposed positions of FA'S and HA's of microbial origin: whereas
incubation period o 4 davs
I
I
60
H/C
I50
I 40
0 50
0 54
0 58
0 62
0 66
0 70
O K
Figure 3. Position in the Van Krevelen diagram of fulvic acids from microbial substrates relative to (a) their molecular weight and (b) their degree of humification.
higher molecular weight FA fractions have wider H:C and 0:C ratios than material of lower molecular weight and are therefore less aromatic and more oxidized in structure (see also Figure 3b), higher molecular weight HA fractions show narrower H C and 0 : C ratios and would consequently have a more aromatic or condensed structure than lower molecular weight material. This could be indicative of a different mechanism of formation of the two types of humic matter, although a difference in their susceptibility toward oxidation cannot, at this stage, be excluded. Figure 3b and Table I indicate for the FA molecular weight fractions of microbial origin a negative correlation Environ. Sci. Technol., Vol. 17, No. 7, 1983
415
Table 111. Correlations between the Elementary Composition of Aquatic Humic Substances and the log Value of Their MolecuIar Weight fulvic acids C
lake river stream swamp '"
-0.97' -0.94' -0.89b - 0.94'
Significant at p < 0.05.
humic acids
H
N
0
C
-0.88b -0.97' -0.93: -0.88
0.32 - 0.90' -0.03
0.97' 0.94' 0.9lC 0.94'
0.84b -0.94' -0.96' 0.98'
-0.78'"
Significant at p < 0.01.
H N 0 '"
4
7
14
28
60
days
days
days
days
days
180 days
-0.30 -0.7ga -0.80'" 0.72'"
-0.62 -0.19 -0.86b 0.8ab
-0.91b -0.88& -0.85b 0.96'
-0.90b 0.09 -0.84b 0.94'
-0.94' 0.34 -0.84' 0.94c
-0.7aa 0.49 -0.84b 0.84b
Significant a t p < 0.05.
' Significant at D < 0.001.
Environ. Scl. Technol., Vol. 17, No. 7, 1983
-0.70a 0.96' 0.97' -0.62
-0.98' - 0.94' - 0.96' -0.93'
-0.94' -0.93' -0.96'
Table V. Elementary Composition (%) of Humic Substances Obtained from Lake and River Waters Sampled from May through Sept C
fulvic acids H N
O
Lake 1.8 49.3
May July Sept
44.8 44.8 45.6
4.1 4.3 4.2
2.0 1.9
May July Sept
41.5 42.1 43.0
4.2 3.8 3.8
2.2 2.1 1.6
Significant a t p < 0.01.
between the H:C ratio and the degree of humification (time of incubation) of the humic matter. Humification is therefore shown to result in a lower H:Cratio and consequently in a more aromatic structure of the humic molecule. As is shown in Figure 3b, the fulvic matter of microbial origin, on maturation, becomes both more oxidized and more aromatic. The changes observed in this figure concord well with the suggestion in ref 2 that during the early to middle stages of humification humic molecules lose aliphatic parts, whereas in a later stage humification is accompanied by dehydrative condensation reactions. From Figure 3, parts a and b, it would appear, therefore, that the degree of aromaticity of the microbially produced FA'S decreases with the molecular weight and the degree of humification. Similar observations were made in the case of certain types of soil humic matter (14, 15). For this reason, the latter authors considered the humification process to consist of progressive degradation of initially formed high molecular weight substances. According to them humification resulted therefore in the formation of lower molecular weight substances. In this sequence of events FA'S would thus mainly arise from degradation processes. In the present investigation the above-mentioned phenomenon was only noticed with respect to FA'S from the microbial substrate. This would suggest that microbial FA'S and aquatic humic matter are fundamentally different substances. Observations on Humic Matter of Aquatic Origin. Although for soil humic matter it is generally accepted that FA'S, containing a higher portion of peripheral aliphatic side chains, are less aromatic in structure and less condensed than HA's (16-18), this is not evident for humic matter of aquatic origin. As Figure 1 indicates, the majority of the FA and HA molecues from the aquatic environments had very similar H:C ratios. The basic structures of these two groups of substances would therefore appear to have a greater similarity than those of FA'S and HA's present in soils (Figure 1). The oxygen content of the aquatic humic matter was normally richer in this element in higher than in lower weight fractions. This is contrary to what has generally been reported for soil humic matter (13-14) and could indicate that aquatic humic substances are less stable (or less stabilized by their environment) than soil humic 416
- 0.90'
0
Significant at p < 0.001
Table IV. Correlations between the Elementary Composition of Microbially Produced Fulvic Acids and the log Value of Their Molecular Weight
C
N
H
49.0 48.3
C
humic acids H N
O
51.7 51.9 52.8
5.0 4.9 4.9
2.1 2.0 2.4
41.2 41.2 40.1
48.5 49.5 51.0
4.8 4.6 4.5
1.9 1.9 1.9
44.9 43.9 42.6
River 52.1 51.9 51.5
matter. The higher molecular weight fractions of the aquatic FA'S in particular had very wide 0 : C ratios surpassing the range observed for soil FA'S (Figure 1). This would mean that in the higher molecular weight range, aquatic FA'S are richer in oxygenated groups than soil FA'S and are therefore probably more hydrophilic in character. This would agree with observations indicating that the high molecular weight aquatic FA'Swere more surface active than their terrestrial counterparts (19). The similar positions occupied by aquatic FA'S of different origin but of comparable molecular weight, as distinct from the quite scattered positions of the various HA fractions, would suggest that environmental differences have less effect on the chemical and physicochemical properties of aquatic FA'Sthan on their HA counterparts. In the aquatic environment, therefore, FA'S as a group, would appear to be more homogeneous than HA's. It is also evident that humic matter of aquatic origin generally has H:C ratios that are above the average obtained for soil HA's and FA'S. This confirms observations by Kalle (20),who found that autochthonous aquatic humus has a lower degree of condensation (aromaticity) than its terrigenous counterpart. With regard to seasonal changes in the elementary composition of the aquatic humic matter, Table V shows that during the course of the summer season the humic material gradually became richer in carbon and poorer in oxygen. The hydrogen content also normally decreased during this period. The observed decreases in H:C and 0:C ratios between May and Sept (Figure 4) would indicate that during this period the aquatic humic matter became more humified. In another context we found in the lake and river water fulvic and humic acids a decrease in the E,:E6 ratio for the period of May-Sept, reflecting likewise an increase in their degree of humification (21). The molecular weight of the humic matter also increased during this period, The river water humic matter appears, on the whole, to be more oxidized than comparable material extracted from the lake water. Furthermore, considering the buffering action of the relatively large volume of the lake water (- 70 000 m3), it stands to reason that the seasonal changes in the humic matter of this environment are
I
260r
I 0 80
I I
08 Lo
0 60
0 70
I
J
0 90
103
o/c
Flgure 4. Seasonal changes In the composition of fulvic and humic aclds from aquatic envlronments as expressed by their position In the Van Krevelen diagram.
smaller than those observed in humic substances from the river. Effect of Ultraviolet Irradiation on the Structure of Humic Matter. The bleaching of humic compounds in the aquatic environment by ultraviolet light was reported by Gjessing (22))who postulated a breakdown of the molecule by oxidation mechanisms probably involving free radicals. To investigate further the effect of this type of radiation on the structure of humic matter by means of the Van ' 0 solutions of nonfractionated Krevelen diagram, 0.057 humic matter from the microbial cultures, sampled after 4 and 60 days of incubation, were irradiated at a distance of 21 cm from 2 GE germicidal lamps (no. G 2518; 25 W), with maximum ultraviolet emission at 253.7 nm. It appears (Figure 5) that the irradiation of the humic matter resulted in a partial oxidation of the material and in a partial loss of its aromatic structure. Recently formed material was more easily attacked than more humified matter. The latter seemed to be more resistant, in particular to the introduction of oxygen into the molecule. FA'S, and in particular recently formed fractions (4 days of incubation), are shown to be more susceptible to degradation by ultraviolet irradiation than HA's. The exposure of FA'S to the ultraviolet light resulted in the formation of highly oxygenated aliphatic compounds (H:C = 2.47; 0:C = 2.03). In summarizing the above findings, the effect of ultraviolet light on organic matter in the aquatic environment would consist of a partial loss of humified organic matter, in particular of recently formed substances, with the result that the more humified material will show a relative increase.
Conclusions For aquatic humic matter carbon, hydrogen, and nitrogen contents normally diminished with increasing molecular weight of the material; for oxygen the reverse was usually true. Aquatic humus has a less aromatic structure than its terrigenous counterpart. Whereas the process of humification normally results in the formation of a more aromatic type of humic matter, for certain types of humus such as FA'S from a microbial culture it would appear that the degree of aromaticity decreases with progressive humification. Aquatic FA'S are richer in oxygenated groups than soil FA'S. Fulvic and humic acids of aquatic origin resemble each other more closely than their terrestrial counterparts. Low molecular weight fractions of aquatic
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Figure 5. Effect of illumination with ultraviolet light on H:C and 0 : C atomlc ratios of mlcrobial fulvic and humic acids with different degrees of humiflcatlon.
fulvic and humic acids would appear to have a very similar basic carbon structure. Whereas higher molecular weight humic matter fractions normally have a more aromatic structure than low molecular weight fractions, in FA'S of microbial origin an increase in aromaticity was noticed toward the lower molecular weight range. The degree of humification of humic matter present in natural waters was found to increase in the course of the summer season. Recently formed humic matter is more sensitive to ultraviolet irradiation than more humified material; FA'S are also more susceptible to it than HA's. Registry No. C, 7440-44-0;H, 12385-13-6;N, 17778-88-0; 0, 17778-80-2.
Literature Cited (1) Van Krevelen, D. W. Fuel 1950,26, 269-284. (2) Kuwatsuka, S.; Tsutsuki, K.; Kumada, K. Soil Sci. Plant Nutr. 1978,24, 337-347. (3) Kononova, M. M. "Soil Organic Matter",2nd ed.; Pergamon: New York, 1966. (4) Kumada, K.; Aizawa, K. Soil Sci. Plant Nutr. 1958, 3, 152-159. (5) Visser, S. A. J . Soil.Sci. 1964, 15, 202-219. (6) Schnitzer, M.; Desjardins, J. G. Can. J . Soil Sei. 1966,46, 237-243. (7) Jackson, T. A. Soil Sci. 1975,119, 56-64. ( 8 ) Saiz-JimBnez, C. An. Edafol. Agrobiol. 1975,34,829-839. (9) Posner, A. M.; Creeth, J. M. J . Soil. Sci. 1972,23,333-341. (10) Safford, H.W.; Stragand, G. L. Anal. Chem. 1951, 23, 520-522. (11) Kumada, K. J. Sci. Soil Man. Jpn. 1955, 26, 19-22. (12) Visser, S. A. J. Environ. Sci. Health, Part A 1982, 17, 767-788. (13) Rashid, M. A.; King, L. H. Chem. Geol. 1971, 7, 37-43. (14) Swigt, R. S.; Thornton, B. K.; Posner, A. M. Soil Sei. 1970, 110,93-99. (15) Inoko, A.; Tawai, M. JARQ 1977,11, 30-35. (16) Felbeck, G. T. Adu. Agron. 1965, 17, 327-368. (17) Mendez, J.; Lojo, M. I. Acta Salmant. Ser. Cienc. 1972,41, 187. (18) Schnitzer, M.; Skinner, S. I. M. Can. J. Chem. 1974, 52, 1072-1080. (19) Visser, S. A. Rev. Fr. Sci. Eau 1982, 1, 285-295. (20) Kalle, K. Oceanogr. Mar. Biol. Annu. Rev. 1966,4,91-104. (21) Visser, S. A., submitted for publication. (22) Gjessing, E. T. "Physical and Chemical Characteristics of Aquatic Humus"; Ann Arbor Science: Ann Arbor, MI, 1976.
Received for review August 23,1982. Revised manuscript received January 24, 1983. Accepted February 21, 1983. Environ. Scl. Technol., Vol. 17, No. 7, 1983
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