# Manure and Compost Sampling Preparation Protocols

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## Manure and Compost Sampling Preparation Protocols

Leopold 2000-42: Optimizing swine hoop manure management for soil quality and crop system peformance

Field 16: moisture, bulk density, pH, EC, total organic C, total N, inorganic N, organic and inorganic P, K, CO2 respiration, and cress seed phytotoxicity bioassay

PFI farms. As above, except only one P analysis (not specified)

Both: 1 analysis per field plot and 1 analysis per compost pile at each sampling time. Compositing as below:

## Procedure

1.Sample collection. Bulk density should be measured in the field using method 2a if possible. For additional analysis a single composite sample should be collected from each compost pile or the manure being spread on each field plot. For each composite, 20 grab samples should be collected (20 trayfuls per plot or 5 depths from each of 4 transects into compost piles) and combined in a 5 gallon bucket. After measuring bulk density of the bucket (see method 2b), mix and subsample to collect a single 0.75 to 1 kg composite sample. Cool sample immediately, or freeze if not homogenized within 24 hours.

2.Measure bulk density:

Alternate methods (in order of preference)
a) Ideally done during field application by striking a manure spreader off level, weighing the load, measuring the spreader and calculating the volume.
b) Place fresh sample in a 5 gallon bucket, tamp 10 times dropped from 6 inches, weigh, measure settled depth and diameter, and calculate volume.
c) Place thawed sample in 2 liter plastic container, tamp 10 times, weigh, measure settled depth and diameter, and calculate volume.

3.Homogenize each sample in Hobart mill. Split sample into 5 bags: 100 g for fresh analysis at LEBRC (pH, EC, moisture); 350 g for cress bioassay at Liebman’s lab; 250 g for $\displaystyle{ CO_2 }$ respiration at Richard’s lab; 100 g for acidification at Richard’s lab and ultimately total C and N analysis at Cambardella’s (20 g of ground material will go to Hort lab for ashing and ICP analysis); and 50 g for KCl extraction at Cambardella’s lab. Refreeze $\displaystyle{ CO_2 }$ respiration, cress bioassay, acidification and KCl samples if those analyses are not going to be done immediately.

4. EC and pH: Measure in triplicate. Add 150 ml deionized water to 30 g fresh sample. Measure pH and EC with electrodes and calibrated meter. See attached method for complete method description.

5. Moisture: Use remainder of LEBRC sample (100 g – 30 g EC & pH). Record tare weight of tin and fresh weight of tin + manure. Dry at 75°C until constant weight (24 hrs?) and record results. See Method 07.09, Test Methods for Compost Analysis for complete method description.

6.Cress bioassay: Need description.

7.CO2 respiration: 1 incubation for each sample. If samples have been frozen, remove 20 g 1 week before pre-incubation and incubate at 37°C for one week, then blend with thawed sample. Adjust moisture to 50% and pre-incubate for 3 days in unsealed plastic bag. Incubate triplicate 25 g samples in 4L glass jars with NaOH traps for 4 days, titrating and replacing NaOH daily. See attached method for complete method description.

8.Acidification for C and N analysis. 100 g sample are placed in 8 x 5 x 2 tupperware trays. Add 200 ml of 0.5 N HCl and stir with stainless spoon until all compost is submerged. Dry in fume hood with fan for 5-6 days. Grind in Retsch mill and analyze in duplicate in Carlo-Erba CHN analyzer. See attached method for complete method description.

9.Inorganic N analysis using KCl extraction: Analyze in triplicate. See attached method.

## Appendix 1

Bulk Density

• Ideal Method
1. This method is performed during field application.
3. Measure the spreader dimensions and calculate volume.
• Fresh Sample Method
1. Place a fresh weighed sample in a 5-gallon bucket.
2. Tamp 10 times by dropping bucket from 6 inches off the ground.
3. Measure the settled depth and bucket diameter and calculate volume.
• Frozen Sample Method
1. Place a thawed weighed sample in a 2-liter plastic container.
2. Tamp 10 times by dropping container from 6 inches off the ground.
3. Measure the settled depth and diameter of container and calculate volume.

## Appendix 2

Sample Homogenization and Distribution

• Materials Needed:
Hobart buffalo chopper
Rubber spatula
5-gallon bucket or other large container
Scissors
Ziploc® bags
Scale
• Setup
1. Thaw frozen samples before chopping.
2. Determine what types of sample analysis will occur after homogenization. Label Ziploc® bags with the proper sample identification and intended analysis or destination.
3. Attach the blade, comb and bowl of the chopper.
4. Latch the chopper cover down. This is a safety feature and the chopper will not run if the cover is not latched.
• Procedure
1. Turn on the chopper and place the sample in the bowl.
2. If the sample contains large pieces of straw or cornstalk it is helpful to cut up these pieces with scissors and only run part of the sample through the chopper at a time. If the sample gets caught around the comb or blade, stop the chopper and unclog the comb; otherwise the blade cannot rotate properly and the sample will get hot.
3. While the bowl is turning, use a rubber spatula to scrape wet material from the bottom and sides of the bowl. Stop the chopper once the large pieces are chopped up and the sample looks homogeneous.
4. Place the chopped material in a 5-gallon bucket and continue chopping the remaining sample. Once an entire sample has been chopped, mix the sample by hand in the bucket.
5. Homogenize the sample thoroughly but quickly to avoid volatilization and changes in sample characteristics.
6. Divide the chopped sample into the proper Ziploc® bags and seal tightly. Place all bags in the freezer unless the analysis will be done immediately.
7. Clean the buffalo chopper after each day’s use and as necessary between samples. Remove the blade, comb and bowl and wash these pieces in the sink. Wipe out the cover of the chopper with a sponge. Dry all parts with a paper towel before reassembling.

## Appendix 3

Electrical Conductivity and pH

• Materials needed:
Scale
Deionized water and water bottle
pH buffers, 7 and 10
Kim wipes
1000-ml beaker
Accumet pH meter
200-ml beakers
Stirring rod
YSI portable electrical conductivity meter
• Sample Preparation:
1. Thaw frozen samples before performing analysis.
2. Use the same sample for pH and electrical conductivity measurements.
3. Weigh 30 g of compost into a 250-ml Erlenmeyer flask.
4. Add 150 ml of deionized water.
5. Swirl the flask to mix and submerge entire sample. Let the flask sit for 30 minutes, swirling occasionally.
6. For electrical conductivity measurements the sample needs to soak in water a total of three hours.
• pH Measurement
1.Turn the pH meter from “Stand-by” to “pH” mode.
2.Set the “Slope” knob to 100 and set the correct temperature.
3.Calibrate the meter using the pH 7 and 10 buffers:
a. Place the electrode in the pH 7 buffer and wait for the reading to stabilize. Then use the “Standardize” knob and set the meter to 7.
b.Rinse the electrode with deionized water, blot dry with a Kim wipe and place it in the pH 10 buffer. Wait for the reading to stabilize and use the “Slope” knob to set the meter to 10.
c.Rinse and blot the electrode and test the pH 7 buffer again. If the meter does not read 7, perform the calibration again.
4.When the calibration is complete, rinse the electrode and place it in the first sample flask. Wait for the meter reading to stabilize and record the pH. Rinse the electrode between samples.
5.When finished, rinse the electrode well and blot dry, then replace the electrode in the storage buffer. Return the meter to “Stand-by” mode.
• Electrical Conductivity Measurement:
1. After three hours, pour only the liquid from the sample flask into a 200-ml beaker.
2. Squeeze as much liquid out of the sample as possible using a glass stirring rod and pour this into the beaker as well.
3. Turn on the YSI portable EC meter. This meter needs no calibration, but make sure it is set to measure electrical conductivity.
4. Hold or suspend the probe in the beaker so that the liquid covers the opening at the top of the probe. The probe should not touch the sides or the bottom of the flask.
5. Wait for the meter reading to stabilize and record the electrical conductivity.
6. Rinse the probe between samples and when finished. Turn the meter off when finished to conserve battery power.

## Appendix 4

Moisture and Volatile Solids

• Materials Needed:
Small aluminum tins
Scale
75 °C oven
550 °C oven
Heavy gloves and metal tongs
• Procedure
1. Thaw frozen samples before performing analysis.
2. Record the ID number and weight of a small aluminum tin.
3. Place 40-50 g of sample into the tin and record sample + tin weight.
4. Dry samples at 75 °C to a constant weight (24 hours).
5. Record the dry sample + tin weight for moisture calculations.
6. Place samples in the muffle ovens set to 550 °C for six hours. Use the heavy gloves and metal tongs to remove hot samples.
7. Record the ash + tin weight for volatile solids calculations.

## Appendix 5

Cress and Ryegrass Phytotoxicity Bioassay

• Materials Needed:
Mason Jars, quart size
Cheese cloth
1 L centrifuge containers
Long stem funnels
250-mL Nalgene bottles
Distilled water
Cress seed (Lepidium sativum)
Annual Ryegrass seed (Lolium multiflorum)
0.45 um Millipore nylon syringe filter
Scintillation vials for extract storage
P5 filter paper (100mm)
50 ml syringe
• Sample Handling:
1. Combine each of the samples from an individual treatment or pile-sampling date into one sample bag that will be used for the bioassay.
2. Homogenize each composite sample with the buffalo chopper (see appendix 2).
3. Determine the dry weight content of each sample (see appendix 4).
• Extraction Procedure - goal obtain 10% aqueous extracts
1. Tare a 1-quart Mason jar on a balance.
2. Place a 70-g dry weight equivalent of as received material into the jar (see calculations below). Add distilled water for a total mass of 770 g.
3. Seal the Mason jar and place in the oscillating shaker at low speed for 15 hours at room temperature.
4. Strain the contents of each Mason jar through 3 layers of cheesecloth and collect the extract in 1-liter plastic centrifuge containers (NSTL room 201).
5. Centrifuge extracts for 30 minutes at 22°C and 3000 rpm (floor centrifuge in NSTL room 201).
6. Decant supernatant into two 250-ml Nalgene® bottles.
7. Store final extracts in 4°C cooler if not used immediately.
grams of desired dry weight equivalent/1-(% moisture /100)=Wet weight needed

Example: (70g) / (1-(50%/100)) = 140g wet material
• Germination Test Procedure:
1. Use distilled water to dilute the 10% extracts to 5% in centrifuge tubes. Centrifuge at 4000 g for 10 minutes at 22ºC (in Agronomy 1515).
2. Place 20 seeds of a species (Lepidium sativum or Lolium multiflorium) onto 2 layers of Fisher® Qualitative P5 filter paper in 100 x 15-mm sterile petri dishes.
3. Filter 2.4 ml of the 5% extract through a 0.45-μm Millipore® nylon syringe filter and apply to the seed-containing petri dishes.
4. Replicate the procedure 5 times in a completely randomized design with a control of 2.4 ml of distilled water.
5. Place the petri dishes in crisper bags with 5 ml of extra water in the bottom of the bags for humidity control.
6. Incubate in alternating conditions of 30ºC (16 hours of light) and 20ºC (8 hours of dark).
7. Arrest growth after 4 days by adding 2 ml of 50% ethanol to each petri dish.
8. Store in 4ºC cooler until ready to be examined.
9. Measure the radicle length of each seed to the nearest millimeter. If the radicle length is less than 0.5 mm or the seed did not germinate at all, then record a length of zero.

## Appendix 6

$\displaystyle{ CO_2 }$ Respiration

• Materials Needed:
Incubator – capable of sustaining temperatures of 35± 2ºC.
Beakers – two 150 ml glass per sample.
Respiration Flask – 4 L Glass Containers that can be readily sealed.
Scintillation vials for $\displaystyle{ CO_2 }$ trap storage
Titration machine (NSTL 201)
Squeeze bottle de-ionized $\displaystyle{ H_2O }$
Spray bottle with de-ionized $\displaystyle{ H_2O }$
Sodium Hydroxide 2N (approximately 2 L for every 25 samples)
Balance (± .01g)
• Sample Handling and Pre-incubation:
To assure test results that represent an approximation of compost material stability, the sample must be in a condition where aerobic microbes flourish. In laboratory condition we can control the moisture and the temperature and to a lesser extent the aeration of the sample of compost in question. A pre-incubation period is necessary for the microbial community of the sample to reestablish its biological activity (after being frozen). Two days will be required to thaw out the samples, determine the moisture content, and make the necessary wet weight adjustments. The pre-incubation period is to last 3 days and finally 4 days are required for the respiration test itself. To standardize the conditions that we are testing the various compost/manure materials under we need to follow the protocol below.
Day One – Thaw out samples in the 4ºC walk-in cooler (if frozen, if not go to day 2)
Day Two – Moisture Determination
In order to standardize the experimental conditions the moisture content of each sample has to be determined (refer to appendix 5, do not add this material back into the sample), so that the moisture content can be adjusted. The remaining material should be placed back in the 4ºC cooler.
Day Three through day five – Moisture Adjustment and Pre-incubation
The goal is to have constant moisture content for all of the samples being tested. We will use 50% moisture on a wet weight basis as the standard. First, the entire sample needs to be weighted in the one-gallon ziploc bag where the pre-incubation will occur. If the moisture content is greater than 50% then the sample should be layout flat with the bag open and in a well aerated area (like under the hood or with a fan blowing in them). If the sample is drier than 50% water will need to be added. Lay the bags out flat with the bag open to the ambient air and add the necessary distilled $\displaystyle{ H-2O }$, using a spray bottle, while the bag is on the balance.
The 50% moisture content weight should be calculated, so that an easy reference point can be used to monitor the moisture adjustment process (remember to exclude the bag weight).
Initial wet weight (g) * 1 – H2O% = Initial dry weight (g)
Initial dry weight (g) * 2 = 50% $\displaystyle{ H_2O }$ content weight
Once the sample has been layout flat within the ziploc bag it should remain flat (samples will ball-up if disturbed, making it difficult to keep them homogenized). Using a clipboard to slide under the bag when moving or weighing will be of help here
This moisture adjustment period and the pre-incubation period occurs simultaneously. Moisture adjustments should be made daily throughout the pre-incubation period.
Day Six through Day Nine – Incubation
At the end of the third day of the pre-incubation period make final moisture adjustments to 50% (assuming that the organic matter respired during the pre-incubation is of no significance).
Transfer 25±2 grams of pre-incubated sample into an incubation vessel (150ml beaker). Record the weight of the beaker and the sample to the nearest 0.01 g. Transfer 20 ml of 2N NaOH into a 150 ml beaker.
Place the NaOH and the compost sample into an incubation vessel. Close the lid tightly and place in an incubator set at 37ºC.
Set up a blank by placing a 20 ml aliquot of 2N NaOH into an incubation vessel without a compost sample.
Record the time and date the first samples were prepared.
With the remaining material the final moisture content and biodegradable volatile solids content will need to be determined. Refer to appendix 5 for description of BVS.
After each day of incubation remove the NaOH traps, transfer each used NaOH trap into a Scintillation vial for storage until titration, and then replace it with a fresh 20 ml aliquot of 2N NaOH. When transferring the used NaOH trap into the Scintillation vial it is important that all of the NaOH be transferred, so a squeeze bottle of distilled $\displaystyle{ H_2O }$ can be used to wash all of the NaOH out of the trap beaker into the vial (the added H2O will not alter the results). Use the manual jug top burette to measure out the 20 ml of 2N NaOH for the traps.
• Titration
Titrations will be conducted on the NaOH traps in NSTL 201 using their titration machine. (see Jody Ohmacht in NSTL 201)
The calculations for interpreting the results will have to be worked out but involve the following; titration results, time of incubation, moisture content, and volatile solids content.

## Appendix 7

Acidification for C and N Analysis

• Materials Needed:
Rubbermaid container, 8”x 5”x 2”
Ziploc® bags
HCl solution, 0.5 N
Larger spoon
Stainless steel spoon
Storage vials
Retsch mill with 0.5-mm
Shallow plastic tub
ring sieve
Paintbrush
Scoopula
Tin boats for weighing
8-mm sieve
1000-ml glass beaker
• Acidification Procedure
1. Add 6 liters of deionized water to the carboy labeled “0.5 N HCl.”
2. Add 260 ml of concentrated hydrochloric acid to the water in the carboy and mix by shaking gently.
3. Place a 100-g sample into a Rubbermaid container. Label the container with the proper sample ID and date that was on the original sample bag. Add 200 ml of 0.5 N HCl to the container.
4. Using a stainless steel spoon, break up the sample and spread it out along the bottom of the dish so the entire sample is submerged.
5. Leave samples in the fume hood until completely dry.
6. Use a spoon or spatula to break up the dry sample and transfer the sample to a labeled Ziploc® bag.
7. Grind the dry sample in the Retsch mill.
• Setup for Grinding
1. The Retsch mill needs to be located in a fume hood with an air nozzle nearby to reduce the amount of dust and facilitate clean-up.
2. Pass the dried sample through the 8-mm sieve to break it up. This allows it to pass through the input funnel on the Retsch mill. Remove all rocks and plastic pieces.
• Grinding
1. Turn on the mill and start the rotor. Allow it to reach full speed before beginning to add sample (14000 rpm).
2. Add sample in small amounts, using the scoopula to clear the input funnel. When the entire sample has gone through the grinder, turn off the rotor.
3. Remove the sample basin and pour the ground material into the glass beaker. Use the brush to dust all material from lid, ring sieve, and basin into the beaker. Mix the sample thoroughly.
4. Transfer a portion of the sample to the labeled storage vial and return the remainder to the Ziploc® bag.
5. Use the air jet to clean all equipment before moving on to the next sample. After a grinding session, or as needed, disassemble parts for cleaning. Wipe off the lid, basin, ring sieve, and rotor parts with a wet paper towel and allow all parts to dry fully before reassembling and resuming grinding.
• Weighing for Moisture
Remove 3-5 g of ground sample for lab moisture determination and dry overnight at 105 °C.
• Weighing for Carlo Erba Analysis
Weigh 5-10 mg of the ground sample on the Perkin-Elmer microbalance into tin boats for total C and N analysis on the Carlo-Erba.
1. Samples first are checked for carbonates. Place some sample in a spot plate, add a few drops of water and ‘stir’ to make a paste.
2. Add a few drops of 1N HCl and if carbonates are present effervescence will be seen.
3. If carbonates are present, weigh the sample into silver boats, treat with 1N HCl and place the open boat in a dessicator to “dry”.
4. If there are no carbonates, weigh samples into tin boats and store in holding trays until analyzed on the Erba.
• Weighing Procedure
1. Sponge down the work area and rinse tools with alcohol before beginning. Repeat this procedure every ten samples.
2. Place boat on sample pan.
3. Lower pan arrest (release brake).
4. Press Autotare (tare). Wait until “INT” is complete and no longer displayed. The display will read 0.00.
5. Raise pan arrest (brake the pan).
6. Remove the boat. ‘Scoop’ some sample into the boat (5-10mg). After a few are done your “eye” will become calibrated to the amount to place in the boat.
7. Fold the boat to a square pillow shape. Some boats will be broken or punctured when first learning the procedure. If so, toss the torn boat and repeat the above procedure.
8. Place the boat containing sample on the pan and lower pan arrest. Get the next boat ready (opening it) while waiting for the weight to stabilize.
9. Record weight on the data sheet and place the boat in the holding tray. Be sure the cup number in the tray and on the data sheet correspond.
10. Repeat above procedure for other samples. Remember to clean work area and tools after doing 10 samples.
11. Keep holding trays in a dessicator until samples are analyzed on the Erba.

## Appendix 8

Inorganic N Analysis

• Materials Needed:
Mason Jars, quart size
No. 40 Whatman filter paper
Long stem funnels
Scintillation vials for extract storage
2M KCl
• Sample Handling
Fresh samples are extracted immediately upon arrival or frozen if extractions are to be done at a later date.
• Setup
1. Perform setup the day before extraction.
2. Make KCl solution and put 800-ml aliquots in the Mason jars.
3. Set the flat lid on the jar and place labels on the jar.
4. Label and weigh moisture pans.
• Extraction
1. Remove samples at least 3 hours prior to extraction time
2. Take 5 sub-samples from each respective bulk sample. Sample size should be between 9.80 and 10.20 g.
3. Remove approximately 30 g for moisture determination and dry overnight at 105 °C.
4. Lay samples horizontally on an Eberbach shaker and shake for ½ hour.
5. Pour the extract through a funnel with filter paper and collect the extract in a 20-ml scintillation vial.
6. Freeze samples until they can be analyzed using a LACHAT flow injection analysis unit.
7. Thaw extracts the day of LACHAT analysis.