Site Description
The study site was a floodplain forest on the east bank of the Ogeechee River, Georgia (32°08'N, 81°23'W) (Fig. 1). At this point, the Ogeechee is a low-gradient (19.6 cm km-1) sixth-order alluvial blackwater river (Wharton, 1970). The river gets its distinctive color from dissolved organic C, leached from sandy soils typical of southeastern Coastal Plain uplands (Meyer, 1986). The Ogeechee River runs 
390 km through the Georgia Coastal Plain to the Atlantic Ocean (Cuffney and Wallace, 1987) and is considered relatively unpolluted (Meyer and Edwards, 1990). Annual flooding occurs during the winter and spring months, primarily from January to April (Benke and Meyer, 1988). Water depths in the floodplain can vary throughout the year from 0 to >2 m (Cuffney and Wallace, 1987). The pH of the river water ranges from 6.6 to 8.1 annually (Cuffney and Wallace, 1987). Average water temperature is 19°C; summer temperatures (June to August) average 28°C (Findlay et al., 1986). Average total P (Pt) concentration in the Ogeechee River is 59 µg L-1, with an average N/P ratio of 10 (Meyer, 1986; Benke and Meyer, 1988).
Soils are loams of the Chastain series (fine, mixed, semiactive, acid, thermic Fluvaquentic Endoaquepts) and silty clay loams of the Tawcaw series (fine, kaolinitic, thermic Fluvaquentic Dystrudepts) (M. Thomas, USDA Soil Scientist, Effingham Co., GA, personal communication). As estimated by loss on ignition (LOI), soil OM content ranges from -1 on levees to as much as 500 g kg-1 in the soils of stagnant back swamps adjacent to the upland (Cuffney, 1988). Higher elevation ridges are dominated by Quercus laurifolia M., Carpinus caroliniana W., Pinus spp., and Arundinaria gigantea W. Muhl.; lower elevation swales are dominated by Quercus nigra L., Taxodium distichum L. Richard, Nyssa sylvatica M., Nyssa aquatica L., Liquidambar styraciflua L., and Sabal minor (Jacquin) Persoon (Radford et al., 1968; Lockaby et al., 1996a).
Experimental Design
The effects of flooding on soil P pool sizes and P availability were addressed in a 6-mo in situ mesocosm study. Mesocosms (n = 24) were constructed from open-ended plastic cylinders (0.45-m diam. and 1.0-m height) inserted into the floodplain substrate in March 1992 to a depth of 0.45 m, in a 30 by 60 m area located near the river (for a more detailed description, see Lockaby et al., 1996a). Mesocosms were arranged in a randomized complete block design with blocking based on surface soil characteristics (i.e., percent OM, extractable P, and pH) (Lockaby et al., 1996a). River water was pumped to the mesocosms at a constant rate according to the following four treatments (n = 4 mesocosms per treatment): (i) continuously floooded for 6 mo (CF); (ii) flooded–drained (FD): flooded for 3 mo and then drained for 3 mo; (iii) periodically flooded (PF): flooded for 2 mo, drained for 1 mo, flooded for 2 mo, and drained for the final month; and (iv) nonflooded control (mesocosm control; MC). Two more sets of mesocosms (n = 4 mesocosms per set) were flooded according to the FD regime, with one set receiving elevated P (+P) and one set receiving elevated N (+N). Constant drip rates with target concentrations of 1 and 10 mg L-1 for PO4–P and NH4–N, respectively, were maintained with peristaltic pumps (for a more detailed description, see Lockaby et al., 1996a). An elevational difference of 10 cm between intake and outflow spouts ensured continuous water movement through each mesocosm, maintaining dissolved O2 levels in overlying floodwaters similar to those of river waters (5–6 mg L-1).
Soil Collection and Analysis
In January 1992, soil samples (n = 20) were collected to a depth of 15 cm from the area where mesocosms would later be established to gain baseline information on soil P pool sizes and P availability. In June 1992 (at the beginning of the study period), the late winter-early spring flooding season had ended and no standing water was present in the mesocosm study area. Immediately following treatment initiation in June 1992, and at monthly intervals thereafter for 6 mo, single soil samples were collected from each mesocosm to a depth of 10 cm. During this period, soil samples (n = 4) were also collected, at random, from locations outside the mesocosms to examine the effects of mesocosm construction on soil P pool sizes and P availability (nonmesocosm control [NC]). All soils were collected using a 5.2-cm-diam. (Al) bulb planter. Soil sampling removed from each mesocosm over the 6-mo study period. Collected soil samples were deposited in polyethylene bags, placed in coolers filled with ice, and stored at 4°C upon returning to the laboratory. Soils were composited, removing coarse leaf and root material, and 10- to 20-g portions were dried to a constant mass at 80°C to estimate gravimetric moisture content. Soils collected in January 1992 were analyzed for pH in a 1:2 slurry of soil/deionized water, soil texture (Bouyoucos, 1962), and percent OM by LOI at 550°C overnight in a muffle furnace (Lim and Jackson, 1982).
All soils were analyzed for P fractions using a modification of the Hedley et al. (1982a)(1982b) fractionation procedure. In this procedure, soil P fractions are sequentially extracted on the basis of their relative solubilities in water, alkali, and acid. Readily extractable forms (e.g., water-soluble, resin-extractable, and NaHCO3-extractable P) are considered immediately available to plants (Agbenin and Tiessen, 1995; Cross and Schlesinger, 1995); less readily extractable forms (e.g., NaOH-extractable P) are considered available to plants over a growing season (Agbenin and Tiessen, 1995); and recalcitrant forms (e.g., NaOH II–extractable P [a second NaOH extraction after ultrasonification of soil particles], hydrochloric acid [HCl]-extractable P, and residual P) are considered available to plants only over long periods of time, if ever (Cross and Schlesinger, 1995).
Duplicate (n = 2) 0.5-g dry weight equivalent (dwe) field-moist subsamples of each soil sample were placed in 50-mL screw-cap centrifuge tubes. Extractions for labile P (resin-extractable, water-soluble, NaHCO3-extractable, and chloroform [CHCl3]-fumigated/NaHCO3-extractable P) were initiated within 30 h of soil collection. In this modified Hedley procedure, the fractionation sequence was initiated with the estimation of microbial biomass P by CHCl3 fumigation (CHCl3–fumigated/NaHCO3-extractable P) to minimize potential negative effects of resin extraction on microbial biomass P. Resin-extractable and NaHCO3-extractable P were determined on separate 0.5-g dwe subsamples (n = 2) of each soil sample, that is, subsamples that did not proceed through the remainder of the Hedley fractionation scheme.
In preparation for soil extraction, AER bags (0.4-g air dried Dowex 1 by 8, >0.450-mm-diam. Cl-form AER beads encased in mesh nylon stocking bags) were converted to 75% HCO3 form by incubating each bag in 30 mL of 0.5 M NaHCO3 for two half-hour sessions, using a fresh solution each time. Soil samples were shaken with resin bags in 30 mL of deionized water for 16 h using a reciprocating water bath shaker. Following shaking, resin bags were removed and supernatants were centrifuged for 20 min at 1200 x g (3000 rpm). The supernatant solution was vacuum filtered through 45-µm membrane filters (Supor 450, Pall Life Sciences, Pensacola, FL) and digested with ammonium peroxydisulfate in an autoclave for 30 min at 120°C and 100 kPa (15 lb in-2) of pressure (Grasshoff et al., 1983) to estimate water-soluble organic P (Po). Resin-extractable inorganic P (Pi) was estimated by incubating resin bags for 16 h in 30 mL of 0.5 M HCl per bag and then shaking for an additional 0.5 h. Incubation is required to release CO2 to prevent subsequent interference with bubble separation of samples during autoanalysis. Following extraction, resin bags were converted back to 75% HCO3 form for reuse.
Sodium bicarbonate–extractable Pi was estimated by shaking 0.5-g soil subsamples with 30 mL of 0.5 M NaHCO3 for 16 h, then centrifuging and filtering as described above. To estimate P released and subsequently readsorbed by soil particles during NaHCO3 extraction, separate 0.5-g subsamples (n = 2) were also shaken for 16 h with 0.15 mL KH2PO4 (250 µg P mL-1) and 30 mL of 0.5 M NaHCO3, then centrifuged and filtered as above (Brookes et al., 1982). Both NaHCO3-extractable P and CHCl3-fumigated/NaHCO3-extractable P values were adjusted for P readsorption during extraction following Brookes et al. (1982).
To initiate the Hedley fractionation scheme, soils were treated with 0.5 mL of liquid CHCl3, and centrifuge tubes were then capped, incubated for 16 h under a fumigation hood, uncapped, and then incubated for an additional 2 h. Soils were then shaken with 30 mL of 0.5 M NaHCO3 for 16 h, and centrifuged and filtered as described above. Microbial biomass P was estimated as the difference in 0.5 M NaHCO3-extractable Pi in CHCl3-fumigated versus nonfumigated soils, using a recovery rate (kp) of 0.4, which has been found to represent the average recovery of microbial P by this technique for a wide range of soils (Brookes et al., 1982; Hedley and Stewart, 1982; Walbridge, 1991).
Following CHCl3–fumigation and 0.5 M NaHCO3 extraction, soil residues were sequentially extracted with (i) 30 mL of 0.1 M NaOH (NaOH I P); (ii) 30 mL 0.1 M NaOH following 2 min of ultrasonification (with centrifuge tubes in ice) (NaOH II P); and (iii) 30 mL of 1.0 M HCl (HCl P). Following the 16-h extractions, all extracts were centrifuged and filtered as described above. Sodium hydroxide I and II extracts were centrifuged at 4°C for 90 min to facilitate filtration. All extracts were adjusted to pH 2.5 and analyzed for Pi by the method of Murphy and Riley (1962), using a Technicon Autoanalyzer II (Technicon Instrument Corporation, Tarreytown, NY) (method number 696-82W; Bran and Luebbe, 1989). Colored extracts were subsequently reanalyzed for potential color interference after removal of ascorbic acid from the autoanalysis reagent stream (i.e., preventing formation of the molybdate blue color in the presence of Pi). Samples registering above the newly established (lower) baseline were noted and differences were attributed to color interference and were factored into concentration calculations. The Po concentration of 0.5 M NaHCO3 extracts (including CHCl3-fumigated/NaHCO3-extractable P) and 0.1 M NaOH extracts was estimated as the difference between Pt following autoclave digestion of supernatants, using methods described for determinating water-soluble Po and supernatant Pi concentrations. Microbial Po was estimated as the difference between CHCl3-fumigated/NaHCO3-extractable Po and NaHCO3-extractable Po. Hydrochloric acid supernatants were not analyzed for their Po content because previous investigators have found Po concentrations in these extracts to be negligible (Hedley et al., 1982a). Following HCl-extraction, residual soils from the fractionation sequence were digested in a Technicon block digester (Technicon Instrument Corp., Tarreytown, NY) with concentrated sulfuric acid (H2SO4) and 30% hydrogen peroxide (H2O2) for at least 30 min at 360°C (Haynes, 1980; Lowther, 1980). The resulting supernatant was analyzed for Pt content (residual P) using a Technicon Autoanalyzer II (method number 696-82W; Bran and Luebbe, 1989). Nonextracted soils (duplicate 0.200-g subsamples) were digested similarly to estimate Pt, for comparison with the sum of the Hedley fractions. Digests of soils collected in January 1992 were also analyzed for total N content (method number 696-82W; Bran and Luebbe, 1989).
In Situ Anion-Exchange Resins
Anion-exchange resin bags (8.0 g of air dried Dowex 1 by 8, >0.450-mm-diam. Cl-form AER beads encased in nylon stocking material and converted to 75% HCO-3 form using methods described above) were buried and replaced in each mesocosm (n = 1 per mesocosm) at monthly intervals as an in situ estimate of P availability. Resin bags (n = 4) were also buried and replaced at monthly intervals in soils outside the mesocosms. Resins exchange HCO-3 for PO3-4 in the soil solution in a manner similar to plant roots; P availability indexed by in situ resin bags has been shown to correlate positively with P availability to plants (Walbridge, 1991; Giblin et al., 1994). Phosphate was extracted from resin bags using methods described above for 0.4-g resin bags, using 100 mL of extracting solution per resin bag. Daily P supply to in situ AER (µg d-1) was calculated as the amount of P absorbed by each resin bag divided by the number of days it was buried in the soil. Total P supply to in situ AER is the amount of P absorbed by each resin bag during the time it was buried in the soil.
Statistical Analyses
The method of restricted maximum likelihood (PROC MIXED) was used to examine differences in soil P pool sizes and P availability as a function of time (SAS, 1996). Autocorrelation was taken into account using a repeated measures statement within the procedure. Individual significant differences between sampling periods within each treatment were identified using the method of least squares means with a Tukey adjustment (SAS, 1996). Differences in soil P pool sizes and P availability as a function of treatment were examined using ANOVA (SAS, 1996). Tukey's studentized range simultaneous comparison procedure was used to identify significant differences between treatments within each sampling period (SAS, 1996). In situ AER P data were log-transformed before statistical analysis to meet the assumption of normality required by the ANOVA method. Differences in total P supply to in situ AER in treatment versus control soils were examined using ANOVA for unbalanced sample sizes (PROC GLM) (SAS, 1996). The method of ANOVA with contrast statements was used to compare microbial biomass P in common flooded treatments versus controls for each month of flooding (SAS, 1996).
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