Journal of Environmental Quality 30:668-674 (2001)
© 2001 American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America
TECHNICAL REPORT
WETLANDS AND AQUATIC PROCESSES
Hydrologic Influence on Stability of Organic Phosphorus in Wetland Detritus
H.K. Pant and
K.R. Reddy
Univ. of Florida, Wetland Biogeochemistry Lab., Soil and Water Science Dep., Institute of Food and Agricultural Sciences, 106 Newell Hall, P.O. Box 110510, Gainesville, FL 32611-0510
Corresponding author (krr{at}gnv.ifas.ufl.edu)
Received for publication April 21, 2000.
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ABSTRACT
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Accretion of organic matter in wetlands provides long-term storage for nutrients and other contaminants. Water-table fluctuations and resulting alternate flooded and drained conditions may substantially alter the stability of stored materials including phosphorus (P). To study the effects of hydrologic fluctuation on P mobilization in wetlands, recently accreted detrial material (derived primarily from Typha spp.) was collected from the Everglades Nutrient Removal Project (ENRP), a constructed wetland used to treat agricultural drainage water in the northern Everglades. The detrital material was subjected to different periods of drawdown and consecutive reflooding under laboratory conditions. The 31P nuclear magnetic resonance (31P NMR) spectroscopy analysis revealed that sugar phosphate, glycerophosphate, polynucleotides, and phospholipids (glycerophosphoethanolamine and glycerophosphocholine) were the major forms of P in the detrital material. After 30 d of drawdown, polynucleotides were reduced to trace levels, whereas sugar phosphate, glycerophosphate, and phospholipids remained the major fractions of organic P. Microorganisms seemed to preferentially utilize nucleic acid P, perhaps to obtain associated nutrients including carbon and nitrogen. At the end of the 30-d reflooding period, cumulative P flux from detritus to water column accounted for 3% of the total P (
15 d of drawdown) and further decreased to 2% at 30 d of drawdown, but increased to 8% at 60 d of drawdown. The drawdown (
30 d) not only reduced P flux to the water column, but also increased the humification and microbial immobilization of P. Excessive drawdown (60 d), however, triggered the release of P into the water column as the water content of detritus decreased from 95 to 11%.
Abbreviations: DOP, dissolved organic phosphorus EAA, Everglades Agricultural Area ENRP, Everglades Nutrient Removal Project NMR, nuclear magnetic resonance SRP, soluble reactive phosphorus STA, stormwater treatment area TP, total phosphorus
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INTRODUCTION
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PHOSPHORUS retention by wetlands includes plant uptake (Greenway and Woolley, 1999), microbial immobilization (Newbold et al., 1983; Reddy et al., 1999), precipitation in the water column (Reddy et al., 1987), and surface adsorption on minerals (Zurayk et al., 1997). These processes could be combined into two distinct P retention pathways, namely, sorption and burial in treatment wetlands (Reddy et al., 1999). When plants and microbes die off, a portion of the P contained in detrital tissue is recycled within the wetland and released back into the water column. The remaining refractory detrital tissue eventually undergoes humification and becomes soil organic matter.
Accretion of organic matter has been reported as a major sink for P in wetlands (Craft and Richardson, 1993; Reddy et al., 1993). Wetlands tend to accumulate organic matter because of the production of detrital material from biota and their suppressed rates of decomposition (DeBusk and Reddy, 1998) due to reducing conditions. The genesis of this new soil is a relatively slow process. With time, productive wetlands accumulate organic matter (which ultimately forms peat) that has different physical, chemical, and biological characteristics from the underlying soils. Eventually, the organic matter gets compacted to form new soil, perhaps with different P mobilization characteristics from the original soils.
Research has shown that draining and subsequent reflooding of organic soils increases P flux into the water column (Reddy, 1983; Martin et al., 1996; Olila et al., 1997). Depending on soil characteristics, drawdown may consolidate the detrital material and increase the stability of inorganic P through precipitation reactions. Frequency or length of drawdown period may influence microbial immobilization and humification of newly accreted organic material and decrease P flux to the water column. Water-level drawdown of lakes and wetlands is one of the techniques used to consolidate detrital material to improve water quality (Dooris et al., 1982; Dierberg and Williams, 1989; Kadlec and Knight, 1996). Drawdown of constructed wetlands can help to consolidate detrital material and accelerates solid buildup (Coveney et al., 1994). However, little is known about the effects of drawdown on P distributions in various pools and their stability. Thus, the objectives of this study were to determine the influence of water-level drawdown on (i) the stability of different organic P compounds in detrital material and (ii) P retention and release characteristics of detritus.
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MATERIALS AND METHODS
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Site Description
The Everglades Agricultural Area (EAA) in southern Florida is a large (240000 ha) productive drainage basin with sugarcane (Saccharum officinarum L.) as a major crop. The Everglades Forever Act (EFA Section 373.4592, Florida Statutes) was enacted by the Florida Legislature in 1994 to restore and protect the ecological integrity of the Everglades, and authorized a number of measures including the construction of several stormwater treatment areas (STAs) as a buffer to treat the drainage water discharged from the EAA. The Everglades Nutrient Removal Project (ENRP) is one of the several STAs built to reduce P loading to the northern Everglades (Fig. 1)
. The ENRP is a constructed wetland (1545 ha) built on agricultural land formerly used to grow sugarcane. The ENRP is located in southern Florida at 26°38' N and 80°25' W. The ENRP is fed with P-enriched runoff from the EAA, whose function is to reduce P concentrations to acceptable levels prior to release southward to Water Conservation Areas (WCAs) (Walker, 1995). Water from the S-5A drainage basin (which drains the northeastern part of the EAA) is first pumped into a buffer cell of the ENRP and then distributed to two independent parallel treatment-trains (Cells 1 and 3, and 2 and 4), which are separated by a transverse levee. Treatment Cells 1 and 2 remove the bulk of the P while Treatment Cells 3 and 4 provide final polishing of the outflow water. The representative samples of suspended and/or semisuspended detrital material were obtained from areas adjacent to inflow (Cell 1), dominated by cattail (Typha domingensis Pers. and Typha latifolia L.).

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Fig. 1. A schematic system plan of the Everglades Nutrient Removal Project's (ENRP) stormwater treatment areas (STAs)
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Phosphorus Flux from Detrital Matter
Homogenized (using a blender) detrital material (300 g moist weight; 4.6 cm depth) was placed in 30 wide-mouth (9 cm i.d.) bottles. The bottles were subjected to five different drawdown periods (0, 7, 15, 30, and 60 d) by allowing them to dry in an incubator at 30°C. At the end of each drawdown period, detritus samples from three bottles were subjected to a P fractionation scheme, while the second set of three bottles were reflooded with 300 mL (4.6 cm water depth) filtered (through a 0.45-µm filter) outflow water (containing 1 µg P L-1; collected at the ENRP) for 30 d. To monitor P flux from detrital material, 25 mL water was sampled from the water column at 3, 7, 15, 22, and 30 d after reflooding. To correct for the water loss due to evaporation, the bottles were weighed at each sampling occasion prior to sampling and replenished with deionized distilled water. Similarly, the sampled water was replenished with 25 mL of the ENRP water after each sampling. One portion of the water sample was acidified with a drop of concentrated H2SO4 to determine total phosphorus (TP) and the other was filtered through a 0.45-µm filter to determine soluble reactive phosphorus (SRP). Thereafter, both the acidified and the filtrate samples were frozen until they were analyzed for P.
Chemical Analysis
Phosphorus Fractionation
Both the flooded and nonflooded detrital materials were fractionated for the determination of labile and nonlabile P pools using the P fractionation scheme as described by Ivanoff et al. (1998). Detritus material (0.5 g dry wt. basis) was extracted with 25 mL 0.5 M NaHCO3 in an end-over-end shaker for 16 h. The suspensions were centrifuged at 5000 x g for 15 min and filtered through a 0.45-µm filter. The filtrates were stored at 4°C for SRP and TP analysis. The residues were extracted with 25 mL 1 M HCl for 3 h. Thereafter, the suspensions were centrifuged and filtered as described previously. The supernatants were saved for P analysis and the residues were extracted with 25 mL 0.5 M NaOH for 16 h. The extracts were then centrifuged and the supernatant filtered through a 0.45-µm filter, and saved for further analysis. The residues were then analyzed for TP.
A portion of the NaOH extract was acidified to pH 0.2 with a few drops of concentrated H2SO4 (two drops per milliliter of sample), then centrifuged and filtered through a 0.45-µm filter as described above. The supernatants (fulvic acid fraction) were analyzed for TP and the precipitates (humic acid fraction) were discarded. However, P content in the precipitates was calculated from the difference between NaOH-TP and fulvic acid-TP.
To determine microbial P, 0.5 g detritus (dry wt. basis) was weighed in a 50-mL centrifuge tube and incubated with 2 mL chloroform (CHCl3) with an air-tight cap on for 24 h. Afterward, the CHCl3 was evaporated by leaving the cap off in a fume hood for 24 h. The CHCl3treated detritus was then extracted with 0.5 M NaHCO3 as described above. The difference between P extracted from CHCl3treated and untreated detritus was considered as total microbial P to avoid overestimation, although it has been suggested that 0.5 M NaHCO3 extracts only about 40% of the microbial P from fumigated samples (Stevenson, 1986).
Soluble reactive P in all the extracts was determined using an automated ascorbic acid method (Method 365.1; USEPA, 1983). Total P in the extracts was also determined by the above method after persulfate digestion (Method 365.1; USEPA, 1983). Dissolved organic phosphorus (DOP) was calculated from the difference between TP and SRP. Total P in detrital material and the residues of the sequential extracts was determined using the method of Anderson (1976). Total carbon (TC) and total nitrogen (TN) in detrital material were determined using a CNS Analyzer (Carlo Erba Model NA-1500, Milan, Italy).
Fractionation by Gel Filtration and 31P Nuclear Magnetic Resonance Spectroscopy Analysis
Detritus material (20 g dry wt. basis) was extracted with 200 mL demineralized water by shaking in an end-over-end shaker at 20 ± 2°C for 18 h. The suspensions were centrifuged for 20 min at 6000 x g. The residues were then extracted twice with 80 mL 0.4 M NaOH for 4 h each. After each extraction, the suspensions were centrifuged as described above. The supernatants were combined and subjected to gel filtration.
The NaOH extract was fractionated using a G-25 Sephadex column (with a fractionation range of 100 to 5000 mol. wt.; dry bead diameter = 20 to 80 µm; bed volume = 4 to 6 mL g-1; column volume = 75 mL; Amersham Pharmacia Biotech, Piscataway, NJ) as described by Pant et al. (1999). The extract (20 mL) was pipetted onto the top of the column and eluted with demineralized water by pumping at a rate of 0.6 mL min-1. Eighty, 3-mL fractions were collected using a fraction collector. The fractions containing NaOH were separated using a litmus paper test. To check for loss of any forms of P, the NaOH fractions were tested for the presence of P. No NaOH was found up to the 49th fractions and no P was found after the 49th fraction. The fractions free from NaOH were pooled and concentrated (10 times) in a vacuum rotatory evaporator at 35°C.
Four milliliters of the concentrated extracts were scanned in a 12-mm tube at 121.4688 MHz on an NT 300 31P NMR Spectrometer (Nicolet Magnetics, Fremont, CA) (Buszko et al., 1998) using a 90° pulse with a 3.0 s delay and a sampling interval of 0.0000622 s. To obtain an acceptable signal to noise ratio and avoid possible changes in signals, a minimal number (1000) of scans were collected (Pant et al., 1999). The chemical shifts were determined with respect to an external standard of 85% phosphoric acid. The identification of peaks of different P compounds in NMR spectra was done by comparing chemical shifts of the peaks with those of the references reported by Gadian et al. (1979) and Pant et al. (1999). The relative composition of different P forms was not determined because of somewhat noisy signals; however, the signals are presented to observe the distinct changes induced by water-level drawdown. Some resonance is sensitive to pH (Navon et al., 1977; Preston, 1996) and salt concentration (Gadian, 1982). Thus, to avoid misinterpretation of 31P NMR spectra, samples were also spiked with 0.1 mL (10 mg P mL-1) of pyrophosphate (Na4HP2O7) as an internal standard.
Data Analysis
Unless otherwise stated, all experiments were carried out in triplicates and means were reported. All the data were subjected to two-way analysis of variance (ANOVA) using SAS Windows Version 6.12 (SAS Institute, 1996). Statistical significance was tested using least significant difference (LSD) at the p < 0.05 level.
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RESULTS AND DISCUSSION
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Influence of Drawdown and Reflooding on Phosphorus Pools
The detrital material contained 445 g kg-1 C, 30 g kg-1 N, and 781 mg kg-1 P. No significant effect of water-level drawdown was observed on C to N and C to P mass ratios (Table 1). However, the C to P ratio in detritus increased upon reflooding following the drawdown because of the P release to the water column. Regarding the apparent increase in TP in detrital matter until the 15-d drawdown and consecutive reflooding, and the decrease in the case of 30- and 60-d drawdowns and consecutive reflooding, they may indicate removal of P from the water column, possibly due to sorption and microbial consumption, and desorption and mineralization, respectively.
The drawdown of detrital material reduced the water content of the material from 95 to 11% over a period of 60 d. The drawdown significantly affected the P distributions in some of the chemical pools (Table 2). Readily labile P (NaHCO3extractable detrital P prior to fumigation) was not affected by the drawdown, whereas microbial P (moderately labile P; the difference in NaHCO3extractable P before and after CHCl3fumigation) was significantly affected. Microbial P was increased until 30 d of drawdown, followed by sharp decrease at 60 d of drawdown. The increase in microbial activities until 30 d of drawdown was expected because of the decrease in inundation (reduction in water content from 95 to 85%); however, further decrease of water content to 11% at 60 d of drawdown may have induced water stress on microbes. Wet conditions limit microbial activity by restricting O2 diffusion (Merila and Ohtonen, 1997; Gulledge and Schimel, 1998), whereas dry conditions are known to reduce microbial activity by inducing physiological water stress (Conant et al., 1998; Gulledge and Schimel, 1998; Murphy et al., 1998). Microbial biomass is a relatively labile constituent of organic matter (Jenkinson and Ladd, 1981) as well as a key component for P mineralization (Brookes et al., 1984) by the production of phosphatases (Cole et al., 1978; Pant et al., 1994a,b). Microbial P could represent a substantial portion of total P in soils (Anderson and Domsch, 1980), sediments (Davelaar, 1993), and detritus of wetlands and could play a crucial role in P retentionrelease phenomena in wetlands.
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Table 2. Effects of drawdown only, and drawdown and consecutive reflooding of 30 d on P distributions in detrital material
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A significant increase in relatively nonlabile inorganic P (HCl-extractable P) was observed as the drawdown increased (Table 2). This may suggest that mineralization of organic P, and subsequent adsorption and precipitation of mineralized P by cations including Ca and Mg (co-occurrence of mineralizationsorption phenomena) as the drawdown period extended. A steady increase in humified P (NaOH-extractable P, i.e., fulvic acid + humic acidassociated P) until 30 d of drawdown, but a decrease at 60 d of drawdown, may also support the co-occurrence of mineralizationsorption phenomena. As the drawdown period extended, the amount of high-molecular-weight organic P (humic acidassociated P) increased steadily until 30 d of drawdown, unlike low-molecular-weight organic P (fulvic acidassociated P).
Upon reflooding, the effect of drawdown on microbial P became insignificant, though changes in microbial P occurred (Table 2). A 53% decrease in microbial P was observed in detrital material at 30 d of drawdown, whereas a 100% increase was observed at 60 d of drawdown. This may emphasize the critical role of water content of detritus on microbial proliferation. Overall, no changes in the percentage composition of labile P occurred in detrital matter due to drawdown and consecutive reflooding, which may suggest that drawdowns could be used to stabilize P in wetlands without increasing the labile P pool. However, a steady increase in low-molecular-weight organic P (FA-P) was observed as the residual P diminished, unlike the high-molecular-weight organic P (HA-P). This may suggest a faster conversion of residual P to FA-P than conversion of FA-P to HA-P under flooded conditions compared with dry conditions. Moreover, a slow decomposition of high-molecular-weight compounds to low-molecular-weight compounds under flooded conditions may have caused the apparent decrease in HA-P in some cases. The smaller the detrital particles, the lower the C to P or N to P ratio (i.e., a higher P content in fulvic acids than humic acids) (Mostajir et al., 1998). A steady decrease in recalcitrant P (residual P) until 30 d of drawdown, but an increase at 60 d of drawdown (Table 2), perhaps indicates a typical P cycle in detrital material of wetlands. The mineralization and microbial accumulation of P occur as the inundation decreases. Similarly, precipitation and formation of occluded P could also occur as the water content is drastically reduced. This may emphasize the role of microbial activities in redistribution of P into different forms (Hedley et al., 1982).
Influence of Drawdown on Phosphorus Forms
The TP in the NaOH extracts was 60 to 85% of the TP of the detrital matter. 31P NMR spectroscopy revealed that before drawdown, 0.4 M NaOH extracts of detrital material consisted of various P forms including, glucose-6 phosphate (G6P), glycero-P (gly P), polynucleotides (poly N), and phospholipids (glycerophosphoethanolamine [GPEA] and glycerophosphocholine [GPC]) (Fig. 2)
. As the drawdown proceeded to 7 d, the relative composition of nucleoside monophosphate (NMP) increased while that of the polynucleotides decreased, and GPEA remained unchanged. At 15 d of drawdown, however, the gly P became the dominant form of P. As the drawdown proceeded to 30 d, NMP and poly N were reduced to trace amounts while phosphoenolpyruvates (PEP), GPEA, G6P, and gly P remained as major P forms. At 60 d of drawdown, however, PEP levels were reduced and GPEA became the dominant form of P. The changes observed in P forms due to water-level drawdown, perhaps, are closely related with the microbial activities and associated abiotic factors. It is apparent from the study that microbes preferentially utilize nucleic acidbound P, perhaps to obtain associated nutrients (carbon and nitrogen). Various chemical fractionation techniques have been used to quantify different pools of P for their stability determination (Hedley et al., 1982; Ivanoff et al., 1998). This study indicated that compounds may be refractory at one time and release P at another, thus, the speciation of organic P could substantially enhance its stability determination.

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Fig. 2. Effects of drawdowns on different phosphorus forms in NaOH extracts as detected by 31P nuclear magnetic resonance spectroscopy. G6P, glucose 6-phosphate; polyN, polynucleotides; PEP, phosphoenolpyruvates; NMP, nucleotide monophosphate; GPEA, glycerophosphoethanolamine; glyP, glycerophosphate; GPC, glycerophosphocholine. The values in parentheses are approximate chemical shifts
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Influence of Drawdown on Phosphorus Flux upon Reflooding
As the drawdown period extended up to 30 d, the P flux from detritus to the water column decreased upon reflooding; however, the flux was triggered at 60 d of drawdown (Fig. 3)
. At the end of the 30-d reflooding period, cumulative P flux to the water column was 3% of TP at
15 d drawdown, and 2% of TP at 30 d of drawdown, while the flux increased to 8% at 60 d of drawdown. Most P flux to the water column occurred within 3 d of reflooding, as expected, because a rapid flux of nutrients (including P) from decomposing detritus is known to occur in the initial phase as the solubilization of accumulated amounts take place (Ogwada et al., 1984). Thereafter, however, net immobilization of P occurred for up to 30 d of reflooding following the drawdown of
30 d. At 60 d of drawdown, however, the P flux into the water column steadily increased for the first 3 wk of reflooding and leveled off on the fourth week. It is known that air-drying of soils can kill microorganisms (Sparling et al., 1985; Van Gestel et al., 1993), and microbial cells added to soils undergo rapid mineralization and release P (Kapoor and Haider, 1982; Lethbridge and Davidson, 1983). Moreover, organic anions (e.g., citric, oxalic, malic, tartaric, lactic, etc.) liberated during decomposition of organic matter could increase P solubility by competing with phosphate ions for adsorption sites (Lopez-Hernandez et al., 1986; Traina et al., 1986) or decrease P solubility through chelation reactions (Harter, 1969; Le Mare, 1982; Tiessen et al., 1984). Similar phenomena are also expected to occur in detritus. The detrital material may not have the abundance of mineral P to use up the liberated organic anions; however, dissolved cations could chelate them. Thus, organic acids may have been one of the contributing factors in reducing P flux to the water column until 30 d of drawdown. However, at 60 d of drawdown, both the death of microbes and nonavailability of organic acids may have triggered the P flux. Though the accretion of organic matter in wetlands is a major sink for P (Craft and Richardson, 1993; Reddy et al., 1999), prolonged drying of wetlands could induce a substantial internal P load.

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Fig. 3. Effect of drawdowns on phosphorus flux from detritus to the water column during 30 d of reflooding
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In general, and especially at 60 d of drawdown, a decrease in DOP occurred during 30 d of reflooding, in contrast to an increasing trend of SRP (Fig. 4)
. This may indicate a rapid turnover of DOP. The reason is not known; however, the above observations together with 31P NMR analysis suggest that the DOP fraction may be utilized preferentially by microbes to obtain other nutrients, including carbon and nitrogen. A sudden decrease in DOP at 7 d of reflooding of detritus subjected to 60 d of drawdown was perhaps due to its maximum consumption by microbes attaining a normal microbial proliferation after a water stress condition (water content reduced to 11%). Thereafter, an increase in DOP at 15 d of reflooding could simply be due to slow dissolution of consolidated detritus upon reflooding. As expected, a decrease in particulate P upon reflooding was also observed as the drawdown period extended because of the consolidation of detritus along with the reduction in its water content. The observations may suggest that the drawdown of detrital material not only reduced P flux to the water column, but also reduced the turbidity of water.

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Fig. 4. Changes in soluble reactive phosphorus (SRP) and dissolved organic phosphorus (DOP) in the water column during 30 d of reflooding (60-d drawdown treatment). DOP = total dissolved phosphorus (TDP) - SRP
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CONCLUSIONS
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This study showed that a reduction in water content due to drawdown is critical, as far as the P flux to the water column from detrital material upon reflooding was concerned. Up to 30 d of drawdown had a remarkable effect on reduction in SRP flux to the water column from detritus material, but had no significant effect on the proportion of labile P after reflooding. This may suggest that drawdown of detrital material not only helped its consolidation but also reduced SRP flux to the water column. However, the reduction in water content needs to be monitored more frequently to avoid an increase in the P flux due to overdrying. The changes in P distributions in various chemical pools, and the relative composition of different P forms as induced by drawdowns is complex and dynamic. This could either increase or decrease SRP flux to the water column upon reflooding, depending upon the extent of drawdown.
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NOTES
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This work was partially funded by South Florida Water Management District, West Palm Beach, FL. Florida Agric. Exp. Stn. Journal Ser. no. R-07844.
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