The Science of the Total Environment, 81/82 (1989) 143-156 143 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands

SPECTROSCOPIC AND COMPOSITIONAL COMPARATIVE CHARACTERIZATION OF I.H.S.S.

REFERENCE AND STANDARD FULVIC AND HUMIC ACIDS OF VARIOUS ORIGIN

1 2 1 1 N. SENESI , T.M. MIANO , M.R. PROVENZANO and G. BRUNETTI

1 Istituto di Chimica Agraria, Universit~ di Bari, Bari (Italia)

2 Istituto di Chimica Agraria e Forestale, Univ. di Basilicata, Potenza (Italia)

SUMMARY

Eleven standard and reference fulvic acids (FA) and humic acids (HA) of aquat-

ic and terrestrial origin from the collection of the International Humic Substan-

ces Society have been studied by chemical and spectroscopic methods. Aquatic HA

appear richer in 0 and lower in C than terrestrial HA, while the opposite is gen-

erally true for the corresponding FA. The N content of aquatic samples is always

much lower, and C/N ratio and E4/E 6 ratio much higher than those of terrestrial

samples. The infrared spectra of all samples but soil HA are qualitatively simil-

ar one to another, but they differ mainly in the relative intensity of carboxyl,

aromatic and aliphatic group bands. Fluorescence of terrestrial HA occurs at

higher wavelengths than terrestrial FA and aquatic HA and FA in both emission and

excitation modes, while synchronous-scan spectra appear more resolved and inform-

ative. ESR spectra are featured by resonances consistent with conjugated semiqui-

none free radicals and Fe(III) ions, the latter resonance being generally more

intense for terrestrial than for aquatic samples.

INTRODUCTION

A primary purpose of the International Humic Substances Society (IHSS), as

stated in the Bylaws, is to "establish and maintain a collection of standard and

reference samples of humic substances" (ref. i). Up-to-date, this task has been

successfully accomplished by the isolation of a number of terrestrial and aquatic

fulvic acids (FA) and humic acids (HA) of various nature and origin (ref. 2),

which are accessible to researchers worldwide. Unique features of the collection

are the carefully chosen and specifically designated source materials, and the

carefully controlled and IHSS-supervised, fully documented isolation and purific-

ation of FA and HA (ref. 2). Furtherly, the availability of the bulk solid source

materials allows the comparison of different extractive methods.

One of the major benefits that results from the existence of this collection

is the possibility of critically compare the experimental results of humic subst-

ances researchers worldwide. The basic characterization of the samples, according

0048-9697/89/$03.50 © 1989 Elsevier Science Publishers B.V.

144

to a protocol suggested by the IHSS (ref. 3), is currently in progress. A variety

of methods, including elemental and functional group analysis, component analysiw

molecular weight and size determination, ash analysis, and numerous spectroscopic

techniques, are being applied. Some analytical data, mostly referring to element

composition and NMR spectra of river FA and HA are already available (refs. 3,4).

The objective of this paper is to present results on the preliminary characte-

rization of eleven standard and reference FA and HA by elemental analysis and UV-

visible, infrared, fluorescence and electron spin resonance spectroscopies, also

in comparison with corresponding properties of well characterized FA and HA of

similar nature and sources.

MATERIALS AND METHODS

Standard and reference FA and HA were obtained from the IHSS, designated as

referred in Table i. The aquatic samples consist of reference and standard FA and

HA from stream water from the Suwannee river, sampled near Fargo, Georgia, U.S.A.

and Nordic aquatic reference FA and HA from North Sea, Norway, both isolated by

XAD-8 resin technique (ref. 5). The terrestrial samples consist of reference FA

and HA isolated from soil from a mollic epidedon near Joliet, Illinois, U.S.A.,

and peat obtained at Belle Glade Research Station, Everglades, Florida, U.S.A.,

and reference HA from leonardite from an oxidized coal seam at the Gascoyne Mine,

Bowman County in southeastern North Dakota, U.S.A.. No FA is available from the

leonardite because of the very small amount present. HA and FA were extracted

from the bulk-solid earth materials by a 20-step procedure based upon NaOH extra-

ction that was formulated by an international group of humic substances resear-

chears in 1981, on the basis of information then available, as the most suitable

for these materials and for the specific purposes of the establishment of the

IHSS collection (ref. 2). All final materials were obtained in homogenized, +

freeze-dried H -form (ref. 2).

Carbon, hydrogen, and nitrogen were determined by a Perkin Elmer Model 240/A

C,H,N-Analyzer. Sulfur content was measured by BaC12-AgN03, back-titration of

HNO3-digested samples. Oxygen was calculated by difference: %0=IO0-%(C+H+N+S).

UV-visible spectra were obtained over a range of 200 to 700 nm using a Perkin

Elmer Model Lambda 15 UV-vis spectrophotometer on 0.05 -N NaHCO 3 solutions at a

concentration of 50 mg/L for FA and 33 mg/L for HA and a pH between 8 and 9.

-i Infrared (IR) spectra were recorded in the 4000 to 500 cm wavenumber range

145

using a Perkin Elmer Model 399B IR spectrophotometer equipped with a Perkin Elmer

Data Station 3600, on KBr pellets obtained by pressing, under reduced pressure,

uniformly prepared mixtures of 1 mg sample and 400 mg KBr, spectrometry grade,

with precautions taken to avoid moisture uptake.

Fluorescence spectra were recorded using a Perkin Elmer LS-5 Luminescence

spectrophotometer equipped with a Perkin Elmer Data Station 3600, on aqueous sol-

utions of each sample at a concentration of I00 mg/L and adjusted at pH = 8 with

0.05 N NaOH. Emission spectra were recorded over the range 380 to 550 nm at a

fixed excitation wavelength of 360 nm. Excitation spectra were obtained over a

scan range of 270 to 500 um, by measuring the emission radiation at a fixed wave-

length of 520 nm. Synchronous-scan excitation spectra were obtained by scanning

simultaneously both ~ and ~ over a range of A 290 to 550 nm, while keep- exc em exc

ing a constant wavelength difference, /kA = ~ - ~ = 18 nm, which provided the em exc

best resolution.

Electron s~in resonance (ESE) spectra were obtained at room temperature on

solid samples packed in quartz ESR tubes using a Bruker ER 200D-SRC ESR spectro-

meter operating at X-band frequency with i00 kHz magnetic field modulation. Field

range scanned were 5000, i000 and lO0 Gauss (G) with a modulation amplitude of

50, i0, or 6.3 G, respectively. The microwave frequency was 9.78 GHz and a micro-

wave attenuation of 13 dB (corresponding to a microwave power of i0 mW) was used

throughout the experiments. ESR parameters, i.e. spin concentration, signal line-

width, g-values and coupling constants, have been calculated from the experiment-

al spectra according to standard procedures and equations (refs. 6,7), while

their physical significance and chemical information they can provide on humic

substances and their metal complexes have been recently reviewed (ref. 7).

RESULTS AND DISCUSSION

Elemental analysis

The major elemental composition, atomic ratios and ash contents of FA and HA

examined are presented in Table i, together with corresponding data furnished on

the same samples by the IHSS (ref. 4). The trend of our data generally agrees

with that of IHSS data. Although, we find often little lower C and H contents and

C/N ratios and higher N and Q contents and O/C ratios than those provided by the

IHSS (ref. 4), while our S percentages result lower for river FA and HA and

higher for terrestrial samples. No significant difference is observed in the

146

0

4n

o

0

%

4-~ e.

4-~ r" 0 0

,.ffl

g 0

%

o

0

ffl

E 0

o

0 o

4.~ e-,

O ~ O ~ O O O o o o o o 0 4 o ~ d d d d d d ~ ~

• o ° , • . . o .

O ~ O ~ O O ~ O

• CO 0 oj

~ d ~ d d d d S g CO CO O~ CO ~ ~ CO "q" LO

d d d S d ~S S d ~S

O O O O ~ O O ,~ ,-I • ° ° • • • ° • •

• ° o ° • ° ° , •

dS ~,-i ~ ~4 d ~4d

~" O CO OJ C~l ['~ C~J 'q" D-

• • 0 • ° 0 ° • •

O O O O ,-~ ~ O O O

CO gO ~O 03 O ~D C0 kO CO

• ° ° • ° ° • • •

O O O O O C.J O,J 04 OJ

t~ 03 CO 'q" ,-~ C,J ,-I LO '~" O C~J ,-~ C'J '~" ~ 0 0 C O L D

4 4 4 4 ~4 ~44 ~4~4

0 0 0 0 0

.,~

i i ° o o

O ~ O ~ 0 0 0 o o o ~ o 0 4 0 4 ~ 4 d d d ~ d d ~ d ~ d ~

0 0

dd Fd ~ ~S ~d ~

d d S S S S o d o S S

~ 0 ~ ~ ~ ~ ~

~ 0 ~ ~ O ~ ~ O ~

S o S S S ~ o S S ~ d

~ 4 ~ 4 d 4 ~ d 4 d ~

$ 4 d 4 S g ~ ~ ~ d

= 5 ~ N = = S o ~ o o o

N ~ 5 5 ~ N ~ ~

0 0 0 •

c~ ,.Q

I1)

I

.p

~,

I rll

ffl 0

0

0

o

0

0

0 0

4~

E o

4

0

0

~g

H

E 0

ffl 4.~

~2 o

147

elemental composition between reference and standard river FA or HA. In general,

FA show C, H, N, S and ash contents lower and 0 contents, C/N and O/C ratios

higher than those of the HA from the same source, while no trend is observed for

C/H values. The differences observed are more pronounced between terrestrial FA

and HA than between aquatic FA and HA. This behaviour is typical of terrestrial

and aquatic FA versus their HA counterparts (refs. 8-12). Our data for Suwannee

river samples depart from the general trend in that H content is higher for FA

than for HA, and C and 0 percentages and O/C ratio do not differ sensibly between

FA and HA.

Data in Table 1 also show that the C content of river and nordic aquatic FA is

a little higher than that of soil and peat FA, in agreement with literature data

(refs. 8,10-12), whereas the opposite is true for corresponding HA. The H content

of FA follows the order: river > soil > nordic > peat, while terrestrial HA are

always richer in H than aquatic HA. The N content of aquatic FA and HA samples is

much lower and C/N ratio much higher than that of their terrestrial counterparts,

with the exception of leonardite HA. When compared to literature data (refs.8-12~

the N content of Suwannee river, and especially of nordic aquatic FA and HA, ap-

pear to be particularly low and the C/N ratio high, while the opposite is true,

even if at a lower extent, for soil and peat FA and HA. Aquatic HA show an 0 con-

tent and O/C ratio higher than those of terrestrial HA, in agreement with the

literature (ref. 12). The values of these two parameters do not vary much between

FA samples, while lower values are generally reported for river and marine FA

versus soil FA (refs. 8,11,12).

Surprisingly, very high ash contents have been determined in the Suwannee

river HA and peat FA, differently from ash around I% measured in other samples.

UV-visible spectroscopy

The UV-visible spectra of all FA and HA examined are featureless and monoton-

ically decreasing with increasing wavelength, with a more or less pronounced

shoulder at about 270 nm exhibited by all HA (Fig. 1).

The slope of the absorption curves as measured by the ratios of absorbance at

465 and 665 nm, i.e. the E4/E 6 ratio, has been traditionally suggested to be inv-

ersely related to the degree ~f condensation of aromatic groups (aromaticity) and

more recently, to particle size and/or molecular weight (ref. 8). The higher

E4/E 6 ratios measured for aquatic FA, and particularly HA, with respect to those

148

3.0

d e b,c o

~ 1.2 o

~ 0.6

0.0

3.0 a

b

0.6

0.0 36o 560 ~oo

Wavelength (nm)

360 560 Wavelength (nm)

~oo

Fi E . i. UV-visible spectra of FA (left) and HA (right): Suwannee river (a, stand-

ard; al, reference), nordic aquatic (b), soil (c), peat (d), leonardite (e).

of their terrestrial counterparts (Table l) are in general agreement with data in

the literature (refs. 8,9,11,12), and suggest a lower degree of condensed aromat-

ic systems and/or smaller particle sizes or molecular weights for aquatic with

respect to terrestrial FA and HA.

Infrared spectroscopy

The IR spectra of the FA and HA examined (Fi E . 2) are, in general, similar one

to another in the position of principal absorptions, but differences of various

entity are apparent in the relative intensity of some bands in dependence of the

origin and nature of the sample. The IR spectra of FA samples of any source may

be tentatively classified as type II spectra, while those of HA samples strictly

resemble type I spectra, with the exception of the soil HA spectrum which more

resembles type III spectra (ref. 13).

All the spectra are characterized by a strong band at 3390-3370 cm-l(stretch

of hydrogen-bonded OH), which appears relatively more intense for HA than for FA

of any origin, and broadens widely towards lower wavelengths (aromatic C-H stret-

-i chin E modes) for soil and, especially, peat FA. A peak at 2920 cm (stretch of

aliphatic C-H) is apparent with decreasing intensity, in the order, for soil,

leonardite and peat HA, while it appears only as a shoulder in the spectra of the

remainin E FA and HA samples. A strong band at 1715-1710 cm-l(c=o stretch of carb-

oxyl and carbonyl groups) is common to the spectra of all samples but soil HA,

-1 where it appears only as a deep shoulder. A very intense band at 1615-1610 cm

-1 (mainly aromatic C=C and CO0- group vibrations), which broadens towards 1650 cm

-i (amide I band), characterizes the soil HA spectrum. The band at 1615 cm is also

present, very intense but narrower, in the IR spectra of the remainin E HA samples

149

0

I

4000 2000 I000 500 4000 2000 i000 500 Wavenumber (cm -I) Wavenumber (cm -I)

Fig. 2. IR spectra of FA (left) and HA (right): Suwannee river (a, standard; a I,

reference), nordic aquatic (b), soil (c), peat (d), leonardite (e).

while it shows a much reduced relative intensity in the spectra of river and nor-

dic aquatic FA, and is present only as a shoulder in peat and soil FA spectra. -i

Only the spectrum of the N-rich (about 5%) soil HA shows a weak peak at 1520 cm

which is typically attributed to C=O vibrations of amide groups (amide II band).

Additional features common to all the spectra are the band of medium-weak intens-

ity at about 1385 cm-l(o-H deformation and C-O stretch of phenols, CO0- group

vibrations, C-H deformation of -CH3), and the broad, intense absorption centered

around 1220-1200 cm-l(mainly attributed to C-O stretch and O-H deformation of

COOH groups). An intense band at 1040 cm-l(alcoholic and polysaccharide CO

stretch and OH deformation and, eventually, Si-OH bend in silicate impurities) is

typically present in the spectrum of soil HA, while only a faint shoulder is

shown at this wavenumber by almost all the remainin E samples. Minor absorptions -i

appear as shoulders or weak bands in all the spectra in the region below 900 cm

(out-of-plane bendin E of aromatic C-M, vibrations of aliphatic -CH 2- chains,etc.

150

Fluorescence spectroscopy

Fluorescence emission spectra of all samples (Fig. 3) are characterized by a

broad band centered around A =510 nm (green) for soil, peat and leonardite HA, at

471 nm for river and nordic aquatic HA, and between 457 and 465 nm (violet) for

all FA. Some samples also show shoulders at lower wavelength, e.g. soil HA at

about 460 nm, or higher wavelength, e.g. river HA at about 510 nm. This behaviour

is confirmed by the corresponding fluorescence excitation spectra (Fig. 4) which

show patterns better resolved than the emission spectra and consist of two main

peaks at 450 and 467 nm and a less intense peak at 390 nm for terrestrial HA, and

a main peak between 388 and 395 nm, with minor peaks or shoulders at 440 and 466-

467 nm, for aquatic HA and all FA. Pronounced broad shoulders are shown by soil

461

400 440 480 520 550

Wavelength (nm)

Fig. 3. Fluorescence emission spectra of

Suwannee river FA (a,st.;al,ref.) and HA

(a',st.;a~,ref.),nordic aquatic FA and HA

(b,b'),soil FA and HA(c,c'),peat FA and HA

(d,d'),leonardite MA (e).

HA around 320 nm and river FA at

about 360 nm. Maxima for terrestri-

al HA are well differentiated from

those of the corresponding FA in

both the emission and excitation

modes, while aquatic FA and HA sam-

ples are characterized by much

closer peaks in both modes. Data

for aquatic humic substances fall

close to the upper limit of the

range of values reported in the

literature (refs. 9,10,14), while

those for terrestrial samples are

very similar to reported data

(refs. 15-19).

Although the exact nature of the

fluorescing groups is still far

from being clarified, fluorescence

at the higher wavelengths, typical

of terrestrial MA (refs. 15-18), is

generally attributed to either

highly substituted aromatic nuclei,

possibly bearing at least one elec-

tron-donating group, and/or to con-

1 5 1

4~

4~

0

o

o

ID >

3 8 8 3 9 3 1

I i i i

300 350 400 450 500 300 350 400 450 500

Wavelength (nm) Wavelength (nm)

Fig. 4. Fluorescence excitation spectra of FA (left) and HA (right): Suwannee

river (a, standard; al, reference), nordic aquatic (b), soil (c), peat (d),

leonardite (e).

jugated unsaturated systems capable of high degree of resonance (refs. 15-17). On

the other hand, terrestrial FA and, typically, aquatic FA and HA are characteriz-

ed by fluorescence spectra showing maxima at lower wavelengths (refs. 9,10,14,18~

indicating the presence of simpler structural components, i.e. low degree of ar-

omatic polycondensation and low levels of conjugated chromophores. Fluorophore

groups derived by polycondensation between carbonyls and amines, i.e. Schiff-base

systems, and lignin-derived phenolic structures where the OH is conjugated

through the benzene ring with a carbonyl, have been indicated as possible respon-

sible for the fluorescence of humic substances in the region 470-440 nm, in the

emission mode, and at A=390 nm in the excitation mode (refs. 17,20). Coumarin-

like structural units originating from lignin ( A max = 465-470 nm, A max = em exc

387-390 rim), as well as flavonoid moieties ( A max = 520 run, A max = 370 nm) em exc

have also been suggested as possible contributors to fluorescence of river aquat-

ic humic substances (ref. 14).

Fluorescence spectra obtained in the synchronous excitation mode (Fig. 5) ap-

pear for most FA and some HA samples well resolved and differentiated enough in

the position and relative intensity of the peaks to permit a clear distinction

152

c H

o

o o

o

>

o

I~ ~ ~ 1 / i i i

300 350 400 450 500 Wavelength (nm)

\

\ 55O

4 7 2

. - e _ 2 - ~ , , ,

300 350 400 450 500 Wavelength (nm)

550

Fig. 5. Fluorescence synchronous-scan excitation spectra of FA (left) and HA

(right): Suwannee river (a, standard; a l, reference), nordic aquatic (b), soil

(c), peat (d), leonardite (e).

o c o

o

a i

. c

300 400 500 550

Wavelength (nm)

Fig. 6 . Differential fluorescence synchro-

nous-scan excitation spectrum (c) of

Suwannee river FA (a) versus HA (b).

between FA and HA f~om the same

source and FA and HA of different

origin. Although the principal

maxima are localized almost at the

same positions as in the emission

and excitation spectra, their rel-

ative intensity and the presence

of additional minor peaks or

shoulders may furnish further inf-

ormation on the relative concentr-

ation of various fluorophores in

each sample.

One example of differential

synchronous excitation spectrum is

given in Fig. 6 for the Suwannee

river FA versus HA. The pattern

153

clearly indicates how the FA fluoresces predominantly below 450 nm, with a diffe-

rential-maximum around 395 nm, and HA fluoresces mostly above this value, with a

broad differential-maximum between 465 and 500 nm.

Electron spin resonance spectroscopy

Wide scan range (5000 G) ESR spectra of FA and HA examined (Fig. 7) are relat-

ively simply-shaped and substantially similar one to another, although differen-

ces are apparent in the relative intensity of the two principal resonance lines

and in the presence of minor resonances. In general, the ESR spectra of terrestr-

ial FA and HA appear simpler in their patterns than those reported in the

literature (ref. 7), probably because of the extended purification to which the

samples here examined have been subjected.

All the spectra are dominated by a strong, very narrow symmetrical resonance,

typically found in ESR spectra of FA and HA of any nature and origin (ref. 7).

The physical parameters of this signal, referred in Table i, have been calculat-

ed from the enlarged (i00 G) spectra of the region, which appear devoid of any

hyperfine structure. The g-values, similar for all the samples, and linewidths, a

little larger for FA than for corresponding HA, are consistent with organic free

radicals of semiquinone nature conjugated with an aromatic network (ref. 7). Free

radical concentration is similar in FA samples and always lower than in the cor-

responding HA. Aquatic HA, however, contain about one half free radicals than

terrestrial HA, which show the highest concentration in the leonardite HA sample.

The g-values we measure are within the range of reported values (ref. 7), but

differ sensibly from the atypical g-values provided for Suwannee river samples by

IHSS (ref. 4). linewidths are a little larger, except for soil FA, than those

reported in the literature (ref. 7), but lower than those for Suwannee river sam-

ples provided by IHSS (ref. 4). Spin concentrations we measure are definitely

lower, except for peat HA, than the values referred in the literature for samples

of the same nature and origin (refs. 4,7).

The other feature common to all the spectra is an asymmetrical line centered

at g = 4.2-4.3, with a linewidth ranging from 60 to ii0 G, often observed in FA

3+ and HA of any origin and generally attributed to high spin Fe ions in inner-

sphere complexes in a crystal field of rhombic symmetry (ref. 7). The relative

intensity of this signal is generally much lower in FA than in the corresponding

HA and in aquatic FA and HA than in their terrestrial counterparts (Fi E . 7). The

154

~a ) ~ G

8 t

F2" \

2o¢ G 3000 G F#*

•

OI~ANIC FREE c ' ~ "~ RADt~ILL

FREE / RADICAL

f j /

i

Fig. 7. ESR spectra (scan range , 5000 G) of FA (left) and HA (right): Suwannee

river (a, standard; al, reference), nordic aquatic (b), soil (c), peat (d), ! leonardite (e). Spectra labelled a', al, b', c', d', e' have been recorded at a

gain 3-to 80-fold higher than that of base-spectra.

ESR spectra of all HA and peat FA are characterized by a weak resonance at about

3+ g = 9-10, also consistent with Fe ions in sites of near rhombic symmetry (ref.7).

All ESR spectra exhibit an apparently unresolved broad feature at g = 3-2,

shaped for aquatic samples differently than for terrestrial samples, which prob-

ably consist of an envelope of several resonances arising from various magnetic-

3+ 2+ 2+ 4+ ally-interacting ions, such as Fe , Cu , Mn , V (ref. 7). When recording

this portion of the ESR spectrum at a gain one to two orders of magnitude higher,

155

5oG

f Fig. 8. ESR spectrum (scan range, i000 G)

of leonardite HA, recorded at a modulation

amplitude 4+ of 40 G and a gain of 2 x i0

(a, V pattern?) or 2 x i0 (b, free rad-

ical signal), and at a modulatio~ amplit-

ude of I0 G and a gain of 5 x i0 (c, sup-

erhyperfine structure from N ligands co-

ordinating a paramagnetic metal ion?).

a poorly-resolved, unidentifiable

structure is observed for soil and

peat FA and peat HA (Fig. 7c",d").

In the case of leonardite HA, the

lO00-fold gain spectrum of the ex-

panded (scan range, i000 G) region

around g = 2 exhibits peaks which

may correspond to part of the oc-

tuplet-patterns of the parallel

and perpendicular components from

4+ V ions (Fig. 8) (ref. 7). The

same spectrum, measured at a lower

modulation amplitude (i0 G), dis-

plays a series of narrow peaks

(splitting constants 16 G) at a

g-value slightly greater than 2,

of difficult assignement, but ten-

tatively consistent with a super-

hyperfine structure from a number of N ligand atoms coordinating a paramagnetic

2+ metal, such as Cu ion.

CONCLUSIONS

In general, the IHSS FA and HA samples examined in this work can be distingui-

shed sufficiently according to their nature and origin by elemental and spectro-

scopic analysis, although a number of properties are common to various aquatic

and terrestrial FA and/or HA. Furthermore, the samples exhibit relevant similar-

ities, but also some significant differences in their compositional and spectro-

scopic properties with respect to those reported in the literature for FA and HA

of similar origin and nature. All comparison, however, must be viewed with preca-

utions, particularly when the extraction and purification methods are different,

e.g. alkaline versus resin extraction.

REFERENCES

1 International Humic Substances Society, Bylaws, Art. II, Section 3, 1982, 198~

2 P. MacCarthy, R.L. Malcolm, M.H.B. Hayes, R.S. Swift, M. Sehnitzer and W.L.

156

Campbell, Establishment of a collection of standard humic substances, IHSS

Commun. to Members, 1985.

3 International Humic Substances Society, List of characterization, Commun. to

Purchasers of Standard and Reference Humic and Fulvic Acids, 1985.

4 International Humic Substances Society, Sample of analytical data for IHSS

standard and reference humic and fulvic acids, Commun. to Members, 1987.

5 E.M. Thurman and R.L. Malcolm, Preparative isolation of aquatic humic substa-

nces, Environ. Sci. Technol., 15 (1981) 463-466.

6 J.E. Wertz and J.R. Bolton, Electron Spin Resonance: Elementary Theory and

Practical Applications, McGraw-Hill, New York, 1972.

7 N. Senesi and C. Steelink, Application of ESR spectroscopy to the study of

humic substances and their interactions with organic xenobiotics and metal

ions, in: MB.H. Hayes, P. MacCarthy, R.L. Malcolm and R.S. Swift (Eds.),

Humic Substances in Soil, Sediment and Water: Structure and Interactions,

Wiley, New York, in press.

8 M. Schnitzer, Humic substances: chemistry and reactions, in: M. Sohnitzer and

S.U. Khan (Eds.), Soil Organic Matter, Development in Soil Science 8,

Elsevier, Amsterdam, 1978, pp. 1-64.

9 J.R. Ertel and J.I. Hedges, Bulk chemical and spectroscopic properties of mar-

ine and terrestrial humic acids, melanoidins and catechol-based synthetic pol-

ymers, in: R.F. Christman and E.T. Gjessing (Eds.), Aquatic and Terrestrial

Humic Materials, Ann Arbor Science, Ann Arbor, MI, 1983, pp.143-163.

I0 N. Pax~us, Studies on aquatic humic substances, Diss. Thesis, Dept. Analytical

Marine Chemistry, Chalmers Univ. of Technology, Goteborg, Sweden, 1985.

ii G.R. Harvey and D.A. Boran, Geochemistry of humic substances in seawaters, in:

G.R. Aiken, D.M. McKnight, R.L. Wershaw and P. MacCarthy (Eds.), Humic Subst-

ances in Soil, Sediment and Water: Geochemistry, Isolation and Characterizat-

ion, Wiley, New York, 1985, pp.233-247.

12 E.M. Thurman, Organic Geochemistry of Natural Waters, Nijhoff-Junk, Dordrecht,

1986, pp. 273-361.

13 F.J. Stevenson and Gob, Infrared spectra of humic acids and related substance~

Geochim. Cosmochim. Acta, 35 (1971) 471-483.

14 R.A. Larson and A.L. Rockwell, Fluorescence spectra of water-soluble humic

materials and some potential precursors, Arch. Hydrobiol., 89 (1980) 416-425.

15 B.K. Seal, K.B. Roy and S.K. Muckherjee, Fluorescence emission spectra and

structure of humic and fulvic acids, J. Indian Chem. Soo., 41 (1964) 212-214.

16 C. Datta, K. Ghosh and S.K. Muckher~ee, Fluorescence excitation spectra of

different fractions of humus, J. Indian Chem. Soc., 48 (1971) 279-287.

17 K. Ghosh and M. Schnitzer, Fluorescence excitation spectra of humio substance~

Can. J. Soil Sol., 60 (1980) 373-379.

18 S.A. Visser, Fluorescence phenomena of humic matter of aquatic origin and mi-

crobial cultures, in: R.F. Christman and E.T. G~essing (Eds.), Aquatic and

Terrestrial Humic Materials, Ann Arbor Sci., Ann Arbor, MI, 1983, pp. 183-202.

19 G.G. Choudry, Photophysioal and photochemical properties of soil and aquatic

humic materials, Residue Reviews, 92 (1984) 59-112.

20 R.W.P.M. Laane, Comment on the structure of marine fulvic and humic acids,

Marine Chemistry, 15 (1984) 85-87.