Long-Term Inter-site Decomposition Experiment Team
Meeting the challenge of long-term, broad-scale ecological experiments
Study Supplies and Procedures
We get plenty of questions regarding our methods.
Here are the answers to the most common questions.
For the bottoms of our litterbags we used white polyester broadcloth to minimize loss through sifting through the mesh. This material has approximately 55 micron mesh, and is commonly available in fabric stores. We do not wash either the mesh or the broadcloth prior to use.
We sew our bags with polyester core cotton covered thread that is commonly available in fabric stores here, leaving one end open for filling.
The Staples are for the SALCO model 51-FS stapler also known as the Rapid 51. Order a stapler or two to use, as the staples do not fit common staplers. The staples are Code "2 1/4 Monel" with a leg length of 5/32" and they come in boxes of 5000 with price breaks for quantity. These are designed for photographic film splicing and editing.
We individually number our litter bags by attaching racetrack-shaped aluminum numbered tags we purchase through Forestry Suppliers http://www.forestry-suppliers.co). Pin flags are also available from this company.
UV Resistant Plastic Cable Ties (4 inch length (10cm) with a width of about 3 mm.) are used to attach the aluminum tags and can be purchased at most electronics or automotive stores.
We use Size 18 Nylon braided Seine Twine to tie our litterbags into harvest lines in the field. This string is strong and durable, resisting 10 years of exposure to the elements without weakening. String that lasts will increase the likelihood of bag recovery years down the road. It is available at most hardware stores and can be found on the internet with a search for "Seine Twine".
We employ a seamstress to assemble our bags as they usually have the equipment to be very efficient. The mesh is cut to 20cm X 20cm squares and the broadcloth is cut to 22cm X 20 cm. Our seamstress uses a serger (although normal sewing is adequate) to sew three sides of the square closed with polyester core thread. The open side of the square coincides with the 2cm extension of the broadcloth. This flap is tightly folded back over the open side, after filling, and stapled closed with 5-6 Monel staples.
To attach the aluminum number tag we punch a 3 mm. hole through one of the sewn closed corners of the fabric (about 2 cm from the edge, inside the stitching line) with leather punch pliers. We pass a 4 inch UV resistant plastic cable tie through the hole and the aluminum tag hole and close the cable tie, leaving a loop of at least 1-2 cms in diameter to allow easy passage of a string, during field installation. Take care to avoid two numbered tags from sticking together. It will save frustration later if the tags and cable ties are attached in the same way on all the bags, such as the cable tie threaded up from the bottom with tag numbers up. You will spend less time flipping tags to read them later if you have been consistent here.
We collect senescent leaves by hand, only gathering those that are ready to fall. This is time consuming. Evergreens can sometimes be shaken to release their needles onto tarps, and it is often possible to strip the brown needles off lower branches if the collection is properly timed. We often gather broadleaves by running our hands over branches with leaves that look ready, and only collect those which easily release. Freshly fallen leaves are sometime obvious enough to allow collecting them from the ground without collecting older leaves inadvertently. Collect much more than you think you will need because by the time you realize you do not have sufficient material the good collecting time is past, and additional collection will delay everything while the new leaves dry.
Once back at the lab we air dry the leaves in mesh racks lined with brown craft paper with air circulation on all sides. If the collection has had significant contamination we will go through it to remove twigs, fruits, and non-target material. If the collection quality is high the cleaning can be done at the same time the bags are being weighed and filled. Every day or two the trays of drying leaves are fluffed and turned to encourage even drying.
After a couple of weeks the leaves have dried to a fairly uniform moisture content. We mix all the leaves of a species in a large container and get a bulk weight of the total to determine the target weight to fill each litter bag. The total weight is divided by the total number of bags to be filled plus an identical portion for measuring moisture content every 10 - 15 samples. For example a total weight of 1250 grams intended to fill 100 litterbags would need 10 moisture samples. So the total weight divided by 110 gives 11.36 g. per sample. Taking into account the loss of weight due to discarded contamination, I would aim for a target weight of 11 g. per litterbag or moisture sample. Since each litterbag's filling weight is recorded individually it is not critical that each bag is filled identically and so we aim for target weight +/- 1% which in this example would be +/- 0.1 g.
We use an electronic balance, with an accuracy of .01 gram, connected to a computer, and hand record each weight on paper as well, for back-up. Depending on the total amount of leaves collected and the size/bulk of the leaves we put a target weight of 5 - 20 grams per litter bag. Throughout the process we are careful to avoid breaking the leaves unnecessarily, usually only breaking large leaves to achieve our target weight per bag. When large leaves are broken care is taken to avoid filling bags with disproportionate loads of petioles or leaf blades, as these portions can decompose at wildly different rates.
During the bag loading process the target weight of leaves is placed into a weighing tray on the balance, and the weight is recorded. After recording the weight the material is placed carefully into the correctly numbered litterbag, taking care not to miss any of the material resulting in an incorrect weight. The litterbag is then stapled closed with 5-6 Monel staples. Every 10 to 15 bags a similar portion of leaves is weighed and placed in a small brown paper bag labeled with the species and number of the moisture sample. The moisture samples are oven dried and the average moisture conversion factor (dry weight/wet weight) is used to adjust the leaf weight of every litter bag to a calculated initial dry weight for each. The moisture content samples can be used, after they have been dried and weighed, to provide the material for initial condition nutrient sampling.
Throughout the loading process, during transport, and as the bags are placed in the field care should be taken to keep the 1 mm. mesh side up, and avoid unnecessary jostling, both of which can sift material out of the litter bags, biasing the results. This is a particular problem with small conifer needles. With very small conifer needles which were difficult to keep within the litterbags we have gone to the length of placing each litterbag within a ziplock bag until field placement, and then weighing the needles which remained within the ziplock bag, and subtracting this weight from the initial weight to correct for this loss.
Once all bags are filled we organize the samples in the lab prior to field installation. We call this process "shopping". Data sheets are made up to record the tag numbers of each bag as it is assigned to a harvest time and site. Our "shopping lists" are designed to hold all the samples destined for a single time/place (harvest) and have a column of treatments and adjacent blank lines on which to write the tag number of the bag randomly assigned to that harvest. At this stage we usually have seine twine cut to the correct length on which to thread the litter bags of a single harvest line. With the litter bags in a stack or in an overlapped line we pass the string through the cable tie loop, from one end to another, tying a good knot to the last bag cable tie. We leave 30 cms. of string extending past the last bag with which to tie the line to a pin flag in the field. The remaining free end of the string is used to wrap the entire stack of litter bags, all 1 mm. mesh side up, into a secure bundle.
Alternatively, we lay the bags out on the ground and string them in the field. Our philosophy is to maximize efficiency in the field by doing everything possible in the lab. This helps us use the daylight and time in the field to the fullest.
When the group of samples for a harvest are assembled they are placed, all 1 mm. mesh sides up, in a properly sized brown paper bag, labeled with the harvest designation, and then tightly folded closed, and stapled shut, to resist shifting during transport.
To cut the seine twine to length efficiently we have developed a system. First the desired length is determined by multiplying the number of bags on the line by 25 cms. And then adding 50 cms for the terminal end tie. This sort of spacing allow about 4 bags per meter, and if they are placed alternately left and right of the string the spacing is not crowded. On a lab bench we duct tape two wooden dowels projecting horizontally approximately 25 cm out over the floor. The distance between the dowels is ½ the target length of the string sections. We tie one end to a dowel and then wind the string, without slack but not tightly, from one dowel to another until there are 30 - 40 loops between the dowels, ending at the same end as the knot. We use a soldering iron with a sharp work tip to slice through all the strings on the knot end dowel. Keeping the strings slightly apart will keep them from melting together. The severed strings fall to the floor and are cut to the correct length and melted at the ends so they do not unravel. Strings that do melt together will usually separate with a slight tug.
In the field the area of installation is cleared of large branches and the harvest bags are placed out on the ground, about one meter apart, in a line perpendicular to the direction in which the lines will be deployed. Each paper bag is carefully opened, samples removed, and the binding string is unwound and spread out in the direction of the line. The 30 cm of string beyond the knot is tied to a pin flag, by wrapping the string tightly around the metal pin 5-6 times and then tying three half hitches. This knot holds the pin flag tightly and even if the flag is pulled from the ground the string should remain connected, facilitating recovery. The pin flag is thrust halfway into the ground, anchoring the string, allowing the litterbags to be pulled away and spread along its length, leaving 30-50 centimeters for the terminal tie down. The string should be slack since animals can tug at the line when walking through. In areas where many animals are likely to pass we have lightly placed down branches across and on top of the harvest lines to discourage the passage of foot-dragging animals. The free end of the string is tied to another pin flag in the same manner as the first, and then pinned deeply into the ground. At this time we check the numbers of all the litterbags against our record on the shopping list to make sure we have no typos.
On sloping ground, or if there is a concern over disturbance, the individual litterbags can be knotted to the harvest line. This is easily done by pushing a loop of the string about 8 cms. through the cable tie loop, placing a finger though the string loop and twirling it 4-5 times and then pulling the string loop over the aluminum tag until the string can be pulled tight on the cable tie. This will keep the individual litterbag anchored to a point on the harvest line without allowing the bag to slide along the string.
We check each litterbag tag on the harvest line against a list of the tag numbers we expect to harvest. With scissors or pruners we cut the line and check off each bag as we place it into a gallon-size zip lock plastic bag. In the field the ziplocks are squeezed flat of air, sealed, and put into a larger bag labeled with the site and time. At the lab the litterbag is refrigerated until processing. During processing scissors are used to cut the three sewn borders of the bag, just inside the stitch line. The litterbag is folded open and the material is examined to remove new roots and contamination which is obviously not the original filling. After removal, the wet weight of the material is recorded and the material is placed in a labeled small brown paper bag and then dried at 55 degrees C., until a stable weight is reached. In order to reduce error associated with variability of paper bag weights, and thereby not have to open the bag and empty the contents to obtain the dry weight, we pre-weigh empty paper bags into weight classes which vary by only 0.05 grams. Using these bags with reduced variability allows us to use a dried, empty, and stapled bag to tare the scale. The oven dry weight is then recorded and the material is processed for chemical analysis. Ash-free dry weight of both initial and final material should be used to correct for mineral soil contamination allowing a more accurate mass loss calculation.
If you have additional questions please contact me
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