Note: Experimental Design for Study B has been revised. See Addendum to the Ninth Work Plan
Note: Schedule for Study B has been revised. See Addendum to the Ninth Work Plan

Collaborating Institution
Oregon State University

Martin Fitzpatrick
Carl Schreck

Objectives

1) To determine the duration of 17a-methyltestosterone persistence in pond sediment after dietary treatment of tilapia with 17a-methyltestosterone.
2) To determine if pond soil from CRSP site(s) contain detectable amounts of 17a-methyltestosterone.
3) To determine if sex differentiation in tilapia can be affected by culture in systems that contain 17a-methyltestosterone-contaminated soil.

Significance
Treatment of tilapia fry with 17a-methyltestosterone (MT)-impregnated food for producing all-male populations is a common practice throughout the world. All-male populations offer the production advantage of enhanced growth potential (Green et al., 1997). However, significant “leakage” of MT into the pond environment may occur from uneaten or unmetabolized food. This leakage poses a risk of unintended exposure of hatchery workers as well as of fish or other non-target aquatic organisms. Furthermore, in some countries, pond sediments are dredged and used to prepare “soil” for crop production thereby spreading the risk of exposure to MT to terrestrial systems.

In the Eighth Work Plan, we conducted an experiment to examine the fate of MT in “model” ponds. Newly released (10 days post-fertilization) tilapia fry were stocked at 0.3 fish/cm2 into 3.7-L jars that contained 3 L of water and 3 cm of soil. The fish were fed either control food or MT-impregnated food at 60 mg/kg food for 28 days. Water samples were collected prior to stocking the fish and at 7, 14, 21, 28, 35, 42, and 49 days after the onset of feeding; soil samples were collected prior to stocking the fish and at 28, 35, 42, and 49 days after the onset of feeding. Water and soil samples were analyzed for the concentration of MT by radioimmunoassay. Although the levels of MT in the water peaked at an average of 1.7 µg/L at 14 and 21 days after the onset of feeding, the concentration decreased to background level by 35 days after the onset of feeding (1 week after the end of treatment with MT-impregnated food). In contrast, the levels in the soil were 1.8 µg/kg at 28 days after the onset of feeding with MT-impregnated food and remained detectable in the soil at between 0.8 and 1.6 µg/kg through 49 days (3 weeks after ending treatment with MT-impregnated food). Thus, MT persists in the sediments for a considerable time after the end of treatment, posing a potential health risk to workers and an exposure risk to non-target fish and other organisms. Tilapia may disturb sediments when they build nests or search for food, leading to resuspension of MT from the soil into the water column. Thus, “rotating” the pond use from fry production to rearing or breeding will not reduce the risk of re-exposure.

The proposed work will examine if:

1) MT persists in the soil of “model” ponds for up to 3 months after cessation of treatment,
2) MT is detectable in soil from ponds at one (or more) CRSP sites, and
3) MT-contaminated soil affects sex differentiation in tilapia (i.e., are sediments acting as an MT reservoir?).

Anticipated Benefits
An assessment of MT persistence and fate in the soil will provide information on the potential risks of using MT-feeding technology to workers and to non-targeted organisms. Furthermore, determining when MT no longer can be detected in the pond environment will help to establish additional safety guidelines for the use of MT.

Collaborative Arrangements
The collection of soil samples for Study 2 will involve collaboration with personnel from the Africa Project.

Experimental Design
Analytical procedures for measuring MT were developed in the Eighth Work Plan and consist of a specific radioimmunoassay (RIA) for quantifying MT in soil.

Experiment A: Detection of MT in soil after treatment with MT food
Site: Oregon State University, Corvallis, OR

Laboratory Facilities: Oregon State University—Two aquaria containing a total of two males and six females for production of fry, 3.7-L chambers (containing 3 L of dechlorinated oxygenated water and 3 cm of clay substrate collected from Soap Creek fish ponds); 75-L tanks within a recirculating water system for growing fry after hormonal treatment; Waters 660E Multisolvent Delivery HPLC system for separation and detection (by ultraviolet light absorption) of MT. MT radioimmunoassay (RIA) for quantifying MT concentration.

Culture Period: 180 days for offspring.

Stocking Rate: 47 fry/container (corresponds to a stocking rate of 3,000 fry/m²).

Water Management: Water temperature will be maintained at 28-30°C. Every week, one-half of the water will be exchanged in the 3.7-L containers for new aerated water; in the recirculating system, complete water exchange is estimated to occur every 120 min.

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 60 mg MT/kg food for 28 days; and 2) fry fed regular feed. Each group will be replicated. Water samples (25 ml) will be collected on Days 0, 28, 56, 84, and 112 from each 3.7-L container (prior to any exchange with new water); soil samples (3-5 g) will be collected on Days 0, 28, 56, 84, and 112; 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. Fish will be removed from the 3.7-L containers on Day 28 and placed in separate 75-L tanks within a recirculating system for the grow-out phase. 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: H₀1: MT is non-detectable in water at any time during and 1 week after treatment of tilapia fry with MT-impregnated food; H₀2: 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, statistical 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.

Schedule: Data collection, 10/98-3/99; technical report, 7/99.

Study B: Detection of MT in pond soil from a CRSP site
Site: Sagana Station, Kenya; and Oregon State University

Note: Experimental Design for Study B has been revised. See Addendum to the Ninth Work Plan
Note: Schedule for Study B has been revised. See Addendum to the Ninth Work Plan

Pond Facilities: Two 200-m² ponds that are being used in other studies for fry production (sex-reversed and untreated fry) will be used.

Culture Period: 180 days for offspring.

Stocking Rate: 3,000-5,000 fry/m² in each hapa.

Water Management: Static ponds.

Other Inputs: none.

Test Species: Nile tilapia (Oreochromis niloticus).

Sampling Plan: The study consists of analysis of soil samples from pond(s) in normal use at the Sagana station. Sampling will be from a pond that is in use or has been used for sex inversion of tilapia fry. Soil samples (3–5 g) will be collected from beneath the hapas used for feeding fry MT-impregnated food and at 5, 10, and 15 m from the hapas. If possible, soil will be sampled on Days 0, 28, and 56 of feeding to encompass the conditions prior to, at the end of, and 4 weeks after treatment with MT. Soil samples will include the top 3 cm of soil within the ponds. All samples will dried at the Station and stored in whirl-paks before shipment to the US for analysis of MT. The samples will be extracted with ether and MT measured by radioimmunoassay (as described in Experiment A). At the end of the 6-month grow-out period, a subsample (n = minimum of 50) of tilapia will be examined to determine if the treatment with MT resulted in masculinization.

Statistical Methods and Hypotheses: H₀1: MT is non-detectable in pond soil at any time or location within the pond during or after treatment of tilapia fry with MT-impregnated food. This is a descriptive study and therefore, statistical analysis is unnecessary for testing the null hypotheses (i.e., detection of any amount of MT in substrate will be sufficient for rejecting the null hypotheses). Sex ratios will be compared between MT-fed and control (or a theoretical 50:50 male:female population) groups by Chi-squared test.

Deliverables: A technical report.

Schedule: Data collection, 9/98–9/99; technical report, 12/99.
If possible, other CRSP sites will also be screened for the presence of MT in soil sediments.

Experiment C: Impact of MT-contaminated soil on tilapia sex differentiation
Site: Oregon State University, Corvallis, OR

Laboratory Facilities: Oregon State University—two aquaria containing a total of two males and six females for production of fry, 3.7-L chambers (containing 3 L of dechlorinated oxygenated water and 3 cm of clay substrate collected from Soap Creek fish ponds); Waters 660E Multisolvent Delivery HPLC system for separation and detection (by ultraviolet light absorption) of MT. MT radioimmunoassay (RIA) for quantifying MT concentration.

Culture Period: 365 days.

Stocking Rate: 47 fry/container (corresponds to a stocking rate of 3,000 fry/m²).

Water Management: Water temperature will be maintained at 28–30°C. 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 treatments: 1) fry cultured for 28 days in jars containing soil contaminated with MT; and 2) fry cultured for 28 days in jars containing untreated soil. To make the experiment realistic, contamination of the soil will be achieved by cycling 3 sets of fry through the common MT treatment (i.e. each cycle will consist of fry fed 60 mg MT/kg food for
28 days). Water from both treatments will be exchanged weekly without disturbing the soil. Each treatment will be triplicated. Soil samples (3-5 g) will be collected at the end of each cycle (i.e., every 28 days) and at the end of the 28-day exposure period, when the fry will be moved to 75-L tanks for the grow-out phase. At the end of the 6-month grow-out period, the tilapia will be killed to determine if the exposure to MT-contaminated soil resulted in masculinization.

Statistical Methods and Hypotheses: H₀1: The sex ratios of fry cultured in MT-contaminated and in untreated soil will not differ. Sex ratios will be compared between MT-fed and control groups by Chi-squared test.

Deliverables: A technical report. The results of the investigations outlined in this work plan will be synthesized into recommendations concerning the use of 17a-methyltestosterone for masculinization of tilapia.

Schedule: Data collection, 10/99–3/00; technical report, 7/00.

References
Green, B.W., K.L. Veverica, and M.S. Fitzpatrick, 1997. Fry and fingerling production. In: H. Egna and
C. Boyd (Editors), Dynamics of Pond Aquaculture, CRC Press, Boca Raton, FL, pp. 215-243.