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Fima_22_123.236_242.tpNorth American Journal of Fisheries Management 22:236–242, 2002 ᭧ Copyright by the American Fisheries Society 2002 In-Transit Oxytetracycline Marking, Nonlethal Mark
Detection, and Tissue Residue Depletion in Yellow Perch
Department of Wildlife and Fisheries Sciences, South Dakota State University, Brookings, South Dakota 57007-1696, USA South Dakota Department of Game, Fish, and Parks, 4500 South Oxbow Avenue, Sioux Falls, South Dakota 57106-4114, USA Abstract.—Trapping and transfer of juvenile and adult potential use of TC compounds for fish marking, yellow perch Perca flavescens is conducted to fulfill over 100 studies have reported using TC or OTC perch stocking needs in South Dakota. To determine the for 58 species. Modes of induction were immer- utility of oxytetracycline (OTC) hydrochloride markingas an assessment tool for yellow perch stockings, we sion, injection, or feeding. Common objectives investigated a transfer tank marking protocol, compared were to evaluate stockings or to validate age and OTC mark detection in dorsal spines with that in sagittal growth. Most studies reported results for juvenile otoliths, and assayed the depletion of OTC residues in fish; little information exists concerning the effi- adult yellow perch muscle tissue. Juvenile yellow perch cacy of immersion marking of adult fish. Overall, were immersed for 4, 6, or 8 h in 500 or 700 mg OTC/ striped bass Morone saxatilis (e.g., Secor et al.
L at 18ЊC in transfer tanks. Acute mortality was lessthan 1% at 8 h. At 3 months postimmersion, fluorescent 1991), red drum Sciaenops ocellatus (e.g., Murphy marks were detected on all spine sections and otoliths and Taylor 1991), American shad Alosa sapidis- from fish treated for 6 h or longer. Mark quality was sima (e.g., Hendricks et al. 1991), and walleye observed to be slightly better in juvenile dorsal spines Stizostedion vitreum (e.g., Kayle 1992) were some than in otoliths. Adult yellow perch were immersed for of the most commonly marked fishes in those stud- 6 h in 600 mg OTC/L at 19ЊC. Marks were initially ies. In the Midwest, walleye is the most frequently detected at otolith margins at 9 d postimmersion but werebest detected on all otoliths after 51 d postimmersion.
marked sport fish species because of the need to High pressure liquid chromatography of adult yellow quantify stocking contributions. Walleye research- perch muscle tissue indicated that OTC was rapidly de- ers commonly report using 500–700 mg OTC/L in pleted; the acceptable tolerance level of 2 g total OTC a sodium-phosphate-buffered solution for an im- residue/g for human consumption was reached by about mersion period of about 6 h (e.g., Brooks et al.
2 h postimmersion. The nonlinear relation between total 1994; Fielder 1994; Lucchesi 1999). There is con- residue (OTC base and 4-epioxytetracycline) and timewas defined by the equation log siderably less known about OTC applications to other popular coolwater species, such as yellow Intensive trapping and transfer of juvenile and Chemical marking by immersion is a suitable adult yellow perch is done to fulfill stocking needs approach for projects that require large numbers in South Dakota. The time and personnel expense of marked fish. In particular, fluorochromes in used for trapping, transferring, and stocking does the antibiotic group containing tetracycline not readily allow for additional effort associated (TC; C22H24H2O8) and oxytetracycline (OTC;C with holding fish at a marking facility. Stress-re- 22H24N2O9) provide good per formance. Addi- tionally, the hydrochloride form commonly pre- lated mortality associated with higher tempera- ferred for marking is highly soluble in water (1 tures in late summer and early fall, when transfer g/mL; Budavari et al. 1996). The TC family of operations occur (along with other logistics of antibiotics is prepared from cultures of various hauling operations) requires that fish be collected Streptomyces species that fluoresce yellow to and transferred as quickly as possible. Thus, a suc- green under ultraviolet light (Mitscher 1978).
cessful protocol for in-transit marking would ex- Since Weber and Ridgway (1962) described the pedite trapping and stocking operations. Addition-ally, catchable-sized yellow perch often composea high proportion of stocked perch, and informa- * Corresponding author: michael[email protected] tion concerning OTC residues in edible tissue is Received March 20, 2001; accepted July 12, 2001 lacking. Unkenholz et al. (1997) did not detect residues in juvenile fish at 110 d postimmersion A 12 h light:12 h dark photoperiod was maintained but did not define the minimum time required to meet the tolerance level for human consumption.
OTC residue analysis of adult yellow perch.— Therefore, our primary objectives were to evaluate Adult yellow perch were collected from a rearing an OTC marking protocol in transfer tanks and to pond and transferred to an indoor 1,250-L fiber- conduct temporal assays of OTC residues in adult glass raceway to investigate the depletion of OTC yellow perch muscle tissue. Second, we compared in muscle tissue. The fish were immersed in a cal- marks produced by OTC in dorsal spines with culated concentration of 600 mg OTC hydrochlo- those produced in otoliths to investigate a non- ride/L buffered to a pH of 7.2 with sodium phos- phate dibasic for 6 h. Approximately 80 mL ofsilicon-based surfactant was added to reduce foam caused by aeration. At 6 h, normal water flows Transfer-tank marking protocol for juvenile yel- were resumed and the OTC-treated waters were low perch.—Age-0 yellow perch were collected in flushed through an active carbon filter.
September with trap nets from semi-permanent Following marking, the fish were maintained in wetlands used as monoculture rearing ponds. Fish the raceway and fed a prepared salmonid grower were placed in 757-L transfer tanks containing diet to satiation by delivering the ration three times lake water treated with calculated solutions (98.4% daily with a 24-h belt feeder. During the holding active OTC) of either 500 or 700 mg OTC hydro- period, water quality was maintained with a flow chloride/L. The OTC was first dissolved in 15 L rate of 3.75 L/min and supplemental aeration. A of pond water and buffered to neutral pH with 13 h light:11 h dark photoperiod was maintained sodium phosphate (dibasic; Na2HPO4). Tank mix- throughout the study. The marking and holding ing was done with 12-V DC agitators, and aeration was supplemented with pure oxygen. A silicon- Nine treated fish were subsampled at preselected based surfactant was added to reduce foam. Fish time intervals (1–1,080 h) and sacrificed for tissue were transported and held in the transfer tanks for analyses. The samples were stored in a dark en- up to 8 h. Temperature, dissolved oxygen (DO), vironment and frozen atϪ20ЊC, pending prepara- and pH were monitored every 30 min during the tion and analysis. Individual fish from each treat- immersion period. Budavari et al. (1996) and Doi ment were prepared by removing the fillet (skin and Stoskopf (2000) indicated that the potency of off, no bones). Heads were stored frozen and intact OTC decreases with increasing temperature and until otolith removal. For each time period, three pH; the half-life potency at pH 7.0 is 14 d and 26 composite samples were formed, each containing h for aqueous OTC solutions at 25ЊC and 37ЊC, muscle tissue from three fish. Composite tissue respectively. Our marking solution was at 18ЊC samples were separately homogenized in trichlo- and a pH of 7.3 and thus should have retained high roacetic acid and acetate buffer. Tissue samples potency for the 8-h marking period. Also, loss of and OTC-treated waters were analyzed with high potency in aqueous OTC solutions exposed to light pressure liquid chromatography (HPLC), with indicates that photoreduction further facilitates the quantitation levels of 0.05 g OTC/g for fish tissue breakdown of OTC (Mitscher 1978; Doi and Stos- and 0.01 g OTC/mL for water (Houglum and kopf 2000). Therefore, tank hatch covers remained Larson 1999). The precision of the HPLC method closed except when subsampling fishes.
is about threefold better than that of the traditional At 4, 6, and 8 h during the immersion period, microbial inhibition assay for OTC in fish tissue 25 fish were randomly collected from each tank (Stehly et al. 1999). Control fish samples were and given an identifying fin clip to denote the im- determined to contain 0.00 g OTC/g tissue. An mersion time and marking concentration. After re- untreated fish sample spiked with 0.17 g OTC/g ceiving the fin clip, fish were transferred to a 938- tissue was determined to contain that concentra- L circular tank and held for 3 months. During the holding period, good water quality was maintained Mark detection.—For mark evaluation, juvenile with a flow rate of 3.75 L/min and supplemental and adult fish were euthanatized and sagittal oto- aeration; water temperature was maintained near liths were removed by dissection of the frontal 24ЊC. Fish were fed to satiation once each day with bone. Otoliths were dried and mounted (concave a prepared salmonid grower diet (BioDiet, War- side down) on glass slides with cyanocrylate and renton,Oregon) for the first 30 d and then switched allowed to dry in a dark environment for 24 h.
to a diet of fathead minnows Pimephales promelas.
During mark evaluation, otoliths were periodically sanded with wet 1,000-grit sandpaper between subsequent analyses. Comparisons of mark quality viewings to facilitate mark detection.
among OTC concentrations and immersion periods Juvenile yellow perch dorsal spines were ex- were done with analysis of variance (ANOVA).
cised at the base of the articulating process at the For calcified structures, comparisons were made inception of the pterygiophores. Spines were among treatments (OTC concentrations and im- stored in scale envelopes until they were pro- mersion periods) with ANOVA. Otolith and dorsal cessed. To prepare the spines for mark detection, spine mark quality were compared with the paired the second and third dorsal spines were cut away t-test. Bonferroni adjustments were used for si- and placed on an acetate slide. Cellophane tape multaneous comparisons (SYSTAT 1999).
was placed across the two spines to allow slicing Nonlinear regression models were developed to without displacing sections. A Dremel tool, describe the relationship between OTC tissue res- mounted to an articulating base, was equipped with idue (g OTC/g tissue) and depletion time. All a cut-off wheel (no. 409) to section spines. Be- statistical analyses were conducted with SYSTAT ginning just distal to the basal process, cross sec- (1999); an alpha level of 0.10 was used for all tions approximately 1 mm thick were made. Two sections were removed from the dorsal spine andplaced on a drop of cyanocrylate on a glass mi- Results and Discussion
croscope slide. Spine cross sections were viewed immediately after mounting. However, if the cross Approximately 2,400 juvenile yellow perch (89 section was too thick or uneven, the mount was mm mean total length, range ϭ 76–94 mm) were stored in the dark until the cyanocrylate was suf- successfully marked with OTC during the transfer ficiently dry, at which time the section was ground tank experiment. Management of water tempera- with the flat surface of the cut-off wheel.
ture and DO concentration in transfer tank water The equipment configuration used for mark de- is frequently the primary concern when transport- tection on spines and otoliths from juveniles was ing fish. Thus, as temperature increased and ox- ygen saturation declined, we added 23 kg of equipped with a 100-W ultraviolet (Hg arc) light bagged ice to each tank at 3 h. By floating bagged source and fluorescent detection accessories (i.e., ice, we were able to maintain tank water temper- dichroic mirror/interference blue filter cube, 505 ature within 2ЊC of the initial water temperature.
dichroic mirror, 450–495-nm excitation filter, and Oxygen was maintained at greater than 90% sat- 515 interference [IF] barrier filter). Otoliths from uration by controlling temperature and by agitation adult fish sampled for OTC residue analysis were and diffusion of 0.5 L pure O2/min. The pH of the viewed with a Nikon E400 compound microscope OTC-treated tank waters did not vary from 7.3 equipped with a 100-W ultraviolet (Hg arc) light during the marking period. Acute mortality (8 h) source and fluorescent detection accessories (i.e., was determined to be 0.49% and 0.74% for the B3 filter cube, 505-nm dichroic mirror, 420–490- 500-mg and 700-mg OTC/L treatments, respec- nm excitation filter, and 520 IF barrier filter).
tively. We believe this low mortality was accept- Two readers independently examined and scored able because mortality was generally up to 1% both structures for mark presence and quality.
during previous yellow perch trap-and-transfer ef- Mark quality of otoliths and dorsal spines was de- forts without a holding period. Overall, the pro- fined on a rank scale of 0–3 (0 ϭ no mark; 1 ϭ tocol used for OTC-marking of yellow perch in barely detectable; 2 ϭ easily detected, but partial transfer tanks proved to be relatively simple, re- mark or mark was not brilliant; and 3 ϭ bright, quiring little effort beyond a normal trap-and- well-defined, continuous mark). The relative dis- tance of the mark from the otolith margin wassubjectively defined on a rank scale of 0–5 (e.g., Otolith and Dorsal Spine Mark Efficacy 0 ϭ mark on the margin, 5 ϭ mark farthest from Although variable in quality, fluorescent marks were detected in all treatments investigated in this Data analysis.—Paired t-tests were conducted to study. We detected no consistent difference between determine whether differences in rank assignments the mark ranks assigned by readers for otoliths from of mark quality consistently occurred between juvenile yellow perch (P ϭ 0.89). Overall, immer- readers (Conover and Iman 1981). When no sig- sion time (P ϭ 0.06) proved to be a greater influence nificant difference (␣ ϭ 0.10) was detected, data on mark detection and quality in juvenile otoliths were pooled and the mean ranks were used for than did concentration (P ϭ 0.47), likely because TABLE 1.—Mean ranks (0 to 3) for mark quality of oto- TABLE 2.—Mean ranks for mark quality (0 to 3) of oto- liths and dorsal spines from juvenile yellow perch im- liths and relative distance (0 to 5) of the oxytetracycline mersed in 500 (300 in solution, 567 total) or 700 (279 in (OTC) mark from the otolith margin. Otoliths were ac- solution, 796 total) mg oxytetracycline/L for durations of quired from adult yellow perch (5/d, N ϭ 55) and im- 4, 6, or 8 h. Twenty-five fish were sampled for each treat- mersed in 600 (197 in solution, 587 total) mg OTC/L for ment combination. Row P-values indicate the probability 6 h. Mean distance ranks followed by the same lowercase of a significant difference between calcified structures letter are not statistically different (␣ ϭ 0.10) based on a the solute components of the two treatment con-centrations were similar. Within the 500-mg OTC/L treatment, mark quality did not significantly vary (P ϭ 0.15) among immersion times, but it did slightly increase with immersion time (Table 1).
Otoliths were extracted from 153 adult yellow Within the 700-mg OTC/L treatment, mark quality perch (167 mm mean total length, range ϭ 129– differed little after 6 h (P ϭ 0.33). These results 202 mm) used for a time series residue analysis.
are similar to observations made by Unkenholz et Otoliths were collected (n ϭ 5 per period) from 9 al. (1997), where readers were able to detect visible d after the marking period (216 h postimmersion) marks in 100% of juvenile yellow perch fingerlings through day 63 (Table 2). Marks were consistently immersed for a minimum 6 h in 534-mg and 748-mg detected on all adult otoliths, but there was no detectable difference (P ϭ 0.91) in the mark qual- Similar patterns in the quality of marks were ity found among the time series samples. Although observed for dorsal spines (Table 1). Mark quality there was minimal observed change in mark improved over time (P ϭ 0.07), more so than with brightness over time, greater distances between the increased concentration (P ϭ 0.82). Within the mark and the margin allowed quicker detection.
500-mg OTC/L treatment, mark quality increased We were unable to quantify body and otolith with time, but not significantly (P ϭ 0.33). Like- growth because of advanced fish size and the short wise, within the 700-mg OTC/L treatment, mark duration of the experiment. Although subjective, quality increased with time, but not significantly we were able to assign a rank score for the relative (P ϭ 0.23). Fluorescent marks were detected on distance of the mark from the otolith margin.
all spines from fish immersed for at least 6 h in Based on those scores, there was a significant (P Ͻ 0.001) increase in relative distance with time Assays of OTC-treated transfer tank waters sam- (Table 2). Thus, although marks were marginally pled at the midpoint (4 h) revealed soluble con- detectable in adult otoliths at 9 d, the likelihood centrations of 300 (567 mg/L total in solution and of detection would be greater after at least 51 d residual) and 279 (796 mg/L total) mg OTC/L for postimmersion, particularly for an inexperienced the 500- and 700-mg OTC/L treatments, respec- tively. A number of factors, such as water hard- The HPLC analysis of OTC-treated water (600 ness, level of mixing, and OTC activity, could have mg OTC/L) from the adult marking experiment influenced the amount of OTC detected in the so- showed 197 mg OTC/L in solution and 587 mg lutions. Additionally, the actual amount of OTC OTC/L total. Again, previously mentioned factors extracted from the solution by yellow perch would may have influenced the amount of OTC in so- be difficult to accurately assess. We suspected that lution. Water hardness was 380 mg/L as CaCO3.
both moderately high water hardness (ϳ440 mg/ Several calcified structures (e.g., dentary and TABLE 3.—Results of high pressure liquid chromatog- maxillary bones, spines, fin rays, vertebrae, and raphy analysis of OTC residues in yellow perch muscle.
teeth) have been evaluated for OTC or TC marks.
Adult yellow perch were immersed in 600 mg OTC/L for Retention of marks in external structures such as 6 h. Epi-OTC (4-epioxytetracycline) is one of the most scales did not exceed 3 months in walleyes common breakdown products of OTC. Each concentration (Brooks et al. 1994), but marks were present in represents a composite sample of nine fish.
the scales of red drum after 10 months (Bumgard- ner 1991). Because tetracycline antibiotics are sen- sitive to natural light, internal bony structures are not as subject to degradation (Muth and Bestgen 1991). As such, the most common calcified struc- tures examined for marks are sagittal otoliths. Tet- racycline fluorescence has been observed in the growing surfaces of all internal bones except the There are several benefits to using sagittal oto- liths for mark detection. Otoliths are among the first calcified tissues formed in fish (McElman and Balon 1979). Otoliths do not appear to be reab- sorbed during periods of stress, they are easilyremoved, and they have the added benefit of show-ing daily growth rings for analysis of growth in The current Food and Drug Administration (FDA) young fishes (Taubert and Coble 1977; Miller and tolerance level for total tetracycline residues in the Storck 1982; Schmidt 1984). The major disadvan- muscle tissues of various animals, including sal- tage of using otoliths is that fish must be sacrificed monids and channel catfish Ictalurus punctatus, is 2.0 g OTC/g (21 CFR 556.500). The acceptable The detection of marks (presence or absence) daily intake is 25 g/kg of body weight/d.
was similar between otoliths and dorsal spines (Ta- The total OTC residue (OTC base and epi-OTC ble 1); thus, spines provide a nonlethal alternative [4-epioxytetracycline]) decreased below 2.0 g to sacrificing fish for removal of otoliths. In yellow OTC/g within a few hours (Table 3). A nonlinear perch, the first dorsal fin is supported by 13–15 model (loge[total OTC] ϭ 0.960 Ϫ 0.389·loge[time], spines, while the anal fin has two spines (Craig r2 ϭ 0.99) predicted that 2.0 g total OTC residue/ 1987). The spines retain essentially the same form g was present at 2 h postimmersion. A nonlinear throughout life, with cross sections resembling a mammalian heart with unequal lobes. Spine (loge[OTC base] ϭ 0.932 Ϫ 0.499·loge[time], r2 ϭ growth is accomplished by seasonal deposition of 0.93) describing the residueϪtime relation sug- tissue on the margin that is proportional to otolith gested that 1.6 g OTC base/g was present at 2 h growth. In other spines, such as pectoral spines, deterioration around the lumen may obscure part Before OTC immersion marking and stocking of the first annual mark in older fish, which could evaluations of edible-sized fish can occur in public present a problem in detecting marks. Therefore, waters, it is necessary to know the persistence of dorsal or anal spines would likely be more appro- OTC in muscle tissues. However, most available priate for detection of marks in long-term assess- information on OTC residue depletions is based ments. Additionally, the dark tegument on the dor- on feeding or intraperitoneal or intramuscular in- sal fin likely limits photodegradation of the mark.
jection. Current FDA guidelines regarding OTC Further research is warranted on mark retention residues in food fish are specifically for feed use and detection in spines. Additionally, we encour- in salmonids and ictalurids. Mandatory withdrawal age the development of new HPLC protocols for times from OTC-medicated feed are 7 d for Pacific the detection of OTC in spine tissue. This would salmon Oncorhynchus spp. and 21 d for other sal- enable managers to quantify OTC concentrations, monids and for ictalurids (21 CFR 558.450). Un- likely allowing modifications to marking proto- kenholz et al. (1997) reported no detectable OTC residue in muscle at 110 d in yellow perch im- mersed in 309, 534, or 748 mg OTC/L. Our results The HPLC analysis showed that OTC residues indicate that edible tissues from adult yellow perch were rapidly depleted from muscle tissue (Table 3).
immersed in OTC under a similar protocol (i.e., 600 mg OTC/L, 6 h, 19ЊC) do not exceed the FDA Conover, W. J., and R. L. Iman. 1981. Rank transfor- tolerance level following a 2-h depletion period.
mations as a bridge between parametric and non- At higher temperatures, depletion time should de- parametric statistics. American Statistician 35:124–133.
crease. Therapeutic studies have reported rapid Craig, J. F. 1987. The biology of perch and related fishes.
clearing of OTC residues from muscle tissue with higher temperatures (e.g., Xu and Rogers 1994).
Doi, A. M., and M. K. Stoskopf. 2000. The kinetics of Overall, the OTC-marking protocol used for yel- oxytetracycline degradation in deionized water un- low perch in transfer tanks proved to be relatively der varying temperature, pH, light, substrate, and simple, requiring little additional effort beyond a organic matter. Journal of Aquatic Animal Health normal trap-and-transfer episode. The primary Fielder, D. G. 1994. An evaluation of the suitability of concern would be the loss of work time while fish marking walleye fry and fingerlings with oxytet- are held in the marking solution. Although suffi- racycline. South Dakota Department of Game, Fish cient marks were obtained at 6 h of immersion, and Parks, Fisheries Completion Report 94-15, we recommend that further research be directed toward the use of potentiators. Potentiators may Hendricks, M. L., T. R. Bender, and V. A. Mudrak. 1991.
accentuate OTC uptake and possibly reduce im- Multiple marking of American shad otoliths with mersion time, but investigators have reported var- tetracycline antibiotics. North American Journal of iable degrees of success (e.g., Weber and Ridgway Hettler, W. F. 1984. Marking otoliths by immersion of 1967; Scidmore and Olson 1969; Odense and Lo- marine fish larvae in tetracycline. Transactions of gan 1974; Hettler 1984; Wahl and Stein 1987).
the American Fisheries Society 113:370–373.
Lastly, we recommend that investigators quantify Houglum, J. E., and R. D. Larson. 1999. Liquid chro- OTC depletion times, particularly when reporting matographic determination of oxytetracycline res- on edible-sized fishes. This information would al- idue in fish tissue and in water. U.S. Department of low personnel to minimize holding times of OTC- Health and Human Services Laboratory Information marked fishes stocked in public waters.
Kayle, K. A. 1992. Use of oxytetracycline to determine the contribution of stocked walleye fingerlings.
North American Journal of Fisheries Management We thank the numerous graduate students and technicians at South Dakota State University that Lucchesi, D. O. 1999. Evaluating the contribution of provided assistance with field and laboratory stocked walleye fry and fingerlings to South Dakotafisheries through mass-marking with oxytetracy- work. We also thank D. Willis, D. Isermann, and cline. South Dakota Game, Fish and Parks, Progress several anonymous reviewers for providing in- sightful comments on earlier drafts of this man- McElman, J. F., and E. K. Balon. 1979. Early ontogeny uscript. Funding for this project was provided by of walleye, Stizostedion vitreum, with steps of sal- South Dakota State University and the South Da- tatory development. Environmental Biology of kota Department of Game, Fish and Parks through Federal Aid in Sport Fish Restoration Project Miller, S. J., and T. Storck. 1982. Daily growth rings in 1565. This manuscript was approved for publi- young-of-the-year largemouth bass. Transactions ofthe American Fisheries Society 111:527–530.
cation by the South Dakota Agricultural Experi- Mitscher, L. A. 1978. The chemistry of the tetracycline ment Station as Journal Series Number 3232.
antibiotics. Marcel Dekker, New York.
Murphy, M. D., and R. G. Taylor. 1991. Direct vali- References
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O Evangelho Segundo Tomé PRÓLOGO Estas são as sentenças ocultas que o Jesus vivo pronunciou e Judas Tomé, o Gêmeo registrou. 1 E ele disse: "Quem quer que descubra a interpretação destas sentenças não provará a morte". 2 Jesus disse: "Que aquele que procura não deixe de procurar até que encontre. Quando encontrar, ficará perturbado. Quando estiver perturbado, ficará