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REPRODUCTION CONTROL RESEARCH
Detection of Masculinizing Agents in the Pond Environment
The revisions to this work plan are limited to Reproduction Control Research 3A.
Detection of MT in Aquarium Water after Treatment with MT Food
Reproductive Control Research 3A
Operative period for revised experimental design: 3/97 – 12/97
Site: Oregon State University, Corvallis, Oregon
Laboratory Facilities: Oregon State Unversity—two aquaria containing a total of two males and six females for production of fry, 3L–chambers with soil substrate; Waters 660E Multisolvent Delivery HPLC system for separation and detection (by ultraviolet light absorption) of MT. MT radioimmuno-assay (RIA) for quantifying MT concentration.
Culture Period: 180 days for offspring.
Stocking Rate: 47 fry/container (corresponds to a stocking rate of
3000 fry/m2).
Water Management: Water temperature will be maintained at 28-30C. Every week, one-half of the water will be exchanged for new aerated water.
Other Inputs: None
Test Species: Nile tilapia (Oreochromis niloticus ), Ivory Coast strain.
Sampling Plan: The experiment consists of two groups: 1) fry fed MT at 60mg MT/kg food for 28 days; and 2) fry fed regular feed. Each group will be replicated. Water samples (25ml) will be collected on Days 0, 7, 14, 21, 28, and 35 from each tank (prior to any exchange with new water); 1-2g of substrate will be collected on Days 0, 28, and 35; all samples will be extracted on Sep-Pak (water) or with ether (substrate). Samples will be analyzed for steroid metabolites of MT by HPLC and for MT concentration by RIA. The following water quality parameters will be measured weekly: pH, ammonia, nitrates, and temperature. At the end of the 6-month grow out period, the tilapia will be killed to determine if the treatment with MT resulted in masculinization.
Statistical Methods and Hypotheses: Ho 1: MT is non-detectable in water at any time during and 1 week after treatment of tilapia fry with MT- impregnated food; Ho 2: MT is non-detectable in substrate at any time during and 1 week after treatment of tilapia fry with MT-impregnated food. This is a descriptive study, and therefore statisical analysis is unnecessary for testing the null hypotheses (i.e. detection of any amount of MT in water or substrate will be sufficient for rejecting the null hypotheses). Sex ratios will be compared between MT-fed and control groups by Chi-squared test.
Deliverables: A technical report and possibly a journal article.
Schedule
Data collection, 3/97–12/97; technical report, 3/98.
