Materials and Methods
Raceways and Filtration We housed 76 bonytail chub at the University of Arizona’s Environmental Research Laboratory, (UA ERL) in four independent, independently indoor re-circulating raceways (1.22 m x 3.43 m x 0.51 m) all housed in a room with some degree of climate control. Each 2,500L raceway was constructed of marine plywood coated with fiberglass sheeting (Walker, et al. 2009). Filtration in each raceway included consisted of a swimming pool filterpressurized sand filter for removal of solid wastes, and a gravity-fed, reverse-flow, fluidized sand filter. that provided surface area for denitrifying bacteria. This filtration system removed solid waste from the raceways and provided surface area for nitrifying bacteria that converted potentially toxic forms of reduced nitrogen to non-toxic oxidized forms. We provided filtration to avoid low dissolved oxygen concentrations and toxic levels of un-ionized ammonia.
We randomly assigned two raceways as control and two raceways as treatment. Control raceways contained tap water treated with by carbon filtration and reverse osmosis (RO); treatment raceways contained 100% secondarily treated sewagewater collected from the Santa Cruz River near Tucson, Arizona. effluent. We added a commercially-available salt mixture used in the aquarium trade aquarium salt and sodium bicarbonate to control waterwater used in control tanks to parallel match the pH and conductivity of treatment waterwater used in treatment tanks as closely as possible. We provided each raceway with bubble-type, diffuse aerators to keep maintain or enhance dissolved oxygen levels. high enough for fish survival. We replaced about one third of the water in each raceway every two weeks by back-flushing through the filtration system. Back-flushing filters was necessary to clear filters of solid waste build up. Between back-flushing events, we added water in each tank to compensate for evaporative loss. We measured temperature, pH, specific conductivity, and dissolved oxygen (percent saturation and mg/L) using a Hydrolab Surveyor 4 Datasonde daily to insure water conditions were safe for fish and were consistent as possible among raceways.
We used a submersible pump to collect water used in treatment raceways We collected secondarily treated sewage effluent from the Santa Cruz River near Tucson Arizona approximately 400m within a quarter of a mile of thebelow the outfall of the Rodger Road Waste Water Treatment Plant (WWTP), on the Santa Cruz River, Tucson, AZ (Figure needed). Water was transported from the Santa Cruz River and stored at the UA ERL in 2,650L stainless steel-lined containers. We used a sump pump to collected treatment effluent, and transported and stored effluent in large sterilized 52,000L aluminum containers.
Fish Introduction and Care
Bonytail chub were transported from Dexter National Fish Hatchery, Dexter New Mexico, to the Environmental Research Laboratory on March 13, 2007. We anesthetized fish in 38L aquaria containing 10L of tap water treated by carbon filterationed and RO reverse osmosis water mixed with a 2:1 ratio of sodium bicarbonate and MS-222 (tricaine methanesulfonate). We sexed and inserted passive integrative transponder (PIT) tags below the epidermis on the ventral side, posterior to the pelvic fin and anterior to the anal fin of each fish. We revived fish in 38L aquaria that contained water from their home respective raceways, salt, and aeration. We randomly distributed equal numbers of male and female bonytail chub to each of the 4 raceways.
We housed bonytail chub in rRaceways were constructed in an heavily insulated room, inside a freestanding building. We kept fish under controlled daylight and temperature conditions. Over two and a half years, daylight photoperiod and temperature were controlled to mimic approximate natural seasonal changes for the region; we keptand conditions were kept consistent among all raceways. Water temperature was controlled by air temperature (A/C and passive evaporative cooling pre-chillers) and in-raceway chiller units. During videotaping, day light (14 hr daylight, 10 hr dark) and temperature (average 24.850 – 24.910C) were kept constant. Fish were fed ad libitum once a day, in the morning, and food was kept consistent among all raceways. We cleaned raceways each morning by moving cover and substrate placed in the raceways in-raceways cover-substrates for to netting and sweeping of any solid wastes that had not been removed by water flow.
Each raceway was covered with 1.8 cm plastic mesh fitted to a frame made of PVC to prevent fish escape by jumping. We covered each raceway with an out-of-raceway cover to prevent the possibility of fish escaping raceways by jumping. Out-of-raceway covers consisted of a large PVC frame covered with 1.8 cm plastic mesh. We constructed five variations of in-raceway substrates and covers, and introduced them to tanks to find a cover-substrate that fish would use. Substrates needed to function as a combination of habitat enrichment, cover, and egg/feces traps. The first substrate, designed as spawning substrate, was a stainless-steel pan (dementions?) with holes drilled into the bottom. The bottom was covered with rocks held in place with silicone. Each pan was covered with a ½” PVC frame covered with 1.8cm mesh, held in place with a large, all-plastic bungee cord (Figure needed). The second structure, designed as spawning substrate, was made of coconut fiber. We constructed a ½” PVC frame (66cm x 50cm), covered with approximately 5 cm thick coconut fiber sheet, and held in place with zip ties (Figure needed). The third substrate, designed as cover and spawning substrate to mimic an undercut bank, was a ½” PVC trapezoid box frame (53cm x 53cm x 36cm x 16 cm) with a floating cover frame (53cm x 33cm). The trapezoid box frame was covered with 0.4cm mesh and the floating cover was covered with coconut fiber, attached with zip ties (Figure needed). When we introduced the fish to all three of the substrates, fish swam away and stayed in a crowded swimming ball in the corner furthest away from the introduced substrate. With acclimation fish eventually came out of the corner, sometimes taking days, but treated the substrates no different from any other section of the tank. The fourth structure, designed as spawning substrate, was a ½” PVC frame with 22cm PVC legs at each corner. We covered the substrate with 1.8cm plastic mesh (figure needed). When we introduced the fourth structure, fish swam underneath the substrate. The last substrate was a variation of the fourth structure. For the final substrate, we stacked two standing frame structures and covered them with a combination of mesh sizes (1.8 and 0.4cm), leaving a box structure the fish could swim into (figure needed). The bonytail chub’s response to this structure was immediate. All fish swam into the structures and were reluctant to leave the structures. We used the final in-raceway cover-substrate for the remainder of the experiment.
Growth Rate and Condition Factor We used an UWE HS-3000 digital scale to weigh and measure bonytail chub twice during the experiment; at the time of during introduction into the raceways and at the end of the experiment. Fish were anesthetized, weighed and measured, and revived in 38L aquaria. We computed the Fulton Condition Factor (C = (W/L3) X 10,000) for each fish at each time period.
Behavioral Observations
We used Wisecomm CCD cameras and an X (getting brand from Chris Goforth) DVR to videotape bonytail chub in each control and treatment raceway. Videotaping allowed us to observe and analyze fish behavior without any observer effects (citation needed). We mounted video cameras to the ceiling, 1.8m above each raceway. We painted a 20 cm black strip in two places on each raceway frame to use for distance scaling in later spatial analysis. We placed a divider into the raceway to prohibit fish movement into the first third of the raceway, leaving them X (need to measure) space. Prohibition of fish movement was necessary to insure that all areas of the tank could be viewed by cameras overhead, and aided in acclimation of fish to in-tank structures. We videotaped fish in raceways from September 22, 2009 to November 5, 2009. We provided fish with two in-raceway cover-substrates for two weeks and one in-raceway cover-substrate for the remaining four weeks of the experiment. The majority of our analyses included the reaction of fish to a limited resource, the in-tank cover-substrates. To insure the cover was first a resource, and secondly limiting, we initially provided two substrates to demonstrate that all fish would use the substrate if given the opportunity. We then removed one of the covers for the remainder of the behavioral trials, limiting the cover resource. We tapedfilmed fish in three different time blocks, 0730-1030 hrs., 1430-1500 hrs., and 1830-1900 hrs. Temperature in all raceways varied throughout the day (0.5 to 2.0 degrees C), and time blocks were chosen to reflect the daily minimum and maximum temperature. Personnel entered the room only once a day, for feeding and cleaning, to avoid any unscheduled disturbance and observer effects. Video recordings were downloaded and stored onto a laptop computer and external hard drive.
In-Raceway Disturbance
We created in-raceway disturbance in all raceways each morning during feeding and cleaning. Each morning we entered the insulated room at 0900 hr to feed fish, record physio-chemical readings, and clean raceways. We cleaned raceways buy removing the out-of-raceway cover over the raceways and then moving the in-raceway cover-substrate to net and sweep solid wastes from underneath and around raceways. After cleaning, we replaced both in-raceway cover-substrates and out-of-raceway covers to the original position. We randomly chose days from two conditions; seven days from the two cover-substrate condition and 14 days from the one cover-substrate conditions. We videotaped from 0730-1030 in order to observe and analyze bonytail chub during the morning disturbance regime.
We used previously taped video to examine the differences between treatment and control fish while fish they experienced in-raceway disturbance. We counted and recorded the number of fish out of the in-raceway cover-substrate five minutes before, mid-disturbance, and five minutes after in-raceway disturbance. We started counting fish in and out of the cover-substrate as soon as the in-raceway cover-substratethis was placed back into its original position after cleaning, marking our mid-disturbance measure. We counted the number of fish noses, assuming that fish with exposed heads may feel more vulnerable, than fish that were inside the cover-substrate but may be exposing their tails (Figure needed?).
These measures were designed We used cover and substrate to show two phenomena; first that in-raceway cover-substrate was a limited resource, and secondly to examine any differences between treatment and control fish that had experienced an in-raceway a disturbance.
Average Nearest-Neighbor and Distance to Substrate
We randomly selected 14 days of tape from the one single in-raceway cover-substrate condition. We analyzed still frames of video from all three time blocks; 0830 hr., 1440 hr., and 1840 hr. We pre-marked fish noses with a colored dot on still frame pictures using the Roxio Photosuite 8 computer program. Pre-marking pictures increaseed the ease and accuracy of finding fish in still frame pictures in later analyses. We counted the number of fish out of the in-raceway cover-substrate for later fish density analyses.
We used Arc GIS version 9.3.1 to compute two spatial measures; average nearest-neighbor and average distance to cover-substrate. The average nearest-neighbor is a measure of spatial clumping on a two dimensional plane. Distance to the closest neighbor was measured for each individual fish outside of the in-raceway cover-substrate, and the average for all fish at each time was recorded (figure needed). Average distance to the cover was computed by measuring the distance between each individual fish and the closest point to the in-raceway cover-substrate,. and tThe average distance for each time and randomly selected day was recorded (Figure needed). Arc GIS measured in standard units of measures, which were converted to metric units. Arc GIS spatial measures are generally used for landscape scale projects, but we were able to use it on a smaller scale by adjusting scaling using the 20 cm black strip that was visible in the picture.
Activity Level and Chasing
We randomly selected 14 days of video tape from the one single in-raceway cover-substrate condition. We counted the number of fish out of the in-raceway cover-substrate for each time block. We tracked the movement of two randomly selected target fish on video from each of the three time blocks; 0830 hr., 1440 hr., and 1840 hr. We played video files on a laptop computer using VideoViewer software compatible with the X DVR. We recorded the total time the target fish was out of the in-raceway substrate-cover cover-substrate for up to five minutes. We placed a sheet of glass over the computer monitor and marked the placement of the target fish every two seconds with a dry erase marker, connecting each successive mark to show a line of the fish movement. We used a commercial map measurer to measure the distance of the line created by the target fish while moving outside of the in-tank cover-substrate. We used both measures, time out of cover-substrate and distance traveled, to give us a measure of swimming activity (distance traveled/time).
We recorded number of chasing or paired swimming events of each target fish to quantify any possible aggressive interactions between fish pairs. (Figures needed-citation needed for paired swimming)
Statistical Analysis
To be filled in after results section is completed.