The following protocol for DECOTAB preparation and deployment has been adopted from Kampfraath et al. (2012).

1. Equipment and materials

• Drying oven (standard 40 - 50 °C, but up to 70 °C possible)
• Analytical balance (± 0.1 mg)
• 2 L Erlenmeyer flask
• Stir/heat plate
• Stir bar
• Moulds (standard multi-well polycarbonate mould (15-mm diameter, 5-mm height), but alternative shapes and sizes possible)
• Purified Agar
• Powdered cellulose
• Ascorbic acid
• Coarse mesh bags (4 mm mesh size) for field study
• Fine mesh bags (50-500 μm mesh size) for field study
• Invertebrates for laboratory study

2. Experimental procedures

2.1. Standard DECOTAB Preparation

1. Boil 1 L of deionized water with 20 g purified agar for 3 min (100 ºC).
2. Cool solution down to 60 ºC while stirring continuously on hot plate.
3. While stirring add 60 g of powdered cellulose and 10 mg ascorbic acid as antioxidant to the solution (see section 3.2 for alternative DECOTAB types).
4. Pour solution into the moulds.
5. Remove DECOTABs from the moulds after 7 minutes of cooling and store in a closed container at 7 ºC. To prepare more DECOTABs, steps 4 and 5 can be repeated while the solution is kept at 60 ºC and stirred continuously.
6. Dry a subset of 20 DECOTABs per type at 40 - 50 ºC for 2 days, and weigh on analytical balance to determine initial DECOTAB dry mass.
7. Tips
   1. Agar solution is ready when it becomes transparent.
   2. Do not let agar cool below 50 ºC, since it will then solidify, do not reheat the solution when cooled below 50 ºC.
   3. DECOTABs can be stored up to three weeks in a closed container in the fridge without noticeable decay or dehydration. Sterilization of DECOTABs (e.g. submersion in ethanol) and storage in deionized water can substantially prolong this period.

2.2 Alternative DECOTAB types

The standard composition (described in section 3.1 standard DECOTAB preparation, step 3) can be altered by exchanging cellulose for natural organic matter (1) or plant litter (2), or by adding specific substances (3) to the DECOTABs. A number of example procedures for alternative DECOTAB types are described below, but other combinations or options are also possible. 

1. Natural organic matter

1. Collect sediment of study area, or use bulk standardized garden soil.
2. Re-suspend and separate by weight to obtain sediment-free organic matter.
3. Dry organic matter at 40-50 ºC for 2 days.
4. Sieve (500 μM - 2 mm) organic matter to discard coarse material and obtain material large enough to be retained within agar matrix
5. Substitute all cellulose by organic matter (Hunting et al., 2016).

1. Plant litter
   1. Collect fresh plant material.
   2. Rinse, dry at 40-50 ºC for 2 days
   3. Grind/Mill plant material to powder.
   4. Substitute all cellulose by powdered plant material (Hunting et al., 2016; Vonk et al., 2016).
2. Add specific (hydrophobic) substances

1. Add PUFA; e.g 0.40 g L^-1 linoleic acid (Vonk et al., 2016) in accordance with the linoleic acid content of submerged plants (Van Ginneken et al., 2011). 
2. Add antibiotic; e.g. 60 mg L^-1 chloramphenicol (Kampfraath et al., 2012).
3. Add fungicide; e.g. 21 mg L^-1 natamycin (Pedersen, 1992).

2.3 Deployment and retrieval

1. Deployment of DECOTABs in the laboratory or in the field:
   1. Laboratory: place DECOTABs under controlled conditions in microcosms or mesocosms inoculated with microorganisms and/or stocked with invertebrate species. A deployment time of 21 days is recommended, or less when observed consumption rates are high.
   2. Field: place DECOTABs in coarse and fine mesh bags in a randomized order at 5 m interval on the sediment of the water body. Check DECOTAB mass regularly, and retrieve when a substantial (approximately 50%) DECOTAB mass is decomposed.
2. Rinse the DECOTABs carefully after removal from the laboratory or the field.

Dry DECOTABs at 40 - 50 ºC for 2 days, and weigh on analytical balance to determine final DECOTAB dry mass.

2.4 Analysis

Decomposition rates can be calculated as a function of time by the exponential decay model (k). Alternatively, microbial decomposition and invertebrate consumption can be estimated as a function of time by a linear mass loss model:

Microbial Decomposition (MD) = (D_i - D_f) / t

Invertebrate Consumption (IC) = ((D_i - D) / t) - MD                              

In which microbial decomposition (MD) for each DECOTAB type (mg) is the difference between the weight of the initial DECOTAB (D_i) and the weight of the corresponding DECOTAB deployed in fine mesh bags (D_f) at the end of the exposure period. In the field, invertebrate consumption (IC) for each DECOTAB type (mg) is the difference between the weight of the initial DECOTAB (D_i) and the weight of the corresponding DECOTAB deployed in coarse mesh bags (D_c) at the end of the exposure period, and subtract the mass loss of the DECOTAB in the fine mesh bags (MD). Results can be expressed as mg dry mass loss over the deployment time (t) (e.g. in days).

References

Hunting, E. R., Vonk, J. A., Musters, C. J. M., Kraak, M. H., & Vijver, M. G. (2016). Effects of agricultural practices on organic matter degradation in ditches. Scientific Reports, 6, 21474.

Kampfraath, A. A., Hunting, E. R., Mulder, C., Breure, A. M., Gessner, M. O., Kraak, M. H., & Admiraal, W. (2012). DECOTAB: a multipurpose standard substrate to assess effects of litter quality on microbial decomposition and invertebrate consumption. Freshwater Science, 31(4), 1156-1162.

Pedersen, J. C. (1992). Natamycin as a fungicide in agar media. Applied and Environmental Microbiology, 58(3), 1064-1066.

Van Ginneken, V. J., Helsper, J. P., de Visser, W., van Keulen, H., & Brandenburg, W. A. (2011). Polyunsaturated fatty acids in various macroalgal species from north Atlantic and tropical seas. Lipids in Health and Disease, 10(1), 104.

Vonk, J. A., Van Kuijk, B. F., Van Beusekom, M., Hunting, E. R., & Kraak, M. H. (2016). The significance of linoleic acid in food sources for detritivorous benthic invertebrates. Scientific Reports, 6, 35785.