A.

FIELD SAMPLING

1.

Plot and Transect Setup in the Field

a.

Choose the area and direction for the 1 km transect:

• Identify the north with a GPS
• Face the north direction
• Keep the transect perpendicular to the north
• Record GPS data for each plot

b.

Laying out each plot (10 m x 10 m) using ropes based on Pythagoras’s theorem (a2 + b2 = c2):

• Use three ropes preferably non-stretchable, and waterproof of about > 1 cm thickness
• Two of the ropes should be 10 m long with overlaps of ~15 cm at each end of the rope to facilitate handling. Also mark the middle of the rope at 5 m
• Use bright color paint or permanent marker to ensure visibility especially in dense vegetation
• The third rope should be 14.1 m in length (still with the overlap for handling). This represents the hypotenuse to help achieve perfectly square plots from (at right angle). (See Fig. 1).
• Mark the position of each plot and the four subplots with wire flag

Fig. 1

c.

Setting up sampling positions in each plot:

• Take a GPS reading at each plot (A, B, C & D) at the left (90o angle) corner of the plot when facing the north
• In the plot diagram below (Fig. 2), 1, 2 & 3 represent the PVC ring positions for CO2 measurement and are also sampling positions for vegetation and organic matter (partially decomposed surface residue)
• Soil samples for bulk density are taken around the perimeter of core 2
• Ten soil samples are taken randomly from each plot to be used as bulk soil samples
• Sub samples for soil moisture evaluation are taken from the bulk soil samples back in the lab

Fig. 2. Sampling positions for soil, vegetation, litter, and CO2 in a plot

2.

Sampling Vegetation and Organic Matter

a.

Carefully place a PVC ring (10 cm diameter, 5 cm depth) on the soil surface

b.

Cut off all plant material within the circumference of the PVC ring with a pair of clippers

c.

Place the plant material in a pre-labeled plastic bag (in case it rains in the field. A paper bag is used for drying the samples back in the lab)

d.

Collect all the organic materials on the soil surface within the circumference of the ring (green leaves are sorted out as accidental harvesting)

e.

Place a block of wood on the PVC ring and push it into the soil uniformly until about 1 cm of the ring is protruding above the soil surface (Fig. 3).

f.

Use a wooden or rubber mallet if necessary to achieve the right depth

g.

Repeat steps ‘a’ to ‘g’ for positions 1, 2 & 3, and in plots A, B, C & D (Fig. 3).

3.

CO2 Measurement Using the PP Systems EGM-4

a.

Before taking the first reading, allow the soil to settle for at least an hour after pushing the PVC rings into the soil

b.

Follow the EGM-4 operation menu (Appendix A) for CO2 measurement

c.

Record data on a data sheet manually in the field as a backup in case there is a problem downloading the data

d.

Take three to four readings in a day

e.

Leave at least 2 hours between each sampling

f.

Download the data every day in the lab by following the EGM-4 operation menu (Appendix A)

g.

Bring an external battery and connection cord to the field with the EGM-4 system in case the internal battery runs out in the field

h.

Recharge the EGM-4 and external battery in the lab if you plan to use them again the next day

4.

Soil Moisture Measurement

a.

Pre-weigh 4 moisture tins + lids

b.

From each of the 4 bulk soil samples, (after each bag has been gently mixed), weigh out approximately 60 g of soil into a pre-weighed moisture tin +lid 

c.

Immediately weigh the tin + lid + field moist soil

d.

Put the tin + soil into an oven at 105° C for 24 hours

e.

Weigh the oven-dry soil + tin + lid

f.

Soil moisture is calculated on a g/g and % basis where

Soil moisture (%) = ((fresh soil weight – dry soil weight) /dry soil weight) x 100

5.

Bulk Density Measurement

a.

Soil for bulk density determination is taken from within 15 cm perimeter of the PVC ring at position 2 for each plot using a 5 cm x 5 cm soil core

b.

*If you use another core size, always calculate the volume of the core used for sampling to compute the bulk density

c.

Soil sampling depth:  0 - 5 cm

d.

Trim soil with a knife where necessary, to the size of the sampling core

e.

Empty the soil into pre-weighed and labeled soil cans

f.

With a spatula or table knife, carefully clean all the soil in the core into the soil can

g.

Put the lid on immediately to avoid moisture loss

h.

Weigh the tin + lid + fresh soil immediately on arrival in the lab

i.

Put into an oven at 105o C for 24 hours

j.

Weigh oven-dry soil

k.

Calculate soil bulk density using the relationship: 

Soil Bulk Density (g/cm3)= Mass of oven dry soil (g) ÷ total volume of soil (cm3)

6.

Bulk Soil Sample Collection

a.

Sampling depth: 0 – 10 cm

b.

10 random sampling positions within each 10 m X 10 m plot

c.

Soil sampling is done with a core (5 cm in diameter)

d.

The 10 cores of soil are put into a labeled plastic bag and immediately stored in an ice chest with blue ice in the field

e.

There should be approximately 1 kg of bulk soil from each plot

f.

The soil corer needs to be carefully cleaned between samples using a table knife

g.

On return to the laboratory, the soil sample in each bag is gently mixed by hand from the outside of the bag before taking sub-samples for analyses

7.

Sub-Sampling for Each PI for Invertebrate, Chemical and Molecular Analyses

Bulked soil samples are divided into the following sub-samples for separate analyses

a.

Diana Wall Lab (450 g of soil) for: 

1. Soil samples for nematodes counting: 100 g
2. Soil microbial biomass: 20 g
3. Potential rates of decomposition: 50 g
4. Mineralizable C & N: 30 g
5. Total C, N, P: 10 g
6. Soil moisture content: 60 g

b.

Richard Bardgett Lab (70 -150 g of soil) for: PLFA.

c.

In addition, two samples from each bulked soil sample will be processed in the field lab (200 g for Diana Wall Lab for morphology analysis and 200 g for Jim Garey Lab for molecular analysis). The protocol for processing these samples in the field lab is:

1. Soak soil samples in 95% ethanol
2. Stir the soil 
3. Pour liquid off through 4 pre-wetted mesh screens (2mm, 1mm, 0.5mm, 0.1mm)
4. Back wash the soil with 95% ethanol from each screen into pre-labeled plastic vials (50 ml for Jim Garey, 250 ml for Diana Wall).

Flowchart of soil sub-sampling and analyses

8.

Equipment List for Field Trips (Not Included)

B.

DETAILED FIELD AND LAB PROTOCOLS IN THE APPENDICES (Not Included)

Appendix

Protocol

Reference

A

EGM-4 (Environmental Gas Monitor) Operating Menu for CO2 Measurement in the field

EGM-4 Environmental Gas Monitor For CO2, Operator Manual, Version 4.12, 2002 PP System

B, C

Sugar Centrifugation Methods for Extracting Nematodes in Fine and Coarse Textured Soils

Wall & Virginia 1993

D

Counting Invertebrates

E

C and N Mineralization

LECO CHN-1000

F

Potential Rates of Decomposition

Wardle et al 2004?

G

Microbial Biomass

Parkinson & Paul 1982

H

Total C and N

Carlo-Erba Instrument

I

Total P

Kuo, 1996

J

Molecular Analyses