Environ. Sci. Technol. 1993, 27, 1052-1059
Effects of Dissolved Humic Substances on the Speciation of Iron and Phosphate at Different pH and Ionic Strength Roger I. Jones,'9+ Peter J. Shaw,t and Henk De Haad
Institute of Environmental and Biological Sciences, Lancaster University, Lancaster, LA1 4YQ U.K., and Netherlands Institute of Ecology, Centre for Limnology, Nieuwersluis, The Netherlands
The effects of dissolved humic substances (DHS) on the speciation of iron and phosphate at different pH in waters of contrasting ionic strength and DHS content were studied by Sephadex gel filtration following incubation of filtered (0.2 pm) epilimnetic lake water samples to which both 55FeC13.6H~0and 3zP043-had been added. The simultaneous movement of 55Feand 32Pto fractions of greater apparent molecular size depended on the presence of DHS and diminished in response to decreasing pH. In the absence of significant quantities of DHS, much of the added 55Feprecipitated from the incubation mixture or was retained within the gel column. 3zP043added to clear water of high ionic strength underwent minor transformations to large apparent molecular size,while the 3zP043recovered in eluates from experiments with clear waters of low ionic strength was dependent on pH and did not undergo transformation, the remainder being coprecipitated with 55Feand retained within the ne1 column. Introduction
The prediction of primary production in freshwaters is fundamental to the management or recovery of lake water quality. It is generally considered that the availability of phosphate determines levels of productivity in many northern temperate lakes. However, models relating phytoplankton biomass or production to external phosphorus loading (e.g., refs 1 and 2) have been reported to overestimate biomass development in dystrophic lakes. This overestimate may arise because of the reduced availability of key nutrients due to abiotic interactions with dissolved humic substances (3). Some studies have been made of the influences of dissolved humic substances (DHS) on planktonic P uptake and metabolism ( 4 , 5 ) ,but the influence of DHS on the movement of key nutrients such as phosphorus and iron from epilimnetic lake water into planktonic organisms is not fully understood. In this context, the underlying abiotic mechanisms of DHS-nutrient interactions are of fundamental importance. It has been demonstrated that PCh3--P may associate with DHS in the presence of iron (6-10) and that this association can be reversible (8). Furthermore, exposure of complexes of DHS, phosphate, and iron to light has been reported to result in the release of bound Pod3- (6, 8,9).Such results lead to the hypothesis that the formation of DHS-Fe-PO2- complexes results in decreased instantaneous availability to algae of P043--P, while maintaining a pool of potentially available dissolved organic P043--P. It is generally considered that the interaction between DHS and Fe involves the weakly acidic carboxylic and phenolic functional groups contained within DHS (11). Thus, the ionic composition and pH of humic lakes can +
t
Lancaster University. Centre for Limnology.
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Envlron. Scl. Technol., Vol. 27,No. 6, 1993
be expected to be key factors in the complexation and release of Fe and Po43-. Consequently, variation of the pH and/or the ionic composition of humic lakes may affect the speciation and cycling of Fe and P04%and hence their availability for uptake by planktonic organisms. Numerous softwater lakes with varying DHS contents are known to have undergone substantial pH shifts in recent decades due to anthropogenic acidification. The variable response of the plankton community in these lakes to acidification may be in part due to alterations in the DHS-FWPO~~interactions. We have examined the effecta of pH and ionic strength on these interactions using a double isotope labeling technique introduced in a previous paper (IO). Materials and Methods
The study lakes were selected to provide samples of contrasting ionic composition and DHS content (see Table I). Thus, comparisons between humic lake water samples and clear water samples, each of high or low ionic strength, indicate the modification of abiotic interactions involving Fe and P attributable to the presence of DHS. Similarly, comparison of humic lake waters of low and high ionic strength indicates the influence of ionic composition on these interactions. Surface water from Seathwaite Tarn having low ionic strength and negligible apparent quantities of DHS (see Table I) thus served as a "control" against which to assess the effects of increasing either ionic strength (Malham Tarn) or DHS (Mekkojiirvi) or both together (Tjeukemeer). Experimental procedures were designed to maintain the properties of DHS in natural surface waters. The potential effects of experimental handling on the properties of DHS (12)were minimized by the avoidance of concentration or chemical fractionation procedures. Natural water samples were used in all experiments after vacuum filtration at -80 kPa through 0.2-pm Anodisc membrane filters (not prerinsed) toremove the bulk of particulate material. NaN3 was added to a final concentration of 0.02% to prevent microbial activity (see ref lo), which increased sample ionic strength by 3 mM. Surface water samples were filtered and sterilized with NaN3 as soon as possible after sampling (hours to days) and then stored in darkness at 4 "C. Subsamples were taken prior to the addition of NaN3 for major ion analysis [cation analysis by PerkinElmer AAS; anion analysis by Dionex ion chromatography; bicarbonate analysis by the method of Mackereth et al. (13)l to enable calculation of ionic strength (I). The pH responses of filtered and sterilized samples to additions of 0.05 M H2S04were tested, enabling manipulation of pH at intervals of 0.5 pH unit in the range pH 4-7. The 10-mL pH-adjusted samples were spiked with 128 kBq 3zP043(Mekkojiirvi and Tjeukemeer) (carrierfree, sp act. 338 TBq mol-I; Amersham International) or 12.8 kBq 32P043(Seathwaite Tarn and Malham Tarn) (carrier-free, sp act. 314.5-337.5 TBq mol-l; DuPont) and 71.0 kBq 55FeC13 (all samples) (sp act. 37-1850 MBq m g l 0013-936X/93/0927-1052$04.00/0
0 1993 Amerlcan Chemlcal Soclety
are expressed as percentages of the total measured activities applied to the columns. The locations of measured radioactivities and optical densities in the elution profiles are described by the partition coefficient Kav(17): ir - 11' o 'e 0.084 0.021 0.800 0.500 E254 K,, = 4167 8485 445 ionic strength 430 vt - VI3 1183 13.7 1290 27.2 [calciuml where V, is the elution volume, V , is the void volume, and 99 34.1 530 45.1 [magnesium] Vt is the total volume. 148 213 1700 93.5 [sodium] 15 11.5 290 32.0 [potassium] An appreciable proportion of the 32P043and 55Fe3+ 115 2440 405 35.7 [chloride] added to subsamples of Mekkojlirvi surface water under147 17.3 860 46.0 [sulfate1 went transformations and eluted as material of greater 2340 0.0 1840 225 [bicarbonate] apparent molecular size (Figure 1).These transformations 1.4 29.9 10.6 [iron] were sensitive to pH. At higher pH (Figure If and g), the a All concentrations are in pM. E254 is UV absorbance at 254 nm, radioactivity was distributed between three distinct peaks 10-mm light path. associated with material at the void volume position, Kav = 0 (peak I); material eluting at intermediate Kav values Fe; Amersham International). The spiked samples were (peak 11);and material at Ka, = 1(peak 111). The quantities incubated in darkness at room temperature (ca. 20 "C) for of 32P and 55Fe activities associated with peaks I and I1 24 h. decreased markedly in response to falling pH, to the extent Transformations of the added radioisotopes were assesthat negligible activities were observed in peaks I and I1 sed by application of 2 mL of the incubation mixtures to at pH 4.08 (Figure la). At the same time, 32Pactivity Sephadex G-100 gel filtration columns. To prevent associated with peak I11 increased as pH decreased. The distortion of the configuration of the DHS applied to the K,, value at which maximum activities in peak I1occurred columns, the potential effects of an ionic strength differalso responded to pH, rising from Kav = 0.4 at pH 7.03 ential (14) were minimized by using an eluant with ionic (Figure lg) to Kav = 0.8 at pH 5.12 (Figure IC),indicating strength equal to that of the samples (obtained by additions a decrease in the apparent molecular size of the material of 10 mg mL-1 NaCl to 0.02% NaN3 in milli-Q water). with which maximum peak I1 activities were associated. Similarly, the potential effects of a pH differential (15) The distributions of UV-absorbing material in the Mekkowere minimized by matching eluant pH to sample pH by jlirvi sample appeared as two distinct peaks, one in the dropwise additions of 0.1 M HzS04. Eluants were degassed region of K,, = 0 and the remaining material occurring as prior to use in all experiments. Flow rate was controlled a single broad peak with maximum optical density in the using a peristaltic pump (LKB Bromma Varioperpex 11) Kav= 0.7-0.9. At higher sample pH (Figure If and region at 1mL min-l. The Sephadex columns were calibrated g) a 'shoulder' was observed on the leading edge of the using Blue Dextran 2000 and 32P043-.Black PVC was larger absorbance peak. As pH decreased, the quantity wrapped around the columns to prevent the possible effects of UV-absorbing material at Kav= 0 diminished, while the of exposure to light on the processes under investigation. shoulder was incorporated into the larger absorbancepeak The concentration of DHS ([DHSI) in the eluate was and ceased to be distinguishable. The larger absorbance estimated by continuous monitoring of optical density at peak as a whole also shifted toward higher K,,, indicating 254 nm (E254) using a Pharmacia-LKB UV-1 optical unit decreasing apparent molecular size of this material with with a 10-mm flow cell. Numerous studies have demondecreasing pH. UV-absorbance of unfractionated lake strated that this parameter can be used to provide a water was only reduced by
