Molecular characterization of dissolved organic matter in freshwater wetlands of the Florida Everglades

Water Research 37 (2003) 2599–2606

Molecular characterization of dissolved organic matter in freshwater wetlands of the Florida Everglades X.Q. Lua,b, N. Maiea,b, J.V. Hannac, D.L. Childersa,d, R. Jaffe! a,b,* a

Southeast Environmental Research Center, Florida International University, Miami, FL 33199, USA b Department of Chemistry, Florida International University, Miami, FL 33199, USA c ANSTO NMR Facility, Materials Division, Lucas Heights Research Laboratories, Private Mail Bag 1, Menai NSW 2234, Australia d Department of Biological Sciences, Florida International University, Miami, FL 33199, USA Received 31 July 2002; received in revised form 20 December 2002; accepted 4 February 2003

Abstract In this study, the molecular composition of dissolved organic matter (DOM), collected from wetlands of the Southern Everglades, was examined using a variety of analytical techniques in order to characterize its sources and transformation in the environment. The methods applied for the characterization of DOM included fluorescence spectroscopy, solid state 13C CPMAS NMR spectroscopy, and pyrolysis-GC/MS. The relative abundance of proteinlike components and carbohydrates increased from the canal site to more remote freshwater marsh sites suggesting that significant amounts of non-humic DOM are autochthonously produced within the freshwater marshes, and are not exclusively introduced through canal inputs. Such in situ DOM production is important when considering how DOM from canals is processed and transported to downstream estuaries of Florida Bay. r 2003 Elsevier Science Ltd. All rights reserved. Keywords: DOM; Humic substances; Synchronous fluorescence; Solid state marshes

1. Introduction Environmental restoration of the Southern Everglades, including the Everglades National Park (ENP) Panhandle, is part of a larger effort to restore the hydrologic regime of the greater Everglades watershed and Florida Bay [1]. For the past 95 years, Florida Everglades marshes have been subjected to numerous anthropogenically induced changes in their landscape ([1,2] and others). Approximately 65% of the original extent of the Everglades has been drained for agricultural and residential purposes through the construction of B2400 km of canals and levees. Historical water inputs *Corresponding author. Environmental Geochemistry Laboratory, Southeast Environmental Research Center, Florida International University, University Park, OE 147, Miami, FL 33199, USA. Tel.: +1-305-348-2456; fax: +1-305-348-4096. E-mail address: jaffer@fiu.edu (R. Jaff!e).

13

C CPMAS NMR; pyrolysis-GC/MS; Freshwater

into the Everglades have been severely affected during this time, resulting in significant and detrimental changes to this ecosystem. However, water management practices and annual rainfall have substantially increased water inputs in some areas since 1992 [1]. There is some concern that this increased water input may have an effect on nutrient loading to the oligotrophic freshwater wetlands of the Southern Everglades and to Florida Bay. The ENP Panhandle landscape is a small watershed of the greater Everglades that drains into northeastern Florida Bay estuary ([2], Fig. 1). Freshwater inputs to the Panhandle are controlled by water management of the C-111 Canal. The canal controls flows in the ENP Panhandle, flooding in southern Miami-Dade County, maintains a head against salt water intrusion into the Panhandle and supplies water to this ecosystem [2]. In an effort to increase sheet flow through Panhandle marshes to northeast Florida Bay, the southern berm of

0043-1354/03/$ - see front matter r 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0043-1354(03)00081-2

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Fig. 1. Map of sampling sites.

the C-111 was removed in 1997, permitting water to flow south whenever water levels in the canal exceed bankfull. Most nitrogen (N) and phosphorus (P) in the freshwater Everglades environment are in an organic form [3]. Because suspended particulate concentrations in these environments are very low, most of this organic matter is associated with the dissolved organic matter (DOM) fraction. A significant portion of the labile DOM fraction may be remineralized by microbes to inorganic constituents depending upon ambient nutrient status [4] and chemical bioavailability [5]. Bacterial utilization of DOM, and subsequent grazing on these bacteria by protists and microzooplankton (the microbial loop), is an important alternative pathway in many aquatic food webs. Fully understanding the dynamics of DOM cycling in aquatic environments requires detailed knowledge of the sources, transport and transformation of this material. However, the diversity of aquatic habitats in Everglades wetlands, as well as significant seasonal variations in the hydrology and primary productivity within this system, further complicates this task. Everglades DOM sources are likely associated with local plants, such as mangroves, sawgrass, aquatic

macrophytes and periphyton. Leachates from wetland soils and canal inputs are also likely DOM sources. However, detailed molecular characterization of this DOM has received little attention. Therefore, the main objective of this study was to characterize the molecular compositional changes of Everglades’ surface water DOM on a spatial and temporal scale. Our goal was to better understand the source strengths, transport and fate of humic and nonhumic DOM components, using a suite of analytical methods [6–8].

2. Materials and methods 2.1. Study area The general study area is located in the Southern Everglades, particularly in the C-111 Basin of the ENP Panhandle, South Florida, USA (Fig. 1). Water entering the Panhandle from the C-111 canal first flows through freshwater marshes dominated by Cladium jamaicense (sawgrass), followed by a region dominated by dwarf

X.Q. Lu et al. / Water Research 37 (2003) 2599–2606

Rhizophora mangle (red mangrove, [9]), and ultimately, through mangrove creeks into northeast Florida Bay [1]. The freshwater marsh is interspersed with Eleocharis spp. (spikerush) and periphyton assemblages [9] which exist throughout the freshwater Everglades. Wetland soils are mostly calcareous marls (freshwater) and peaty marls (estuarine, [9]).

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HS were operationally defined as the DOM fraction, which was retained by the XAD-8 resin, while the unretained fraction represented the NHS. The HS concentration was calculated by difference between the measured DOC concentration of the original water samples and that of the NHS fraction. 2.5. Molecular characterization

2.2. Sample collection and treatment A variety of surface water samples were collected from a transect within the C-111 Basin. This transect consisted of four sites (Fig. 1), starting at the C-111 canal (Site C-111), followed by sites W1, W2 and W3 located about 50 m, 2 km and 4 km downstream from the canal, respectively. Site C111 is located in the center of the C-111 Canal, W1 and W2 are located in sawgrass marsh environments, and W3 was at the sawgrass–mangrove ecotone. These sites were sampled at the end of the dry season (June 25th), early wet season (August 25th), after a hurricane (October 26th), and at the end of the wet season (December 11th) of 1999. Two liters of surface water were collected at each site, placed in prewashed polyethylene bottles and stored on ice before being taken to the laboratory for analysis. The water samples were filtered through pre-combusted (470
C for 8 h) 0.7 mm Whatman GF/F glass fiber filters. Immediately after filtration, DOC concentration and UV–visible and fluorescence analyses were performed. A 1.5 l volume was concentrated using rotary evaporation, frozen, freeze dried under vacuum and stored for 13C-NMR and py-GC/MS analyses. For the biomass DOM leaching experiments, an approximately equal mass (wet weight) of yellow leafs of the red mangroves, life blades of sawgrass and seagrass, and floating periphyton mat samples were rinsed with doubly distilled water and placed individually in amber glass bottles in doubly distilled water, and allowed to leach for 24 h within a refrigerator to minimize bacterial activity. Biomass leached DOM was isolated and analyzed by solid state 13C CPMAS NMR as described below. 2.3. DOC analysis DOC concentrations were measured by Pt-catalyzed high temperature combustion (680
C), using a Shimadzu TOC-5000A total organic carbon (TOC) analyzer, coupled to a non-dispersive infrared CO2 detector. All chemicals used were AR grade and were purchased from Fisher (Pittsburgh, PA), Sigma, (Bellefonte, PA), and Supelco (St. Louis, Mo). 2.4. Separation of humic substances Humic substances (HS) and non-humic substances (NHS) were isolated as described in the literature [10].

Fluorescence spectra were obtained in 1 cm quartz fluorescence cells at room temperature (B20
C), using a Perkin Elmer LS50B spectrofluorometer equipped with a 150-W Xenon arc lamp as the light source. Slit widths were set at 8 nm for the emission monochromator. Fluorescence emission spectra were obtained at wavelengths ranging from 300 to 700 nm with an excitation wavelength at 370 nm. Synchronous fluorescence spectra were recorded with a constant offset (dl ¼ 30 nm) between excitation and emission wavelengths and 10 nm slit widths [11]. Synchronous fluorescence spectra were background corrected against Milli-Q water (Millipore), after correction for optical density differences using a method similar to that described in the literature for fluorescence emission spectra [6]. Ultra-violet and visible measurements of DOM were carried out with 1 cm quartz UV– visible cells at room temperature (B20
C), using a Shimadzu UV-2101PC UV–visible double beam spectrophotometer. Milli-Q water was used as a reference. All DOM samples were characterized by solid-state 13 C cross-polarization, magic-angle-spinning nuclear magnetic resonance (13C CPMAS NMR) on a Bruker CXP-90 NMR spectrometer operating at a 13C frequency of 22.65 MHz and at an MAS rate of 3.5 kHz. Pulse widths of 3.5 ms were used with a 2 s recycle time and a 1 ms contact time. Spectra were collected in 1 K points, zero filled to 4 K, and Fourier transformed with a line broadening factor of 50 Hz. The relative quantities of each carbon type or functionality present in the DOM samples were estimated by direct integration and comparison of the different spectral regions of each spectrum. Pyrolysis gas chromatography mass spectrometry (py-GC/MS) was performed on a CDS Pyroprobe 2000 interfaced to a Hewlett Packard 5890 gas chromatograph and a HP 5973 mass selective detector. The pyrolysis was performed in a helium atmosphere heating the sample from 300
C to 650
C at a rate of 20
C/ms, holding the maximum temperature for 20 s. Published collections of py-GC/MS data of natural organic matter [8,12] were used for source assignment purposes. The peak areas of all pyrolysis products were integrated to calculate the relative proportions (%) of the corresponding four major classes detected in the DOM samples.

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of humic-like substances such as tannins. In contrast, the NHS fraction most likely consisted of highly functionalized, plant-derived compounds, and microbially derived leachates [13], such as hydrophilic acids, carbohydrates and proteins. The concentrations of HS and NHS throughout the transect (Fig. 2b and c), showed slightly higher values for the W2 and W3 sites compared to C-111 and W1, particularly for the June sampling, suggesting that the freshwater marsh environment was a source of both HS (and HS-type OM) and NHS. The significantly larger DOC concentration for the W2 and W3 sites during the June sampling is likely caused by the DOM leaching resulting from the rehydration of soils, senescent plant materials and partially dried periphyton mats during the early stages of the wet season. Fluorescence spectroscopy can be useful in DOM source determinations [6,14]. In this study, both fluorescence emission and synchronous fluorescence spectroscopy were used in DOM source assessments (Figs. 3 and 4). The fluorescence index, f450 =f500 ; defines DOM of allochtonous (terrestrial) and autochtonous (microbial) origin with end-member values of about 1.4 and 1.9, respectively [6], and f450 =f500 values determined in this study (mean of 1.5870.07) are shown in Fig. 3a. Overall, these values were intermediate between the terrestrial and microbial end-members, and suggest a mixed terrestrial–microbial (probably periphyton) DOM source. It is important to point out that this index was developed for the characterization of fulvic acids [6] and may not properly account for the contribution of NHS. Synchronous fluorescence spectra were similar for all samples (Fig. 4) but showed differences in relative intensity of four characteristic peaks at about 285, 350,

3. Results and discussion The concentrations of DOC, HS and NHS and the %HS are shown in Fig. 2 for a series of surface water samples collected in 1999 at the C-111 basin transect. Both spatial and seasonal variations were observed over the period of this investigation. For example, differences in DOC concentrations (Fig. 2a) were observed at the sampling sites, both spatially and seasonally, suggesting that the canal was a source of DOM but also that DOM was produced as water flowed through the marshes. DOC leached from periphyton and sawgrass (all sites), mangrove leaves (site W3), as well as from senescent plant materials, detritus and soils may explain the increase in the concentrations of DOC that we observed along the sampling transect. In addition, variations in DOC concentrations at different sampling times suggested that DOC concentrations were diluted by rain during this investigation. While DOC concentrations were highest at stations W2 and W3 during the June sampling event, the values were noticeably lower after the rainy season started. DOC concentrations were particularly low after the passage of Hurricane Irene during the month of October. Notably, Hurricane Irene was responsible for over 40 cm of rain in about 36 h, and mean water levels increased by nearly 50% during this event. Variations in the molecular composition of the DOM reflected these concentration changes, as shown below. The %HS ranged from 54.2% to 79.5% with an average of 67.6%, while the percent of non-HS ranged from 20.5% to 45.8% with an average of 32.4%. The high %HS would suggest high amounts or organic-rich soil-derived fulvic and humic acids, but marsh plants may also contribute to the HS pool through the leaching W1

W2

W3

15

NHS (mgC L )

20 -1

-1

DOC (mgC L )

C111

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10 5

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-1

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15

%HS

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70 60

0

50 June '99 Aug. '99 Oct. '99 Dec. '99

(b)

80

Sampling date

June '99 Aug. '99 Oct. '99 Dec. '99

(d)

Sampling date

Fig. 2. Seasonal variations in the carbon concentration of: (a) dissolved organic carbon (DOC), (b) humic substances (HS), (c) nonhumic substances (NHS), and (d) %HS along the C-111 basin sampling transect.

X.Q. Lu et al. / Water Research 37 (2003) 2599–2606 C111

W1

W2

W3

f450 / 500

1.8

1.6 1.4 1.2

(a)

June '99

Aug. '99

Oct. '99

Dec. '99

June '99

Aug. '99

Oct. '99

Dec. '99

%peak I

45 40 35 30

(b)

25

Emission Intensity (arbitrary unit)

Fig. 3. Seasonal variations in the fluorescence index, f450=500 (a), and the relative abundance (%) of Peak I (285 nm) in the synchronous fluorescence spectra (b), along the C-111 basin sampling transect.

125 Peak II 100 75

Peak I

Peak III

50 Peak IV 25 0 -25 250

300

350

400

450

500

550

Excitation wavelength (nm)

Fig. 4. Typical synchronous fluorescence spectrum of C-111 basin water samples after optical density correction (dl ¼ 30 nm).

385 and 458 nm. This is most likely caused by different concentrations of fluorophores in the DOM. Peak I, reported in the range from about 250 to 300 nm, has been assigned to the presence of NHS, specifically protein-like materials [14,15]. In addition to possible DOM source differences, the lmax for Peak I can also depend on the dl used for the synchronous fluorescence [14]. In our study, the lmax was most commonly observed at about 285 mn, and this wavelength was used throughout the study to characterize Peak I. This peak was very abundant in the spectrum of DOM leached from periphyton (data not shown), and from mangrove leaves and sawgrass [11]. Peaks II and III are characteristic for fulvic acids, whereas peak IV is

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associated with the presence of humic acids [16]. Therefore, synchronous fluorescence has the potential to provide useful information regarding the relative abundance of protein-like vs. humic-like DOM (i.e. relative abundance of Peak I, %285 nm, vs. the total abundance of Peaks I–IV). In general terms, the relative abundance of this protein-like signal increased from the canal and W1 sites to the W2 and W3 sites, except for the June sampling event (Fig. 3b), suggesting autochtonous inputs of this potentially labile/bioavailable DOM fraction at the more remote freshwater marsh sites. This effect was not observed for the June sampling, where the relative increase of protein-like DOM components is skewed due to the simultaneous production of soilderived humic materials during soil re-hydration. Our general observations suggest that canal waters are likely to contain more photo-degraded and bio-degraded DOM due to longer residence times compared to the freshwater marsh sites, which appear to receive an important input of autochtonous (non-canal-derived) DOM. This protein-like material could be a key component fuelling the microbial loop in these highly oligotrophic freshwater wetlands. The solid state 13C CPMAS NMR spectra of the DOM samples collected from the C-111 transect and some reference DOM leached from local biomass are shown in Figs. 5a and b, respectively. Spectral features were summarized in four groups by carbon type and/or functionality as %alkyl C (0–50 ppm), %O-alkyl C (50– 110 ppm), %aromatic (arom) C (110–160 ppm) and %carbonyl (carb) C (160–210 ppm; [17] and the references therein). The NMR-based DOM composition is shown on a relative (%) and absolute (mgC/l) scale in Figs. 6a and b, respectively. The absence of a sharp peak at around 32 ppm, which is commonly seen for soil organic matter, suggested that this DOM does not contain significant amounts of long aliphatic C chains that derive from biopolymers such as cutin and suberin. Thus, the alkyl C for all the samples is considered to be highly branched. The %O-alkyl C consistently increased along the transect for the four sampling periods, which confirms the production of NHS DOM components, particularly carbohydrates, at the marsh sites (W2 and W3). Since all the plant-leached DOM samples contained a high %O-alkyl C, it is suggested that the DOM in the canal sample is highly degraded, while biomass-derived carbohydrates were introduced at the freshwater marsh sites. DOM leached from mangrove and sawgrass showed two distinct peaks in phenolic region (140–160 ppm; Fig. 5b) that are typical for tannins [18]. However, these compounds seem to be highly modified in the surface water samples, since these spectral features were quite small. In general, the abundance of aromatic C was low (10–19%) and showed little variation along this transect (Fig. 6a), while the %carbonyl C, although less consistent across

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Fig. 5. Solid-state 13C CPMAS NMR spectra of surface water DOM samples for June 1999 along the C-111 basin sampling transect (a), and for biomass-leached DOM samples (b).

the sample set, presented a decreasing trend from the canal site to the marsh sites. Oxidative degradation of organic materials is generally accompanied by the generation of carboxyl groups. Thus the lower %carboxyl C in the DOM collected from W2 and W3 may indicate that DOM at these sites is less biodegraded than that at the canal site. When looking at the concentrations of each C species in each site (Fig. 6b), O-alkyl C concentrations were always 1.3–2.3 times higher (on average) at the fresh water marsh sites (W2 and W3) than at the canal sites (C-111 and W1), while other C species did not show any appreciable trend. This result confirms the generation of polysaccharides in the fresh water marsh. Finally, molecular parameters frequently used as source and diagenetic indicators in solid-state 13C NMR studies of natural organic matter [17,19,20] such as the O-alkyl C/alkyl C ratio and the arom C/(alkyl C+O-alkyl C+arom C) ratio (also referred to as aromaticity), confirmed the above suggestions. The

O-alkyl/alkyl ratio is consistently higher at stations W2 and W3 than at stations C-111 and W1 (Fig. 7a), suggesting a higher degree of degradation at the later. In agreement, although with more scatter, the aromaticity values were generally lower at sites W2 and W3 compared to C-111 and W1 (Fig. 7b). Overall, the NMR data show an increment in the relative abundance of the polysaccharides along this transect for all sampling periods. This evidence for higher relative abundance of NHS in the marshes compared to the canal site clearly confirms our previous suggestions. In addition, the higher aliphatic and aromatic character of the canal site suggests that the canal-derived DOM is more diagenetically reworked compared to that of the freshwater marsh sites. Overall, 58 different pyrolysis products were identified for the pyrolysis-GC/MS experiments, which presented evidence for the presence of proteins (Pr), carbohydrates (Ps), lignins (Lg) and polyhydroxy-aromatics (PHA) in the DOM samples [8,12,20]. For example, some major

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and characteristic Pr-derived pyrolysis products were 2, 5-dimethyl-1H-pyrrole, pyridine, while Ps-derived products were dominated by methyl- and/or hydroxyl-2cyclopenten-1-ones. Lg produced 2,3- and 2,4-dimethyl phenols, 4-ethyl phenol, while PHA-derived pyrolysis products were methyl- and di-methyl-1H-indenes among others. With the exception of the transect samples from August 1999, where the variations in Pr, Ps and Arom (Lg+PHA) were not significant (Fig. 8), all other sampling events showed a consistent increase in the Ps,

June '99

Aug. '99

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Dec. '99 Alkyl C

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O-Alkyl C

%

Aromatic C 30

Carbonyl C

20

C111 W1 W2 W3 C111 W1 W2 W3 C111 W1 W2 W3 C111 W1 W2 W3

10

(a) 8

June '99

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and to a lesser extent the Pr contribution and a decrease in the Arom contribution along the transects. These results agree with the synchronous fluorescence and the 13 C-NMR results, confirming that carbohydrates and to some extent proteins were produced as water masses flowed from the canal through freshwater wetlands.

4. Conclusions A combination of analytical techniques was used to characterize DOM in surface water samples collected from freshwater marshes of the Southern Everglades, and provided spatial and temporal information on the composition and source of these materials. This study clearly revealed that, while some DOM entered the system via canal inputs, significant amounts of DOM, including carbohydrates, were produced in situ by soil and plant leaching. The important ecological implication of this finding is that carbohydrates and protein-like compounds, in the C-111 basin are derived from a combination of canal and freshwater marsh inputs. It is clear, that internal wetland processes are important to

Dec. '99

60

June '99

Aug. '99

Oct. '99

Dec. '99 Pr Ps

50

Arom

4

%

mgC L-1

6

40

2

30

20

Fig. 6. Seasonal variation in the 13C CPMAS NMR-based DOM composition (a) and concentration (b), along the C-111 basin sampling transect.

C111 W1 W2 W3 C111 W1 W2 W3 C111 W1 W2 W3 C111 W1 W2 W3

(b)

C111 W1 W2 W3 C111 W1 W2 W3 C111 W1 W2 W3 C111 W1 W2 W3

0

Fig. 8. Seasonal variation in the py-GC/MS based DOM composition, along the C-111 basin sampling transect.

25

1.25

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O-alkyl C / alkyl C

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1

0.75

0.5

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20

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June '99

Dec. '99

(b)

Aug. '99

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Sampling date

Fig. 7. Seasonal variation in the 13C CPMAS NMR-based O-alkyl C/alkyl C ratio (a) and the aromaticity (b), along the C-111 basin sampling transect.

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landscape-scale DOM dynamics in the Southern Everglades, which has significant implications to the assessment of the effects of canal inputs on the biogeochemistry of organic nutrients in this system.

Acknowledgements The authors thank the Southeast Environmental Research Center (SERC) and the Department of Chemistry for supporting this project. Special thanks to the staff of the FIU Wetland Ecosystem Ecology Laboratory for assistance with sample collection and Dr. R. Jones for DOC analyses. This project was partially funded by the South Florida Water Management District through contract No. FR-1960, and by the National Science Foundation as part of the FCE-LTER program (DEB-9910514). SERC contribution #194.

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