Pd/a Crsp Seventeenth Annual Technical Report

The objective of the study was to determine if methyltestosterone (MT) can be detected in the treatment environment and, if so, for how long after treatment. Oreochromis niloticus fry with a mean initial length of 9.5 mm were stocked into adjacent cages in an earthen pond at Auburn University, Alabama, at 2,000 fry cage-1 and fed for 28 days a feed containing 60 mg MT kg-1 or a non-treated feed. At the end of the controlled feeding period both sets of fish were harvested, and growth and survival were determined. Fish were returned to their respective hapas and fed a non-hormone treated feed for additional growth until a mean total length of approximately 5 cm was reached. A sample of 100 fish from each hapa was preserved in 10% formalin and the sex was determined. Water and soil samples from the treatment pond were taken prior to, during, and after the hormone administration period. Water samples (collected approximately 10 cm below the surface) were taken weekly from within the cage receiving hormone-treated feed and the cage receiving non-treated feed. At the same time intervals water samples were taken at 2, 5, and 10 m from the cage receiving hormone-treated feed. Soil samples were taken of the upper 5 cm of soil at the same locations at which water samples were taken; samples were collected from under the cages and from the pond bottom at the distances specified above. Soil and water samples were analyzed at Oregon State University. There was no evidence that MT altered the sex ratio of non-target tilapia held in the same pond as and confined near fish receiving MT. The treated population was 91% male, 5% female and 4% intersex. Fish held in an adjacent cage approximately 50 cm away and fed a nonhormone-treated diet had a sex ratio of 48% males and 52% females. Mean MT concentration in the water sampled within MTtreated or non-treated cages did not differ (P = 0.14). Pretreatment MT concentration in the water column was 8.0 ± 5.7 pg g-1, and values within the treatment cage were similar except for one sample during the treatment period. The radioimmunoassay when used with soil cross-reacted with other materials in addition to MT. Pretreatment soil samples from the pond, which had no previous history of MT administration, had a concentration of 875 ± 147 pg g-1. The highest concentration of MT indicated (1,417 pg g-1) was from a soil sample beneath the cage receiving the non-treated feed. SEVENTEENTH ANNUAL TECHNICAL REPORT 58 sets of fish that had been given the hormone-treated feed and were successfully sex-reversed. Abucay et al. (1997) found that reusing static water aquaria that had been used to hold tilapia fry that were given an MT-treated feed for 25 days could alter sex ratios. When a second group of fish were stocked into such aquaria and given a non-hormone-treated feed, the sex ratio was skewed. They also found that when an all-female set of fry were stocked into a cage in an aquarium and an MT-treated feed was added to the bottom of the aquarium where the fish had no access to it, the sex ratio became skewed to males. In a laboratory setting, Fitzpatrick et al. (1999) detected MT in the water and soil of containers in which tilapia were fed an MT-treated feed during a treatment period of four weeks. Concentrations of MT in the water had decreased to within background levels by one week after the end of treatment, while measurable levels above background in the soil persisted for four weeks post-treatment. MT is susceptible to breakdown when exposed to light or high temperatures (McEvoy, 1997). Both fungi and bacteria can metabolize exogenous steroids. Many different steroid metabolism reactions, including the metabolism of MT, are possible in bacteria (Schubert et al., 1972; Jankov, 1977), as well as metabolism of steroids to CO2 and H2O (Sandor and Mehdi, 1979). In an outdoor pond setting where fish are held in cages, the combination of light, temperature, and microbial degradation may result in a rapid breakdown of MT. This study evaluated the concentrations of MT in soil and water when tilapia were given a diet containing MT for a 28-day period when cultured in cages in an outdoor earthen pond. The effect on the sex ratio of non-treated tilapia held in an adjacent cage was also examined. METHODS AND MATERIALS Oreochromis niloticus fry were obtained from a mass spawning of brooders in an earthen pond and graded to obtain fish with a mean initial length of 9.5 mm. Fish were stocked into adjacent cages in a 400-m2 earthen pond at Auburn University, Alabama, at 2000 fry cage-1 and fed for 28 days a feed containing 60 mg MT kg-1 or a non-treated feed. Fish were fed at 25% body weight the first week; thereafter, feeding was reduced by 5% of body weight each week. Fish were sampled weekly during the feeding period, average weights were determined, and feeding rates were adjusted daily based on observed growth rates. At the end of the controlled feeding period both sets of fish were harvested, and growth and survival were determined. Fish were returned to their respective hapas and fed a non-hormone-treated feed for additional growth until a mean total length of approximately 5 cm was reached. A sample from each hapa of 100 fish was preserved in 10% formalin and dissected. Gonads were removed, lightly stained with fast green, squashed to facilitate microscopic examination, and the sex was determined. The entire gonad was examined and those containing both ovarian and testicular tissue were classified as intersex; gonads having only one form of tissue were classified as male or female, corresponding to the type of tissue observed. Water and soil samples from the treatment pond were taken prior to, during, and after the hormone administration period. Water samples (collected approximately 10 cm below the surface) were taken weekly within the cage receiving hormonetreated feed and the cage receiving the non-treated feed. At the same time intervals, water samples were taken at 2, 5, and 10 m from the cage receiving hormone-treated feed. Soil samples were taken of the upper 5 cm of soil at the same locations at which water samples were taken; samples were collected from under the cages and from the pond bottom at the distances specified above. All soil and water samples were frozen soon after collection and held frozen for shipment. Soil and water samples were analyzed at Oregon State University following the procedures of Fitzpatrick et al. (1999). RESULTS AND DISCUSSION There was no evidence of MT altering the sex ratio of non-target tilapia held in the same pond and confined near fish receiving MT. Treatment of O. niloticus fry for 28 days with MT at 60 mg kg-1 of diet was effective in altering the sex ratio. The resultant population was 91% male, 5% female, and 4% intersex. The control group of fish held in an adjacent cage approximately 50 cm away and given a non-hormone-treated diet had a sex ratio of 48% males and 52% females. The lack of intersex fish in the non-treated fish is further evidence that MT was not released into the environment at a rate to alter sex ratios. There was little to no evidence of MT escapement from the treatment cage environment. Mean MT concentrations in the water sampled within MT-treated or non-treated cages did not differ (P = 0.14). Pretreatment MT concentration in the water column was 8.0 ± 5.7 pg g-1, and values within the treatment cage were similar during the treatment period with the exception of one sample. A concentration of 161 ± 28 pg MT g-1 was detected in this sample, and it is thought that this was caused by a particle of feed collected in the sample. MT concentrations in the water from the treatment cage for the 20-day post-treatment period averaged 9.7 ± 4.1 pg g-1. The mean concentration of MT detected 2 to 10 m from the treatment cage was 9.7 pg g-1. Concentrations of MT within the control cage averaged 9.2 pg g-1 for the study period. Concentration of MT in water or soil did not increase in relation to the increased MT input over time associated with feeding (Figure 1). On the last day of hormone treatment, 43.3 g of feed 0 20 40 60 80 100 120 140 160 180