Phosphorus speciation in a eutrophic lake by 31P NMR spectroscopy

Accepted Manuscript Phosphorus speciation in a eutrophic lake by

31 P NMR spectroscopy

Emily K. Read, Monika Ivancic, Paul Hanson, Barbara J. Cade-Menun, Katherine D. McMahon PII:

S0043-1354(14)00434-5

DOI:

10.1016/j.watres.2014.06.005

Reference:

WR 10716

To appear in:

Water Research

Received Date: 6 January 2014 Revised Date:

28 May 2014

Accepted Date: 3 June 2014

Please cite this article as: Read, E.K., Ivancic, M., Hanson, P., Cade-Menun, B.J., McMahon, K.D., 31 Phosphorus speciation in a eutrophic lake by P NMR spectroscopy, Water Research (2014), doi: 10.1016/j.watres.2014.06.005. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Orthophosphate monoesters

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Lake Mendota, WI, USA

Orthophosphate diesters

Sampling locations PP Particulate phosphorus DP Dissolved phosphorus

PP DP

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Yahara River

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Polyphosphate

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Phosphonate

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Pheasant Branch

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31P NMR Chemical shift (ppm)

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ant Branch i nflow Pheas

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Yahara R

iver ou

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Phosphorus speciation in a eutrophic lake by 31P NMR spectroscopy

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Emily K. Read*1,2, Monika Ivancic3, Paul Hanson2, Barbara J. Cade-Menun4, and Katherine D.

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McMahon5,6

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Cary Institute of Ecosystem Studies, Millbrook, NY, USA

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Center for Limnology, University of Wisconsin-Madison, Madison, WI, USA

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Department of Chemistry, University of Vermont, Burlington, VT, USA

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Madison, WI, USA

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and Agri-Food Canada, Swift Current, Saskatchewan, Canada

Civil and Environmental Engineering Department, University of Wisconsin-Madison,

Department of Bacteriology, University of Wisconsin-Madison, Madison, WI, USA

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*Corresponding author. Permanent address: Center for Limnology, University of Wisconsin-

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Madison, 680 N. Park St., Madison, WI, 53706. Email: [email protected]. Phone: 1 (231)

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557 2949

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Contributors (Required): EKR collected and analyzed samples, interpreted data, and wrote

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manuscript; MI performed nuclear magnetic resonance spectroscopy, data processing, methods

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development, and wrote manuscript; and BCM, KDM, and PCH contributed to methods

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development, data interpretation, and wrote manuscript.

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Abstract For eutrophic lakes, patterns of phosphorus (P) measured by standard methods are well documented but provide little information about the components comprising standard operational

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definitions. Dissolved P (DP) and particulate P (PP) represents important but rarely characterized

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nutrient pools. Samples from Lake Mendota, Wisconsin, USA were characterized using 31-

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phosphorus nuclear magnetic resonance spectroscopy (31P NMR) during the open water season

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of 2011 in this unmatched temporal study of aquatic P dynamics. A suite of organic and

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inorganic P forms was detected in both dissolved and particulate fractions: orthophosphate,

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orthophosphate monoesters, orthophosphate diesters, pyrophosphate, polyphosphate, and

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phosphonates. Through time, phytoplankton biomass, temperature, dissolved oxygen, and water

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clarity were correlated with changes in the relative proportion of P fractions. Particulate P can be

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used as a proxy for phytoplankton-bound P, and in this study, a high proportion of polyphosphate

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within particulate samples suggested P should not be a limiting factor for the dominant primary

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producers, cyanobacteria. Hypolimnetic particulate P samples were more variable in composition

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than surface samples, potentially due to varying production and transport of sinking particles.

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Surface dissolved samples contained less P than particulate samples, and were typically

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dominated by orthophosphate, but also contained monoester, diester, polyphosphate,

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pyrophosphate, and phosphonate. Hydrologic inflows to the lake contained more orthophosphate

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and orthophosphate monoesters than in-lake samples, indicating transformation of P from

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inflowing waters. This time series explores trends of a highly regulated nutrient in the context of

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other water quality metrics (chlorophyll, mixing regime, and clarity), and gives insight on the

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variability of the structure and occurrence of P-containing compounds in light of the phosphorus-

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limited paradigm.

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Keywords

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eutrophication, phosphorus, 31P NMR, cyanobacteria, Lake Mendota

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-Variations in time and space indicate major transformations of P-containing compounds

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-P-speciation was correlated with temperature, dissolved oxygen, and phytoplankton

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-Cyanobacteria biomass was positively correlated to in-lake polyphosphate and phosphonate

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1.

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Introduction Phosphorus (P) enrichment is widely recognized as the cause of aquatic eutrophication

(Schindler et al., 2008), and for more than a century, nutrient management to enhance water

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quality has occurred in the Lake Mendota, Wisconsin watershed (Brock, 1985). Despite

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sustained efforts, progress in reducing P loads has not occurred (Jones et al., 2010). Phosphorus

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enters Lake Mendota by external loading by rivers and runoff (Soranno et al., 1997), internal

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loading from the anoxic hypolimnion and sediment (Holdren and Armstrong, 1980; Soranno et

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al., 1997), and atmospheric deposition (Mahowald et al., 2008; Soranno et al., 1997). While total

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P (TP) remains elevated in eutrophic systems, dissolved reactive P (DRP) routinely falls below

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the limit of detection in surface water, due to rapid uptake by phytoplankton (Currie and Kalff,

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1984; Hudson et al., 2000) and reduced solubility under oxic conditions. Low measurable

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concentrations of DRP during summertime stratification coincide with harmful algal blooms

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throughout the open water season in eutrophic systems, including Lake Mendota (Lathrop,

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2007).

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Orthophosphate (OrthoP) is the preferred P substrate for both phytoplankton and

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bacterioplankton in aquatic systems (Rigler, 1956; Wetzel, 2001), but more complex P-

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containing compounds can also be used by bacteria and algae (Cotner and Wetzel, 1992; Løvdal

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et al., 2007). Complex dissolved P forms may be undetected by standard water quality

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measurements, but have been confirmed and characterized in freshwaters (e.g., Nanny and

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Minear, 1997; Cade-Menun et al., 2006; Reitzel et al., 2006). Phosphorus-31 nuclear magnetic

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resonance spectroscopy (31P NMR) is a tool to identify and quantify complex P-containing

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molecules to address questions related to the transformation of P (Cade-Menun, 2005). TP and

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DRP are well documented for freshwater systems, but the dynamics, structure, and concentration

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of the unreactive P-containing compounds in eutrophic lake water, as can be characterized by 31P

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NMR, are largely unknown.

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The chemical makeup of aquatic particulate P (PP) has likewise rarely been characterized in aquatic systems (but see Cade-Menun et al., 2006; Ding et al., 2010a). For routine water

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quality monitoring, total particulate P (PP) can be measured by standard methods (TP of acid-

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digested 0.45 um filter, following filtration of water sample), or calculated as the difference

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between TP and total DRP. Particulate P in eutrophic systems can represent a large proportion of

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total P, but like dissolved ‘unreactive’ P, the characteristics of PP through time and space in

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eutrophic lakes are unknown. Characterization of marine dissolved P (DP) and PP using 31P

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NMR has provided insights about the transformations between the two fractions, and it is

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speculated that the biological community composition may drive the composition of marine PP

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(Paytan et al., 2003). In systems dominated by cyanobacteria the characteristics of PP could be

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used as a proxy for internal phytoplankton P, and may point to physiological nutrient status. For

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a system in which cyanobacterial blooms abound, phytoplankton nutrient status may reflect the

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temporal dynamics of P limitation for the dominant primary producers in the system, which in

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turn could be used to inform nutrient management with the goal of reducing eutrophication.

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Understanding how bioavailable forms of P, not only those measurable by standard methods, vary through time and space is critical for aquatic scientists and lake managers

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interested in the prediction and prevention of harmful algal blooms. In this study of Lake

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Mendota, a dimictic eutrophic temperate lake, we used 31P NMR spectroscopy to characterize the

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dynamics of DP and PP at two discrete depths (0.5 and 14 m) from May through October of

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2011, and at two hydrologic inflows. Epilimnetic P-limitation was expected to coincide with

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reduced concentrations of labile DP and PP compounds (pyrophosphate (PyroP) and

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orthophosphate esters). We expected phytoplankton-bound P to reflect extended P limitation,

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indicated by limited internal P-storage compounds (i.e., polyphosphate (PolyP)). Physical and

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steric constraints were expected to make particulate P more recalcitrant and less variable through

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time than dissolved fractions. To interpret the spatio-temporal patterns of P speciation in terms of

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important water quality indicators including chlorophyll-a concentration, dissolved oxygen,

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clarity, temperature, and phytoplankton biomass, we analyzed water quality data sampled

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concurrently by the North Temperate Lakes Long Term Ecological Research program (NTL

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LTER, lter.limnology.wisc.edu). Our observations allow qualitative conclusions about the

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transformations P-containing molecules through space and time.

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2.

Methods

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2.1

Site description

Lake Mendota (43°06′24″ N 89°25′29″ W) is a calcareous eutrophic lake located in south

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central Wisconsin, in a 686 km2 watershed dominated by dairy, cattle, and forage crop

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agriculture. The lake has a surface area of 36 km2 with mean and maximum depths of 12 and 25

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m, respectively. The mean hydrological residence time is ~ 4.5 years and the lake has three main

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hydrologic inflows and one outflow. Lake Mendota is dominated by cyanobacteria biomass

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(typically Microcystis and Aphanizomenon) from mid-June through the fall turnover event.

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Physical, chemical, and biological variables concurrent with 31P NMR sample range were

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collected and analyzed by the NTL LTER program using standard methods (NTL-LTER, 2011a,

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2011b, 2011c). These variables include temperature, dissolved oxygen, clarity (Secchi depth),

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integrated surface (0-2m) chlorophyll-a, phytoplankton biomass microscopic counts, historical

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colorimetric DRP, and historical acid-digested colorimetric TP. Subsets of these data are shown

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in Figure 1; the complete dataset is provided in the Supplemental Materials in tabular format.

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We sampled at approximately biweekly frequency from May 2011 through October 2011

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at the location of maximum depth from Lake Mendota, and on three occasions (July and October

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2011 and March 2012) from two major hydrologic inflows to the lake: Pheasant Branch

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(sampled at Century Avenue overpass, 43°6'25.2" N, 89° 28' 58.8" W) and the Yahara River

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(sampled at Highway 113 overpass, 43°9'3.6" N, 89°24'7.2" W) (Table 1). Samples were

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collected at these locations under a range of flow conditions (low, average, and high flow), from

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72-110 cubic feet per second (cfs) for the Yahara River to 2-4 cfs for Pheasant Branch.

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On Lake Mendota at the geographic location of maximum lake depth, samples were drawn from 0.5 m and 14 m using a horizontal Van Dorn sampling device, and from 0.5 m depth

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at the center of the channel from the two inflowing rivers using a grab sampler. Surface (0.5m)

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and 14m sampling depths always occurred in the upper, mixed portion of the water column and

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well below the thermocline, respectively. For convenience, we refer to these locations as

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epilimnion and hypolimnion hereafter, although they refer to discrete depths sampled within

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those layers, not an integrated sample of the entire layers.

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Water was stored in acid-washed bottles on ice until further processing. Approximately

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8-10 L were collected from each site/depth. Within one hour of sample collection, water was

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filtered through 0.45-µm nitrocellulose filters; filters were pre-soaked in de-ionized water from

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between 1-3 h prior to filtration. Filtrate was immediately frozen in acid-washed ice cube trays at

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-20 oC and filters were stored at -20 oC, both remaining frozen until further processing. Prior to

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extraction, filtrate was concentrated to dry solids by lyophilization. A sample with an original

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volume of 8 L typically resulted in ~ 2.5 g of dry solids. Colorimetric determination was used to quantify DRP (after 0.45 µm filtration), and total

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and total dissolved P (TP and TDP, after potassium persulfate acid digestion) using the

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ammonium molybdate method (Murphy and Riley, 1962), with ascorbic acid reductant. All

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reported values reflect mean value of three replicates. Iron (Fe) and manganese (Mn)

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concentrations were determined for a subset of samples (epilimnetic and hypolimnetic particulate

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samples prior to extraction and in concentrated NMR-ready solute) using inductively coupled

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plasma optical emission spectroscopy (ICP OES) performed by the Wisconsin State Lab of

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Hygiene (SLOH) analytical laboratory (Table 1). The Fe concentration never exceeded the limit

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of detection for the SLOH ICP OES (0.1 mg Fe L-1) in samples prior to concentration, nor in the

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~3000 fold concentrated samples (data not shown). For that reason, we considered only P/Mn

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ratios and the potential effect of these paramagnetic ions on T1 relaxation times. Although we did

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not analyze T1 relaxation times explicitly for this study, for most samples (>75%) analyzed for

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Mn and Fe, a T1 relaxation time of 5s should be adequate for quantitative spectra for a variety of

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P-compounds (McDowell et al., 2006). When inadequate T1 relaxation time is used, it is advised

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to interpret peaks by presence/absence, rather than quantitatively. In this study, we approach

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spectra interpretation quantitatively. However, our findings do not rely on quantitatively

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determined patterns only: important patterns are robust to interpretation by peak

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presence/absence as well.

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2.3

Extraction and preparation

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Particulate P (PP) samples were extracted in 50 mL acid-washed falcon tubes with 15 mL each of 0.5 M NaOH and 0.1 M EDTA for 16 h at room temperature on a shaking plate.

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Following shaking, tubes were centrifuged for 20 min at 2000 rpm; supernatant was decanted

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and filtered through pre-soaked 0.45-µm nitrocellulose filter (Millipore) and frozen at -80 oC.

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After freezing, samples were lyophilized, and solids were stored at -20 oC until final preparation

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for NMR experiments.

Following filtration, dissolved P (DP) samples were frozen at -20 oC. Frozen samples

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were lyophilized in acid-washed freeze dry flasks; solids were collected after all water was

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removed, and were stored at -20 oC until extraction.

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Lyophilized DP solids were pre-extracted: solids were dissolved in 15 mL 0.1 M EDTA and shaken for 15 min, then centrifuged at 2000 rpm for 10 min (Hupfer et al. 2004). Supernatant

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was decanted and discarded, and remaining solids were extracted in the same manner as the

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particulate fraction: samples were extracted with 15 mL each of 0.1 M EDTA and 0.5 M NaOH

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for 16 h, on a shaker plate. Samples were then centrifuged at 2000 rpm for 10 min and liquid was

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decanted and frozen. Following freezing, samples were lyophilized and stored at -20 oC until just

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prior to NMR experiments. EDTA pre-treatment and extraction has the potential to decrease the

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amount of P extracted from sediments (Ding et al. 2010b). However, Ding et al showed that for

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calcareous sediments, the effect of EDTA pre-treatment was minimal. Although we did not

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explicitly test the effect of EDTA pre-treatment, the potential for loss via EDTA pre-extraction

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warrants consideration upon interpreting NMR results.

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Lyophilized PP and DP samples were re-suspended in D2O, 0.5 and 1 M NaOH, 0.1 M

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EDTA, and H2O following methods of Cade-Menun et al. (2006) to a total volume of 1.4 - 2.4

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mL or ~ 4 mL, depending on the use of a 5-mm or 10-mm NMR probe. Freeze-drying and

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subsequent dissolution has a risk of loss of organic P due to incomplete dissolution (Xu et al.

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2012); however, our lyophilized samples dissolved completely at this step. In some cases, extra

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D2O was added to 5-mm samples in order to decrease the viscosity to allow for adequate

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shimming. The 31P NMR experiments were conducted within 1 h of sample preparation.

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2.4 31P NMR Experiments and data processing

Particulate P spectra were acquired on a 500 MHz Varian Unity spectrometer equipped

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with a 5-mm NMR broadband probe and DP spectra on a 360 MHz Bruker Avance spectrometer

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equipped with a 10-mm NMR broadband probe. The data was acquired at 24 °C, using a 90°

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pulse, a 0.70 s acquisition time and a 4.5 s relaxation delay, with 20 Hz spinning (5-mm probe)

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and 12 Hz spinning (10-mm probe). The number of scans varied from 1200 (PP) to 11000 (DP).

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A spectral width of 24554 Hz or 121 ppm was used for the data acquired on the 500 MHz Varian

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Unity spectrometer, while a spectral width of 17483 Hz or 120 ppm was used for data acquired

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on the 360 MHz Bruker Avance spectrometer. Data were processed using the MestReNova

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NMR processing program, applying a 7 Hz exponential for full spectral integrations and a 2 Hz

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exponential for integration of specific regions (7 ppm to 2.5 ppm and 2.5 ppm to -4.5 ppm).

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For external reference shift standardization, samples were spiked with a small amount of

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PO4 at the end of the NMR experiment after adequate signal from sample was achieved, and this

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peak was referenced to 6.0 ppm as an internal standard. Adenosine monophosphate (AMP) was

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also added to several samples to confirm peak assignment of this compound. Preliminary

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standardization for all samples was done using D2O, which typically resulted in assignment of

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the orthophosphate peak within +/- 0.2 ppm from 6.0ppm.

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Peak assignments were made using 31P NMR chemical shifts of OrthoP (6 ppm),

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pyrophosphate (PyroP, -4 ppm) and polyphosphate (PolyP, -17 to -22 ppm), phosphonate (Phos,

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18 to 22 ppm), and orthophosphate monoesters (MonoP, 5.9 to 3.6 ppm) and diesters (DiP, 2.5 to

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-1 ppm), which are well established in the environmental chemistry literature from river, lake,

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and marine samples (e.g. (Cade-Menun et al., 2006; Kolowith et al., 2001)). The relative

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proportions of P species were estimated by integration of 31P NMR spectral peaks. For relative

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proportions of polyphosphate and pyrophosphate, we combined peak height into one class

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(PyroP+PolyP), as PolyP can be hydrolyzed to pyrophosphate during alkaline extraction (Cade-

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Menun et al., 2006).

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2.5 Regression Analysis

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In order to quantify the relationships between P species in Lake Mendota and associated environmental variables, we performed regression analyses on the time series of PP in the

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epilimnion and hypolimnion of Lake Mendota. We regressed each P fraction against eight

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environmental variables hypothesized to be related to P dynamics. Depth-specific environmental

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variables included in the regression analysis were temperature; dissolved oxygen; Secchi depth;

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Chl-a concentrations from 0-2m integrated sampling; and log10-transformed biomass of five

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phytoplankton groups: Cyanophyta, Cryptophyta, Chrysophyta, Chlorophyta, and

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Bacillariophyta, all analyzed from a 0-8m integrated sample (NLT-LTER, 2011c). Because

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phytoplankton biomass data contained zeros, log10(x + 0.0001), where x = biomass,

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transformation was applied to biomass data. Regressions included environmental variables were

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collected, on average, within of three days of NMR sample collection; all data used in

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regressions can be found in Table 1 or Supplemental Table 2. Due to a low number of samples

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collected and analyzed for Lake Mendota DP samples, and for PP and DP of hydrologic inflows,

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the regression analysis only included PP samples, for which a suite of concurrent environmental

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variables were available.

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Because the dataset had a temporal component, we tested for autocorrelation for each pairwise regression using the ccf function in R and discarded pairs with significant

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autocorrelation (R2 >0.6) before subsequent regression analysis. Data were screened for

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normality by visual inspection using the qqnorm function from the “stats” R package (R Core

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Team, 2014). We report linear coefficient of determination (R2) and significance values (p-

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values), which were calculated in the R programming environment (R Core Team, 2014) using

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the cor function from the “stats” R package. In order to correct for type 1 errors associated with

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multiple pairwise comparisons for each NMR species, we applied a Bonferroni, shown in

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Equation 1:

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Eq. 1

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where p-valueB is the Bonfoerroni corrected significant level, p-value is the uncorrected statistic,

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and n is the number of pairwise comparisons made (n=8 for all P species analyzed); all p-values

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reported have been Bonferroni-corrected.

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3.

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3.1

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Results

Colorimetric determination of lake phosphorus Timing and magnitude of TP and DRP trends for epilimnetic and hypolimnetic samples

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were characteristic of historical patterns in this lake (Brock, 1985; NTL-LTER, 2011;

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Supplemental Figure 1 and Supplemental Table 1). Surface (0.5 m) TP and DRP concentrations

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decreased from May through September, with epilimnetic DRP falling below limits of detection

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from day 179 (July 12, 2011) through day 264 (September 21, 2011; Figure 1a). Epilimnetic TP

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and DRP both increased significantly by day 278 (October 5, 2011), following a drop in air

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temperature and an extreme wind event, which resulted in thermal mixing of the lake. Mean

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epilimnetic TDP increased from 15 µg L-1 on day 123 to 65 µg L-1 on day 153, after which it

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remained at or near the limit of detection from days 193-264. Epilimnetic TDP increased to 41

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µg L-1 by day 278. Hypolimnetic (14 m) TP and DRP both increased from day 123 (May 3,

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2011) through day 264 (September 21, 2011), with DRP comprising the bulk of TP at 14m

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(Figure 1a). Hypolimnetic TDP increased monotonically from day 123 through day 264, from 21

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to 295 µg L-1, and decreased again after thermal mixing to 115 µg L-1 on day 278. The difference

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between TDP and DRP, termed dissolved unreactive P (DURP), was estimated for both

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epilimnetic and hypolimnetic samples. Epilimnetic DURP was <10 µg L-1 for all observations,

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while hypolimnetic DURP ranged from <10 to 33 µg L-1.

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3.1

Lake particulate phosphorus

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Twenty PP samples from the epilimnion (0.5m) and hypolimnion (14m) of Lake Mendota from day 123 (May 3, 2011) through day 278 (October 5, 2011) were analyzed by 31P NMR

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spectroscopy (Table 1, Figures 1 and 2). Total PP (following acid digestion) ranged from 10 to

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35 µg P L-1 (TPP, Table 1). Total P following extraction and preparation (NMR TP) ranged from

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1 to 27 µg P L-1, with extraction efficiencies of 4-99%. Inorganic P, the sum of orthophosphate

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(OrthoP), polyphosphate (PolyP) and pyrophosphate (PyroP) accounted for between 31-69% of P,

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while organic P compounds accounted for the remainder. For both the epilimnetic and

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hypolimnetic samples, PyroP+PolyP and phosphonate represented relatively greater proportions

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of P present during mid- summer through fall (after day 180), particularly for epilimnetic

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samples (Figure 1b). The maximum fraction of PyroP+PolyP was observed in the epilimnion on

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day 248 (42%), while the maximum fraction observed in the hypolimnion occurred two weeks

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later on day 264 (56%). Orthophosphate monoesters (MonoP) and diesters (DiP) represented

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significant fractions of PP for both epilimnetic and hypolimnetic samples (11-44% and 0-31%,

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respectively). Example spectra from each sampling location are shown in Figure 2.

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Regression analysis revealed significant correlations between epilimnetic and hypolimnetic PP and environmental variables (Table 2). Epilimnetic PyroP+PolyP was

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significantly correlated to Bacillariophyta biomass (R2=-0.88, p<0.01) and temperature (R2=0.78,

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p<0.1). Hypolimnetic Phos was significantly correlated to dissolved oxygen (R2=-0.82, p<0.1)

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and Cyanophyta biomass (R2=0.78, p<0.1). Hypolimnetic MonoP was significantly correlated to

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dissolved oxygen (R2=0.80, p<0.1), Bacillariophyta biomass (R2=0.85, p<0.01), and Cyanophyta

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biomass (R2=0-.78, p<0.1). For both the epilimnion and the hypolimnion, Phos and PyroP+PolyP

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was consistently positively correlated to Cyanophyta biomass and negatively correlated to

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Bacillariophyta, although not all correlations were statistically significant (Table 2). For

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epilimnetic and hypolimnetic OrthoP and MonoP, the opposite trend was observed: positive

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correlations with Bacillariophyta and negative correlations with Cyanophyta, although not all

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statistically significant, were present.

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3.2

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Lake dissolved phosphorus Dissolved P, when detected, varied in the relative proportion of dominant P species

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(Table 1). Inorganic P accounted for all P detected in several samples (day 179 epilimnetic and

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hypolimnetic samples), while on day 278, only organic P (phosphonate) was observed in the

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hypolimnetic DP sample. Day 206 epilimnetic and day 278 hypolimnetic samples contained the

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greatest diversity of compounds (Table 1). Day 278 hypolimnetic P more closely resembled lake

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and river particulate samples, unlike day 206 epilimnetic signature, for which monoesters were

310

conspicuously missing, while OrthoP, DiP, and PyroP+PolyP were present.

311 312

3.3

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Phosphorus from hydrologic inflows

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For the Yahara River and Pheasant Branch, typical flow conditions on day 208 (July 28, 2011), base flow conditions on day 264 (September 21, 2011), and spring runoff conditions on

315

day 67 of 2012 (March 7, 2012) were characterized using 31P NMR. DRP ranged from below

316

limit of detection (<10 µg L-1) to 41 µg L-1 and TP ranged from 35-276 µg L-1 (Table 1).

317

Inorganic P comprised the largest fraction of detected PP in Pheasant Branch (60-82%), while

318

inorganic P from the Yahara represented 35-47% of P detected. MonoP was the most dominant

319

form of organic P of the Yahara samples (28-48% of total P), and represented 9-28% of TP from

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Pheasant Branch samples. Particulate phosphonate was detected once, on day 67 (March 7, 2012)

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in the Yahara River, under spring runoff conditions.

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Dissolved P was detected in a subset of samples: day 209 and 67 (2012) from the Yahara and day 67 (2012) from Pheasant Branch (Table 1). On day 209, DRP and TDP were near or

324

below the limit of detection, and no DP was detected in in the NMR experiment. For day 67,

325

characterizing spring runoff, 100% of P detected by NMR from the Yahara River was in the

326

OrthoP region, while for Pheasant Branch, P was comprised of OrthoP (66%) and MonoP (34%).

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On day 67 at the Yahara River, phosphonate was detected in the dissolved fractions.

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4.

Discussion

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In 2011, standard measurements of TP and DRP were characteristic of a stratified eutrophic lake, and consistent with historical patterns in Lake Mendota, WI (Supplemental

332

Figure 1). 31P NMR, in contrast, revealed a diverse and dynamic landscape of dissolved and

333

particulate P-containing compounds (Table 1 and Figure 1). Six classes of P compounds were

334

observed in Lake Mendota and its inflows: inorganic OrthoP, PolyP, and PyroP; and organic

335

Phos, MonoP, and DiP. The characteristics of each sample fraction (dissolved or particulate)

336

varied across time, depth and location, suggesting biogeochemical transformations across P

337

pools. We investigated transformations between dissolved and particulate fractions at single

338

locations over time, transformations between inflow and in-lake P, differences across depths, and

339

environmental correlates. Although this dataset does not permit calculation of fluxes between P

340

pools or across space, data on the characteristics of P standing stocks in the context of well

341

established ecosystem processes provide a rich and relevant perspective on P nutrient limitation

342

and bioavailability in Lake Mendota, WI.

345

4.1

Temporal dynamics of DP

Although 31P NMR DP samples were concentrated ~ 1000 fold, for samples in which P

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measured by colorimetry was below the limit of detection (e.g., day 206 and 234 epilimnetic

347

samples) no dissolved P was detected by 31P NMR. Dissolved P samples by NMR showed

348

OrthoP typically dominating hypolimnetic samples, and one epilimentic sample early in the

349

season. After turnover, however, epilimnetic DP increased and included several P species (Table

350

1). The difference between DP at the surface and 14m depths on this day (278) is surprising

351

considering the proximal (~2 days prior) whole-lake mixing event. Hypolimnetic P was likely

352

the source of the diverse P compounds found in the epilimnion after mixing, but the processes by

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which either particulate P became dissolved, or by which OrthoP was transformed to more

354

complex molecules, is unknown.

356

4.2

Dissolved and particulate P interactions

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Phytoplankton play a key role in Lake Mendota’s biogeochemistry, and the biomass of

358

the most dominant phytoplankton was significantly correlated to key P concentrations through

359

time (Table 2). Phytoplankton community succession after ice-off is characterized by four

360

stages: diatom (Bacillariophyta) dominated, zooplankton-dominated clear water phase,

361

cyanobacterial (Cyanophyta) dominated, and finally a diatom-dominated period prior to ice-on

362

(Brock, 1985; Supplemental Table 2). In living cells, P may be present as nucleic acids (diesters

363

MonoP and DiP), lipids (diesters MonoP and DiP), lipopolysaccharides (MonoP and DiP

364

degradation products), and cytoplasmic solutes (OrthoP, soluble PolyP and PyroP, and others).

365

Sinking marine PP has been shown to be comprised of phytoplankton-derived detritus, and

366

generally reflects marine phytoplankton PP speciation (Paytan et al., 2003), and here we assumed

367

PP from in-lake samples to be biogenic: epilimnetic (0.5m) samples likely representative of

368

living cells, and hypolimnetic (14m) samples are likely comprised of detritus of phyto- and

369

bacterioplanktonic origin. Particulate P, and presumably internal phytoplankton P, was dynamic

370

through time in Lake Mendota (Figure 1b). During 2011, PolyP+PyroP and phosphonate were

371

the most variable fractions of PP in both surface and hypolimnetic samples (Figure 1b), and both

372

of these fractions were significantly correlated to key environmental variables. Below, we

373

discuss the ecological relevance of these two P species and implications for water quality of

374

Lake Mendota.

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376 377

4.2.1

Polyphosphate The large contribution of PyroP+PolyP to total particulate epilimnetic P from day 123

through day 248 is compelling in light of the dogma of Lake Mendota as a P-limited system

379

during summertime stratification in the epilimnion (Brock, 1985). PolyP and PyroP can be

380

accumulated intracellularly by all organisms as energy or nutrient storage molecules, and are

381

known to serve a variety of intracellular functions including metabolism, stress response, and

382

structural function (Brown and Kornberg, 2008). Conventional knowledge of both natural and

383

engineered systems views cellular accumulation of PolyP indicative of ‘luxury uptake’ of P,

384

thought to occur in the presence of P following periods of P starvation (Hupfer et al., 2007). This

385

model of bacterial PolyP metabolism, in which accumulation of PolyP is associated with excess

386

external P concentrations, is harnessed to remove P from wastewater, and is also thought to be

387

important in aquatic systems under nutrient-replete conditions, as protection for future nutrient-

388

limiting conditions (Hupfer et al., 2004; McMahon and Read, 2013). In contrast, PolyP

389

accumulation has also been associated with osmotic stress in both cultured phytoplankton and

390

natural samples, and the concept of nutrient limitation as the primary cause of intracellular PolyP

391

accumulation is an area of debate (e.g., see Feuillade et al., 1995; Kornberg et al., 1999; Cade-

392

Menun and Paytan, 2010).

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PolyP and PyroP were positively, but not signifincantly, correlated to Cyanobacterial

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biomass, but this fraction was negatively correlated to spring- and fall-dominant phylum

395

Bacillariophyta (Table 2), and positively correlated to temperature (Table 2). For Lake Mendota,

396

increasing concentrations of intracellular PyroP+PolyP in epilimnetic samples contradicts the

397

notion of P limitation for phytoplankton. Although strong evidence for P limitation on long

398

timescales exists (Schindler et al., 2008), the importance of N and P co-limitation also has wide

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support (Conley et al., 2009). The results of the present study suggest that regardless of the

400

mechanism (luxury uptake, N stress response (Kornberg et al., 1999), or otherwise),

401

phytoplankton are not so limited by P as to prevent accumulation of PolyP. Phosphorus is

402

targeted as the main cause of surface water eutrophication at the state and regional level, but

403

significant reduction of P input to the system is still required to reduce nuisance algal blooms and

404

improve water clarity (Lathrop et al., 1998). Until in-lake levels of P approach the threshold of

405

limitation for primary producers, additional research to identify and control alternative limiting

406

nutrients (e.g., N) for noxious cyanobacteria could have important implications for water nutrient

407

and water quality management.

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PyroP+PolyP concentrations were variable in the hypolimnion, and were negatively correlated to Bacillariophyta biomass, but not significantly. Ranging from 0-56% contribution,

410

the source of this P component could be sinking phytoplankton-derived detritus enriched in

411

PyroP+PolyP; although no correlations were observed, which could be due to a lag in epilimentic

412

phytoplankton production and transport to the hypolimnion. Phytoplankton production and

413

senescence occur at varying rates over the open water season, as indicated by chlorophyll-a

414

concentration, dissolved oxygen, and clarity (Figures 1c and 1d; Brock, 1985). Variable

415

production, degradation, and transport by entrainment or rapid deepening of the thermocline due

416

to storm or wind events could result in time-variant transport of PyroP+PolyP to the hypolimnion.

417

Once in the hypolimnion, PolyP and PyroP are rich energy substrates for heterotrophic bacteria,

418

and could be rapidly degraded under hypolimnetic anaerobic conditions (Hupfer et al., 2007).

420

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4.2.2 Phosphonates

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421

Phosphonates were detected primarily in particulate samples during the second half of the sampling range, and were correlated with Cyanophyta biomass and dissolved oxygen in both the

423

epilimnion and hypolimnion, but only significantly in the hypolimnion (Table 2). Positive

424

correlation of Phos with Cyanophyta biomass indicate that the source of phosphonate detected in

425

particulate samples may be cyanobacterial production (Dyhrman et al., 2009; Namikoshi and

426

Rinehart, 1996). Similar to marine systems (Dyhrman et al., 2007), internal and external sources

427

of phosphonate have not been described in detail for freshwater systems, but given the statistical

428

relationship between Phos and cyanobacterial biomass, further study of the potential production

429

of phosphonates within the lake is warranted. External loading is another possible source of

430

phosphonates, potentially entering the lake as agricultural chemicals such as glyphosate bound to

431

suspended sediments.

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Phosphonates were detected in only two dissolved samples: epilimnetic DP from day 278 (October 5, 2011), and DP from the Yahara river on day 67 (March 7, 2012). Sources of

434

dissolved phosphonate may include biogenic sources (cyanobacterial excretion or cell lysis) or

435

loading from external synthetic sources (i.e., glyphosate application to residential land or

436

agricultural crops). Synthetic phosphonate, such as glyphosate, and its degradation product

437

aminomethyl phosphonic acid (AMPA), can be distinguished from naturally occurring

438

phosphonate, such as 2-aminoethyl phosphonic acid (AEP), by chemical shift (B. J. Cade-Menun,

439

unpublished data). In the two DP samples in which phosphonate were detected, the chemical

440

shift of phosphonate peaks were consistent with synthetic sources of phosphonate as glyphosate

441

(17.058 ppm) and its breakdown product AMPA (19.824 ppm), rather than naturally occurring

442

phosphonate. However, we did not confirm the identification of these peaks with spiking

443

experiments, and so can only speculate on the assignment of these peaks to specific P

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compounds. Further research is warranted to specifically identify these P forms. There is

445

evidence for greater recalcitrance of Phos in marine environments as compared to MonoP and

446

DiP (Clark et al., 1998), but phytoplankton utilization of phosphonate is also documented

447

(Beversdorf et al., 2010; Lipok et al., 2007; Lipok et al., 2009). After dissolution of PP due to

448

cell lysis or excretion, the complex P-containing compounds detected in PP samples likely

449

undergo rapid microbial mineralization, explaining the very limited detection of phosphonates in

450

dissolved samples.

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4.3

Hydrologic inflows

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For 2011 samples, differences between P concentration and speciation in hydrologic inflows and lake samples were notable. Dilution, sedimentation of particles, OrthoP uptake by

455

primary producers and microbial heterotrophs, and enzymatic hydrolysis of OrthoP from more

456

complex molecules all may contribute to differing P signatures. The residence time of Lake

457

Mendota is ~5 years (Brock, 1985), and on an annual basis, inflow volume from the Yahara

458

River and Pheasant Branch represents < 20% of total lake volume; thus dilution of river P is an

459

important factor responsible for differences between in-lake and river P characteristics. Notably,

460

phosphonate was only observed in the Yahara River and only on a single occasion, during the

461

spring runoff event occurring on March 7, 2012 (day 67). Phosphonate accounted for 16% of PP

462

and 100% of DP on this date, with peaks characteristic of synthetic glyphosate and its breakdown

463

product, AMPA (B. J. Cade-Menun, unpublished data). Thus, river Phos may have agricultural

464

herbicide sources, but this conclusion is based on a small sample size, infrequent detection, and

465

was not confirmed with detailed spiking experiments. River samples tended to contain more

466

MonoP and less PyroP+PolyP than lake samples. Uptake of OrthoP and mineralization of

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MonoP by in-lake plankton, followed by storage as PyroP+PolyP may be an important

468

transformation process, and confirms bioavailability of DURP. The differences between river

469

and in-lake P characterization, together with the relatively long residence time, indicate that in-

470

lake uptake, transformations, and production of P are more important to in-lake P signature than

471

loading from hydrologic inflows.

472

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The hydrologic inflows sampled during this study were physically distinct: 1) the Yahara River is a 4th order stream while Pheasant Branch is 3rd order, resulting in the Yahara delivering

474

~ 20-100 times greater mean discharge to Lake Mendota than Pheasant Branch; and 2) Pheasant

475

Branch underwent fish habitat restoration at the sampling site prior to and during sampling

476

events. Pheasant Branch was modified for a shallower, wider, braided channel to enhance aquatic

477

macrophyte and fish spawning habitat. Although erosion-control devices including geo-

478

membranes and hay berms were placed during restoration, the impact of riparian construction on

479

water quality was apparent: PP from Pheasant Branch captured on filters in the lab qualitatively

480

different (darker in color) than from the Yahara, likely because of higher concentration of

481

sediments suspended as a result of construction.

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Differences in PP speciation between the hydrologic inflows were evident (Table 1). In

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particular, OrthoP dominated Pheasant Branch PP, while MonoP dominated the Yahara River PP.

484

The Yahara and Pheasant Branch River PP were more dominated by non-OrthoP forms of P than

485

river PP speciated in a previous study (Cade-Menun et al., 2006). Although OrthoP was on

486

average the most abundant form, MonoP, DiP, and PyroP+PolyP accounted for overall greater

487

fractions in these systems than those observed in the Pee Dee River, South Carolina, by Cade-

488

Menun and others (2006). Generally, Pheasant Branch more closely resembled the Pee Dee

489

River than the Yahara River. DP in both inflows was low (< 10 µg L-1) on the three sampling

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dates, and when detected by 31P NMR spectroscopy, was variable in composition (Table 1).

491

Urban and agricultural construction runoff is regulated at the regional level to protect

492

downstream water quality (Jones et al., 2010), and because our sampling occurred during

493

construction, we have an interesting glimpse at the nature of construction-affected river PP.

494

Construction runoff at Pheasant Branch may have resulted in increased OrthoP loading to Lake

495

Mendota.

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498

5.

Conclusions

The application of 31P NMR at the temporal and spatial resolution used in this study is

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unprecedented for freshwater aquatic systems and provides a detailed account of the dynamics of

500

P-containing compounds in a eutrophic lake. Regression analysis with biogeochemical factors

501

provided insight into potential mechanisms for the P dynamics observed, including

502

phytoplankton dynamics, water temperature, dissolved oxygen, and water clarity.

503

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Generally, PP samples were comprised of phosphonate, MonoP, DiP, OrthoP, and PyroP+PolyP. Epilimnetic PP samples were more consistent in the relative proportions of the

505

compound classes than hypolimnetic samples, which could be due to consistent buildup of

506

epilimentic P in biomass coupled with differential degradation of sinking materials. Particulate

507

phosphonates in the epilimnion and hypolimnion were positively correlated with cyanobacterial

508

biomass. Epilimnetic and hypolimnetic DP contained all of the compound classes found in

509

particulate samples, but were typically dominated by OrthoP, and in some cases contained no

510

detectable P. Hydrologic inflows from 3rd and 4th order streams entering the lake were distinct

511

from each other: one site was undergoing extensive habitat restoration construction during the

512

sampling period, and typically contained significantly more OrthoP as a relative proportion of

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513

total PP detected. River DP was generally much lower in concentration than PP and was

514

dominated by OrthoP. River P signatures varied from in-lake characterization, indicating major

515

transformations occurring to externally-loaded P. Our hypothesis that during periods of low DRP, concentrations of labile dissolved and

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516

particulate P would be reduced and that some standing stock of dissolved recalcitrant P would be

518

maintained through the open water season was not supported by the data. In contrast, we found

519

very low or undetectable DP, while PP concentration increased consistently through time, likely

520

due to rapid scavenging and accumulation of dissolved P by phytoplankton. Epilimnetic

521

particulate P, which steadily increased in PyroP+PolyP abundance during summer stratification,

522

was comprised of cyanobacterial biomass, and indicates that epilimnetic phytoplankton growth

523

may not be P limited, as is frequently assumed for this system. Negative correlation of PolyP +

524

PyroP with OrthoP, and positive correlation to TPP and cyanobacterial biomass, Low DP

525

concentrations taken together with elevated PolyP and cyanobacterial biomass, indicates that

526

dissolved P is in high demand and thus low concentration, during summertime stratification. If P

527

does not limit harmful algal blooms in Lake Mendota, identification of the factors that

528

effectively control growth is essential for prevention of water quality degradation, given limited

529

resources dedicated to protecting and improving surface waters in Wisconsin.

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531

Acknowledgements

532

EKR and KDM were supported by an NSF CAREER award (CBET 0738039) and the National

533

Institute of Food and Agriculture, United States Department of Agriculture (ID number

534

WIS01516). Routine water quality monitoring data included in this manuscript were generated

535

by NTL LTER, supported by the National Science Foundation under Cooperative Agreement

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#0822700. NMR data were acquired on a 360MHz Bruker Avance funded by NSF CHE-

537

9709065 (1999) and on a 500MHz Varian Unity spectrometer funded by NSF CHE-9629688

538

(1998). We are grateful to Kevin Kauffman and the Wisconsin State Lab of Hygiene for iron and

539

manganese analysis; and to Luke Winslow, who provided assistance with the graphical abstract.

540

Many thanks to Jay Hawley, Aaron Besaw, James Mutschler, Tingxi Zhang for laboratory

541

assistance, sample collection, and sample preparation, and to William Songzoni and Ryan Batt

542

for thoughtful input on the manuscript.

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ACCEPTED MANUSCRIPT

Rigler, F., 1956. A tracer study of the phosphorus cycle in lakewater. Ecology 37, 550–562.

641 642 643 644

Schindler, D.W., Hecky, R.E., Findlay, D.L., Stainton, M.P., Parker, B.R., Paterson, M.J., Beaty, K.G., Lyng, M., Kasian, S.E.M., 2008. Eutrophication of lakes cannot be controlled by reducing nitrogen input: results of a 37-year whole-ecosystem experiment. Proceedings of the National Academy of Sciences of the United States of America 105 (32), 11254–8.

645 646

Soranno, P., Carpenter, S.R., Lathrop, R., 1997. Internal phosphorus loading in Lake Mendota : response to external loads and weather. Methods 1893, 1883–1893.

647 648

R Core Team, 2014. R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. http://www.R-project.org/.

649 650

Wetzel, R.G., 2001. Limnology: Lake and River Ecosystems, 3rd ed. Academic Press, San Diego, CA.

651 652 653

Xu, D., Ding, S., Li, B., Jia, F., He, X., Zhang, C., 2012. Characterization and optimization of the preparation procedure for solution P-31 NMR analysis of organic phosphorus in sediments. J Soils Sediments. 12:909-920.

M AN U

SC

RI PT

640

AC C

EP

TE D

654

29

ACCEPTED MANUSCRIPT

Table 1. 31P NMR P speciation and extraction efficiency of water samples collected in-lake and from hydrologic inflows to Lake Mendota, WI. Phosphonate

656

(Phos), orthophosphate (OrthoP), orthophosphate monoesters (MonoP), orthophosphate diesters (DiP), pyrophosphate plus polyphosphate (PyroP + PolyP) were

657

discriminated and quantified using 31P NMR. Total phosphorus (TP), total particulate phosphorus (TPP), total dissolved phosphorus (TDP) and dissolved reactive

658

phosphorus (DRP) were measured colorimetrically after acid digestion (for TP, TDP, and TPP). NMR extract was subsampled prior to experiment for TP

659

analysis by ICP OES (NMR TP). Extraction efficiency (%) was calculated as [NMR TP/TPP or TDP]. Inorganic phosphorus (Pi) and organic phosphorus (Po)

660

are shown as a percent of total P observed in NMR analysis. The ratio of phosphorus to manganese (P/Mn) of the NMR-ready extract is shown for lake

661

particulate samples.

662 Lake Particulate Phosphorus

Depth

ID

(m)

Day of Date

Ortho

Mon

P

oP

Phos Year

Extraction

PyroP +

TPP

DRP

NMR TP

PolyP

(µg/L)

(µg/L)

(µg/L)

DiP

0.5

05/03/11

123

0

27

26

0.5

05/18/11

138

0

24

24

0.5

06/01/11

152

0

35

0.5

06/28/11

179

0

38

0.5

07/12/11

193

5

41

0.5

07/25/11

206

47

0.5

08/09/11

53

0.5

51

0.5

P/ Po

Efficiency

Pi (%)

Mn (%)

(%)

41

16

15

31

18

n/d

n/d

42

58

n/d

35

23

17

35

35

20

55

41

59

2.9

17

23

31

30

18

56

5

27

46

54

3.5

30

35

9

25

23

< 10

21

91

56

44

48.9

27

21

14

33

30

< 10

27

89

60

40

26.8

16

11

24

22

27

20

< 10

9

45

38

62

13.0

221

3

9

31

21

36

13

< 10

10

77

45

55

3.4

09/05/11

248

15

9

19

16

42

17

< 10

9

53

51

49

2.6

09/21/11

264

0

15

38

10

37

24

< 10

7

29

51

49

1.6

AC C

EP

20

TE D

Sample

M AN U

SC

RI PT

655

30

ACCEPTED MANUSCRIPT

0.5

10/05/11

278

17

11

36

8

27

33

< 10

18

55

38

62

3.3

22

14

05/03/11

123

0

24

43

12

21

31

13

11

35

46

54

1.2

27

14

05/18/11

138

0

25

39

18

17

34

38

1

4

42

58

1.7

30

14

06/01/11

152

0

31

44

15

10

27

60

05

54

41

59

5.2

32

14

06/12/11

165

14

14

17

0

55

14

107

14

98

69

31

2.1

36

14

06/28/11

179

0

37

33

30

0

19

131

14

75

37

63

1.7

40

14

07/12/11

193

18

19

17

14

32

42

14

07/25/11

206

22

25

20

27

7

54

14

09/05/11

248

17

11

18

52

14

09/21/11

264

18

3

11

61

14

10/05/11

278

0

35

27

Day of Location

Date

ID

Ortho

Mon

P

oP

Phos Year

Yahara

07/28/11

209

0

43

Yahara

09/21/11

264

0

44

Ph. Branch

09/21/11

264

62

Ph. Branch

03/07/12

63

Yahara

03/07/12

SC

99

51

49

16.7

14

224

10

71

31

69

0.9

M AN U

18

26

14

248

11

79

37

63

39.8

11

56

<10

292

6

100

59

41

28.9

17

21

33

102

19

58

56

44

1.0

PyroP +

TPP

DRP

NMR TP

PolyP

(µg/L)

(µg/L)

(µg/L)

Extraction Efficiency

Pi (%)

Po (%)

(%)

28

41

15

17

276

< 10

n/d

n/d

45

55

23

48

18

12

112

< 10

n/d

n/d

35

65

0

49

28

12

12

48

17

n/d

n/d

60

40

67

0

64

9

9

18

273

41

n/d

n/d

82

18

67

16

15

28

10

32

35

3

n/d

n/d

47

53

AC C

49

171

DiP

EP

Sample

18

28

TE D

River Particulate Phosphorus

RI PT

60

663

31

ACCEPTED MANUSCRIPT

664 Lake Dissolved Phosphorus

PyroP + ID

NMR

Extraction

TP

Efficiency

(µg/L)

(%)

20

n/d

n/d

100

0

< 10

< 10

n/d

n/d

0

0

< 10

< 10

n/d

n/d

0

0

Day Depth (m)

Date

of

Phos

OrthoP

MonoP

TDP

DiP PolyP

(µg/L)

Year

DRP

Po Pi (%)

(µg/L)

(%)

0.5

06/28/11

179

0

100

0

0

0

55

0.5

07/24/11

206

0

0

0

0

0

65

0.5

08/22/11

234

0

0

0

0

0

58

0.5

10/05/11

278

20

29

24

12

6

48

41

n/d

n/d

43

57

64

14

06/28/11

179

0

100

0

0

0

141

131

n/d

n/d

100

0

56

14

07/24/11

206

0

87

0

7

7

229

224

n/d

n/d

94

6

66

14

08/22/11

234

0

100

0

0

0

225

192

n/d

n/d

100

0

59

14

10/05/11

278

0

100

0

115

102

n/d

n/d

100

NMR

Extraction

TDP

DRP TP

Efficiency

(µg/L)

(%)

Sample Location

Date

of Year

57

Yahara

07/28/11

209

75

Yahara

09/21/11

264

74

Ph. Branch

09/21/11

264

Phos

OrthoP

M AN U

TE D

AC C

ID

Day

EP

River Dissolved Phosphorus

MonoP

SC

69

0

51

RI PT

Sample

0

PyroP DiP

+ (µg/L)

Po Pi (%)

(µg/L)

PolyP

(%)

0

0

0

0

0

12

< 10

n/d

n/d

0

0

0

0

0

0

0

37

< 10

n/d

n/d

0

0

0

0

0

0

100

24

17

n/d

n/d

100

0

32

ACCEPTED MANUSCRIPT

68

Yahara

03/07/12

67

100

0

0

0

0

< 10

< 10

n/d

n/d

0

100

67

Ph. Branch

03/07/12

67

0

66

34

0

0

53

42

n/d

n/d

66

34

RI PT

665

AC C

EP

TE D

M AN U

SC

666

33

ACCEPTED MANUSCRIPT

Table 2. Regression analysis of NMR P fractions (columns) with environmental variables (rows); linear coefficient of determination is reported with Bonferroni-

668

corrected significance value indicated with asterisk for p<0.10 (*) and p<0.01 (**). Non-auto correlated results shown only.

OrthoP -0.47 0.47 0.16 -0.29 0.33 0.26 -0.29 -0.22 -0.25

MonoP -0.57 0.30 -0.32 0.40 0.34 0.49 -0.06 0.49 -0.35

EP

MonoP -0.66 0.80 * 0.11 -0.12 0.85 ** 0.28 -0.23 0.09 -0.78 *

AC C

Temperature (14m) Dissolved oxygen (14m) Secchi depth Chl-a (0-2m integrated) log (10) Bacillariophyta log (10) Chlorophyta log (10) Chrysophyta log (10) Cryptophyta log (10) Cyanophyta

Phos OrthoP 0.39 -0.04 -0.82 * 0.44 -0.07 0.07 0.01 -0.02 -0.67 0.63 -0.35 0.50 0.39 -0.53 -0.15 -0.21 0.78 * -0.22

TE D

Hypolimnion

DiP -0.11 -0.32 0.62 -0.55 0.40 -0.33 0.13 -0.23 -0.41

PyroP + PolyP 0.78 * -0.26 -0.01 -0.11 -0.88 ** -0.72 0.02 -0.24 0.38

M AN U

Temperature (0.5m) Dissolved oxygen (0.5m) Secchi depth Chl-a (0-2m integrated) log (10) Bacillariophyta log (10) Chlorophyta log (10) Chrysophyta log (10) Cryptophyta log (10) Cyanophyta

Phos 0.39 -0.26 -0.39 0.52 -0.15 0.25 0.25 0.20 0.62

SC

Epilimnion

RI PT

667

DiP 0.19 -0.37 -0.56 0.08 0.01 0.03 -0.25 -0.07 0.27

PyroP + PolyP 0.16 -0.18 0.20 0.05 -0.57 -0.30 0.37 0.16 0.12

669

34

8 4 0

Hypolimnion (14m)

TP PP DRP

μg PL-1 8

18 12 6

0 140 160 180 200 220 240 260 Day of year Fall mixis Onset of stratification

RI PT

140 160 180 200 220 240 260

18 12

6

PolyP PyroP DiP MonoP Ortho Phos 12 8 4

DO (mg L-1)

140 160 180 200 220 240 260

140 160 180 200 220 240 260

M AN U

10

DURP

SC

Fraction PP 12

DO (mg L-1)

14

0

TE D

30 24 18 12 6

140 160 180 200 220 240 260

Chl−a (μg L-1)

0

1

0.5

Temp (C)

μg PL-1 Fraction PP

0.5

EP

D

140 160 180 200 220 240 260

AC C

C

300 200 100 0

1

Temp (C)

B

80 60 40 20 0

Secchi (m)

A

ACCEPTED MANUSCRIPT

Surface (0.5m)

0 140 160 180 200 220 240 260 Day of year Fall mixis Onset of stratification

Temp DO

Secchi depth Chl−a

Figure 1. Temporal dynamics of phosphorus from epilimnion (0.5m) and hypolimnion (14m) depths in Lake Mendota, WI. A: Temporal dynamics of total phosphorus (TP) and dissolved reactive phosphorus (DRP) and, by subtraction, particulate (PP: TP-total dissolved P) and dissolved unreactive P (DURP) shown for epilimnetic (left) and hypolimnetic samples (right).B: Fraction of extracted epilimnetic (left) and hypolimnetic (right) particulate P (PP): phosphonate (Phos), orthophosphate (OrthoP), orthophosphate monoesters (MonoP), orthophosphate diesters (DiP), pyrophosphate (PyroP) and polyphosphate (PolyP) were discriminated and quantified using 31P NMR. C: Mean epilimentic and hypolimnetic temperature (C) and dissolved oxygen (DO, mg L-1) D: Secchi depth (m) and average Chl-a concentration (μg L-1) for 0-8m integrated samples.

ACCEPTED MANUSCRIPT

Orthophosphate (OrthoP)

Orthophosphate monoesters (MonoP)

Phosphonates (Phos)

RI PT

Orthophosphate diesters (DiP)

Polyphosphate (PolyP)

SC

Pyrophosphate (PyroP)

PP

M AN U

DP

PP

TE D

DP

PP

AC C

EP

DP

24

22

20

18

16

14

12

10

8

6

4

2

0

PP DP

-2

-4

-6

-8

-10

-12

-14

-16

-18

-20

-22

Chemical shift (ppm)

Figure 2. 31P NMR sample spectra from particulate (PP) and dissolved (DP) phosphorus samples from Lake Mendota and hydrologic inflows indicated by location, date, and day number.

-24

Lake Mendota epilimnion 28 June 2011 (179)

Lake Mendota hypolimnion 25 July 2011 (193)

Pheasant Branch 07 Mar 2012 (67)

Yahara River 28 July 2011(196)

Lake Mendota Total Phosphorus: 0m depth ACCEPTED MANUSCRIPT

Lake Mendota Dissolved Reactive Phosphorus: 0m depth 0.15

0.20

Year

Year

1995

1995

1996

1996

1997

Dissolved Reactive P (mg/L)

1998 1999

0.10

1997 1998

0.15

1999

2002 2003 2004 2005

0.05

2000 2001 2002 2003

0.10

2004

2006 2007 2008 2009

0.05

2010 2011 0.00 100

200

300

Day Number

0

Lake Mendota Dissolved Reactive Phosphorus: 14m depth Year

0.4

200

Day Number

2005 2006 2007 2008 2009 2010 2011

300

Lake Mendota Total Phosphorus: 14m depth Year

M AN U

0.5

100

SC

0

RI PT

2001

Total P (mg/L)

2000

1995 1996 1997

Dissolved Reactive P (mg/L)

0.3

1995 1996

0.4

1997

1998

1998

1999

1999

2001 2002 2003

0.2

2004

TE D

2005 2006

2000

Total P (mg/L)

2000

0.3

2001 2002 2003 2004 2005

0.2

2006

2007

0.1

2007

2008 2009 2010

2008 2009

0.1

2010

0.0 120

160

200

2011

EP

2011

Day Number

240

280

120

AC C

Lake Mendota Dissolved Reactive Phosphorus: 20m depth

200

Day Number

240

280

Lake Mendota Total Phosphorus: 20m depth

Year

Year

1995

1995

1996 1997 1998

1997 1998

1999

1999

2000

2000

2001

0.4

1996 0.6

2002 2003 2004 2005

Total P (mg/L)

Dissolved Reactive P (mg/L)

0.6

160

2001 2002

0.4

2003 2004 2005

2006

0.2

2007 2008

0.0 0

100

200

Day Number

300

2006 2007

0.2

2008

2009

2009

2010

2010

2011

2011 0.0

0

100

200

Day Number

300

ACCEPTED MANUSCRIPT

SC

M AN U

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 14 14 14 14 20 20 20 20 20 0 0 0 0 0 0 0 0 0

TP (mg/L) DRP (mg/L) 0.084 0.098 0.063 0.072 0.053 0.06 0 0.046 0 0.03 0.003 0.032 0.002 0.023 0 0.02 0 0.025 0 0.057 0.025 0.113 0.079 0.146 0.11 0.142 0.122 0.093 0.134 0.112 0.214 0.197 0.307 0.262 0.312 0.284 0.084 0.218 0.184 0.375 0.372 0.343 0.312 0.52 0.468 0.123 0.056 0.119 0.065 0.093 0.076 0.106 0.087 0.114 0.095 0.097 0.068 0.088 0.069 0.068 0.023 0.056 0

TE D

AC C

1995 1995 1995 1995 1995 1995 1995 1995 1995 1995 1995 1995 1995 1995 1995 1995 1995 1995 1995 1995 1995 1995 1995 1995 1996 1996 1996 1996 1996 1996 1996 1996 1996

DayNumber Depth 129 143 157 172 187 199 214 226 242 254 268 284 298 314 340 157 187 214 242 129 157 187 214 242 100 108 122 135 149 162 178 190 204

EP

Year

RI PT

Supplemental Table 1. Historical record of total phosphorus (TP) and dissolved reactive phosphorus (DRP) from Lake Flagged (suspect) data have been removed. Source: NTL-LTER, 2011. Chemical Limnology Dataset. In: North Temperate Lakes Long Term Ecological Research Prog http://lter.limnology.wisc.edu/

SC

0 0.002 0 0.004 0.089 0.126 0.109 0.1 0.102 0.151 0.238 0.255 0.272 0.158 0.238 0.378 0.52 0.126 0.099 0.098 0.096 0.103 0.105 0.081 0.043 0.062 0.022 0.011 0.015 0.005 0.065 0.147 0.153 0.099 0.116 0.119 0.208 0.286 0.346 0.234 0.146

M AN U

0.035 0.032 0.014 0.029 0.116 0.137 0.132 0.111 0.121 0.169 0.239 0.301 0.275 0.19 0.248 0.368 0.569 0.134 0.148 0.134 0.123 0.12 0.155 0.11 0.068 0.083 0.054 0.035 0.063 0.038 0.126 0.168 0.154 0.137 0.123 0.128 0.214 0.301 0.407 0.151 0.177

TE D

0 0 0 0 0 0 0 1 14 14 14 14 20 20 20 20 20 20 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 14 14 14 14 20 20

EP

218 232 249 260 290 313 330 37 162 190 218 249 37 162 190 218 249 313 99 111 122 133 148 162 176 190 205 218 230 245 274 303 334 14 48 162 190 218 245 48 162

AC C

1996 1996 1996 1996 1996 1996 1996 1996 1996 1996 1996 1996 1996 1996 1996 1996 1996 1996 1997 1997 1997 1997 1997 1997 1997 1997 1997 1997 1997 1997 1997 1997 1997 1997 1997 1997 1997 1997 1997 1997 1997

RI PT

ACCEPTED MANUSCRIPT

SC

0.324 0.52 0 0.67 0.156 0.102 0.091 0.111 0.041 0.087 0.083 0.078 0.028 0.017 0.003 0.001 0.002 0 0.009 0.104 0.137 0.158 0.12 0.115 0.42 0.002 0.128 0.284 0.3 0.022 0.54 0.72 0.123 0.106 0.094 0.096 0.088 0.086 0.076 0.05 0.02

M AN U

0.336 0.575 0.591 0.721 0.161 0.131 0.138 0.144 0.084 0.096 0.091 0.086 0.152 0.076 0.045 0.025 0.027 0.021 0.05 0.12 0.148 0.144 0.122 0.14 0.487 0.031 0.157 0.292 0.337 0.494 0.658 0.759 0.137 0.14 0.115 0.106 0.109 0.106 0.097 0.081 0.074

TE D

20 20 20 20 20 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 14 14 14 14 20 20 20 20 20 20 0 0 0 0 0 0 0 0 0

EP

190 218 245 274 334 72 85 100 114 131 148 159 173 187 204 214 230 245 271 300 327 37 159 187 214 245 114 159 187 214 245 300 53 88 102 116 130 146 158 172 187

AC C

1997 1997 1997 1997 1997 1998 1998 1998 1998 1998 1998 1998 1998 1998 1998 1998 1998 1998 1998 1998 1998 1998 1998 1998 1998 1998 1998 1998 1998 1998 1998 1998 1999 1999 1999 1999 1999 1999 1999 1999 1999

RI PT

ACCEPTED MANUSCRIPT

SC

0.005 0 0.005 0.003 0.025 0.096 0.097 0.128 0.111 0.229 0.282 0.346 0.232 0.211 0.095 0.112 0.171 0.341 0.47 0.522 0.656 0.096 0.096 0.101 0.089 0.053 0.055 0.028 0.058 0.07 0.054 0.049 0.002 0 0 0 0.056 0.113 0.123 0.091 0.071

M AN U

0.049 0.037 0.04 0.034 0.064 0.136 0.115 0.132 0.14 0.229 0.297 0.169 0.24 0.237 0.113 0.129 0.202 0.375 0.527 0.537 0.71 0.131 0.116 0.117 0.111 0.094 0.072 0.059 0.08 0.093 0.107 0.079 0.047 0.048 0.04 0.042 0.084 0.138 0.137 0.111 0.092

TE D

0 0 0 0 0 0 0 1 14 14 14 14 20 20 20 20 20 20 20 20 20 20 20 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 14

EP

200 214 228 242 270 299 326 25 158 187 214 242 25 53 102 130 158 187 214 242 270 299 326 24 73 101 116 130 143 158 171 185 199 213 229 241 269 298 333 59 158

AC C

1999 1999 1999 1999 1999 1999 1999 1999 1999 1999 1999 1999 1999 1999 1999 1999 1999 1999 1999 1999 1999 1999 1999 2000 2000 2000 2000 2000 2000 2000 2000 2000 2000 2000 2000 2000 2000 2000 2000 2000 2000

RI PT

ACCEPTED MANUSCRIPT

SC

0.178 0.218 0.413 0.097 0.166 0.09 0.055 0.082 0.125 0.233 0.412 0.538 0.56 0.178 0.123 0.12 0.091 0.047 0.053 0.071 0.065 0.072 0.06 0.003 0.017 0.003 0.002 0 0.049 0.125 0.118 0.087 0.177 0.239 0.349 0.225 0.117 0.054 0.108 0.212 0.317

M AN U

0.194 0.239 0.439 0.115 0.2 0.114 0.103 0.099 0.144 0.259 0.472 0.58 0.577 0.209 0.138 0.134 0.142 0.098 0.089 0.082 0.084 0.097 0.091 0.063 0.026 0.028 0.035 0.032 0.099 0.146 0.136 0.115 0.202 0.262 0.369 0.251 0.133 0.09 0.136 0.193 0.347

TE D

14 14 14 20 20 20 20 20 20 20 20 20 20 20 20 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 14 14 14 14 20 20 20 20 20 20

EP

185 213 241 24 59 73 101 130 158 185 213 241 269 298 333 17 66 100 113 127 141 155 169 183 197 211 225 239 269 295 330 155 183 211 239 17 66 113 127 155 183

AC C

2000 2000 2000 2000 2000 2000 2000 2000 2000 2000 2000 2000 2000 2000 2000 2001 2001 2001 2001 2001 2001 2001 2001 2001 2001 2001 2001 2001 2001 2001 2001 2001 2001 2001 2001 2001 2001 2001 2001 2001 2001

RI PT

ACCEPTED MANUSCRIPT

SC

0.431 0.35 0.603 0.118 0.117 0.068 0.056 0.054 0.064 0.073 0.063 0.051 0 0

M AN U

0.475 0.582 0.619 0.155 0.132 0.104 0.096 0.075 0.083 0.093 0.08 0.07 0.034 0.02 0.024 0.025 0.019 0.024 0.124 0.102 0.142 0.16 0.214 0.104 0.076 0.1 0.166 0.369 0.499 0.571 0.106 0.101 0.091 0.083 0.078 0.048 0.052 0.036 0.022 0.021 0.024

TE D

20 20 20 20 20 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 14 14 14 20 20 20 20 20 20 20 20 20 0 0 0 0 0 0 0 0 0

EP

211 239 269 295 330 87 98 114 126 140 154 169 182 196 207 224 238 266 294 322 169 196 224 87 114 140 169 196 224 266 294 322 21 93 104 118 133 160 174 188 205

AC C

2001 2001 2001 2001 2001 2002 2002 2002 2002 2002 2002 2002 2002 2002 2002 2002 2002 2002 2002 2002 2002 2002 2002 2002 2002 2002 2002 2002 2002 2002 2002 2002 2003 2003 2003 2003 2003 2003 2003 2003 2003

RI PT

ACCEPTED MANUSCRIPT

0 0 0 0.1 0.086 0.123 0.14 0.177 0.067 0.054 0.072 0.147 0.324 0.464 0.487 0.082 0.085 0.078 0.068 0.046 0.031 0.04 0.006 0.002 0 0.003

SC

0.003 0.007 0 0.018 0.104 0.095 0.072 0.151 0.212 0.076 0.05 0.039 0.112 0.24 0.354 0.479 1.44 0.105 0.099 0.097 0.061 0.039 0.046 0.05 0.045 0.049 0.034 0.015 0 0 0 0 0.028 0.102 0.103 0.081 0.209 0.097 0.047 0.069 0.128

M AN U

0.021 0.02 0.017 0.04 0.126 0.121 0.089 0.175 0.225 0.091 0.079 0.057 0.135 0.284 0.387 0.52 0.6 0.133 0.118 0.113 0.113 0.085 0.066 0.07 0.067 0.074 0.062 0.041 0.029 0.026 0.038 0.021 0.062 0.121 0.119 0.097 0.235 0.104 0.084 0.093 0.139

TE D

0 0 0 0 0 0 14 14 14 20 20 20 20 20 20 20 20 20 20 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 14 14 14 20 20 20 20

EP

216 230 245 272 300 329 160 188 216 21 104 133 160 188 216 245 272 300 329 34 89 103 119 131 145 159 173 188 201 216 229 251 279 308 159 188 216 34 103 131 159

AC C

2003 2003 2003 2003 2003 2003 2003 2003 2003 2003 2003 2003 2003 2003 2003 2003 2003 2003 2003 2004 2004 2004 2004 2004 2004 2004 2004 2004 2004 2004 2004 2004 2004 2004 2004 2004 2004 2004 2004 2004 2004

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ACCEPTED MANUSCRIPT

SC

0.169 0.429 0.49 0.532 0.096 0.097 0.098 0.091 0.096 0.082 0.053 0.031 0 0 0 0.004 0 0.002 0.14 0.133 0.124 0.173 0.231 0.251 0.108 0.104 0.096 0.179 0.313 0.392 0.512 0.632 0.14 0.132 0.135 0.124 0.112 0.088 0.105 0.085 0.049

M AN U

0.182 0.464 0.536 0.552 0.119 0.121 0.135 0.114 0.201 0.112 0.082 0.075 0.067 0.041 0.029 0.023 0.031 0.029 0.158 0.154 0.142 0.198 0.281 0.297 0.137 0.136 0.113 0.179 0.344 0.468 0.577 0.682 0.155 0.16 0.15 0.144 0.135 0.115 0.129 0.091 0.078

TE D

20 20 20 20 20 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 14 14 14 14 20 20 20 20 20 20 20 20 20 20 0 0 0 0 0 0 0

EP

188 216 251 279 308 31 105 115 129 143 157 171 186 199 213 227 241 269 306 334 157 186 213 241 31 105 129 157 186 213 241 269 306 334 65 94 107 122 135 150 163

AC C

2004 2004 2004 2004 2004 2005 2005 2005 2005 2005 2005 2005 2005 2005 2005 2005 2005 2005 2005 2005 2005 2005 2005 2005 2005 2005 2005 2005 2005 2005 2005 2005 2005 2005 2006 2006 2006 2006 2006 2006 2006

RI PT

ACCEPTED MANUSCRIPT

SC

0.016 0.003 0 0 0 0.004 0.053 0.118 0.114 0.136 0.205 0.272 0.386 0.117 0.11 0.209 0.293 0.478 0.667 0.55 0.117 0.114 0.115 0.112 0.105 0.083 0.055 0.06 0.061 0.046 0.018 0 0 0 0 0 0.025 0.119 0.126 0.138 0.181

M AN U

0.058 0.042 0.036 0.027 0.018 0.026 0.089 0.147 0.135 0.152 0.235 0.306 0.37 0.138 0.136 0.24 0.325 0.535 0.669 0.617 0.139 0.129 0.133 0.127 0.129 0.115 0.08 0.082 0.074 0.074 0.067 0.026 0.031 0.023 0.026 0.025 0.051 0.152 0.144 0.16 0.201

TE D

0 0 0 0 0 0 0 0 0 14 14 14 14 20 20 20 20 20 20 20 20 20 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 14 14

EP

177 191 205 219 233 249 275 303 331 163 191 219 249 107 135 163 191 219 249 275 303 331 3 50 92 106 120 134 149 162 176 190 204 218 232 247 274 302 330 162 190

AC C

2006 2006 2006 2006 2006 2006 2006 2006 2006 2006 2006 2006 2006 2006 2006 2006 2006 2006 2006 2006 2006 2006 2007 2007 2007 2007 2007 2007 2007 2007 2007 2007 2007 2007 2007 2007 2007 2007 2007 2007 2007

RI PT

ACCEPTED MANUSCRIPT

SC

0.276 0.266 0.114 0.144 0.086 0.084 0.203 0.394 0.524 0.577 0.66 0.119 0.125 0.122 0.024 0.011 0.024 0.058 0.053 0.043 0.034 0.007 0.003 0.003 0 0 0.125 0.09 0.184 0.289 0.332 0.157 0.027 0.053 0.182 0.321 0.454 0.626 0.638 0.11 0.127

M AN U

0.305 0.297 0.131 0.171 0.118 0.105 0.241 0.394 0.553 0.616 0.687 0.138 0.147 0.145 0.141 0.029 0.037 0.072 0.075 0.13 0.152 0.053 0.05 0.038 0.029 0.039 0.147 0.114 0.214 0.341 0.374 0.186 0.136 0.075 0.209 0.381 0.529 0.676 0.665 0.138 0.152

TE D

14 14 20 20 20 20 20 20 20 20 20 20 20 0 0 0 0 0 0 0 0 0 0 0 0 0 0 14 14 14 14 20 20 20 20 20 20 20 20 20 0

EP

218 247 3 50 106 134 162 190 218 247 274 302 330 44 105 119 133 149 161 175 189 203 217 231 249 273 303 161 189 217 249 44 105 133 161 189 217 249 273 303 21

AC C

2007 2007 2007 2007 2007 2007 2007 2007 2007 2007 2007 2007 2007 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2009

RI PT

ACCEPTED MANUSCRIPT

SC

0.133 0.116 0.075 0.116 0.085 0.052 0.038 0.016 0 0 0.002 0.034 0.139 0.128 0.107 0.174 0.053 0.279 0 0.113 0.127 0.137 0.268 0.428 0.486 0.543 0.144 0.112 0.014 0.018 0.014 0.03 0.04 0 0 0 0 0 0 0 0.026

M AN U

0.165 0.148 0.126 0.147 0.11 0.155 0.125 0.106 0.07 0.044 0.053 0.075 0.164 0.153 0.13 0.2 0.291 0.316 0.025 0.071 0.149 0.169 0.312 0.468 0.539 0.601 0.181 0.136 0.12 0.103 0.063 0.056 0.063 0.043 0.034 0.046 0.038 0.032 0.034 0.029 0.066

TE D

0 0 0 0 0 0 0 0 0 0 0 0 0 0 14 14 14 14 20 20 20 20 20 20 20 20 20 0 0 0 0 0 0 0 0 0 0 0 0 0 0

EP

105 117 131 148 160 174 187 201 217 231 245 273 301 337 160 187 217 245 21 105 131 160 187 217 245 273 301 32 88 103 116 130 144 158 172 186 200 215 228 242 278

AC C

2009 2009 2009 2009 2009 2009 2009 2009 2009 2009 2009 2009 2009 2009 2009 2009 2009 2009 2009 2009 2009 2009 2009 2009 2009 2009 2009 2010 2010 2010 2010 2010 2010 2010 2010 2010 2010 2010 2010 2010 2010

RI PT

ACCEPTED MANUSCRIPT

SC

0.062 0.106 0.17 0.198 0.248 0.104 0.028 0.025 0.189 0.268 0.376 0.46 0.555 0.061 0.071 0.007 0.016 0.031 0.055 0.041 0 0 0.003 0 0 0.04 0.069 0.093 0.169 0.209 0.267 0.207 0.009 0.043 0.149 0.301 0.46 0.581 0.551 0.07

M AN U

0.094 0.13 0.201 0.226 0.275 0.128 0.07 0.058 0.223 0.316 0.445 0.493 0.617 0.108 0.093 0.066 0.058 0.064 0.074 0.066 0.031 0.022 0.018 0.016 0.02 0.088 0.113 0.116 0.195 0.224 0.288 0.23 0.068 0.076 0.177 0.361 0.476 0.607 0.58 0.114

TE D

0 14 14 14 14 20 20 20 20 20 20 20 20 20 0 0 0 0 0 0 0 0 0 0 0 0 0 14 14 14 14 20 20 20 20 20 20 20 20 20

EP

313 158 186 215 242 32 103 130 158 186 215 242 278 313 19 108 122 137 151 164 192 206 216 234 255 276 304 164 192 216 255 19 108 137 164 192 216 255 276 304

AC C

2010 2010 2010 2010 2010 2010 2010 2010 2010 2010 2010 2010 2010 2010 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011

RI PT

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT

tive phosphorus (DRP) from Lake Mendota, WI.

AC C

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ng Term Ecological Research Program. Madison, WI.

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2.5 2.3 6 7.2 3.3 3.3 2.6 3.3 1.8 2 2.2

SC

M AN U

TE D

AC C

2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011

Day Number Depth (m) Temp (C) DO (mg/L) Secchi m 122 0.5 7.95 12 137 0.5 11.15 10.15 151 0.5 14.95 8.1 164 0.5 18.85 10.3 178 0.5 20.55 9.3 192 0.5 25.8 11.6 206 0.5 27.35 10.3 216 0.5 28.1 8.4 234 0.5 25.1 9.1 255 0.5 22.5 11.8 276 0.5 15.9 8.6 122 14 7.8 11.7 137 14 10 9.1 151 14 11.9 7 164 14 12.9 6.6 178 14 13.2 4 192 14 13 0.3 206 14 13.3 0.1 216 14 13.8 0.1 234 14 13.5 0.2 255 14 13.8 0.1 276 14 15.6 6.6

EP

Year

RI PT

Supplemental Table 2. Ancillary physical, chemical, and biological water quality data. Flagged (suspect) data have been removed. Sources: NTL-LTER, 2011a. Physical Limnology Dataset. In: North Temperate Lakes Long Term Ecological Researc NTL-LTER, 2011b. Chemical Limnology Dataset. In: North Temperate Lakes Long Term Ecological Resear NTL-LTER, 2011c. Biological Limnology Dataset. In: North Temperate Lakes Long Term Ecological Resear http://lter.limnology.wisc.edu/ *Depth 0.5m is an average value taken from measurements/samples collected at 0m and 1m discrete depths.

Chlorophyll a (ug/L 0-2m integrate 13.4 4 0.45 1.8 2.7 5.85 8.7 6.2 8.3 9.3 16.05

ACCEPTED MANUSCRIPT

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kes Long Term Ecological Research Program. Madison, WI. akes Long Term Ecological Research Program. Madison, WI. akes Long Term Ecological Research Program. Madison, WI.

m and 1m discrete depths.

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BacillariophytaChlorophyta (mg biomass/L, (mg Chrysophyta biomass/L, 0-8m integrated (mg Cryptophyta 0-8m biomass/L, sample) integrated (mg Cyanophyta 0-8m biomass/L, sample) integrated (mg 0-8m biomass/L, sample) integrated 0-8m sample) integrated sample) 6.1342 0.1135 0.0287 0.1633 0.0741 1.0498 0.0321 0.0195 0.0534 0.0473 0.3511 0.0031 0.0002 0.0117 0.0163 0.0002 0.0157 0.0005 0.0065 0.7414 0.0011 0.0227 0.0000 0.0038 0.6604 0.0000 0.0178 0.0156 0.0054 4.6434 0.0563 0.0393 0.0114 0.0212 19.9906 0.0000 0.0128 0.0022 0.0065 0.4262 0.0000 0.0052 0.0104 0.0479 0.9814 0.0000 0.0042 0.0135 0.1176 0.7011 0.0060 0.1147 0.0026 0.1310 0.7193

RI PT

ACCEPTED MANUSCRIPT

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0-8m integrated sample)