Using ArcMap software, extract or construct the open-water shapefile corresponding to the targeted study wetland. This shapefile may be extracted from existing publicly accessible wetlands data such as the National Wetlands Inventory data layer (U.S. Fish and Wildlife Service, 2020), or it may be created manually by constructing a polygon in reference to aerial imagery. For either method, select data or imagery collected as close to your sampling time as possible to ensure desktop-sourced wetland boundaries are representative of current conditions.

Navigate to the “create fishnet” tool (“Toolboxes” → “Data Management Tools” → “Sampling” → “Create Fishnet”). Using the measuring tool, determine whether or not the distance between the northern and southern shoreline is longer than the distance between the eastern and western shoreline; if longer, transects should run in an east–west direction (n rows = 9, n columns = 1, where n is number), and if shorter should run in a north–south direction (n rows = 1, n columns = 9).

Use the “clip” tool (“Toolboxes” → “Analysis Tools” → “Extract” → “Clip”) to clip the transects to the wetland outline.

Each transect will contain one survey station. The survey stations are in two strata: either less than 5 meters (m) of the innermost ring of emergent vegetation near shore or 50 m from the innermost ring of emergent vegetation (see example in fig. 1). If the wetland is small, it is possible to modify the strata according to proportional distances between the vegetation zone and wetland center. Use a random-selection technique (such as a coin flip or a random number generator) to select the starting transect and shoreline to alternate between strata.

Create survey point shapefiles by creating a new feature class for point features and manually placing them in the appropriate positions along the transects.

Assign a unique identifier to each survey station that includes the wetland name and station number.

Print a copy of the map, and load survey station shapefiles onto a Global Positioning System as waypoints, ensuring survey points are clearly labeled with a unique identifier on both.

After loading the boat with necessary supplies (appendix 1), use the field Global Positioning System (GPS) device to navigate to a preprogrammed survey station.

Upon arriving at the survey station, prepare to take a benthic sweep. To collect the benthic sample, use the unmodified dip net. Plunge the net to the wetland bottom in a vertical position to avoid capturing invertebrates on the way down. Note the water depth by using depth increments marked on the handle and record it on the macroinvertebrate sampling data sheet (appendix 5). If water depth exceeds the length of the dip-net handle, either attach an extension to the handle or move along the transect toward shore until reaching an appropriate depth. If the GPS coordinates changed from the predetermined point because of depth or accessibility limitations, record the actual GPS coordinates for repeating samples or spatial analyses.

While keeping the net perpendicular to the wetland bottom, sweep horizontally for 0.5 meter (m), which can be referenced on your boat for improved accuracy. Skim the net along the top layer of sediment to the depth at which target organisms reside (generally a few centimeters deep) for 0.5 m. Take care to keep the handle vertical (that is, straight). Then, raise the net out of the water while keeping it in a vertical position to avoid capturing water column invertebrates.

Once the net emerges from the water, rotate it into a horizontal position. If vegetation and other organic debris are hanging over the net’s rim, place 50 percent of the vegetation/debris into the net and discard the rest. While keeping the dip-net rim out of the water, rinse sediment and fine organic matter from the sample by gently dipping the bell of the net in and out of the water, or by moving it from side to side slightly below the water’s surface. Splash the edges with water to flush all materials to the bottom, allow water to drain out, and place the contents into an appropriately labeled benthic sample bottle. Benthic samples can either be stored in separate bottles for each survey station or composited into the same bottle(s) for wetland-level estimates. Each bottle should contain an external and internal label. Internal labels must be written on an ethanol-resistant paper with an ethanol-resistant writing utensil (such as a graphite pencil), and external labels should be secured with clear packing tape. Immediately pour 95-percent ethanol into the bottle to an approximate volumetric ratio of 1 part sample to 2 parts ethanol.

An optional step for wetlands with sufficient water depth and depending on research objectives is to prepare to take a water column sweep. Shift the boat slightly (1–2 m) away from the disturbed area. To collect the water column sample, plunge the modified dip net to a depth equal to the total water depth minus the benthic net height. Shake out the net to ensure the mesh is free of the frame. Move the net sideways to undisturbed water and raise it vertically through the water column. Once the net emerges, while keeping the rim out of the water, splash the edges with water to flush all materials to the bottom and place the contents into an appropriately labeled water column sample bottle; like the benthic samples, water column samples can either be stored in separate bottles for each survey station or composited into the same bottle(s). Immediately pour 95-percent ethanol into the bottle to an approximate volumetric ratio of 1 part sample to 2 parts ethanol.

Repeat steps 2–5 for each survey station and ensure both field data sheets (appendix 5) have been completed before leaving the wetland.

Supplies

Construction of sieve

The sieve is composed of a stainless-steel mesh secured with staples between two wooden frames; this provides depth above and below the mesh to contain the sample and allow water to rinse through. Wood should be treated with a sealer to prevent water damage. The sieve shown in figure 8.1A–C measures about 29 cm long by 34 cm wide by 7.5 cm high.

Construction of grid

The overlying, removable grid is composed of a wooden frame containing a simple 2 x 2 wire grid to define the subsampling areas. The grid frame shown in figure 8.1D–F measures about 33 cm long by 38 cm wide.

Put on gloves and safety goggles, open the sample bottle, and carefully decant ethanol from the sample by pouring the fluid through a small 500-micrometer sieve held inside a funnel over a labeled ethanol waste container. Repeat this step for the remaining bottles of the same sample. Thoroughly backwash/rinse the small sieve into the sample bottle to remove any organisms or debris that may have been collected on the sieve mesh while decanting.

If the contents of one sample are stored in more than one sample bottle, pour the contents of all bottles into a single appropriately sized tub. Rinse the inside of the sample bottles over the tub using water to collect any organisms or debris remaining inside. Pull the internal label out of the sample using forceps. Rinse the label over the tub using a water wash bottle, and set the label aside. Add enough water to the tub to completely cover all sample materials and thoroughly but gently mix them with a spoon or spatula (fig. 4A).

Once the sample has been mixed, pour its contents onto the subsampling sieve, which contains 500-micrometer stainless steel mesh. Next, gently rinse the sample with water to remove remaining ethanol and fine sediment. Rinse any large rocks or organic materials (whole leaves, twigs, macrophyte mats, and so on) over the sieve, visually inspect them under a lighted 3× microscope to remove remaining attached macroinvertebrates and discard them.

Place the subsampling sieve inside a tub filled with water to a level slightly above that of the sample materials; this allows the macroinvertebrates and debris to become slightly suspended in the water while remaining contained within the sieve frame. Use a spoon, spatula, or long forceps to gently spread the sample material over the sieve mesh as evenly as possible. Once the sample is evenly dispersed across the mesh, lift the sieve out of the water-filled container and place the gridded subsampling frame (four grids) over the sieve’s frame (fig. 4B).

Record the sample identifier information from the sample labels on a macroinvertebrate sample log form (appendix 7).

Use a random number generator to determine the order in which grids will be processed from the subsampling apparatus. Using a plastic spoon, spatula, paintbrush, and scissors (if needed to cut through vegetation), remove all materials (organisms and debris) from the randomly selected grid and place them into a separate container (fig. 4C). Any organism that is lying over a line separating two grids is considered to be on the grid containing its head. If it is not possible to determine the location of the head, the organism is in the grid containing most of its body. Cover the remaining sample in the apparatus with the tub lid or tin foil to prevent the desiccation of remaining macroinvertebrates, and occasionally mist the sample with water to keep it moist.

To maximize the visibility of macroinvertebrates mixed with sample debris, pick the subsampled contents gradually. Transfer 2–3 heaping tablespoons of subsampled materials into a shallow white tray partially filled with water (fig. 4D). Visually inspect the contents once with the naked eye and then again with the 3× lighted magnifier while using forceps to gently remove all macroinvertebrates. Place all macroinvertebrates or select taxonomic groups directly into prelabeled vials with 70-percent ethanol; a single vial may be filled as much as halfway with specimens. Keep track of total individuals collected by using a hand tally counter. Once the tray’s contents have been thoroughly examined and all macroinvertebrates have been removed, remaining materials in the tray may be discarded. Repeat these steps until the subsampled grid’s contents have been processed entirely.

Once processed, if the cumulative analysis of the first grid (25 percent of the sample) yields a representative quantity of macroinvertebrates (this may vary based on project objectives and needs), then subsampling is complete. In some cases, project investigators may wish to incorporate an additional subsampling target, such as a minimum number of macroinvertebrates (for example, 400 macroinvertebrates per composited, wetland-level sample) to ensure representativeness. If so, the grids must be processed, one at a time, until the target is reached. Once you begin processing a grid, that grid must be picked entirely to ensure macroinvertebrate counts are scaled up properly. Record the total number of grids processed and total number of specimen vials used in the macroinvertebrate sample log form (appendix 7) and label the vials with the following information: contents, sample identifier, and number of grids processed.

Once picking is completed and all data have been recorded in the macroinvertebrate sample log form (appendix 7), remaining sample debris may be discarded, and sample bottles can be cleaned. Pour excess water into the sink with a strainer to prevent debris from entering the drain. Dispose of ethanol according to local regulations.