6.4 Humic Acids 



IRINA V. PHRSHINA 



Institute of Global CUimilc and Ecology, State Committee for Hydroineteoroloi^v and Academy of Sciences, Moscow, USSR 



Introduction 



The bulk ofthc dissolved organic mnller in natural waters, 

 which is resistant to biochemical degradation, consists of 

 brown, heterogeneous polymers known as humic acids ( U A"s). 

 These substances ticcount lor about 30-60';^ of the total 

 dissolved organic carbon (DOC) in scawaler (Sluermer & 

 Payne, 197.5; Paxeus, 1985). 



In oceanic areas unaf'tected by freshwater runoff, humic 

 acids are mostly a by-product of algal cell degradation (Harvey 

 etal.. 1988). The concentration of humic acids in seawater is, 

 therefore, related to primary productivity of a region. Recent 

 investigations (Carder el al., 1986, 1989) have shown that 

 phytoplankton and marine humus are only weakly covariant. 

 However, this lack of correspondence is perhaps due to 

 significantly longer residence times for marine humic substances 

 ( Bordovski & I vanenkov, 1 979) relative to the algal population 

 that produced it and the quite variable Hushing and mixing rates 

 for different regions. For this reason pools of marine humus 

 could be indicators of past primary productivity that is not 

 manifested in the present values of productivity and chlorophyll 

 content of the area under consideration. Especially significant 

 correlations between these two parameters could be expected 

 in the highly productive regions such as the Bering and Chukchi 

 Seas. 



Materials and Methods 



Collection and Extraction of Seawater 



.Seawater was collected at depths from 0^ 20 m with a 

 pumping system "Midiya" (Glebov et al.. Subchapter 6..^, this 

 volume). Seawater was pumped through the system of parallel 

 filters (0.5|im pore size membrane) to remove particulate 

 matter, and the filtrate collected in 10-12 plastic vessels 

 (35 liter capacity). When each vessel was full, 70 ml of 

 concentrated HCl was added to it. The acidified seawater 

 (pH 2) was passed through two 1.5 x 15-cm glass columns 

 containing 50 cm' of Amberlite XAD-2 resin at a flow rate of 

 2 l/h per column. Each column extracted 200-250 1 of seawater 

 in each 48 h. Prior to use, the resin was cleaned as described 

 by Gomez-Belinchon et al. (1988). 



Isolation of the E.xtracted Humic Acids 



Columns were first rinsed with 5 6 I of distilled water to 

 remove salts. The collected HA's were elutcd with 500 ml of 

 concentrated NH, solution. To prepare the columns for reuse, 

 they were then eluted with acetone (500 ml), ethanol (800 ml ), 

 and distilled water (8-9 1 ). The regenerated resin was then used 



to extract the next portion of seawater. The ammonia cluent 

 was concentrated to dryness in a rotary evaporator. The crude 

 HA's were dried in a desiccator over P^O,. 



Samplini^ I'rocedure for llwirometric Analysis 



The samples of natural water were taken with a 5- 1 Niskin 

 bottle from the standard depths within the photic /.one 

 (Table 1). The samples were stored in 100-200 ml glass bottles 

 at about -5°C. 



Apparatus 



Infrared (IR) spectra were recorded on an UR-20 instrument 

 (GDR). 



Ultraviolet (UV) spectra were recorded on a Hitachi 

 spectrophotometer, model 100-60. Quart/cells with I cm path 

 length were used. 



Fluorescence spectra (excitation and emission spectra) 

 were recorded on Jasko spcctrofluorimetcr, inodel SP 240. 

 Excitation and emission spectra were collected with 1 0-nm slit 

 widths I'or both monochromators. Spectra were not corrected 

 for wavelength dependence of monochromator throughout or 

 PMT response. For all fluorescence measurements, 1 -cm path 

 length quart/ cells were used. 



Chemicals 



All chemicals were of analytical grade. 



Preparation of the Standards 



An aliquot amount of the dried HA (-20 mg) was weighted 

 and redissolved in distilled water (-20 ml). The aqueous HA 

 solution was filtered to remove insoluble residue and added 

 with distilled water to 25 ml. The insoluble residue was dried 

 and weighed. Concentration of the stock solution of HA's was 

 determined by subtraction of the weight of the dry residue from 

 the initial aliquot amount of the HA samples. 



Calibration Curve Technique 



The prepared stock solutions were used to plot the 

 calibration curves. Standard solutions were excited at 3 1 5 nm 

 and emission spectra were recorded from 350 to 500 nm. The 

 relative intensity of the standard solution was registered at the 

 maximum of emission spectra and plotted against the 

 concentration. The plot of relative intensity versus the 

 concentration showed a linear relationship from at least 

 0. 1 ppm up to 20 ppm. This straight line relationship was valid 

 forall of the standard curves. Obtained calibration curves were 

 used for evaluating the concentration of humic acids in the 

 seawater samples. 



231 



