Dedicated To The Tribal Aquaculture Program

December 2005-Volume 54

Topics Of Interest:

*    The Ashland FRO is Now Equipped to Read OTC Marks

*    New Oxytetracycline Approval for Skeletal Marking

*  Calcein for Skeletal Marking - Promising New Technology

*    Formalin Treatment System Without the Stink

The Ashland FRO is Now Equipped to Read OTC Marks !

The focus of the next  article  was intended for those Tribal
fish hatchery programs that are currently, or are considering the use of Oxytetracycline (OTC) to mark fish for later identification.

The use of OTC will hopefully serve as an inexpensive fish marking tool  that will allow future assessments efforts (for fishery managers) to verify the recruitment levels of stocked fish that "originated from hatchery programs."

The treatment procedure with OTC involves keeping the fish in a small holding tank containing 700 parts per million of OTC for 7-8 hours. During the treatment period, the OTC is incorporated into the bony structures of the fish. When these structures (otoliths) are viewed using a microscope and ultraviolet light, the presence of an OTC mark will be noted as a yellow-gold band within the otolith.

Tribal fish hatchery programs interested in utilizing this fish marking technique (within the northern Michigan, Wisconsin and Minnesota area) may want to contact the Ashland FRO for services in applying or determining the presence of OTC marks from sampled fish.  Depending on work load, the Ashland FRO may be able to provide this service under a reimbursable agreement.

New Oxytetracycline Approval for Skeletal Marking
The information that follows was obtained from the Aquatic Animal Drug Approval Partnership (AADAP) Program located in Bozeman, MT.

Phoenix Scientific, Inc. (St. Joseph, Missouri) received an approval for their Oxytetracycline HCl Soluble Powder343 for skeletal marking in finfish fry and fingerlings (Abbreviated New Animal Drug Application No. 200 247). Currently (29 July 2005) Phoenixs product is available over the counter with a revised label claim, which includes poultry, cattle, sheep, swine, bees and skeletal marking in finfish. Phoenix Scientific directly markets a small portion of the product under their AmTech Group Inc. label, with the remainder of the drug being private labeled for authorized distributors. For the names of distributors in your region, or any other information regarding this product, contact Phoenix Scientific at 1-800-759-3664.  Buffering Oxytetracycline Hydrochloride Immersion-marking Solutions with Sodium Phosphate Dibasic Daniel Carty, Jim Bowker, Molly Bowman, and Bonnie Johnson; USFWS AADAP Program; 4050 Bridger Canyon Road, Bozeman, Montana 59715, USA.

 

Introduction

Oxytetracycline hydrochloride (OTC-HCL) water soluble powder is approved in the U.S. for use in the skeletal marking of finfish fry and fingerlings by immersion at concentrations of 200-700 mg/L active OTC for 2-6 h. This compound is acidic, and therefore OTC-HCL solutions usually need to be buffered to prevent or minimize mortality in treated fish. Anhydrous sodium phosphate dibasic (SPD) is commonly used as a buffer, probably because it is relatively safe to humans and because its buffering effects are relatively easy to control.  The Michigan Dept. of Natural Resources (MDNR) has recently published step-by-step guidelines for immersion-marking walleye fry and fingerlings with OTC-HCL and detecting resultant marks (Fielder 2002).

Building on MDNRs solution-preparation and buffering information, we conducted a study to show (a) pattern and magnitude of pH decrease when OTC-HCL is incrementally added to a source water to produce a 700-mg/L active OTC solution and (b) pattern and magnitude of pH increase when SPD is incrementally added to buffer a 700-mg/L active OTC solution to pH 7.0.

Methods

Test articles used in our study were (a) Oxytetracycline HCl Soluble Powder-343 and (b) sodium phosphate dibasic. We used five source waters, ranging from a cold, highly buffered, hard water to a cool water with no buffering capacity and no hardness. Initial pH values of the five source waters ranged from 7.1 to 7.9. Two 2-gal replicate water samples were collected from each source water, and each sample was processed as follows. First, fourteen 0.5-g aliquots of OTC-HCL were sequentially added to the water sample to achieve a non-buffered, nominal 700-mg/L active OTC solution. Second, ten 1-g aliquots of SPD were sequentially added to the resultant solution to buffer it to pH 7 (note: to enhance solubility of SPD, each aliquot of SPD was dissolved in 10 mL of microwave-warmed source water before adding it to the solution). The pH of the water sample was measured after each aliquot of OTC-HCL or SPD was added and dissolved. Replicate pH measurements were averaged for each source water to facilitate comparisons of pH change among source waters.

Results

In all five source waters, pH decreased incrementally as aliquots of OTC-HCL were added to achieve non-buffered, 700-mg/L active OTC solutions. Decreases observed in the two waters with high natural buffering capacity (cold spring and cold/warm spring waters) were more gradual and of less overall magnitude (2.3 and 2.6 pH units, respectively) than decreases observed in the two waters with moderate natural buffering capacity (cold/warm spring water diluted 1:1 or 1:2 with distilled water) or in distilled water (4.4, 4.5, and 4.4 pH units, respectively). In addition, minimum pH values observed in cold spring and cold/warm spring waters (5.3 and 5.2, respectively) were much higher than the minimum pH values observed in cold/warm spring waters diluted with distilled water or in distilled water (3.5, 3.1, and 2.7, respectively). Finally, the active OTC concentration at which pH was first observed to be unsuitable for aquatic life (pH ≤ 6.5, according to the U.S. Environmental Protection Agency) was highest in cold spring and cold/warm spring waters (250 mg/L in both), intermediate in cold/warm spring waters diluted with two levels of distilled water (150 and 100 mg/L, respectively), and lowest in distilled water (50 mg/L).  In all five source waters, pH increased incrementally as aliquots of SPD were added to buffer nominal 700-mg/L active OTC solutions to pH 7. Increases observed in cold spring and cold/warm spring waters were more gradual and of less overall magnitude (1.8 and 1.9 pH units, respectively) than increases observed in cold/warm spring waters diluted with distilled water or in distilled water (3.6, 4.0, and 4.4 pH units, respectively). However, regardless of source water, 4-5 g of SPD (a 0.6-0.7:1 ratio with OTC-HCL) were needed to buffer pH to levels suitable for aquatic life (pH ≥6.5), and 8-9 g of SPD were needed to buffer pH to 7.0 (a 1.1 - 1.3:1 ratio with OTC-HCL).

Finally, regardless of source water, maximum pH values achieved were never higher than
7.1, even after all 10 g of SPD had been added to a 700-mg/L active OTC solution.

Discussion and Conclusions

Our results demonstrated that the natural buffering capacity of source water substantially affects pattern and magnitude of pH change when OTC-HCL is added to water to achieve a non-buffered, 700-mg/L active OTC solution, and when SPD is subsequently added to buffer such a solution to pH 7.0. Natural buffering capacity can best be estimated by measuring total alkalinity, as opposed to measuring total hardness. Total alkalinity and total hardness will be similar if limestone is the source for both. However, these two water quality parameters can differ considerably in soft waters that have high alkalinity, e.g., soda lakes or in hard or soft waters that have low alkalinity, e.g., some ground waters or already acidic waters. The American Fisheries Society uses the following criteria to characterize source waters based on total alkalinity: (a) minimally acceptable buffering = 20 mg/L; (b) poorly buffered < 25 mg/L; (c) moderately buffered = 25-75 mg/L and (c) highly buffered > 75 mg/L.

Therefore, we suggest measuring the total alkalinity of a source water before preparing and buffering an OTC-HCL solution, and we agree with Fielders (2002) advice to measure pH before and during immersion-marking sessions.

In addition, our results indicated that (a) most OTC-HCL solutions will need to be artificially buffered to maintain pH at levels safe for fish and that (b) such buffering is relatively easy to achieve with SPD. For example, in the five source waters used in our study, pH levels ≤ 6.5 were first observed in non-buffered solutions at active OTC concentrations of only 50-250 mg/L far lower than the FDA-approved maximum of 700 mg/L active OTC.

However, both our results showed that an approximate 1:1 ratio (weight:weight) of SPD to OTC-HCL should be sufficient to buffer virtually any 700-mg/L active OTC solution to pH 7.0 and that addition of excess SPD will not increase pH much above 7.0.

It is important to note that overuse of SPD can be toxic to treated fish.

Finally, it should be cautioned that our results and inferences may have limited application to real-world immersion-marking sessions because our study was conducted under controlled laboratory conditions with only two test articles, five source waters, and no fish. However, the information in the MDNR guide (Fielder 2002) can be adapted to a variety of real-world immersion-marking situations.

Therefore, we encourage our readers to read the MDNR guide and to conduct their own experiments to determine how their test articles, source waters and fish interact. Note: The MDNR guide can be obtained in pdf format (3.5 MB) at:
http://www.michigandnr.com/publications/pdfs/ifr/ifrlibra/technical/reports/2002-1tr.pdf.  See below for the abstract, conclusion and image portions of this document.

 

Methodology for Immersion Marking Walleye Fry and Fingerlings in Oxytetracycline Hydrochloride and Its Detection with Fluorescence Microscopy
By:  David G. Fielder

Abstract This manual summarizes the process of conducting walleye stocking evaluations based on immersion marking with oxytetracycline hydrochloride. The summary includes methodology for application of the immersion treatment in the hatchery setting, detection of the mark using fluorescence microscopy and some considerations for planning evaluations based on the technique. The methodology described here was based on available literature and refined to meet the specific needs of the Michigan Department of Natural Resources, Fisheries Division.

Conclusion - Immersion marking with OTC is a substantial advancement in walleye stocking evaluation, but requires skill and expertise to accurately apply and interpret. Critical evaluations of stocking should utilize multiple years and consider additional evaluation techniques.  Publication of evaluation results should discuss marking methods and their effectiveness to help ensure the further evolution of this procedure.

Images - Please click on each picture to enlarge the image.

Under white light, this otolith has been "over" sanded.  Attempting to detect an OTC mark is no longer possible.		Under UV light, a yellow-gold ring is seen at the edges of the otolith from this recently treated fingerling.
Under UV light, a yellow-gold ring is seen at the center of the otolith from these two samples treated at the fry stage.
Under UV light, the presence of a yellow-gold ring is absent from this sample.

Contact Information for the purchase of OTC

 

Calcein for Skeletal Marking - A Promising New Technology
The information that follows was obtained from the Aquatic Animal Drug Approval Partnership (AADAP) Program located in Bozeman, MT.

The calcein story began in 1993 when biologists from the USFWS-Northeast Fishery Center in Lamar, PA (NEFC) canvassed northeast federal fish hatchery managers to determine their needs in the way of new technology for evaluating their particular hatchery products.

Responses from the managers revealed a common need for a new, low-cost tagging or marking technology which would allow a non-lethally detectable mark to be applied to large numbers of small fish simultaneously with some degree of mark longevity.

In addition this mark would need to be easily detectable without the need for complicated equipment and procedures. As a result, the quest for discovery of such technology began with a literature search. This effort led NEFC biologists to hypothesize that calcein dye may have the ability to produce brilliant, long-lasting marks in calcified fish tissues in addition to otoliths.

The first experiment with calcein was initiated at NEFC by exposing Atlantic salmon fry to 24 and 48-hour immersions in calcein concentrations similar to those used with oxytetracycline (i.e., 125-250 mg/L).  Biologists were excited to find brilliant, bright green marks on fin rays and other calcified tissues when viewed under an epifluorescence microscope. Subsequent experiments resulted in improvements in calcein mark application such that the mark can now be applied to thousands of fry simultaneously via osmotic induction in only 7 minutes. Further work at NEFC included a preliminary tissue residue study along with development of a pure grade of calcein by Western Chemical of Ferndale, WA. Work by the AADAP program at Bozeman, led to an Aquaculture Investigational Animal Drug exemption (INAD #10-987) for immersion of fish in calcein (SE-MARK; for details on INAD #10-987 go to http://www.fws.gov/fisheries/aadap/calcein.htm).

As a result studies are being conducted from coast to coast with a variety of fish species to determine the capabilities and limitations of using calcein as another tool in the fish management toolbox. Early in 2005, the USFWS was officially awarded a patent for a hand-held calcein detection device which was developed at NEFC and licensed to Western Chemical, Inc. of Ferndale, WA.  Were happy to say that the calcein-marking story does not end here, but continues with improvements in calcein mark application, and has led to a new round of experimentation with sturgeon, salmonids and striped bass in a project funded with USGS Science Support Program funds. The project is being conducted jointly at the Bozeman FTC/AADAP, Northern Appalachian Research Lab in Wellsboro, PA and NEFC, with much appreciated input and assistance from Ron Secor of Western Chemical, Inc. Western Chemical is the sponsor manufacturer of calcein (SE-MARK). The project involves inducing and evaluating calcein marks administered via fish feed. Once perfected, this technique will hopefully simplify calcein mark application by allowing the mark to be applied to any size fish by offering calcein medicated feed to the target animals for a few days. We are hopeful that this work will lea to another INAD exemption for inducing calcein marks in fish via feed. Preliminary results from these studies are very exciting! Additional information on calcein and calcein studies can be found by contacting Jerre Mohler; Fishery Biologist; U.S. Fish & Wildlife Service; Northeast Fishery Center; Lamar, Pennsylvania and Ron Secor; Western Chemical Inc.; Ferndale, Washington.

 

Formalin Treatment System Without the Stink !
By Steve Turner, Eagle Creek National Fish Hatchery

If your hatchery facility requires the need to treat developing eggs with formalin, you may want to look at the treatment station now in use at the Eagle Creek National Fish Hatchery.  Thanks to the Assistant Hatchery Manager, Steve Turner, the MTAN obtained the following images. 

The concept was designed to keep fumes out of the building by pumping formalin within a closed (recirculating) system.  Visualize a pumping system that is flowing within a loop (once you turn on the pump).  Each stack of eggs has a separate valve that allows the attendant to administer the desired volume of creek water as well as the amount of chemical (formalin) to be used.  The formalin is first pumped into a separate container.  This container then allows the chemical to gradually drip into the top stack of eggs.  Once all the formalin containers are filled, the pump is turned off.  The remaining chemical in the main line, is returned into the chemical drum and the formalin continues to drip down from each of the egg stacks.  This milk jug flow system was originally conceived by Steve Pastor, former Hatchery Assistant Manager with the US Fish & Wildlife Service . The formalin treatment system as shown below was designed and installed by Steve Turner (in 1988) and has proved to be a safe fume free environment for the staff.

This picture shows the formalin barrel with the suction and bypass hose.	The pump is called the "Little Giant." The orange valve that is partly open is a bypass valve. The bypass valve is used to reduce the pressure on the lines when filling the reservoirs with formalin.  This line is routed back into the chemical storage tank.	The orange valve at the top of the picture is partly open and supplies the incubator chemical reservoirs with formalin.
The Technician is opening the chemical reservoir. The reservoir is a small plastic veggie bowl, calibrated for 284 ml of formalin. It has a 20 Ga. hypodermic nettle cap, punctured with a 20 Ga. hole to dispense 284 ml of formalin in 15 minutes. The treatment is 1 - 600.  Respirators are not required for this system but full face protection is recommended.	Each milk jug is calibrated for gpm flow. The proper flow for the treatment is 3 gpm.  The small tupperware containers is for formalin that mixes with the creek water at 1- 600 parts for 15 minutes. It is necessary to have a simple method of calibrating water flow as part of the treatment system to get proper concentration of formalin.	This is the clean water flush pipe that is used to flush out the formalin network.
This picture shows an orange valve for filtered creek water open to the calibrated milk jug.  The creek water is needed to incubate the eggs. We use 3 gpm creek water that flows through the milk jugs and to each stack of eggs.	Notice the exhaust fan located close to the formalin barrel and pump station. This is VERY important for adequate ventilation of the building.	This picture shows the entire treatment network.
Formalin treatment pump station.	Directions have been posted for operating the pump station.