Details of the methods used for collection, preparation, and analysis of samples are included as an aid to the reader. Data are, in varying degrees, operationally defined. An example is the phosphate data in the water chemistry tables. The analyses were done using an ion chromatograph that determines only orthophosphate. Water samples were not analyzed for dissolved reactive phosphate.

Sampling sites were located in Water Conservation Areas (WCA) 2A, 2B, 3A and 3B, the Big Cypress Preserve (BCNP), and in and near the Everglades National Park (ENP) (Figure 1). Within WCA-2A, samples were collected at sites established by the South Florida Water Management District (SFWMD) along two transects referred to as the F and E transects. These transects are approximately 14 kilometers in length and follow a general north-to-south direction and a nutrient gradient. The F transect includes sites F1, F2, F3, F4 and U3, and the E transect includes sites E1, E2, E4, and U1. Sites are approximately evenly spaced along the 14-kilometer gradients. Also, a set of samples was collected along the L-6 canal that forms the northwest boundary of WCA-2A. The sampling site in WCA-3A is referred to as 3A15 and the site in WCA-3B, located near the L-67 canal, is referred to as 3B67. The sites in the BCNP are permanent hydro-stations in the preserve. The hydro-stations in BCNP are sites at which samples are collected for long-term studies. The hydro-stations that were sampled, listed in a general north-to-south direction are: A-1 (located in the northwest corner of the preserve), A2, A3, A12, A13, A5, A11, and Canal 28 (L28) at Interstate 75 (located on the northeast boundary of the preserve). Samples from the Everglades National Park were collected near the Tamiami Canal. Samples also were collected from wetlands close to the boundary of the ENP near the C-111 canal. Table 1 lists the latitude and longitude of each sampling site. Table 2 lists all the sites and gives the month and year the samples were collected and the numbers of samples that were collected at each site.

In March and July 1995, plastic syringes were used to collect triplicate water samples with volumes of approximately 12 mL each from within the periphyton mats and from the water column adjacent to the mats. Replicate samples taken from the same place are labeled a, b, and C. In December 1995, June 1996 and December 1996, water samples were collected filling either a 12 mL borosilicate vials with Teflon-lined cap or 125 mL linear polyethylene bottles approximately 5 cm below the surface of the water column.

Floating mats were the dominant type of periphyton and were sampled if present. At sites E1 and F1 in WCA-2A, and A12 (a cypress dome) in BCNP, the periphyton was present as a felt-like growth attached to dead leaves. Periphyton mats were collected by supporting floating material with an open hand, drawing the material up from the water, and transferring it to either (a) a zip-lock bag (whole mat sample) or (b) a water-tight polycarbonate box (sectioned mat sample). Samples in zip-lock bags were transported on ice to the laboratory. Whole periphyton samples that would be sectioned in the laboratory were frozen in polycarbonate boxes using solid carbon dioxide at the sampling site to preserve sample integrity. These samples were shipped frozen and kept frozen until they were sectioned.

Periphyton samples were collected by different techniques at site A5 in the Big Cypress National Preserve, and at site U3 in WCA-2A, to determine any differences in analytical results. Periphytic material was collected at 60^o intervals on the circumference of a one-meter circle around a stake driven into the marsh sediment. Samples were collected using latex gloves or bare hands. At each location on the circle one sample (a) was collected using gloved hands and transferred to a Teflon container. Another sample (b) was collected using gloved hands and transferred to a zip-lock bag. A third sample (c) was collected with a bare hand and transferred to a zip-lock bag.

In December 1996, detritus samples were collected at 60^o intervals on the circumference of the same one-meter circle that was used for periphyton collection. Detritus is made up of particles from disaggregated periphyton mats. It was collected by skimming an open-gloved hand along the interface between the water column and the bottom sediment.

Water samples were filtered through 0.2 µm polycarbonate filters within 6 hours of collection. Samples were stored at 4^oC until analyzed.

If periphyton samples could be air-dried within a week to ten days of collection, they were refrigerated at 4^oC before drying. Samples stored for longer periods were frozen before air-drying. Macrophytes (large plant parts) were removed from the mats. Periphyton attached to the surface of macrophyte leaves or stems was separated and saved; leaves or stems were discarded. The remaining periphytic material was air-dried in aluminum pans at room temperature in a laminar flow hood. When dry, the samples were ground with an agate mortar and pestle to a particle size of not more than 125 µm. Samples were dried over calcium chloride in a desiccator before weighing. All concentrations are reported on a weight of analyte per gram of dry sample. In general, periphyton samples contained approximately 90-95 percent water.

A band saw was used to section frozen periphyton mats. The frozen whole blocks of periphyton had the approximate dimensions of 10 x 20 x 5 cm. Approximately 2-cm were removed from each of the long ends. Horizontal slabs of the top and bottom were removed from the remaining 10 x 16 x 5 cm block. The middle section of the block was cut into three horizontal sections. Each of the sections was approximately 1 cm thick. For mats that were not fully 5-cm thick, the number of horizontal sections was adjusted so each horizontal section had an approximate thickness of 1-cm.

Measurements of water-column samples were made within 6 to 10 hours of sample collection. A portable pH meter (Hanna Instruments, HI 909025, microprocessor pH meter) was calibrated using standard buffers of pH 7.00 (± 0.02 at 25^oC) and pH 4.00 (± 0.02 at 25^oC) for samples with pH values 7, and using standard buffers of pH 7.00 (± 0.02 at 25^oC) and pH 10.00 (± 0.02 at 25^oC) for samples with pH values 7. The meter was recalibrated after each set of two water column samples. Readings were recorded when the meter reading drifted no more than 0.01 units per minute.

Alkalinity. Alkalinity was determined using a Radiometer ABU93 Autotitration System and Gran titration calculations (Gran, 1952). Samples were diluted 1:10 before titration. In aerobic waters, alkalinity concentrations determined by this method can include not only carbonate, but also ammonia, and some organic acids such as acetate and formate.

Anions. Anions were determined in water column samples using a Dionex Ion Chromatograph. The principle for the method used in Dionex IC equipment was published by Small and others (1975). The specific conductance of the analyte ions is maximized in the weak carbonic acid solution that carries the analytes through the conductivity detector. Representative means and standard deviations for Cl ^-, SO₄ ^-2, and PO₄^3- were 7 ± 0.003 ppm (0.200 ± 0.001 meq L^-1), 9.6 ± 0.04 ppm (0.2000 ± 0.0008 meq L^-1) and 1.9 ± 0.07 ppm (0.0400 ± 0.0014 meq L^-1), respectively.

Cations. Calcium and other cation concentrations in samples acidified with nitric acid were determined using an American Research Laboratories SpectraspanV DCP and direct-coupled plasma atomic-emission spectrometry. Representative means and standard deviations for a mixed standard containing 5.0 ppm (0.25 meq L^-1) calcium and 6.0 ppm (0.261 meq L^-1) sodium were 5.0 ± 0.02 ppm (0.25 meq L^-1 ± 0.005) for calcium and 6.0 ± 0.06 ppm (0.261 ± 0.003) meq L^-1) for sodium.

Microwave Digestion. In preparation for determination of total mercury and phosphorus in the periphyton, samples were digested with concentrated nitric and hydrofluoric acids using the Microwave Sample Preparation System, Model MDS-2100, manufactured by CEM Corporation. The method used is a modification of CEM application note AG-12 (CEM Corporation, 1994). Modifications included the use of approximately 820 watts of power to the magnetron, adding the nitric and hydrofluoric acids in two steps, and the use of boric acid at the end of the digestion to bind unused hydrofluoric acid in the digestion matrix. Certified reference materials, National Institute of Standards and Technology (NIST) SRM 8030 or SRM 8031, were included in each set of digestion samples. These aquatic plant (Lagarosiphon major) and aquatic moss (Platihypnidium riparioides) materials have certified mercury concentrations of 0.34 ± 0.04 µg g^-1 and 0.23 ± 0.02 µg g^-1, respectively (National Institute of Standards and Technology, 1982). Sample data were used from digestion sets in which the analyzed values for the standards included in the set agreed with the certified values for the standards to within 5% of the reported value. Replicate analysis of (n=3) of SRM 8031 had an average concentration of mercury of 0.33 ± 0.02 µg g^-1.

Total Mercury. Total mercury concentrations were determined in microwave digests of periphyton samples using a ThermoSeparation cold vapor mercury analyzer and the instructions of the manufacturer (ThermoSeparation Products, 1994). Blank solutions of 10 percent nitric acid were run between samples.

Phosphorus.The microwave digestion procedure described above converts all forms of phosphorus in samples to orthophosphate. The molybdenum blue method of Murphy and Riley as described in Rand and others (1976) was used to determine orthophosphate concentrations in microwave digests of periphyton samples. Boric acid was added to the samples before the addition of the color reagent to complex any fluoride that was not completely removed by the addition of boric acid during the microwave digestion process. Certified reference material, National Institute of Standards and Technology 1571 (National Bureau of Standards, 1977), orchard leaves, has a certified phosphorus concentration of 2.1 ± 0.1 mg per gram dry weight. A mathematical transformation of phosphate to phosphorus concentrations in samples was used to provide phosphorus concentration data for standards and samples. Replicate microwave digests of this standard gave a concentration of 2.2 ± 0.1 mg phosphorus per gram dry weight (n=5).

Methylmercury. Methylmercury concentrations were determined using the method of Simon (1997). Methylmercury was extracted from samples using supercritical fluid carbon dioxide modified with 5 or 7.5 percent methanol. Certified samples Dolt-2, 0.69 ± 0.05 mg g^-1 ethylmercury as mercury, and Dorm-1, 0.73 ± .06 mg g^-1 methylmercury as mercury (National Research Council of Canada, 1993), were routinely run to insure that the extraction procedure was consistently extracting methylmercury from samples. Average concentrations (n=3) of Dolt-2 and Dorm-1 (n=3) were 0.70 ± 0.02 mg g^-1 methylmercury as mercury, and 0.72 ± 0.01 mg g^-1 methylmercury as mercury, respectively. Methylmercury concentrations were quantified using liquid chromatographic separation and reductive electrochemistry.

Samples were packed in preweighed sample cartridges, reweighed, mixed with granular calcium chloride, and moistened with water approximately 2 hours before being inserted into the extraction system. After bringing the samples to a temperature of 50^oC, the carbon dioxide - methanol fluid was added to the cartridge and a static step when no flow of the fluid occurred lasted for approximately 15 minutes. The exit valve from the sample was opened and approximately 20 milliliters of fluid was used to extract the sample. The extract was collected in approximately 3 milliliters of acetonitrile contained in a separatory funnel with a cold finger in place of a stopper.

A set of 18 spiked periphyton samples was extracted using supercritical carbon dioxide to determine the efficiency of this method for extraction of methylmercury from periphytic material. The spiked concentrations of methylmercury per gram dry weight of periphyton and the efficiency of the recovery of total methylmercury from periphyton samples from three sites, are listed in table 3. The samples were E1 and E4, collected from WCA-2A in August 1995, and A2, which was collected from Big Cypress National Preserve in July 1995. The total mercury concentrations in the periphyton were 0.67, 0.32, and 0.05 µg per gram dry weight of periphyton for samples E1, E4 and A2, respectively. The methylmercury concentrations in the unspiked periphytic material were 0.11, 0.05 and 0.04 µg as mercury per gram dry weight of periphyton.

Carbon and Nitrogen. Carbon and nitrogen concentrations were determined for 0.2-g air-dried, ground samples by flash oxidation of the sample and separation of the gaseous products using either a Carla Erba or Perkin Elmer Instrument. Organic carbon concentrations were determined by analyzing 0.2-g air-dried, ground samples that had been weighed into silver cups and treated with hydrochloric acid fumes in a desiccator. The acid treatment removed carbonate from the samples. Inorganic carbon concentrations were determined by difference. In this scheme, nitrogen concentrations were determined during each run and the average of the two values was reported as the percent nitrogen in the sample.

A standard of plant material with certified concentrations of total carbon and/or organic carbon could not be found. The primary organic compound standard acetanilide has a composition of 71.05% of carbon and 10.36% of nitrogen. Replicate samples provided concentrations of 71.28 ± 0.20 % of carbon and 10.29 ± 0.13 % of nitrogen (n= 5).

National Institute of Science and Technology Standard Reference Material 1571, orchard leaves, is certified for a nitrogen concentration of 2.76 ± 0.05. Replicate samples of orchard leaves gave a concentration of nitrogen equal to 2.72 ± 0.06 % (n=5).