Humic Substances Research Articles

Properties of Humic Substances from the Baltic Sea and Lake Ermistu Mud Monika Ubnerl, 2, Maili Treuman 1, Anu Viitak 1 and Margus Lopp 1.

1 Department of Chemistry, Tallinn Technical University, Ehitajate tee 5, Tallinn 19086, Estonia 2 Health Resort Laboratory, P~irnu College of Tartu University, Ringi 35, P~irnu 80010, Estonia

* Corresponding author (lopp@chemnet.ee)

1 Introduction

Humic substances (HSs) are found in soil, lake and sea sediments, natural waters, river, peat, brown and brown-black coals and other natural materials as a product of microbial degradation, chemical polymerisation and oxidation of organic matter [1,2,3]. HSs play a fundamental role in accumulation and exchange processes of chemical compounds (metals and organic pollutants) in the environment [2].

The classical definitions of HSs are based on aqueous solu- bility. They are defined as highly transformed, amorphous, dark coloured materials. According to their solubility, HSs can be divided into four fractions: a) humic acid (HA; not soluble in water under acidic conditions, but becomes solu-

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Research Articles Humic Substances

ble at a higher pH); b) hymatomelanic acid (HMA; soluble in ethanol); c) fulvic acid (FA; soluble in water at any pH); d) humin (not soluble in water at any pH). HSs sorb peptides, sugars, nucleic acid residues and fats [1,4,5]. The free HSs are directly soluble in alkali, while the bound HSs have to be treated with HCI or some other acid in order to convert them from calcium and magnesium salts into free acids that dissolve in alkali [6].

HSs from the same type of soil are formed under the same environmental conditions. However, distinct compositional differences exist between HSs from different sources (from different types of soils and climate zones). In order to under- stand the major processes and mechanisms in soil and in water, it is obligatory to know the components of HSs [1].

Due to the enormous number of different precursor materi- als, the exact structure of HSs cannot be defined. The pro- posed structures are hypothetical, based on knowledge de- rived from studies of elemental composition, functional group content, degradation products, spectroscopic data, and physi- cal-chemical properties of the HSs [5].

The ability of HSs to bind hydrophobic organic molecules can affect not only their mobility in water but also reduces their bioavailability. This process induces the polymerisa- tion of xenobiotics with HAs and the creation of polymers that are extremely stable in regard to acidic and alkaline hydrolysis, thermal degradation and microbial decomposi- tion [7]. The carboxylic groups in the HS structure unques- tionably have an essential role in the sorption of different inorganic and organic atoms and molecules [8].

HSs are extremely important in the mobilisation and con- centration of toxic metals and radionuclides in the environ- ment. HAs have a strong affinity toward metal cations and can therefore serve as effective chelators for numerous transi- tion metal ions [9]. Due to size changes induced by conforma- tional rearrangement and aggregation/dissociation arising from intermolecular hydrogen bonding, HSs can form soluble com- plexes that can migrate long distances or precipitate, carrying bound cations with them. The migration/precipitation abili- ties depend on the metallic ion, the cation charge, the degree of ionisation of the organic molecule, the ionic strength of the media, and the location of the metal ion [10].

The general type of HS functional groups: oxygen-contain- ing groups including COOH, enolic-, aliphatic-, phenolic- OH and C=O; nitrogen and sulphur containing groups and their content is assumed to influence formation of different metal complexes. HSs may also contain carbohydrates, which may be bound to the central core through H-bonding. Com- plex polysaccharides are known to have an ability to bind inorganic soil particles into a stable aggregate [5].

Estonia is rich in the Baltic Sea and lake fine-grained sedi- ments (mud). Historically, mud has been used in human treat- ment as curative mud and may not be polluted with differ- ent organic and inorganic contaminants. The components, nature and colloidal-chemical properties of the curative mud depend on its deposit area and types. High-molecular HSs are an important part of curative mud and they have a strong effect on its properties [6].

The aim of this work is to characterise and compare HSs, isolated from the Baltic Sea mud (Haapsalu Bay) and from the sediments of Lake Ermistu, and to follow the complexing ability of different HS fractions towards metal ions.

2 Materials and Methods

Resins Amberlite XAD-7 and Amberlite IR-120 were ob- tained from Sigma-Aldrich (USA).

HSs were extracted from the mud of the Baltic Sea (Haapsalu Bay) (SM) and Lake Ermistu (LM). The natural mud (SM wa- ter content 60-70%; LM water content 90%) was extracted with 0.2 mol/L NaOH (1 part of dry SM to 6 parts of 0.2 mol/L NaOH or I part of dry LM to 12 parts of 0.2 mol/L NaOH) at room temperature for 5 h under argon gas. The suspension was centrifuged at 6000G for 30 min. The alkaline extract contained HA, HMA and FA. The origin of the HS fractions is specified by adding a prefix 'Sea' or 'Lake', respectively.

The alkaline extract was acidified to pH 2 by addition of 6 mol/L HCI. After 20 h, this solution was centrifuged and the supernatant containing FA discarded. The sediment (which contained HA and HMA) was repeatedly washed with distilled water until C1- free (control with AgNO3). Solid residue was repeatedly treated with 96 % ethanol to dissolve HMA and separate solid residue (contains HA). Both frac- tions were lyophilised.

The FA solution was passed through the Amberlite XAD-7 resin column. After removal of salts with 0.01 mol/L HC1, the adsorbed FA was recovered by elution with 0.1 mol/L NaOH solution directly onto the strong cation exchange resin bath (Arnberlite IR-120). The FA solution was sepa- rated and lyophilised.

The natural mud was treated with 6 mol/L HCI until pH 2 and shook for 1 h. Suspension stayed overnight and was then centrifuged at 1500G for 15 rain. The centrifuge cake contained acid pre-treated SM and LM which was treated respectively with 0.2 mol/L NaOH and 6 mol/L HCI to sepa- rate HS fractions (Ac.sea HA, HMA, FA and Ac.lake HA, HMA, FA) as stated above.

Elemental analysis of different fractions of HSs (C, H, N} was carried out on the Elemental Analyser PE 2400 Series II CHNS. Dry combustion at 600~ was used for total ash measurement. Oxygen concentrations were calculated from the difference of C, H, N. All chemical analyses were per- formed in triplicate.

Analysis of the metals Mg, Cu, Zn, Fe, Cr, Hg, Pb was per- formed with the atomic absorption spectrophotometer, Pye Unicam SP9/70, with flame technique for Mg, Cu, Zn, Fe, Cr, Pb and with cold vapour technique for Hg. Solutions for the analysis were obtained after HNO 3 digestion of the sam- ples. Concentration of metals in the mud is given for mud dry weight and in humic fractions for dry weight of the cor- responding humic fraction. We dried mud at 110~

Spectroscopic analysis of the samples was conducted on the Kontron Instruments UVIKON 941 PLUS UV/VIS spectro- photometer. UV/VIS spectra were obtained over a range of

JSS - J Soils & Sediments 4 (1) 2004 25

Humic Substances Research Articles

200 nm to 500 nm on 0.001 mol/L N a O H solutions at a concentration of humic fractions of 50 to 100 mg/L. The lyophilised HSs were not used because of possible changes in the structure of HSs on drying. Absorbency values were measured for 250 nm, 280 nm and 365 nm. E2/E3 ratios (extinction ratios at 250 nm vs. 365 nm) were calculated according to [11].

3 Resu l t s and D i s c u s s i o n

3.1 Yield of the extraction humic fractions from the sea and lake mud

We determined the yield of basic extraction in the course of sep~iration (according to the methodology described above) of different HSs components from mud (Table 1).

Table 1 : Yield of the extraction humic fractions from mud g/kg of mud dry weight

HSs SM Ac.SM LM Ac.LM HA 1.5 4.3 28.1 70.6

HMA 0.8 2.8 3.4 4.8

FA 2.4 1.4 2.3 3.6

We found that acid pre-treatment of mud increased the amount of extracted HSs. In the case of SM, the amount of HA in- creased by 2.9 times, HMA increased by 3.5 times, but FA was diminished by 0.6 times. In the LM, the amount of HA increased by 2.5 times, HMA increased by 1.4 times and FA, differently from in the SM, increased by 1.6 times. These re- sults show that HSs in the SM and LM are quite different, and HSs that are bound to the inorganic/organic structure of mud are released in the course of prolonged treatment with acid, which is in good accordance with the early findings [12].

3.2 C, H, N content of HS fractions

The elemental composition, atomic ratios and organic matter contents of the different HS fractions are presented in Table 2.

We performed elemental analysis of the different HS frac- tions. In general, the elemental composition of HSs was quite similar in all studied fractions, although there were signifi- cant differences in Sea and Lake HA fractions. Sea HA had more nitrogen and Lake HA had more oxygen. Literature

data [13] showed that carbon content in HA was higher than in FA. We separated the HM A fraction from HA and found that HMA fractions had the highest carbon content and the lowest nitrogen content. These values were compa- rable with those that were found in [2,6]. Highest carbon content indicated the highest aromaticity (percentage of con- densed structures) of HMA. This result is supported by cal- culated aromaticity of the samples (see 3.4. UV-VIS spectra and calculated aromaticity). Highest oxygen content indi- cated that lake HA contained more heterocyclic structures, as well as carbohydrates, than sea HA. HMA had the low- est nitrogen content, which indicated the lowest content of amino acid residues. Glebova [14] has found that HMA contains more polysaccharides than amino acid residues. These subunits may cause better solubility of HMA in wa- ter as compared with HA.

The acid pre-treatment of the natural SM and LM dimin- ished the content of carbon in most of the HS fractions. The similar effect was also described by Goh and Reid [12]. Ni- trogen content in the Sea HA was ~1.6 times higher than that of Lake HA. After mud acid pre-treatment, nitrogen content in the Sea FA and Lake FA diminished by about two times. This means that amino acid residues, the main source of nitrogen in FA, are released from FA in the course of acid treatment. To characterise elemental composition, atomic ratios are often used (Fig. 1).

It is known that the [H]/[C] ratio is connected with the ratio of aliphatic/aromatic compounds [4]. In our case, the ratio of more than 1.4 indicated the domination of aliphatic groups over aromatics. The highest aliphatic nature was found for

2 1.8

o 1.6 '> 1.4

1.2 1

0.4

o o o o

% " DO A

0.5 0.6 0.7 0.8

O/C

o H A [ ]HMA A F A

0.9 1

Fig. 1 : Atomic ratios [H]/[C] versus [O]/[C] in different humic fractions

Table 2: Elementary composition of HSs in the sea and lake mud

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Research Articles Humic Substances

HA fractions. It is in good accordance with earlier findings [15]. FA has more condensed aromatic structures than HA. The acid pre-treatment increased aromaticity in HS frac- tions. This result is also supported by UV-spectra of the frac- tions (see UV-VIS spectra). The degree of dehydration or oxi- dation of HS can also be estimated from [O]/[C] values [13]. FA has more oxygen containing groups. The acid pre-treat- ment increased the content of these groups.

[N]/[C] ratios in HA and FA are high. The same was found in HA from different sediments of lakes and rivers [15,16,17]. The high [N]/[C] ratio for the sedimentary HSs, in compari- son to soil and water HSs, is due to their precursor materi- als, constituted mainly of phytoplankton [16].

3.3 Metals in mud and HSs fractions

In order to establish the metal distribution of the course of separation and to obtain information about affinity of the cations towards HSs, we determined the content of metals in the mud and its HS fractions. The results are shown in Table 3, Fig. 2, Fig. 3, Fig. 4.

Table 3" Concentration of metals (mg/kg dry weight of mud) in the sea and lake mud

Fig. 3: Concentration of metals in. mg/kg in HMA fractions from natural and acid pre-treated sea and lake mud

In the SM and LM as well as in their HS fractions, we found that the concentration of heavy metals Pb and Cr is lower than <0.08 mg/kg. The concentration of Hg in the LM was 0.30 mg/kg and 0.08 mg/kg in the SM. In general, metal concentration is considerably lower in the LM than in the SM, which is characteristic of the mud of this kind because of the conditions of their genesis.

As can be seen from the data in Fig. 2, Fig. 3, Fig. 4, the concentration of metals vary in different HS fractions.

Fig. 2: Concentration of metals in mg/kg in HA fractions from natural and acid pre-treated sea and lake mud. Cu was not found in the lake mud HA fraction

Fig. 4: Concentration of metals in mg/kg in FA fractions from natural and acid pre-treated sea and lake mud

Sea mud. We observed that Fe and Mg are concentrated into the HA fraction. The acid pre-treatment increased the concentration of Fe, but the concentration of Mg did not change. Cu and Zn were concentrated into the HMA frac- tion, but the acid pre-treatment increased the concentration of Zn and decreased the concentration of Cu. Cu was also concentrated in the FA fraction. However, the acid pre-treat- ment reduced its amount considerably (Cu is extracted).

Lake mud. Mg was concentrated into HA and HMA frac- tions. The acid pre-treatment increased the concentration of Mg in HA and decreased it in HMA. Cu was not found in the HA fraction. Cu and Zn were also concentrated into the HMA fractions. The acid pre-treatment decreased the con- centration of Zn and Cu in this fraction. The concentration of Zn was also considerable in the FA fraction. The acid pre-treatment increased the concentration of Zn.

On the basis of the data presented above, we may consider the following: if we suggest that the differences in the metal cation

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Humic Substances Research Articles

distribution are due to their ability to form complexes with HSs, we may assume that Sea HA forms more stable Fe and Mg complexes and sea HMA contains more stable Zn and Cu complexes. The total metal content in the lake HSs is lower and there are more Mg complexes in HA and HMA fractions. Sea FA binds Cu and Mg better, but lake FA binds Zn.

Comparing the data from Table 1 and Fig. 2, Fig. 3, Fig. 4, we can see that fractions of HSs took very small quantities of metals from natural mud. Sea HSs, as a sum of HA, HMA and FA, binds more Cu, about 3.6%, and Zn, about 1.2% of total Cu and Zn from the sea mud. Lake HSs bind more Zn, about 3.6% of total Zn from the lake mud.

To compare the amount of metals extracted from HA, HMA, FA (with that, remains in HSs fractions), metal concentration of the alkaline extract of the SM was determined (Fig. 5).

100

90

80

70

E 60 f,

50 E

40 c

g a0

20

10

0 r -F - ,

Fe Zn Cu

El Full extract rq HA+HMA+FA

Mg

Fig. 5: Concentration of metals in mg/kg dry weight of mud in the alkaline extract of natural sea mud and in sum of the sea mud humic fractions (HA+HMA+FA)

Alkaline treatment removed 0.8% Mg, 7.9% Cu, 5.2% Zn and 3.8% Fe from the sea mud. We found that, in the HA, HMA and FA fraction (as a sum), there was only 1.7% of Fe, 2.1% of Mg, 23.2% of Zn and 45.2% of Cu left from the total amount of those metals in alkaline extract of mud. This means that Cu is the metal most strongly bound to HSs. The result is in good accordance with the literature data, showing the order for the stability of the metal-hu- mate complexes to be Cu>Zn>Mg>Fe [18].

3.4 UV-VIS spectra and calculated aromaticity

Spectroscopic properties of HS fractions are shown in Table 4.

All humic samples exhibited a featureless increase in ab- sorbance with a decreasing wavelength. The ultraviolet spec- tra of HA, HMA and FA were similar, differing only slightly in optical density. We measured molar absorptivities of the HSs at 280 nm. This wavelength was chosen because r~-~* electron transitions occur in this region of the UV range for phenolic substances, aniline derivatives, benzoic acids, poly- enes, and polycyclic aromatic hydrocarbons. Many of these compounds are considered to be common structural subunits

Table 4: Spectroscopic properties of HS fractions

in the humic matter [11,19]. Since many of these substances are precursors of components of certain types of HSs, molar absorptivity s may serve as a basis for calculation of aro- maticity of humic matter and for evaluation of this origin, using the following equation [19,20]:

Aromaticity = 0.05 s + 6.74, where E is molar absorption, I (molC) -1 cm -1

The molar absorptivity s, on a mole of organic carbon, was calculated instead of amount of humic matter in water (w/v), because of the ash content of the humic material [19].

Peuravuori and Pihlaja [11] found that correlation between the quotient E2/E3 and molar absorptivity at 280 nm was quite moderate for the isolated humic fractions. In our frac- tions, this correlation is moderate too.

As a result, comparing molar absorptivities and calculated val- ues of aromaticity (see Table 4) allows us to evaluate the impact of different sources on the structure and properties of HSs. No sources available to us provide data about the spectroscopic properties of HMA fractions. We found that HMA had the biggest molar absorption and calculated aromaticity.

We examined a possible correlation between calculated aromaticity and the concentration of Zn and Cu in different HS fractions (Fig. 6). The calculated aromaticity value cor- related quite nicely with the amount of Cu and Zn in the fractions. It may be supposed that aromatic structures (such as acids and phenols) are the important chelating substances for those metals.

E g

=o oo

1000

800

600

400

200

0

0

O 5

g ,~ ~,~

10 20 30

aromaticity, %

x Z n S e a H S oZnLakeHS

oCuSeaHS oCuLakeHS

40

Fig. 6: Correlation of concentration of metals (Zn, Cu) in mg/kg in different natural sea and lake HS fractions with calculated aromaticity of those HSs

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Research Articles Humic Substances

We found that aromaticity in humic fractions is grows in the order of HA<FA<HMA. Sea mud HS fractions have higher aromaticity and contain more Zn and Cu. Comparing elemen- tal composition from Table 2 with calculated aromaticity, we see that C content in the organic matter of the humic fraction increases and O content decreases. H M A always contain aro- matic and quinodal cores are bound together through C-C or C-O-C bridges, and contain phenolic hydroxyl, carboxyl groups and short aliphatic side-chains [21]. H M A having the highest aromaticity contain a lot of phenolic hydroxyl and carboxyl groups that may bind Cu and Zn better. HA has a more aliphatic structure which explains the low content of those metals.

4 Conclusions

HSs of the Baltic Sea mu d and the sediment of Lake Ermistu were fract ionated into three fractions: HA, H M A and FA. The ratio of different fractions from Lake HS and Sea HS differ considerably. Also, many characteristics of those cor- responding HS fractions (elementary composi t ion, concen- t rat ion of metals, calculated aromaticity) differ from one another. This means that the origin structure of Sea HSs and Lake HSs also have to differ markedly.

Metals are concentrated variously in different HS fractions. The highest concent ra t ion of Fe, Zn, Cu and Mg was found in the Sea HA and Sea H M A fractions. The lowest concen- t ra t ion of those metals was in the Lake HA and Lake FA fractions. Based on separat ion data, the stability of metal- humate complexes can be ordered as C u > Z n > M g > F e . Cal- culated aromatici ty and the concentra t ion of Cu and Z n are correlated with each other. We found that higher aromaticity induced a higher concent ra t ion of those metals in HS frac- tions. Sea HSs bound Cu and Z n better than Lake HSs. HSs may find use in the env i ronment because of their metal bind- ing properties as hazardous metal concentra t ing/removing agents. The best fraction for that purpose is the Sea HMA.

The Baltic Sea mud and the sediment from Lake Ermistu conta in heavy metals at a very minor level, thus al lowing its use in h u m a n treatment .

List of abbreviations:

HS HA HMA FA SM LM Sea HA Sea HMA Sea FA Ac.sea HA Ac.sea HMA

Ac.sea FA

Lake HA Lake HMA Lake FA Ac.lake HA

Ac.lake HMA

Ac.lake FA

humic substance humic acid hymatomelanic acid fulvic acid mud of the Baltic Sea mud of Lake Ermistu humic acid from the mud of the Baltic Sea hymatomelanic acid from the mud of the Baltic Sea fulvic acid from the mud of the Baltic Sea humic acid from the acid pre-treated mud of the Baltic Sea hymatomelanic acid from the acid pre-treated mud of the Baltic Sea fulvic acid from the acid pro-treated mud of the Baltic Sea humic acid from the mud of Lake Ermistu hymatomelanic acid from the mud of Lake Ermistu fulvic acid from the mud of Lake Ermistu humic acid from the acid pre-treated mud of Lake Ermistu hymatomelanic acid from the acid pre-treated mud of Lake Ermistu fulvic acid from the acid pre-treated mud of Lake Ermistu

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Received: April 17th, 2003 Accepted: November 13th, 2003

OnlineFirst: November 25th, 2003

JSS- J Soils & Sediments 4 (1) 2004 29