Electronic Journal of Polish Agricultural Universities (EJPAU) founded by all Polish Agriculture Universities presents original papers and review articles relevant to all aspects of agricultural sciences. It is target for persons working both in science and industry,regulatory agencies or teaching in agricultural sector. Covered by IFIS Publishing (Food Science and Technology Abstracts), ELSEVIER Science - Food Science and Technology Program, CAS USA (Chemical Abstracts), CABI Publishing UK and ALPSP (Association of Learned and Professional Society Publisher - full membership). Presented in the Master List of Thomson ISI.
Volume 8
Issue 2
Environmental Development
Available Online: http://www.ejpau.media.pl/volume8/issue2/art-27.html


Dorota Gołębiowska, Marta Osuch, Lilla Mielnik, Romualda Bejger
Department of Physics, University of Agricultural, Szczecin, Poland



Optical properties of humic substances (HS) from bottom sediments of two dimictic and meromictic lakes with similar depth (25 m and 29 m) and different thermal circulation and oxygen condition of their water were carried out. Dimictic Marta Lake (ML) and meromictic Czarne Lake (CL) are situated not wide apart, in Drawno National Park, North Poland. Optical analysis: differences UV-VIS - absorption spectra and fluorescence emission spectra were analysed for characterisation of humic acids (HA). HA were taken from bottom sediments of three thermal zones of lakes. All HA from profundal bottom sediments have a bigger UV absorption than HA from littoral. They testify lower degree of oxidative transformation of HA from profundal zone. It was assumed that fluorescence yield of HA from profundal of CL is a few times bigger, than this from profundal of ML. In all humic acids (HA) from profundal zone chromofor (l = 405 nm) and fluorofor (l = 480 nm) originated probably from reduced cytochrom c structure is present. Humic acids molecules are characterised by some exciting and emission centers, which testify their possibility of intermolecular transfer of energy. Coefficients (Kf=IFL/A) proportional to FL yield were calculated. Observed difference of fluorescence yield level indicates the presence of a few time more fluorescent centers in HA from profundal of meromictic than dimictic lake.

Key words: humic acids, bottom sediments, UV-VIS absorption, fluorescence.


Humus substances (HS) are the most abundant organic constituents in environment. They are present in the soil and in the aquatic ecosystems. HS present in the water as dissolved molecules, colloidal suspension and particles of bottom sediments. They are created in the course of decay of remainder of dead organic matter. HS in lakes and lake sediments are originated both from aquatic autochthonous organisms and from organic matter, washed into the lake from surrounding soils.

Humic acid (HA), an important constituent of HS with its specific chemical structure and numerous functional groups, plays substantial role in circulation of elements in nature and determines physical, chemical and biological properties of natural water reservoirs [8, 9, 22]. Aquatic medium in humification process leads to the creation of more homogenous substances in comparison to these in the soil. Concentration of nitrogen in HA from lake sediments is twice bigger than from the soil [7,14]. HS play an important role in light absorption in aquatic ecosystems, which in turn affects the level of primary production.

One of the most important factors determining the humification process and quality of its products is an oxygen, It is red-ox process during which considerable amount of oxygen is used. It may lead to diverse the oxygen concentration in environment, or even locally to its deficit. Humus substances, which are created in such low-oxygen conditions, may have the specific structure. Therefore the composition and properties of humic substances in lake sediments are not only a function of quantity and quality of organic matter but also a function of water aeration [10].

Temperature changes in water profile are one of the main causes of water mixing in lakes and a better aeration of their deeper layers. Among the lakes of various thermal circulation the two types - meromictic and dimictic ones were recognised. Organic matter accumulation rate at the bottom of the lake and its transformation rate depend on trophic status and thermal profile of lake. HA originated from profundal, created often in less oxidative or even reductive medium, characterises lower degree of oxidative degradation, in comparison to ones originated from littoral zone. Spectroscopic techniques, like absorption or luminescence, frequently and extensively have been applied as useful means of characterisation of HS of various origins [1, 5, 6]. Chen et al. [2] and Kumada [13] presented an extensive review of absorption application to humic substance investigation.

Fluorescence phenomenon is characteristic for all humic compounds [3, 17, 19]. Fluorophores "activities" depend on degree of humification [15] and on degree of aggregation of macromolecules of HA as well as on the origin of HA [18].

The aim of this paper is to prove, that humic substances from bottom sediments of two dimictic and meromictic lakes with similar depth and different area, situated not wide apart, may be distinguished by their absorption and luminescence characteristics.


Two lakes of different mixtion - dimictic Marta (ML) and meromictic Czarne (CL) has been chosen for the studies. Mictic types of both lakes were defined and described in works [11,12].

Czarne Lake (CL) is unflowable lake, of area 17.5 ha [4], maximum depth 29 m and visibility of the Secchi Disc 10.8 m. It is situated in syncline of steep slope area. Average oxidation of hypolimnion layer during summer time was 5.4 %, and pH 7.4.

Marta Lake (ML) - unflowable, tunnel-valley lake at maximum depth 26 m and visibility of the Secchi Disc 6 m. Average hypolimnion oxidation during summer stagnation period was 39.8 % and pH 7.4.

Waters of both lakes contain little amount of nutrients and organic matter and they are considered as the oligotrophic lakes [11].

The subjects of studies were HA originated from bottom sediments of both lakes.

Bottom sediments samples were taken with Kajak´s device from an upper layer (up to 35 cm) of three depths representing three zones: profundal (P), sublittoral (S) and littoral (L) (Fig. 1). Five samples taken from each zone were mixed and collected samples were made. Samples were taken during the summer stagnation. All sediment samples were dried in the air and later extracted with 0.1 mol/dm3 NaOH [20]. Carbon content in all solutions was determined according to Orlov and Grindel method [16]. HA were obtained from extract after precipitation with H2SO4 to pH = 2. Fresh HA were purified by HCl-HF lave and dialysed. After purification HA gels were dissolved in Michaelis buffer pH (7.4) and examined by absorption and luminescence methods. UV-VIS absorption spectra were recorded with computed spectrophotometer Specord M-42 Carl Zeiss Jena with ASPECT program. The measurements were performed in concentration 0.1mg C/cm3. All absorption spectra were standardised at l = 230 nm, taking a value of absorption A230 = 2.00. Difference of absorption spectra (D(P-L)/A230 = f(l)) were calculated by subtracting from standardised spectra of profundal humic acid (HA-P), standardised spectra of littoral humic acid (HA-L) and also subtracting from standardised spectra of HA-P standardised spectra of sublittoral HA-S, it is (DA(P-S)/A230=f(l). Q4/6 and Q2/4 absorption coefficients, according to Chen et al. [2] and Kumada [13] were calculated.

Fig. 1. Places of sampling. Cross-section of lake basin along k straight line

Fluorescence emission spectra were drowning with spectrofluorimeter Hitachi F-2000 for solution HA with concentration 0.1mgC/cm3 and absorbency in UV < 0.02. Coefficients (KF = IFL/A) presented fluorescence/absorbency ratio (intensity of Fl at maximum of emission to absorbency at excitation wavelengths), proportional to fluorescence yield were calculated.


Characteristics of total organic carbon content (TOC) for investigated bottom sediments are presented in the Table 1, together with absorption coefficients.

Table 1. Carbon total content (TOC) in bottom lake sediments of three thermal zones: L- littoral: S- sublittoral; P- profundal, and Q coefficients calculated on the base of absorption spectra of solution of HA extracted from the sediments. Q-coefficient presents absorbency ratios at fixed wavelength


Thermal zone

TOC ± S.D.
Bottom Sediments

Absorption Coeffitients
of HA







9.90 ± 1.01





7.05 ± 0.18





26.16 ± 2.85





2.43 ± 0.01





1.97 ± 0.04





12.91 ± 0.23



S.D. - standard deviation;
L - littoral, S - sublittoral, P - profundal


Characteristics of UV-VIS absorption spectra of HA solutions extracted from bottom sediments of profundal, sublittoral and littoral zone of dimictic Marta Lake and meromictic Czarne Lake have been compared.

Values of the absorption coefficients calculated on the base of monotonic absorption spectra of HA have been presented in Table 1. These values are lower when they pass from littoral to profundal. The values of Q4/6 coefficients are characterised by different changes in both lakes. It is known that values of absorption coefficient Q4/6 are negatively correlated with molecular size of HA and that size of molecules is greater when stage of humification process is more advanced [2]. The degree of oxidation and pH of environment are the most important factors responsible for the course of humification. Lower value of the Q4/6 coefficient for HA from profundal of Marta Lake (Q4/6 = 2.9) in comparison to corresponding value of the coefficient in Czarne Lake (Q4/6 = 4.0) as well different course of changes of the coefficients for both lakes clearly shows, that despite of vicinity of both lakes, similar flora, depth, pH of waters and similar shape of bottom of lake in the place of sampling (Fig.1), that the humification process in profundal of both lakes runs differently. Better oxygen conditions exist in profundal of the Marta Lake and they result to creation of more matured HA which are the products of organic matter transformation with higher degree of humification.

To be sure that HA from profundal of Marta dimictic Lake submitted to oxidative transformation in much higher degree than HA originating from meromictic Czarne Lake, analysis of the absorption spectra has been done with the method of subtraction of earlier normalised at L= 230 nm spectra. Differential absorption spectra are presented on Fig. 2.

Fig. 2. Difference of standardised (at l = 230 nm) absorption spectra of HA solutions between HA from dimictic lake (ML):
1 - profundal minus sublittoral, 2 - profundal minus littoral, and between HA from meromictic lake (CL): 3 - profundal minus sublittoral, 4 - profundal minus littoral

In all difference spectra of HA the band at l=405 nm, clearly separated, was observed. It may be attributed to reduced form of cytochrom c - Soret band [21], or pheophytin [10]. In our opinion, absorbing structure must be entirely and substantially included in macromolecule HA, because it is extracted and precipitated from solution together with HA fraction. This structure seems to depend upon oxidative condition of surrounding water. Data on Figure 2 may indicate that at l = 405 nm chromofore either does not exist in HA from littoral and sublittoral zone at all - or it is present there in smaller quantity.


HA obtained from profundal of both lakes have been submitted to fluorescence analysis. Emission spectra of fluorescence exciting by electromagnetic wave of 310 nm, 380 nm, 405 nm, and 440 nm (i.e. four for each of HA examined) have been analyzed. Every of this wavelength generates fluorescence of HA, intensity of which and positioning of maximum in emission spectra changes. Comparison of intensity and placement of fluorescence maximum generated by determined wavelength of exciting light is presented in Fig. 3. It shows that intensity of fluorescence of HA from meromictic Czarne Lake (empty bars) is in all cases higher than corresponding values for HA from dimictic Marta Lake. Analysis of fluorescence emission spectra exciting by different wavelengths has shown also that position of maximum of fluorescence emission in HA spectra from meromictic Czarne Lake are shifted in shorter wavelength direction (hypsochromic shift) except of maximum generated by exciting at l = 380 nm. Position of maximum of emission FL 475 - 480 nm generated by l = 380 nm is the same in HA from both lakes. It is visible that emission spectra at the same exciting wavelength differ only in intensity. It allows supposing, that group responsible for absorption at 380 nm is constant structure element (carcass element) of HA created in both lakes.

Fig. 3. Coefficient KF of fluorescence yield of four exciting centers of HA from profundal Marta Lake (ML) and Czarne Lake (CL) HA from profundal Marta Lake at lex 310, 380, 405 and 440 [nm]. [a.u.] - arbitrary units

Analysis of fluorescence yield is presented in Fig. 4. Yield of HA fluorescence from profundal of meromictic Lake is in all cases many times greater than yield of HA fluorescence from dimictic Lake. Fluorofor absorbing radiation at l = 380 nm and emitting at the band 475 - 480 nm is characterised by the higher yield of emission.

Fig. 4. Intensity of maximum of fluorescence of HA from profundal zone depending on wavelenght of exciting light. [a.u.] - arbitrary units


  1. Difference of monotonic and non-characteristic absorption spectra of humic acids, standardised at l = 230 nm, give useful qualitative information about distinction in internal structure of these compounds.

  2. In all humic acids from profundal sediments, chromofor with absorption at l = 405 nm and fluorofor with emission at l  = 475 - 480 nm originated probably from reduced cytochrom c structure is present.

  3. Humic compounds from bottom sediments are characterised by several fluorescence exciting and emission centers, which testify their possibility of intermolecular transfer of energy.

  4. Intensity and yield of fluorescence of humic acid from profundal of meromictic Lake are distinctly greater then these from dimictic Lake.

  5. Optical characteristics of HS from lakes bottom sediments seem the differences in the values of absorption spectra, fluorescence intensity and yield may be and to prove that good indicator of oxygen conditions in profundal of the lake.

  6. Almost all Fl emission bands of HA from meromictic lake bottom sediments present hypsochromic shift of their maximum in comparison to corresponding maxims of HA from dimictic lake.


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Dorota Gołębiowska
Department of Physics,
University of Agricultural, Szczecin, Poland
Papieża Pawła VI nr 3, 71-459 Szczecin, Poland
email: fizyka@agro.ar.szczecin.pl

Marta Osuch
Department of Physics,
University of Agricultural, Szczecin, Poland
Papieża Pawła VI nr 3, 71-459 Szczecin, Poland

Lilla Mielnik
Department of Physics,
University of Agricultural, Szczecin, Poland
Papieża Pawła VI nr 3, 71-459 Szczecin, Poland

Romualda Bejger
Department of Physics,
University of Agricultural, Szczecin, Poland
Papieża Pawła VI nr 3, 71-459 Szczecin, Poland

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