Variation on the adsorption efficiency of humic substances from estuarine waters using XAD resins

Abstract The XAD8 and XAD4 resins, used in series, show different behaviours in relation to the retention of dissolved organic matter (DOM) from estuarine water samples according to different salinities. For the XAD8 resin, the removal of DOM measured in terms of light absorbance at 250 nm varies from 69.4% ± 0.5 to 51.4% ± 3.5 and for the XAD4 resin it varies from 20.0% ± 1.0 to 25.2% ± 3.0 for salinities ranging from 0.6 to 33.4. The isolation, with XAD8, of DOM from samples increasing in salinity, measured in terms of emission fluorescence intensity at selected wavelengths (410, 440 and 460 nm) for an excitation of 340 nm, decreases. In the same experimental conditions, the increase of fluorescent organic matter (FOM) removed by the addition of XAD4 resin is always greater than 20%. However, addition of NaCl to the sample with the lowest salinity, shows that the retention in both resins is related more to the nature of DOM than to the ionic strength of the sample.


Introduction
The International Humic Substances Society (IHSS) has used XADB resin for the isolation of reference and standard humic substances from freshwater (Thurman and Malcolm, 1981). XAD resins adsorb dissolved organic matter (DOM) according to the hydrophobic nature of its organic components that interact with the resins. Since it is not possible to predict the exact mechanism of adsorption, laboratory studies must evaluate the efficiency of DOM retention on different matrices. Most of the previous work on adsorption efficiency of humics has been carried out using samples prepared with humic substances previously isolated according to the IHSS procedure (Stuermer and Harvey, 1977;Sorouradin et al., 1993;Town and Pow-ell, 1993). These results cannot be extrapolated to obtain the adsorption efficiency of humic substances on XAD resins when using natural water samples. Studies on the adsorption of DOM on XAD resins from estuarine environments under different salinity gradients have not been reported. The present work aims at comparing (using light absorbance and fluorescence) the efficiency of DOM isolation from different estuarine waters using a system of two resins connected in series, XADB followed by XAD4. Such data are important to evaluate the matrix effect on the adsorption efficiency from factors such as salinity and, consequently, to discuss the representativeness of the humic substances obtained.
The procedure for extracting humic substances involves a preliminary stage of acidification to increase the protonation and hydrophobicity of the humic substances and facilitate the adsorption on XAD resins. The degree of protonation 0, (fraction of sites occupied by protons) and the charge Q is related by Bt = 1 -Q/Q,,,, where Q,,, is the maximum (negative) charge of the fulvic acid, i.e. when the sample is fully dissociated (Nederlof et al., 1993). Therefore, at a certain pH the fulvic acids are more negatively charged for high ionic strength. The dissociation is, thus, enhanced with an increasing salt level. Even when all the samples are acidified to the same pH value of 2 before the isolation procedure, the degree of protonation of the humic substances still decreases with increasing salinity (also decreasing the hydrophobicity).
The efficiency of removal on the resins therefore seems to depend on the salinity, even for the same pH.
ity Meter LF 196, and the pH with a pH meter Orion 250A.

XAD Resins
The fines, impurities and unpolymerized monomers were removed with a daily washing of the resins (XAD8 and XAD4) with 0.1 M NaOH and distilled water during 10 days, followed by hot soxhlet extraction (two runs) with methanol, acetonitrile and dietylether (5 days in each solvent) finishing the last run with methanol (3 days) (Santos et al., 1994).
Before the isolation of humic substances, the methanol was removed with distilled water until no absorbance was detected at 220 nm, and the resins were then washed with 0.1 M NaOH followed by 0.1 M HCl and 0.01 M HCl.
Four 13 ml glass columns for each resin (XAD8 and XAD4) were packed with 12 cm3 of resins. All columns were lined with teflon except for the 20 cm tygon portion in the peristaltic pump.
The procedure for the isolation of aquatic humic substances includes delivering 400 ml of the acidified water sample (with HCl to pH = 2), through the XAD8 resin column, collecting the effluent and passing it through the XAD4 resin column, both at a flow rate of 1.7 ml/min. The efficiency of the XAD8 and XAD4 resins was evaluated for a set of four samples for each salinity, measuring A,,, and fluorescence intensities at 410, 440 and 460 nm for A,,, = 340 nm, of the influent and effluent of each column.

Instrumentation
Absorbance readings were performed in a UV-Visible spectrophotometer Shimadzu UV-2101 PC, using a 100 mm quartz cell. The fluorescence measurements, at room temperature, were carried out in a Jasco FP-770 spectrofluorimeter at 410, 440 and 460 nm emission wavelengths and 340 nm excitation (A,,, = 340 nm), using 5nm bandwidths and 1 cm quartz cuvettes. All spectra were obtained in the same conditions, although no calibration of the spectrofluorimeter response and correction for scattering were performed. Salinity was measured in the field with a salinometer WTW Microprocessor Conductiv-The desorption was performed by back-elution with 20 ml ( N three column void volumes; Malcolm et al., 1989) of 0.1 M NaOH.

Results and discussion
In the first isolation, the amount of retained DOM was always higher than in subsequent isolations which means that a fraction of the adsorbed DOM was not eluted with the 0.1 M NaOH solution, and that it is strongly retained in the XAD8 and XAD4 resins. Malcolm (1991) referred to the poor recovery of humic solutes from XAD2 and XAD4 ranging from 75 to 85%. Sorouradin et al. (1993) proposed the use of an alkaline solution of sodium dodecyl sulphate, instead of the traditional 0.1 M NaOH, to enhance desorption efficiency to values greater than 98%. It is well esjablished that XAD4 resins with a pore size of 50 A are inadequate to isolate DOM with a high molecular weight (Aiken et al., 1979) and, therefore, the fraction of adsorbed DOM on the XAD4 resin must be of low molecular weight.  in the amount of FOM, retained on XAD8 resin, with salinity (rh4r0 = -0.836; rAa4a = -0.867; rAdeo = -0.870). The DOM that fluoresces at longer wavelengths (A,, = 460 nm) is more retained in this resin.
The results of Table 3 show that the retention of DOM on XAD4 resin decreases linearly with increasing salinity (rAd10 = -0.927; rh440 = -0.913; r,44ho = -0.904) when the retention efficiency is cal- Table 1 The efficiency of the resin system &AD8 + XAD4) is increased by slightly over 20% (Table 3) by the addition of XAD4. As also observed for the data in the Table 4, the resin system shows a linear decrease (r,,,, = -0.871; rA44o = -0.883; rhbho = -0.857) on the removal of FOM with increasing salinity. As already discussed for the results in Tables 2 and 3 it is interesting to observe that, while XAD8 retains a higher percentage of organic matter fluorescent at longer wavelengths, the XAD4 retains a higher percentage of organic matter fluorescent at shorter wavelengths.
However, the data shown in Table 4 demonstrate that the total retention system (XAD8 + XAD4) does not seem to show clear evi-  dence of fractionation of FOM since there are no significant differences for the retention of FOM at different wavelengths for the same salinity level.
In order to find out whether the obtained results are influenced by salinity or DOM composition, freshwater from Frei-Gil was treated with different amounts of NaCl to obtain salinities of 1.3, 4.8, 23.6, and 32.3. The influence of other seawater ions was not tested.
The results presented in Table 5 apparently show the same trend as observed for the data of Table 1: Table 4 Total FOM of natural water samples, retained on the system of the resins, XAD8 followed by XAD4. The retention (95% confidence intervals) was calculated for the A,, = 410, 440 and 460 nm for an A,,, = 340 nm excitation

Salinity
Retention on (XAD8 + xAD4) resins, (%). the removal of DOM decreasing with salinity in the XAD8 (r = -0.965) and in the system XAD8 + XAD4 (r = -0.9801, and increasing with XAD4 (r = 0.886). However, these results for each resin and for the resins system are not significantly different with an increase in salinity. It is the nature of DOM, associated to different salinities, that plays a major role in the isolation efficiency by XAD resins. FOM adsorption on XAD8, XAD4 and XAD8 + XAD4 in series was also evaluated using the samples described above, with the salinity increased using NaCl.
The results of Table 6 show a total retention percent of fluorescent (A,, = 410, 440, 460 nm; A,,, = 340 nm) organic matter greater than 87.7% with the system of XAD8 and XAD4 resins in series. The XAD4 resin contributes more than 20% of this total. Salinity does not affect the adsorption of FOM on the resins, since no correlation was found between total FOM adsorbed and salinity (rAb,,,rAdet,. Comparison of the results from Table 6 with the freshwater  data from Tables 2-4 shows no significant differences. The ionic strength (NaCl) is not an interferent Table 5 Retention percent on XAD8, XAD4 and @AD8 + XAD4) resins of the sample with different salinities (amounts of NaCI) and the same organic matter content. The retention percent (95% confidence intervals) was calculated on the base of absorbances at 250 nm wavelength on adsorption of natural FOM on XAD8 and XAD4 resins.

Conclusions
For the salinity range S = O-33, the isolation of DOM, characterised by UV absorbance and fluorescence intensity, is increased by more than 20% using a system of XAD8 and XAD4 resins in series, instead of a XAD8 resin only. These results agree with the range of 17-30% obtained by Santos et al. (1994) for freshwater.
In first instance, salinity is thought to be a ruling parameter in the efficiency of humics isolation with the system XAD4 + XADS. The data obtained with natural water samples show a decreasing trend of adsorption of humics, measured as UV and fluorescence radiation, with increasing salinity. However, the XADS and XAD4 show different behaviour patterns concerning the effects of salinity on the adsorption. While adsorption on XAD8 decreases with salinity, the opposite is found for the XAD4 resin, partially compensating the decrease observed in the XAD8 resin. The DOM that fluoresces at longer wavelengths (h,, = 460 nm> is more retained on XAD8 than the one that fluoresces at lower wavelengths. An opposite trend is observed on XAD4, resulting in a bulk of extracted organic matter more representative of humic substances in terms of fluorescence.
An experiment with a freshwater sample to which NaCl was added has shown that salinity may not be the master parameter to explain the decrease in DOM extracted when salinity increases. No clear trend was observed for adsorption of DOM on the resins by adding different amounts of NaCl to the freshwater sample. The change of nature of the DOM appears to be the main reason for different behaviour of the resins adsorption of organic matter from estuarine waters with different salinities.