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Elimination of Methyltestosterone (MT) from Intensive Masculinization Systems: Use of Activated Charcoal in Concrete Tanks 10ER2

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Elimination of Methyltestosterone (MT) from Intensive Masculinization Systems: Use of Activated Charcoal in Concrete Tanks

Effluents and Pollution Research 2 (10ER2)/Experiment and Study/Mexico

Collaborating Institution
Universidad Juárez Autónoma de Tabasco, Mexico
     Wilfrido M. Contreras Sánchez
     Gabriel Márquez Couturier

Oregon State University
     Guillermo R. Giannico
     Carl B. Schreck

Objectives
1) Determine if the 17a-methyltestosterone that escapes to the water after dietary treatment of tilapia fry can be eliminated from intensive masculinization systems.

2) Evaluate the efficacy of a new technology for clean effluents in aquaculture.

Significance
All-male populations are used in tilapia (Oreochromis spp.) aquaculture because the culture of mixed sex populations often results in precocious maturation and early reproduction (Schreck, 1974; Mires, 1995). Furthermore, all-male tilapia populations are desirable because males achieve a larger final size than females (Macintosh and Little, 1995).

Masculinization of tilapia fry by oral administration of 17a-methyltestosterone is considered the most successful method employed; however, under certain conditions this technique is sometimes less favorable. Furthermore, 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 fish or other non-target aquatic organisms, to the steroid or its metabolites.

In recent studies (Contreras-Sánchez, 2001), we found that masculinization of fry through dietary treatment with MT results in the accumulation of MT in sediments and causes the production of intersex organisms and females with altered ovarian development. In systems were substrate was not present, we found higher concentrations of MT in the water and lower (sometimes null) masculinization rates than in those systems with either soil or gravel. We found that charcoal filtration of water from systems where substrate was not present lowers the amount of MT in water to almost background levels and the treatment resulted in almost complete masculinization of all three broods tested (100, 98, and 100% males, respectively). Apparently, the recommended dose of MT for masculinizing tilapia is higher than needed and a significant portion of it separates from the food and remains either in suspension in the water for the short term or persists in the sediments over the long term (Contreras-Sánchez et al., 2001). In the cited study, we recommended the use of activated charcoal filtration systems to eliminate excess MT to increase masculinization, and to prevent potential risks to humans of unintended exposure to MT due to contamination of water and soils in farms.

In Mexico, the use of MT for masculinizing tilapia fry is a new activity. Little is known regarding the use of MT and the scarce information available to hatchery producers and fish farmers does not deal with the potential risks of this practice. In the southeastern region of the country, hatchery production goals have not been reached and the methods used are far from being efficient. Despite almost 30 years of tilapia farming in Mexico, the use of mixed sex populations is still a very common practice and as a result, the productivity of many hatcheries and farms is severely affected.

We propose the use of intensive systems for masculinizing tilapia fry using MT-impregnated food at large scale where the excess of MT is eliminated from the water and the substrate by means of continuous filtration through activated charcoal filters. This method may allow the production of large numbers of all-male populations of tilapia fry using a reliable technique compatible with the proposed Best Management Practices (BMPs) for aquacultural systems. Activated charcoal is cheap and readily available in southern Mexico, and it is well known by aquaculturists that it can be reactivated by sunlight exposure. Sunlight treatment of the activated charcoal may bring another benefit, i.e. the breakdown of MT due to steroid photosensitivity.

If successful, this method can be transferred to tilapia hatcheries that play an important role in poor areas of the states of Tabasco and Chiapas, México. The use of reliable and efficient masculinizing methods in the hatcheries will benefit thousands of small-scale fish farmers who currently see their productivity negatively affected by the use of mixed-sex populations of tilapia. A series of training workshops will be developed and offered to different audiences in the communities of Tabasco and Chiapas to ensure that the above-mentioned methodology is effectively transferred to its final users. Technical workshops will target hatchery managers, extension agents and university students (many of whom will become workshop instructors over time). Public extension workshops will be tailored to the cultural characteristics of the target audience and will be offered to fish farmers, farm workers and selected community leaders.

Anticipated Benefits
The development of clean technologies for aquacultural practices will impact positively the production of sex-reversed tilapia fry.

The use of activated charcoal filters in intensive systems as an alternative treatment for masculinizing tilapia will improve safety in handling masculinizing steroids, produce clean effluents, and potentially increase efficiency of exposure.

Research Design
Experiment A: Elimination of MT from the Water of Intensive Sex-Inversion Systems

Site: Experiments will be conducted at the Laboratory of Aquaculture at UJAT, Tabasco, México.

Methods: Oral administration of MT (dose = 60 mg/kg) in concrete tanks (8 m2).

Laboratory and Pond Facility: Universidad Juárez Autónoma de Tabasco; 1 earthen pond (200 m2) size, 50 net cages (1 m3), 3 concrete tanks (8 m2), 3 grow-out ponds (200 m2), a total of 200 females and 65 males for production of fry.

Culture Period: 3 months or until tilapia reach sampling size (3 cycles)

Stocking Density: 3,000-4,000 fry/m2

Test Species: Nile tilapia (Oreochromis niloticus)

Nutrient Inputs: None

Water Management: Water will be filtered through a 200-l tank containing an activated charcoal filter bed at a rate of 50 l/min. Water will be recirculated through the rearing tanks in all systems. The MT tanks will receive MT-treatment recirculated water; the control tanks will receive control recirculated water. A 25% water exchange will be performed twice a week.

Sampling schedule: The experiment consists of four treatments:

Because of tank availability, each treatment will consist of a single experimental unit. The entire experiment will be repeated three times and used in the data analysis as pseudoreplicates. Water (12 ml) samples will be collected from the sex-inversion tank on days 0, 7, 14, 21, 28, and 35. Five sampling locations will be randomly selected each time. Samples will be frozen (­20°C) and preserved until processing. Activated charcoal used in the treatment will be exposed to direct sunlight for 24 and 48 hours, and samples will be collected at different times for MT detection. All samples will be extracted using ether and the concentration of MT determined by radioimmunoassay (RIA; at OSU). At the end of 3-month grow-out period, a subsample of the tilapia in each experimental unit (100) will be killed with an overdose of anesthetic (MS-222) to determine if the treatment with MT resulted in masculinization. The following water quality parameters will be measured daily: pH, DO, and temperature. Activated charcoal used in the treatment will be exposed to direct sunlight for 24 and 48 hours, and samples will be collected for MT detection.

Statistical Methods and Hypothesis: H01: MT is detectable in water at any time during and 1 week after treatment of tilapia fry with MT-impregnated food independently of the amount of activated charcoal used in the filter. This part of the study is descriptive and therefore, statistical analysis is unnecessary for testing the null hypothesis (i.e. failure to detect any amount of MT in water will be sufficient for rejecting the null hypothesis). H02: Administration of MT feed to tilapia held in systems with different amounts of activated charcoal results in treatments with the same sex ratios. The efficacy of MT treatment will be tested comparing between MT-fed and control treatments by Chi-squared test.

Schedule: Data collection, 7/01-6/02; Technical report, 6/30/02

Study B: Implementation of Safe Intensive Sex-Inversion System in Local Tilapia Farms

Site: Filtering systems will be evaluated at the following farms:

Methods: Oral administration of MT (dose = 60 mg/kg) in concrete tanks.

Laboratory and Pond Facility: All farms have concrete tanks, grow-out ponds (200 m3), and broodstock.

Culture Period: 3 months or until tilapia reach sampling size (3 cycles)

Stocking Density: 3,000-4,000 fry/m2

Test Species: Nile tilapia (Oreochromis niloticus)

Nutrient Inputs: None

Water Management: Water will be filtered through a 200 l tank containing an activated charcoal filter bed at a rate of 50 l/min. Each tank will have its own filter. Water will be recirculated through the rearing tanks in all systems. 25% water exchange will be performed twice a week.

Sampling Schedule: Based on the results from Experiment A, the treatment that results in the maximum percentage of males and the minimum amount of MT in the water will be selected to be used at the farms. Experimental treatments will be as follows:

If possible, replicates will be used. Water (12 ml) samples will be collected from the sex-inversion tank on days 0, 7, 14, 21, 28, and 35. Five sampling locations will be randomly selected each time. Samples will be frozen (­20°C) and preserved until processing. All samples will be extracted using ether and the concentration of MT determined by RIA (at OSU). At the end of 3-month grow-out period, a subsample of the tilapia in each experimental unit (100) will be killed with an overdose of anesthetic (MS-222) to determine if the treatment with MT resulted in masculinization. The following water quality parameters will be measured weekly: pH, DO, and temperature.

Statistical Methods and Hypothesis: H01: MT is detectable in water at any time during and 1 week after treatment of tilapia fry with MT-impregnated food independently of the use of activated charcoal filters. This part of the study is descriptive and therefore, statistical analysis is unnecessary for testing the null hypothesis (i.e., detection of any amount of MT in water or substrate will be sufficient for rejecting the null hypothesis). H02: Administration of MT-feed to tilapia held in systems with activated charcoal results in treatments with the same sex ratios as the controls. The efficacy of MT treatment will be tested comparing between MT fed and control treatments by Chi-squared test.

Schedule: Data collection, 7/01-6/02; Technical report, 6/30/02

Regional Integration
If the results of the Study B are successful, we will exchange information with CRSP Honduras Project researcher Daniel Meyer at Escuela Agrícola Panamericana (Zamorano). We have already discussed with the Peru and Thailand PD/A CRSP research teams our intentions to exchange information among project scientists and for potential technology transfer.

Literature Cited
Contreras-Sánchez, W.M., 2001 Masculinization of Nile tilapia Oreochromis niloticus: II. Efficacy of the masculinizing agent 17a-methyltestosterone in different environments. Chapter 3, Ph.D. dissertation, Oregon State University, Corvallis, Oregon.

Macintosh, D.J. and D.C. Little, 1995. Nile tilapia (Oreochromis niloticus). In: N.R. Bromage and R.J. Roberts (Editors), Broodstock Management and Egg and Larval Quality. Blackwell Scientific, Ltd., Cambridge, Massachusetts, pp. 277­320.

Mires, D., 1995. The tilapias. In: C.E. Nash and A.J. Novotony (Editors), Production of Aquatic Animals. Elsevier, New York, pp. 133­152.

Schreck, C.B., 1974. Hormonal treatment and sex manipulation in fishes. In: C.B. Schreck (Editor), Control of Sex in Fishes. Virginia Polytechnic Institute and State University Extension Division, Blacksburg, Virginia, pp. 84­106.

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