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Ashland FWCO
 Midwest Region
 MTAN LogoDedicated To The Tribal Aquaculture ProgramImage of fish jumping.
    December 2005-Volume 54
http://www.fws.gov/midwest/ashland/mtanhome.html
**Coordinator:
        Frank G. Stone 
        (715-682-6185) Ext.
        202
        U.S. Fish and
        Wildlife Service
        Email: [email protected]**
      
      **Edited
        By:
        Elizabeth W. Greiff 
        (715-349-2195) Ext. 5141
        St. Croix Tribal Nat. Res. Depart.
        Email:  ****[email protected]****** 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."
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. 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 MarkingThe 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 
MicroscopyBy:  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 Phoenix Scientific, Inc. (St. 
Joseph, Missouri)(Web site address not available at this time)
1-800-759-3664
      
      **Omaha 
Vaccine**Product # 08793**Email: [email protected]
1-800-367-4444**
 Calcein 
for Skeletal Marking - A Promising New TechnologyThe 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.
  **Return
to MTAN selection pageReturn to Ashland FRO home pageReturn to Region 3 U.S. Fish and Wildlife Service** Product and company
      names mentioned in this publication are for informational purposes only.
      It does not imply endorsement by the MTAN or the U.S. Government.
 
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