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Terrestrial Methods And Protocols.


TABLE OF CONTENTS

Plant methods.

  1. Biomass Harvest Method
  2. Counting Eriophorum vaginatum Inflorescences
  3. Determining Weight/Tiller in Eriophorum vaginatum
  4. Classification for Undisturbed Tussock Tundra

Soil methods.

  1. Buried Bags and Soil Extraction
  2. Methane production method
  3. Method for Soil Moisture

Field Protocols

  1. Toolik and Sag Fertilizer Needs
  2. Rain Collection Procedures


Plant Methods


Biomass Harvest Method

Field: Samples are obtained by collecting all aboveground biomass and below ground stems in 20 x 20 cm quadrats, located randomly along line transects within the site. Normally, 4 or 5 quadrats are collected along each of 4 transects at each site or treatment. Aboveground biomass is considered "within" the quadrat if it is associated with a meristem that is within the quadrat. Details are given in Shaver and Chapin (Ecological Monographs, 61(1), 1991, pg. 1.)

Lab: Each quadrat is first sorted into species and then into tissue type. Depending on the harvest, tissue types can be broad categories, i.e. above and below, or more detailed, i.e. inflorescences, new growth, old growth, etc. The separated samples are dried for several days at approximately 65 C and then weighed. The dry weight for each quadrat, species and tissue type is recorded. Finally the samples from all quadrats in a transect are combined according to tissue type. Samples are then returned to Woods Hole for nutrient analysis.

Counting Eriophorum Vaginatum Inflorescences

The number of Eriophorum Vaginatum flowering tillers in the plot is counted. At each site there are 6-10 plots which are counted. The count should include all the current year's flowers, including those clipped by ptarmigan but still recognizable by their elongated culms. The sites and site maps are listed in the Eriophorum Vaginatum Flowering notebook. The location and orientation of the plots is described in the site maps.

Determining Weight/Tiller in Eriophorum Vaginatum

This data is collected at the time of peak leaf mass per tiller, in late July or early August. Mature Eriophorum tussocks (Class III tussocks, defined as those with 10-50% cover by other species; Fetcher & Shaver 1982), within 10 m of the permanent Eriophorum flowering plots, are collected. Tussocks which have been sampled previously are rejected. Sampled tussocks are brought back to the lab.

Clumps of 40-60 intact tillers are collected from the centers of the sampled tussocks. Working through the clump, count the number of immature tillers with no green (V0), the number of immature tillers with green (V1), number of current year's flowering tillers, and next-year's flowering tillers encountered before 20 mature tillers are collected. See below for definitions of tiller and flower types. Record the number of immature tillers (both V0 & V1), current year’s flowers (F), next year's flowers (B), and mature tillers (M). Separate the mature tillers from the clump at the point of attachment to the rhizome, clip off dead leaves and tips and place the mature tillers from each tussock in individual bags. Dry the mature tillers at no more then 60 C for a few days and then weigh to 0.001 g accuracy. Box up samples, label well, and return them to Woods Hole.

 

Weight/tiller is defined as the dry mass of the mature tillers divided by the number of mature tillers (usually 20).

The tillering index is defined as the ratio of immature to mature tillers, i.e. (V0 + V1)/(M + F + B).

 

Mature tillers (M): tillers old enough to have accumulated at least one dead leaf.

Immature tillers (V0): tillers recognized by their small size, lack of fully green leaves, and lack of dead leaves.

Immature tillers (V1): tillers recognized by their small size, at least one green leaf, and lack of dead leaves.

Flowers (F): Current year’s flowers.

Buds (B): Next year’s flowers.

Classification for Undisturbed Tussock Tundra

Class I Eriophorum vaginatum tussock 17.5cm diameter, <10% relative cover of moss, shrubs, and Carex.

Class II Eriophorum vaginatum tussock > 17.5 cm diameter, < 10% relative cover of moss, shrubs, and Carex.

Class III Eriophorum vaginatum tussock with 10% and <50% relative cover of moss, shrubs, and Carex.

Class IV Eriophorum vaginatum tussock with 50% and <97% relative cover of moss, shrub, and Carex.

Class V Old Eriophorum vaginatum tussock with <3% of E. vaginatum cover, 97% moss, shrub, and Carex cover. Elevation above surrounding soil/moss surface >5cm.

Class VI Continuous moss cover beneath and between vascular plants, <5cm elevation above immediate surroundings, <1% E. vaginatum relative cover.

Class VII Organic soil with <5% relative cover by green plants and no E. vaginatum.

Class VIII Mineral soil with <5% relative cover by green plants and no E. vaginatum.


Soil Methods


Buried Bags and Soil Extraction

Collecting Cores.

In the field two soil cores are collected in the same micro site within a few cms of each other. Remember to record soil corer diameter . Care should be taken to avoid compacting the core. If there is compaction the core length is measured by measuring the hole depth. Green plants and large roots are removed form the core before bagging the cores in gallon size baggies. If the soil horizons are being separated then each horizon should be measured for length and bagged separately. A dymo label marked with the core number should go in each bag. One core is placed back in its hole and covered with moss and flagged. The other core is brought back to lab for analysis. All notes should go in the field notebook.

Laboratory

1) Weigh each soil core or horizon for bulk density and soil moisture calculations. A separate subsample will be taken to correct wet weight to dry weight. Remember to tare for the bag weight.

2) In a soil pan breakup each soil sample and mix well. Using a knife to cut up the core and then breaking it up with your fingers works well. If there is a lot of material then the core can be subdivided by cutting down the middle and only using half of the core. Subsample the soil as follows:

20g wet weight in into 8 oz.(16 oz. for large samples) deli cups for KCl. extraction.

20g wet weight in into 8 oz.(16 oz. for large samples) deli cups for HCl extraction.

20-100 g wet weight in soil cans for wet to dry weight conversion.

3) KCL Extraction.

Add 100 mls of 2 N KCl to each cup and shake for 4 hours. Remember to run 2 - 4 blank deli cups. After shaking filter the extract through Whatman no. 42 filter paper. The solution is then run for NH4-N and NO3-N. 15 to 20 mls is enough solution for both analysis.

4) HCl Extraction.

Add 100 mls of 0.025 N HCl to each cut of 20 g soil and shake for 5 minutes. Filter through Whatman no. 42 filter paper and then analyze for PO4-P. 10 to 20 mls is enough for the analysis.

5) The wet to dry weight soil subsample is dried for a week at 65 C and weight.

Extract Solutions.

1) 2 N KCl F.W. 74.55

149 g / liter DI water or about 3 kg / 20 liters of DI

Fill a carboy with DI water almost to desired volume (about 10% less). Add a stir bar and start the water stirring. Slowly add the KCl making sure not to stop the stir bar. Stir over night. After the KCl is completely dissolved add DI water to final volume. And remix.

2) 0.025 N KCl F.W. 36.46 Concentration of reagent grade acid - 12.4 N.

2.0 mls / liter DI water

Fill a carboy with DI water almost to desired volume (about 10% less). Slowly add HCl. Mix well and add DI water to final volume and then remix.

Methane production method

Sample preparation

Methane production rates were determined by measuring the amount of methane produced over time in anaerobically incubated subsamples of peat cores (Sass et al., 1990). Cores were taken from wet sedge tundra at the Sag site. The cores were subsampled anaerobically, in a nitrogen-filled glove bag, by homogenizing peat from the whole depth profile. Approximately 25 cc of peat were placed in a 60 ml plastic syringe that was mounted with a three-way stopcock. Often the samples were slurred by adding 5-10 ml of N2 bubbled DI water to the syringe. Then the remaining N2 was purged from the vessel with the plunger. Samples were incubated in a sealed bucket that was flushed with nitrogen. The incubation temperature was either room or in situ.

Sample measurement

The syringes were sampled every 24 hours for approximately 3-4 days. Dissolved methane was liberated by sucking in nitrogen gas (enough to bring the total volume of the syringe to 60 ml) and shaking the syringe vigorously for two minutes (A test to assure that the two minute shake was sufficient was conducted by immediately shaking the sample after the initial shake and measuring the concentration of methane in the headspace). The head space gases were extruded into a separate syringe and injected into either a Shimadzu model 14A or a Carle gas chromatograph with a flame ionization detector (FID). The ratio of gas to sample and resulting methane peak area were recorded. The syringes were incubated further without a head space. This process was repeated to obtain a time series of methane production. Methane concentrations were calculated using peak area of the samples compared to methane standard peak areas. The standards used were from Scotty Specialty Gas. Methane concentrations were calculated as follows:

CH4 (mol/ml peat) = (Standard slope *sample area) * (He added in ml/1000)

(R*T) (Peat vol. in ml)

Where:

Standard slope = The slope obtained from a standard curve

Sample area = The chromatogram peak area of each sample

He added = 60 ml - peat volume

Peat vol = wet volume

R = the ideal gas law constant

T = Temperature in degrees Kelvin

To obtain a methane production rate, the cumulative methane concentrations were plotted against time for each subsample. A linear regression fit was applied to those data, the slope representing the potential production rate.

Soil Moisture

Method description

Cores were taken with a metal corer with a 2.7 cm radius 5 times over the season near the LTER tussock plots. Cores were taken at 6 different sites in both tussock and intertussock micro topographies, see 1992 LTER terrestrial notebook, p.7. In the field whole core length was determined by measuring the depth of the hole. The mineral layer of the actual core was measure, cut off and put back into the tundra. The length of the organic layer was calculated from subtracting the mineral layer length from the whole core length. In the lab, cores were weighed wet and then dried to a constant weight at 60 C. Percent water and bulk density were calculated as follows:

Percent water = 100 x (Wet weight - Dry weight)

Dry weight

Bulk density = Dry weight

Pi x (radius of corer)2 x Length of organic layer

Total Phosphorus

Following is a simple, easy and accurate method for total phosphorus in plants and soils. The basic method is taken form Aspila K.I., H. Agemian and A . S. Y. Chau. 1976. A semi-automated method for the determination of inorganic, organic and total phosphate in sediments. Analyst 101 187-197. The modification of adding Mg(NO3)2 is take from K.R. Ruttenberg. 1990 Diagenesis and burial of phosphorus in marine sediments: implications for the marine phosphorus budget. Ph.D. dissertation. Yale Univ. New Haven, CT.

All samples ground at least to 40-60 mesh, either on a Wiley mill or Bob's roller mill. Weigh about 0.1 gm of sample into a 20 ml glass scint vial. Then add 0.5 mls of 50% w/v Mg(NO2)3 letting the sample soak up the solution but do not let the sample dry completely before ashing or it will ignite and flare up during ashing. Ashed for 2 hours at 550 C (about 1/2 hour to get to temperature and then 1.5 hours at 550). The scint vials can be marked with a diamond tipped pen or other high temperature pens. After cooling add 10 mls of 1 N HCl and vortex to get all sediment into solution. Then shake for 16 hours. If the vials are left over night there is no need to centrifuge or filter the solution.

Dilute subsamples depending on levels expected and measure PO4-P using the method for high concentrations of Harwood, et al. 1969.


Field Protocols


Toolik and Sag Fertilizer Needs


Rate: 10g/m2 N; 5g/m2 P

Fertilizer: 34 - 0 - 0 i.e. 34% nitrogen; 0 - 45 - 0 i.e. 45% P as P2O5 = 19.89%P

 

LTER Sites


 

Tussock

Plot Size: 5 m x 20 m = 100 m2, except Greenhouse and Shade = 2.4 m x 4.8 m = 12 m2

Treatments: (P alone, N alone, N&P, Greenhouse N&P, Shade N&P) x 4 blocks

For nitrogen 8 plots x 100 m2 + 8 plots 12 m2 = 896 m2 26.4kg(58lb) of 34-0-0

For phosphorus 8 plots x 100 m2 + 8 plots 12 m2 = 896 m2 22.5kg(50lb) of 0-45-0

 

Wet Sedge

Plot size: 5m x 10m = 50 m2, except Greenhouse and Shade = 2.4 m x 4.8 m = 12 m2

Treatments: (P alone, N alone, N&P) x 3 blocks

(Greenhouse N&P) x 2 blocks

For nitrogen 6 plots x 50 m2 + 2 plots x 12 m2 = 324 m2 9.5kg(21lb) of 34-0-0

For phosphorus 6 plots x 50 m2 + 2 plots x 12 m2 = 324 m2 8.1kg(18lb) of 0-45-0

 

Heath

Plot size: 5 m x 20 m = 100 m2, except Greenhouse and Shade = 2.4 m x 4.8 m = 12 m2

Treatments: (P alone, N alone, N&P) x 3 blocks

(Greenhouse N&P) x 2 blocks

For nitrogen 6 plots x 100 m2 + 2 plots x 12 m2 = 624 m2 18.4kg(40lb) of 34-0-0

For phosphorus 6 plots x 100 m2 + 2 plots x 12 m2 = 624 m2 15.7kg(34.5lb) of 0-45-0

 

Shrub

Plot size: 5 m x 10 m = 50 m2, except Greenhouse and Shade = 2.4 m x 4.8 m = 12 m2

Treatments: (P alone, N alone, N&P) x 3 blocks

(Greenhouse N&P) x 2 blocks

For nitrogen 6 plots x 50 m2 + 2 plots x 12 m2 = 324 m2 9.5kg(21lb) of 34-0-0

For phosphorus 6 plots x 50 m2 + 2 plots x 12 m2 = 324 m2 8.1kg(18lb) of 0-45-0

 

New LTER Exclosure sites (Started June 1996).


Tussock

Plot Size: 5 m x 20 m = 100 m2

Treatments: (N&P) x 4 blocks

For nitrogen 4 plots x 100 m2 = 400 m2 11.8kg(26lb) of 34-0-0

For phosphorus 4 plots x 100 m2 = 400 m2 10kg(22lb) of 0-45-0

Health

Plot Size: 5 m x 20 m = 100 m2

Treatments: (N&P) x 3 blocks

For nitrogen 3 plots x 100 m2 = 300 m2 8.8kg(20lb) of 34-0-0

For phosphorus 3 plots x 100 m2 = 300 m2 7.5kg(17lb) of 0-45-0

 

Old Toolik Tussock Site


Plot size: 5 m x 20 m = 100 m2

Treatments: (N&P) x 4 blocks

For nitrogen 4 plots x 100 m2 = 400 m2 11.8kg(26lb) of 34-0-0

For phosphorus 4 plots x 100 m2 = 400 m2 10kg(22lb) of 0-45-0

 

Sag Site

Additional treatments: Lime 150 m2; Starch/Sawdust 50 g m2/100 g m2 (dry wt.)

Starch @ 2 boxes/plot=45.5g/m2


Tussock, Heath, Hill Slope, Sedge

Plot size: 2 m x 10 m = 20 m2

Fert. Treatments: (N alone, P alone, N&P) x 2 plots; (lime, starch) x 2 plots

For nitrogen 4 plots x 20 m2 = 80 m2 x 4 sites = 320 m2 9.4kg(20.7lb) of 34-0-0

For phosphorus 4 plots x 20 m2 = 80 m2 x 4 sites = 320 m2 8kg(17.7lb) of 0-45-0

For lime 2 plots x 20 m2 = 40 m2 x 4 sites = 160 m2

For starch 2 plots x 20 m2 = 40 m2 x 4 sites = 160 m2

 

Equisetum

Plot size: 2 m x 8 m = 16 m2

Fert. Treatments: (N alone, P alone, N&P) x 2 plots

For nitrogen 4 plots x 16 m2 = 64 m2 1.8kg(4lb) of 34-0-0

For phosphorus 4 plots x 16 m2 = 64 m2 1.6kg(3.5lb) of 0-45-0

For lime 2 plots x 16 m2 = 32 m2

For starch 2 plots x 16 m2 = 32 m2

 

Willow

Plot size: 2 m x 20 m = 40 m2

Fert. Treatments: (N alone, P alone, N&P) x 2 plots

For nitrogen 4 plots x 40 m2 = 160 m2 4.7kg(10.4lb) of 34-0-0

For phosphorus 4 plots x 40 m2 = 160 m2 4kg(8.8lb) of 0-45-0

For lime 2 plots x 40 m2 = 80 m2

For starch 2 plots x 40 m2 = 80 m2


Totals

 

Toolik LTER

Nitrogen Totals = 2,868 m2 84.3kg(186lb) of 34-0-0

Phosphorus Totals = 2,868 m2 72kg(159lb) of 0-45-0

 

Toolik Old Tussock

Nitrogen Totals = 400 m2 11.8kg(26lb) of 34-0-0

Phosphorus Totals = 400 m2 10kg(22lb) of 0-45-0

 

Sag

Nitrogen Totals = 544 m2 16kg(35lb) of 34-0-0

Phosphorus Totals = 544 m2 13.7kg(30lb) of 0-45-0

Lime = 272 m2

Stach = 272 m2


Grand Totals

Nitrogen = 3,812 m2 * 10 g m-2 = 3,810 g N * 1 g fert/.34 g N = 112,118 g Fert.

95.294 kg Fert * 2.2 = 247 lb 34-0-0 Fertilizer

Phosphorus = 3,812 m2 * 5 g m-2 = 19,060 g P * 1 g fert/.199 g P = 95,779 g Fert.

81.4 kg Fert * 2.2 = 211 lb 0-45-0 Fertilizer

Lime = 272 m2 * 150 g m-2 = 40.8 kg * 2.2 = 90 lb

Starch = 272 m2 * 50 g m-2 = 13.6 kg * 2.2 = 30 lb

Sawdust = 272 m2 * 100 g m-2 = 27.2 kg * 2.2 = 60 lb


Rain Collection Procedures


There are two types of rain collections over the summer season, an event rainfall and a weekly wetfall sample. There is also dryfall that is collected once at the end of the season. All rain is collected at the LTER tussock tundra site across Toolik lake from camp.

An event rainfall is considered over when it does not rain for approximately 6 hours. This definition is not exact.

Weekly rainfall is collected in an Aerochem Metrics wet/dry precipitation collector. The weekly wetfall sample is collected on Wednesdays and mailed to Global Geochemistry from Prudhoe by the Thursday taxi. $3.00 postage.

Address: Stan Shepard
Shepard Analytical Cor.
4545 Industrial St. Unit 5P
Simi Valley, CA 93063

Rainfall, wetfall and dryfall are analyzed for N-NO3, N-NH4, P-PO4 and pH in camp. All supplies have been kept in the Pluck n Suck.

 

Collection:

The wetfall/dryfall collector is put up in the field in the beginning of the season with a solar panel and battery. Dryfall is collected once at the end of the season (See below in Volume section).

Weekly wetfall: Bring up a new bucket and replace the one in the field. Note any bird droppings or other dirt. If bird droppings are present then do not send a sample to Global Geochemistry.

Event rainfall bottles: Each bottles has a letter and a number. When changing bottles match the bottle letter to the funnel letter. Also note droppings or dirt. If the cotton is dirty then change with new polyester cotton. (There is usually some washed and kept in a ziplock kept with the extra bottles).

If bird droppings are present the bottles, funnels or collection buckets must be wiped out, subjected to a short (2 hr) acid wash, and then soaked for over a day in DI.

 

Lab preparations:

All readings are recorded in the lab book on pages setup for the rain. There are columns for volume, pH, and nutrients. At the beginning of each season record funnel and bucket diameter.

Volume:

All scintillation vials and graduated cylinders are rinsed with sample before filling with final sample. Both types of samples are treated the same, except 175-250 mls is saved for sending out to Shepard Analytical from the weekly wetfall. The total amount in each bottle is measured in a graduated cylinder, be sure to record how much you rinsed with!

Dryfall: Add 150-200ml of DI (do not add more because it may dilute the chemistry) to the dryfall bucket at the end of the year. Swish around the water to rinse bottom and sides of bucket. Then treat similar to wetfall but sending what remains after chemistry to Global Geochemistry with the wetfall.

pH:

All scintillation vials and graduated cylinders are rinsed with sample before filling with final sample. The pH probe is calibrated at 7.0 and 4.0. A reading is taken after the pH meter has stopped drifting (2-5 minutes). There is a bottle of old rain water for soaking the pH probe after calibrating it, a 5 minute soak. This leaches the probe for the dilute rain water. Ten mls of sample is used for measuring pH. There are scintillation vials for pH labeled with letters that match the collection bottles which are reused for each collection.

Nutrients:

All scintillation vials and graduated cylinders are rinsed with sample before filling with final sample. At least 20 ml is saved in scintillation vials for PO4, NO3, and NH4 if the nutrients are to be run on the autoanalyzer. If the chemistry has to be run by hand save 10 mls separately for each nutrient.

Rinse rain bottles 3 times with DI water when finished.

 

What to do if not enough sample:

For the wetfall save enough for sending out. Shepard Analytical will be able to use as little as 50 mls.

For the event samples save enough for pH (3 mls) and NO3, NH4 and PO4 (3 mls) to be autoanalyzed.

Please contact arc_im@mbl.edu with questions, comments, or for technical assistance regarding this web site.