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Methods for Experiment 246 -

Experimental establishment and chemical applications.

This experiment utilized 35 plots originally established as prairie at CCESR in 1994 to investigate the effects of plant biodiversity on ecosystem productivity and stability. The plots selected for this experiment met three criteria: (1) they were originally planted with eight prairie species, though not necessarily the same species, (2) a significant proportion of the existing plant community (based on a visual inspection) consisted of C4 grasses and legumes, and (3) different treatments were spatially distributed evenly across the 8 ha experimental field. The experimental
design includes 5 replicates for each of the following treatment combinations: 4 crop treatments (hay, prairie, corn, bare ground) at 2 levels of chemical addition (including tracers, nutrients, and pharmaceuticals). However, no corn control plots were established as corn would never be grown without nutrient addition.

The U.S. Geological Survey, in cooperation with the College of Biological Sciences of the University of Minnesota and the Legislative-Citizen Commission on Minnesota Resources, initiated this study to investigate the effects of land-cover type on the fate and transport of three
surface-applied chemicals of emerging concern. The antibiotics sulfamethazine (SMZ) and sulfamethoxazole (SMX) and the steroidal hormone 17-beta-estradiol (17BE) were applied to the surface of five plots of each land-cover type in May 2008 and April 2009. Current (2013) practices of animal waste application to row-crop systems used for biofuel production can affect surface-water and groundwater quality. Previous research has indicated that perennial crops, such as prairies, have the potential for producing biofuel and reducing flows of water and chemicals to surface water and ground-water compared to row crops. Few studies have documented, in detail, the use of inexpensive analytical methodology for measuring antibiotics and hormones in a variety of environmental sample matrices, such as plant tissues and soils.

The surface-applied chemicals study included 35 plots [11 meters (m) by 11 m] with land-cover types of nonvegetative bare soil, corn, hay, or prairie. Five treatment plots of each land-cover type (20 plots total) received applications of SMZ, SMX, 17BE, and bromide. Background levels of these chemicals in plant tissues, soil, soil water, and groundwater were determined in samples collected from bare soil, hay, and prairie control plots (15 plots total) that did not receive any chemical applications. Background levels of these chemicals in corn plants were determined in samples collected from a single large stand of corn that did not receive additions of these chemicals. Concentrations of SMZ, SMX, and 17BE measured with enzyme-linked immunosorbent assay (ELISA) kits in plant-tissue, soil [0 to 10 centimeters (cm)], soil-water, and ground-water samples collected between October 2008 and October 2009 were used to compare the fate and transport of these chemicals through land-cover types of bare soil and three potential biofuel cropping systems: corn, hay, and prairie.

Additional details about field operations and data processing can be found in: Trost, Jared Jeffrey; Effects of perennial and annual vegetation on a soil water balance and groundwater recharge; 2010; Masters Thesis University of Minnesota Digital Conservancy Permanent URL 2010

All data, methods, and results for well elevations, soil chemistry, water sampling and applications of chemicals can be found in the associated USGS Report: USGS Scientific Investigations Report 2013 - 5202

Land-Cover Effects on the Fate and Transport of Surface-Applied Antibiotics and 17-beta-Estradiol on a Sandy Outwash Plain, Anoka County, Minnesota, 2008 - 2009.

acke246 - Root Ingrowth Biomass

Root Ingrowth Biomass

A root corer was used to create the root ingrowth sampling area in the soil. The core went to a depth of approximately 35 cm. A screen made of hardware cloth was molded around the root corer and inserted into the hole created by the first core. Soil from within the plot was sieved of rocks, live plants and root mass (which was discarded) and then placed in the hole. The soil was packed into the hole until it was level with the surrounding soil and approximately 1 cm of the hardware cloth in the hole was left above soil level.

In order to sample, an initial 1.5? dia core was taken to a depth of 30 cm from within the screened coring area and the contents were put into a labeled plastic bag. The hardware cloth screen was then pulled out of the hole and a reverse corer (a corer with the inside of the bottom edge filed) was inserted in the hole to 35 cm and removed. The screen was then put back into the hole.

The contents removed from the hole with the reverse corer were sifted on 1/4" hardware cloth and poured with a funnel back into the hole. If the hole was not filled up, regular 2? root cores 0-30cm deep from within the plot, but in the 1m buffer area were used.

Within one week of the sampling roots were washed. Soil from each core sample was placed on a screen. Water was then sprayed at a medium speed over the soil (so all of the fine roots wouldn't get blasted through the screen). Once all of the soil was removed, roots were collected from the screen and put into a plastic cup. The cup was filled with water, any clumped roots were separated. Soil and rocks sank to the bottom, roots floated to the top. Roots were skimmed from of the surface of the water. Excess water was squeezed out by hand, roots were placed in labeled paper bags and moved into a drying shed and dried at 40 degrees Celsius. Once the roots were completely dry they were weighed.

acle246 - Biofuels Harvest

Aboveground harvest for biomass for non-corn plots

Objective: Done to estimate harvestable biomass using a variety of techniques. Methodology will allow us to compare the data with past E120 biomass harvest data. Also done to document plot productivity prior to experiment establishment.

Whole plot harvest procedure for non-corn plots?
1. The only biomass that should be included in the whole plot harvest (after 2008) is the center 9x9m area. The plots should have corner markers for both an 11x11m square and an inner 9x9m square.
2. Mow the outer 1m buffer (from the 11x11m posts to the 9x9m posts) with the sicklemower, or any lawnmower and discard this biomass (not kept as the whole plot biomass sample). The sicklemower should be set at about 4? above ground .
3. After the buffer is mowed, measure and record the length of each side of the 9x9m plot to get an accurate estimate of sample area, since the data will be reported as mass/area.
4. Harvest the center 9x9m plot.
a. To begin, sicklemow three evenly spaced transects within the plot.
b. From each transect fill 2/3 of a paper grocery sack with a representative sample of biomass. Each transect will have its own bag. These will be the moisture subsets for the plot.
c. Then, while one person begins to mow the rest of the plot, others may begin raking the cut biomass into pile. The biomass will then be placed in large plastic bags labeled with the plot date and bag number. Once completely cut and bagged, rake again to gather the remaining biomass and place in the bags.
5. To weigh: The person to weigh will stand on the scale and their weight will be recorded. They will then stand on the scale holding the bag and that weight will be recorded with the person?s weight to be subtracted later. The transect moisture bags from each plot are weighed after return from the field and placed in the drying room for 1-2 weeks after which they are weighed dry.

Stubble Harvest:

Objective: Done to estimate the amount of biomass left behind after the whole plot harvest is complete

Done after the whole plot harvest is complete.
1. When ready to clip, again place the fiberglass pole between the strip?s marking flags to make a straight line between the flags of the strip.
2. Begin clipping a strip to the ground the width of the clippers from flag to flag. As you go, pick up the cut biomass and place in a labeled bag.(Try to avoid getting soil in with the biomass)
3. Do the same for the other strip in the plot, and bag the two strips separately.
4. Place the bags in the drying room and weigh them after1-2 weeks.

Stubble harvest notes:

2007: (also referred to as Short Clip) Done immediately following the Sicklemower strip to get an estimate of the amount of biomass lost from harvesting 4? and higher. From the western edge of the mowed 9x.77m rectangle measure 2m in and place the 1 x 0.5m PVC frame. Using a battery operated hand clipper clip all remaining biomass, scrape up by hand, and bag. All biomass was dried and weighed. A fresh percent moisture was not obtained.

2008 and 2009: Two 6 m. strips the width of the hand clippers were harvested for stubble following the whole plot harvest. In 2009, The strips were located, facing the plot number sign, 1.5 m from each corner and 1 m into the plot. The stubble from the strips were bagged separately, dried and weighed

Sicklemower Strip (done in 2007 only): (also referred to as Sicklemower/High on datasheets and bags of biomass) Measure 1m in from the north end of the plot. Using the sicklemower (.77m blade width) set at 4" high, mow a straight line across the plot (9m) to make a 9 x 0.77m mowed rectangle. The sicklemower is made by the Jari company. Rake up all mowed biomass and place in a paper bag. All of the biomass is dried and weighed. A fresh percent moisture was not obtained.

Mulch Mow and Rake: (also referred to as Mow/Rake on datasheets and bags of biomass). This was done on the hay and bareground plots prior to establishment. The plots had to be mowed anyway, so we harvested a portion of the biomass to get another biomass estimate using a different mowing technique. After the plots were mowed with a mulching mower behind a John Deere tractor, a piece of rebar was firmly pushed into the ground approximately 2 m from the south end of each plot on either the west or east side edge. Using a meter tape, an east-west 8.5 m strip was measured and left in place. This measuring tape was used as a guide for raking. A 60cm wire rake was used to rake along the inside of the measuring tape. This area was then raked in one direction trying to attain the most biomass as possible. While raking, attention was paid to the amount of biomass accumulating under the rake. If any biomass started to spill out, the rake would be carefully picked up and cleaned into the pile. This was done along the 8.5m stretch to prevent loss of biomass. After raking, the piles lying next to the measure tape were carefully gathered by hand and placed into a labeled paper bag. All of the biomass was dried and weighed. A fresh percent moisture was not obtained.

All harvest notes:

2007 Notes:
Sicklemower Strip: Done in all hay, prairie, and bareground plots on 9/10/2007 -9/12/2007. Corn plots were harvested this way on 11/19/2007. The mower was set at 4? above ground.
Stubble Harvest: Done in all hay, prairie, and bareground plots on 9/11/2007 ? 9/12/2007
Mulch Mow and Rake: Done in all hay and bareground plots on 9/12/2007
Whole Plot: Done in all prairie plots on 10/26/2007. Done in all corn plots on 11/19/2007.

2008 Notes:
WholePlot Harvest: Done in all hay, prairie, and bareground plots in October 2008. The height of the mower was set at 4?
Stubble Harvest: Done in all hay, prairie, and bareground plots in October 2008

Whole plot and stubble harvests done in conjunction with the Irrigation Nitrogen biofuel harvest experiment.

2009 notes:
Plots were harvested using the whole plot harvest method followed by stubble harvest in early November. The height of the mower was set at about 4?.

Whole plot and stubble harvests done in conjunction with the Irrigation Nitrogen biofuel harvest experiment.

acme246 - Plant species percent cover data

Plant percent cover

The four sample points were located at the corners of the 5 x 5 m inner subplot of each 9 x 9 m plot. From the front of the plot (i.e. the side marked with the plot number), sample points were designated A, B, C, and D in a clock-wise direction, starting with the near right point. At each point sampled, a 1 x 0.5 m quadrat was placed with one corner at the rebar, such that the area within the frame lay entirely within the inner 5 x 5 m subplot and its long side ran parallel to the direction in which the plots are numbered.

We recorded percent cover of all species found within the plot, as well as all other ground cover visible through the vegetation (i.e. bare ground, miscellaneous litter, mosses & lichens). We typically included only rooted species, except in cases where species rooted outside the plot were clearly leaning over or sprawling into the frame (prior to it being placed on the ground)?this rare exception mostly applied to individuals of Lupinus perennis, Stipa spartea, and Baptisia luecantha. Total percent cover in each plot was made to sum to 100, and a lower limit for recorded values was set at 0.1%.

2007 Notes:
Data checked for consistent species spelling, percents adding up to 100 and dates were put in correct format. Plants were identified to species whenever possible. Most ?Festuca sp.? was most likely Festuca ovina and most ?Carex sp.? was likely Carex foena, though it was impossible to accurately key out non-reproductive specimens. All crew members were carefully trained in plant identification before they could assess percent cover on their own or participate in the species inventories.

2008 notes:
We did four percent cover points in each plot, measuring in 2 m each way from the corner posts, and with the long side of the 0.5 x 1.0 m quadrat parallel to the numbered post. The four subplots were labeled A, B, C, and D, with A being to the right of the numbered post, and then proceeding counterclockwise. Percent cover was taken using the palmtops and Clarence Lehman?s program DECLARE. The minimum category for a plant was 0.5%. All species rooted inside the plot were recorded, but nothing that shaded into the plot. Multiple canopy layers were not done?percent cover estimations were done from directly above the plants in the quadrat without moving them. Percent cover always added up to 100%. In addition to all the plant species present, we recorded litter and bare ground.Data checked for consistent species spelling, percents adding up to 100 and dates were put in correct format.

2009 notes:
All 4 points in each of the 35 plots were sampled. Palm pilots were not used; the species and percentages were recorded by hand. As the survey took place in late August, the corn plot percent cover was estimated using the help of a ladder to look down on each of the four points. Data was checked for consistent species spelling and percents adding up to 100.

acne246 - Root harvest biomass

Root harvest biomass

Root biomass in three depths of the soil profile of each plot were sampled. Each plot?s root sample was a composite of six individual cores collected with a 5-cm diameter corer. The six individual cores in bare soil, hay, and prairie plots were taken at equally spaced intervals along a 6-m transect. The six individual cores in corn plots were taken along a 6-m transect that was perpendicular to the direction of the planted rows. Three of the cores in each corn plot were taken in rows and three cores were taken midway between the rows. The field sample comprised soil and roots. Roots were separated from the soil by gently spraying the sample with water on a screen with 1-mm mesh openings. The screen retained the roots as the soil was washed away. After the roots were cleaned of soil, they were dried at 40 ?C for at least 1 week and then weighed.

acse246 - Plant community light interception

Plant community light interception

Light Measurements were taken using Decagon AccuPAR Ceptometers in 4 subplots within each E246 plot. The subplots were located two meters into the plot from the midline on each side and one meter to the right of that midline. All measurements were taken facing SOUTH. Three upper canopy and three lower canopy readings were taken in each subplot per sampling. These three readings are averaged by the Decagon AccuPAR Ceptometers.

acte246 - Soil moisture

Soil moisture - Instrumentation and calibration

Measurments were taken with aTrime FM3 Time Domain Reflectometry (TDR) system, version P3 with T3 tube-access probe (IMKO Micromodultechnik GmbH,
The factory calibration supplied with the instrument produced erroneously high results for our field soil conditions which necessitated an adjustment calculation to the instrument VWC output. The Trime output was calibrated to site specific gravimetric measures of soil moisture. See Appendix 1-Hydrologic Instrument Calibrations and Data Processing of Trost, 2010 (

Soil moisture methods

Time Domain Reflectometry (TDR) access tubes. Permanent 5 cm diameter schedule 40 PVC access tubes were installed to a maximum depth of 220 cm in the soil profile of each plot in May through June of 2008. Installation required repetition of two steps. Step one: insert an auger inside the PVC tube and auger down 15 cm below the leading edge of the PVC. Step two: remove the auger and soil from the tube and tamp the tube in 15 cm to the extent of the augered profile. The leading edge of each PVC tube was beveled inward to minimize compaction of soil outward during the tamping process.

Soil moisture was measured periodically throughout the growing season (May ? October) in 2008 and 2009 at four 17 cm depth increments (0-17, 17-34, 42-59, and 92-109 cm). The TDR probe averages the soil moisture value over the 17 cm interval. Prior to each measurement, the interior surface of each access tube was swabbed to remove excess moisture due to condensation. The instrument?s soil volumetric water content (VWC) output was calibrated to a soil-specific VWC using measurements of VWC (cm3/cm3) calculated gravimetrically from soil samples taken at the same time (refer to Appendix 1 Trost, 2010 ( for detailed descriptions).