3.1 Initial design and setup
Study site selection is based primarily on how accessible the test site is (e.g., availability of a level field of suitable size). Availability and quality of a water supply as well as the potential access to power are important considerations. Security of the study site can also be a factor because mesocosm studies typically run for extended periods (3 or more weeks). Thus, safe access to the site for the setup, experiment and takedown periods is required. Distance from the test site to a suitable location for collection of the test organisms and substrates should also be considered well in advance of deployment. Reconnaissance trip(s) to find the appropriate collection site well before the experiment is highly advisable. All biological collections should be at a reference area with representative diversity of organisms (Fig. 1a).
Our modular mesocosm approach (Fig. 2), was originally designed to be transported to test sites with groups of up to eight (8) replicate streams arranged on treatment tables (Fig. 2a). These treatments were initially attached to supporting wooden pallets for easy transport by forklift (see also Culp et al., 2003 in Assoc. Publications). Early studies using this method focused on effluent gradients from pulp mills and metal mining to assist in the determination of underlying causes of toxicity in these industrial effluents (Environment Canada 2010; 2012). Subsequent experiments have looked at gradients of priority and emerging contaminants to determine safe levels of these substances in the environment or to unravel confounding results due to natural gradients such as nutrient masking of effects in field applications (Alexander et al. 2013).
Each replicate stream has a planar area of 0.065 m2 and a 10-L volume. Each cylinder independently simulates environmental flows while the shorter cylindrical design reduces edge effects on stream periphyton and macroinvertebrate communities (Fig. 3). The rounded edges also allow for reduced wall growth, increased animal movement and better contaminant mixing. Rectangular tanks create backflows and unusual eddies that are widely thought to be more stressful for higher densities or more active organisms. The implications of scale to environmental problems using mesocosms is reviewed in Petersen et al. (2009). Often, however, the weaknesses of the system appear to be directly linked to weaknesses in the study design. This issue is not unique to mesocosm studies.
3.2 Power and Water
River or treatment water is fed into the polyethylene table reservoir before it’s drawn towards the circulation (March) pump (Fig. 4). The water flows through the lower valve of the table reservoir into the March pump where it is pumped into the treatment manifold intake. The second manifold intake is connected to one of the upper valves on the table reservoir which is closed to create system pressure. From the treatment manifold, the water is pushed through the lower hose barbs into each of the stream mesocosms. The water then enters the artificial stream, fills up the cylindrical basin while a motorized stirrer rotates internal paddles to generate instream flow conditions similar to 2nd to 4th order streams. Water then overflows the basin emptying onto the tray where it then drains back into the table reservoir via two cylindrical funnels built into the tray (Fig. 4). Water is exchanged in each replicate stream is typically exchanged every 7-min. The tray discharge then either returns to the reservoir for continued recirculation while the reservoir is completely exchanged every 1-4 hours depending on the temperature needs of the study design. Waste water (either from the table or the reservoir tank) is transported through solid black corrugated loom tubing to a separate wastewater holding tank. Wastewater is then passed through carbon filters (e.g., Culligan Inc., activated carbon filter cylinder, Moncton, NB, Canada) to remove contaminants before any water is discharged to the environment.
3.3 Substrate and benthic macroinvertebrate community collection
Prior to initiating the experiment, benthic substrates are introduced into each replicate stream. Inoculating each stream with a mixture of gravel (fine and coarse) and cobblestones creates an ideal substrate for benthic macroinvertebrates. Gravel and cobble should be collected from a pristine site (e.g., un-impacted location). Mixture of substrate sizes should also reflect the simulated riverine habitat to be tested and preferably be collected near (e.g., downstream) of the benthic macroinvertebrate collection site. Cobble should also be gently hand washed to remove attached invertebrates while maintaining the preexisting periphyton community. This procedure establishes a lotic substrate consisting of a 2-3 cm layer of gravel plus surface cobblestones covered with periphyton similar to the original habitat of the benthic community examined.
3.3.1 Collection of substrates
Use shovels for gravel substrates collection. Fine and coarse gravel are shoveled, sieved and transported to the mesocosm test site. Transfer should be done relatively quickly, as the biofilm on each rock is alive and vulnerable to desiccation or compositional shifts with changing water temperature.
Each replicate stream receives the same volume of fine and coarse gravel of the volume and size measurements below (see Fig. 1c). Mix gently by hand to evenly distribute the amounts of fine and coarse gravel in each replicate stream (Fig. 1d, 3b, 4).
Each replicate stream receives:
250 mL of >2mm to <4 mm of fine gravel
750 mL of >4 mm to <9.5 mm of coarse gravel
Thus, to inoculate 32 streams with substrate:
8-L total, of >2mm to <4 mm of fine gravel is needed (250 ml x 32)
24-L total of >4 mm to <9.5 mm of coarse gravel is needed (750 ml x 32)
3.3.2 Collection of cobble substrates
Typically, in a separate trip, cobbles (5-8 cm in diameter) are collected for each artificial stream. Cobbles will be carefully selected from the stream bottom, gently hand rinsed, and inspected to remove visible invertebrates with a minimum disturbance of the biofilm. Place cobble in a cooler maintaining original rock orientation with algae covered side facing up. Cobble should be covered in water during transport to maintain periphyton community.
We recommend 5 cobbles (approximately 5-8 cm diameter) be placed in each replicate stream on top of the gravel substrate. Systematic allocation of cobbles to each stream is recommended to minimize size bias. Allow 3-5 days for periphyton colonization, dependent on ambient temperatures before inoculating macroinvertebrates to prevent starvation.
3.3.3 Collection and inoculation of macroinvertebrates
The benthic macroinvertebrate assemblage should be collected from a single riffle (e.g., < 10 x 10 m, water depth < 30 cm). Collecting the benthic community from a small defined area reduces the high inherent variability in diversity and abundance. The collection site should be near, preferably upstream of the gravel and cobble collection site (without being in the disturbance zone) as the organisms will already be acclimated to nearby substrate and its biofilm. In our research, we have at times used the same collection site for several years further increasing our understanding of that system and the organisms that reside there as well as facilitating comparisons between experiments over time.
The benthic macroinvertebrate assemblage should also be collected using a sampling device with a known area to facilitate an overstocking of ~10-20% in the event of any mortality due to handling or transport of organisms to the test site (Fig. 1b). We have used U-nets (area = 0.06 m2) for this purpose and have developed a subsampling procedure to inoculate as similar a benthic macroinvertebrate assemblage into each replicate stream at the test site as possible (Fig. 1). This subsampling process has been described in detail elsewhere (e.g., Alexander et al. 2013; see also Associated Publications). In brief, the subsampling procedure consists of collecting a series of U-nets by a team of samplers working together (Fig. 1a). The number of U-nets is determined by the need to overstock by ~10% to offset any mortality of macrobenthos in the transport from the river to the test site. For example, if 100 streams are to be inoculated, 110 U-nets would be required. Both the number of streams to be inoculated and how many subsamples can easily be ‘split’ in the subsampler alter the number of subsamples required (e.g., see 4-way pie plate subsamples in Fig. 1b). As the number of streams used in an experiment depends on the replication required a prioripower analyses is highly recommended to determine whether expected effects would be detectable given the variability in the subsampled community. Please also note, other types of nets can also be used for collections but the relative density of organisms in the test system should not be greater than 20% overstocked compared to the riverine community.
Once macroinvertebrates are collected, sample containers must be kept cool and transported to the mesocosm site as quickly as possible. Minimizing water temperature differences between the river water and the artificial stream water is highly recommended. Once the macroinvertebrates are at the test site, they should be transferred into individual artificial streams using a randomized pattern. It is important to make note of which artificial stream receives which subsample to help with later data interpretation. Turning off the motorized stirrer (Fig 1c,d; 3a,b) also helps invertebrates to settle into the substrate unimpeded during the inoculation transfer. Once macroinvertebrates are introduced, attach emergence traps (e.g., 400 Nitex© mesh, Aquatic Ecosystems Inc., Apopka, FL, U.S.A.) or other barriers to prevent invertebrate escapees (e.g., Fig. 2a). Introduce treatments only after all of the above is functioning as expected. Generally, allow ~2 days for macroinvertebrates to settle before initiating treatment.