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Water Chemistry

Field Collection: Water samples were collected at the historic deep site of each study lake during the summer months (May to September) from 2018-2020. The water chemistry data include samples that were collected by three different programs: a research team at the Iowa State University (ISU) Limnology Laboratory, the Iowa Department of Natural Resources (IDNR) Ambient Lake Monitoring (ALM) Program, and the IDNR Shallow Lakes Monitoring Program. Water samples from all three programs were analyzed by the ISU Limnology Laboratory using the same analytic methods (see below); however, field collection methods different slightly among programs. ALM and Shallow Lakes used an integrated column sampler to sample either the mixed surface layer if the lake was stratified, the top 0-2m of the water column if the lake was mixed, or the top 0-2m of the water column if the thermocline was deeper than 2m. For their research sampling, the ISU Limnology Lab collected both epilimnetic and hypolimnetic water samples. Epilimnetic water samples were taken by lowering the sample bottle up to elbow-depth below the surface of the water (0.25m). The bottle was filled and rinsed with lake water three times before taking the water sample. Hypolimnetic samples were taken using a Van Dorn sampler lowered to 0.5 meters about the sediment-water interface. Sample bottles were rinsed three times with the hypolimnetic water before being filled with sample water. Regardless of program, all water samples were transported and stored at 4 degrees C, and sample analysis began within 24 hours of sample collection.

Soluble Reactive Phosphorus (SRP): samples were filtered (0.45µm GF/C filters) in the lab, preserved with concentrated sulfuric acid to pH 2, and stored at 4 degrees C before being analyzed in duplicate using the molybdenum blue method, modified from Murphy and Riley (1962; Standard Methods 4500-P E) on a Seal Analytical AQ2 Discrete Analyzer

Total Phosphorus (TP): samples were preserved with concentrated sulfuric acid to pH 2 and stored at 4 degrees C before undergoing a persulfate digestion (Standard Methods 4500-P B.5) and being analyzed in duplicate using the molybdenum blue method, modified from Murphy and Riley (1962; Standard Methods 4500-P E) on a Seal Analytical AQ2 Discrete Analyzer

Nitrate (NOx) and Total Nitrogen (TN): Subsamples were filtered (0.45µm GF/C filters) in the lab for nitrate analysis, and all samples were preserved with concentrated sulfuric acid to pH 2. Total nitrogen samples underwent digestion prior to analysis (Standard Methods 4500-N.C). Total nitrogen and nitrate were measured via second-derivative ultraviolet spectroscopy (Crumpton et al., 1992; Childress et al., 1999) using an HP 8453 Spectrophotometer.

Suspended Solids: Total suspended solids were determined by filtering a known volume of sample water (100-200 mL) through a pre-combusted and pre-weighed filter (0.45µm GF/C filters). Filters were dried to a constant weight at 105 degrees C, and this dry weight was used to determine total suspended solids (TSS). The filters were then combusted for 30 minutes at 550 degrees C before being weighed again. Volatile suspended solids (VSS) were determined via loss-on-ignition, and involatile suspended solids (ISS) were assumed to be the difference between volatile and total suspended solids.

Algal Pigments: Water samples were filtered under dark room conditions for analysis of algal pigments chlorophyll-a and phycocyanin. Filters underwent a freeze-thaw cycle and acetone extraction before being analyzed on a fluorometer.

High Frequency Sensors

Temperature Chains: Stings of high frequency temperature sensors were deployed at the deep sites of the study lakes in order to quantify water column thermal structure. We constructed temperature chains by securing HOBO 64K Pendant Temperature loggers on strings that we installed vertically in the water column, secured between a buoy and an anchor. Loggers were placed every 0.5 m and recorded temperature every 30 minutes.

Dissolved Oxygen: HOBO U26-001 Optical Dissolved Oxygen loggers were installed at the surface and 0.5 meter above the sediment water interface for a subset of study lakes and recorded dissolved oxygen concentrations every 30 minutes.

Water Quality: YSI KorEXO Multiparameter Water Quality Sondes were deployed in the surface waters at the deep site of Swan Lake (2018) and South Twin Lake (2019). Sondes recorded chlorophyll-a, phycocyanin, and dissolved oxygen concentrations; dissolved oxygen saturation, water temperature, pH, specific conductivity, and total dissolved solids every 30 minutes

Zooplankton

Field Collection: Zooplankton were collected at the historic deep site of each study lake during the summer months (May to September) from 2018-2020. The zooplankton data include samples that were collected by the research team at the ISU Limnology Laboratory and the IDNR Ambient Lake Monitoring Program. However, all samples followed similar field methods and were counted by the ISU Limnology Laboratory. Zooplankton samples were collected using a vertical Wisconsin net tow (63 micro-meter mesh size) from the surface of the lake to either 1m from the bottom (Carp project samples) or the depth of the thermocline if present or a maximum depth of 9 m (ALM project). Samples were preserved in a 10% formalin solution containing sucrose and later transferred to 70% ethanol.

Counting: For each sample, a 1 mL subsample was taken and a minimum of 60 individual zooplankton were identified to genus for cladocerans and rotifers, order for copepods, and class for ostracods. Copepod nauplii could not be identified to order and were simply identified as nauplii. If less than 60 organisms were in the subsample, a second 1 mL subsample was counted. Zooplankton lengths were measured for up to 25 individuals per taxon per sample to calculate dry mass per liter using length-mass regressions (McCauley, 1984; Dumont et al., 1975).

Benthic Macroinvertebrates

Field Collection: Benthic macroinvertebrates were sampled at 10 sites within each study lake once per summer from 2018 to 2020 using either an Ekman or a Ponar dredge.

Sample Processing: Collected sediments were sieved using 500-µm mesh (APHA, 2005; Baldwin et al., 2018) and preserved in amber bottles with 90% ethanol. Samples from the summer of 2020 were preserved similarly, but a 0.1% Rose Bengal Dye solution was added to assist with later sorting.

Counting: all invertebrates were removed from the sediment and identified to the lowest possible order or family. When possible, a stereomicroscope was used to identify organisms. Worms were identified to subclass and mollusks were identified to family or order. This level of taxonomic resolution has been found to be sufficient to reflect community patterns (Bowman and Bailey 1997). Sorted individuals were used to calculated taxon richness and density (number of individuals/m2).

Macrophytes

Field Sampling: Macrophytes were sampled along transects, perpendicular to the shoreline. Each study lake had 15-25 transects, depending on lake surface area. Each transect had sampling points at every 2-feet of water depth, starting at the shoreline. At each point, a visual assessment of plants within 2m of the boat was completed and a two-side rake was lowered to the bottom and used to collect submersed and floating-leaf macrophytes from an approximately 0.4 m2 area. Species presence and a qualitative estimate of density were recorded at each sampling site. We scored bed density on a scale of 0-3 based on whether the site yielded no plants on the sampling rake or in sight of the boat (score of 0), a few plants on the rake or visible within 2 m of the boat (score of 1), enough plants to fill the rake but leave the tines visible (score of 2), or enough plants to completely fill the rake leaving no part of the metal rake head visible (score of 3). Each transect was sampled until two points had been sampled and yielded no plants on the sampling rake (usually around 6-8 feet).

Identification and classification: Most macrophytes were identified to species, except for the macroalgae Chara spp. and duckweeds (Lemna spp.), which were identified to genus. Macrophytes species richness was summarized at a lake level, but also within functional groups based on growth forms (emergent, floating leaf, and submerged). Emergent macrophytes grow in shallow water, with most vegetative and all reproductive structures above the water. Common examples include cattails (Typha spp.) and bulrushes (Schoenoplectus spp.). Floating-leaf macrophytes, such as water lilies (Nymphaea spp.), are distinguished by leaves and flowers floating on the water surface. Submersed macrophytes spend the majority of their life cycle underwater, though some species may flower above the water surface. Pondweeds (Potamogeton spp.) are a common genus of submersed macrophytes (Dodds & Whiles 2010).

Sediment Core Incubations for Phosphorus Flux Rates

Field sampling: Intact sediment cores were collected at the deep site and 1-2 littoral sites within Swan, North Twin, and South Twin Lakes in June or July of 2019. For each sampling event, four replicate sediment cores were collected at each sampling site using a gravity corer (inner diameter 5 cm). The intact sediment cores and the overlying water were sealed in acrylic core sleeves and transported at 4°C. During sampling, vertical profiles of temperature, pH, and dissolved oxygen were taken using a YSI ProDSS multiparameter sonde.

Core incubation set-up: Back in the laboratory, the cores were exposed to temperature treatments corresponding to conditions at each site at the time of sampling. Temperature treatments were achieved by securing the cores in either a water bath or an incubation chamber. Two of the replicate cores were exposed to oxic conditions while the other two replicate cores were incubated under anoxic conditions. Dissolved oxygen levels were manipulated by bubbling either air mixtures (oxic treatment) or N2 (anoxic treatment) through the water overlying the sediment core. A slow, consistent bubble rate (~1 psi) was used to gently mix the water column without disturbing the sediment surface. After the cores have been installed in the ex situ incubation system, the total height of the water column within each core tube was measured and recorded. A known water volume needed to interpret how the P load in the water column changes over the incubation.

Core incubation sampling: Samples of the overlying water were collected approximately 12, 36, 60, 84, and 96 hours after the initial incubation set-up. For each daily sampling, 85 mL of water was removed for analysis of TP and SRP. An equivalent volume of hypolimnetic water from the corresponding sampling site was used to replace the water sampled. A sample of the replacement hypolimnetic water was also analyzed for TP and SRP daily to account for changes in water column P due to sampling and sample water replacement. SRP samples were filtered (0.45µm GF/C filters) and TP samples underwent a persulfate digestion prior to analysis (Standard Methods 4500-P B.5). Samples were analyzed the same day using a molybdenum blue method, modified from Murphy and Riley (1962; Standard Methods 4500-P E) on an Agilent Spectrophotometer.

Equilibrium Phosphorus Concentration

Sample collection: Sediments were collected from the deep site of each study lake using an Ekman dredge, sealed in plastic bags, and transported and stored at 4 degrees C. All analyses began within 36 hours of sample collection.

Equilibrium Phosphorus Concentration (EPC) Assay: For each lake, small subsamples of fresh sediment were placed into 50 mL polyethylene falcon tubes filled with 40 mL of a phosphate standard solution. For each lake, 10 different solutions were used (every 100 µg/L SRP from 0 to 1000 µg/L). The samples were placed on a shaker table for at least 12 hours and then centrifuged for 30 minutes at 3000 rpm. The supernatant was poured off, filtered, and analyzed for SRP (Standard Methods 4500-P E) on an Agilent Spectrophotometer. The EPC was determined based on the change in phosphorus in the solution per gram dry sediment (estimated based on sediment moisture content). A linear regression was developed for each lake to determine the SRP concentration at which there was no phosphorus exchange between the sediment and the solution (i.e., the equilibrium point).

Sedimentation and Sediment Disturbance

Sediment Traps: Tiered sediment traps were deployed at multiple locations within each study lake (deep site and 1-4 additional littoral sites) to estimate sedimentation rates and the resuspension of bottom sediments. The amount of resuspension can be estimated as the difference in sedimentation between the bottom traps (collecting settling material from the water column and resuspended materials) and the top traps (collecting only sedimentation from the water column). The traps were deployed for 11-15 days in the early season (May) and later in the summer (late July to early August).

Sample Processing: Water samples from each sediment trap were collected and transported back to the laboratory at 4 degrees C. They were filtered for total suspended solids within 36 hours of sample collection. Total suspended solids were determined by filtering a known volume of sample water through a pre-combusted and pre-weighed filter (0.45µm GF/C filters). Filters were dried to a constant weight at 105 degrees C, and this dry weight was used to determine total suspended solids (TSS). The filters were then combusted for 30 minutes at 550 degrees C before being weighed again. Volatile suspended solids (VSS) were determined via loss-on-ignition, and involatile suspended solids (ISS) were assumed to be the difference between VSS and TSS.

Methods References:

APHA. 2005. Standard methods for the examination of water and waste-water, American Public Health Association.

Baldwin, R. C., M. D. Sundberg, T. W. Stewart, and M. J. Weber. 2018. Evaluating alternative macroinvertebrate sampling methodologies in wetlands: Influence of sieve mesh size on relationships between environmental and assemblage variables. Wetlands 38: 677–687.

Bowman, M. F., and R. C. Bailey. 1997. Does taxonomic resolution affect the multivariate description of the structure of freshwater benthic macroinvertebrate communities? Can. J. Fish. Aquat. Sci. 54: 1802–1807. doi:10.1139/f97-085

Childress, C.J.O., W.T. Foreman, B.F. Conner, and T.J. Maloney. 1999. New Reporting Procedures Based on Long-Term Method Detection Levels and Some Considerations for Interpretations of Water-Quality Data Provided by the U.S. Geological Survey National Water Quality Laboratory. U.S. Geological Survey Open File Report 99-193. 24 p.

Crumpton, W.G., T.M. Isenhart, and P.D. Mitchel. 1992. Nitrate and organic N analyses with second-derivative spectroscopy. Limnology and Oceanography 37:907-913.

Dodds, W.K., and Whiles, M.R. Microbes and plants. In: Dodds, W.K., and Whiles, M.R., editors. Freshwater ecology: Concepts and environmental applications of limnology. New York: Elsevier; 2010. pp. 213-218

Dumont, H. J. et al. (1975) The dry weight estimate of biomass in a selection of Cladocera, Copepoda and Rotifera from the plankton, periphyton and benthos of continental waters. Oecologia, 19, 75–97.

McCauley, E. (1984) The estimation of the abundance and biomass of zooplankton in samples. In Downing, J. and Rigler, F. (eds), A manual on methods for the assessment of secondary productivity in fresh waters. Blackwell Publishing Ltd, Oxford (UK), pp. 228–265.

Murphy, J., and J.P. Riley. 1962. A modified single solution method for the determination of phosphate in natural waters. Analytica Chimica Acta 27:31-36.

Standard Methods for the Examination of Water and Wastewater. 1998. 20th Edition. Method 4500-N N.C.

Standard Methods for the Examination of Water and Wastewater. 1998. 20th Edition. Method 4500-P B.5.

Standard Methods for the Examination of Water and Wastewater. 1998. 20th Edition. Method 4500-P E.