Tricaine methanesulfonate is an immersion anesthetic approved in the United States for use in fish; it is commonly used to anesthetize fish for procedures such as manually assisted spawning, weighing, measuring, surgery, transportation, and research.1 The anesthetic effects of this drug are thought to result from sodium channel blockade in neural tissue.2 Tricaine methanesulfonate is a preferred anesthetic for use in fish because it is soluble in water and can be administered by immersion and because concentrations are easily adjustable. Additionally, it produces rapid anesthetic induction and recovery (< 5 minutes each).1,3 Tricaine methanesulfonate is absorbed and primarily cleared across the gill epithelium, although there is also biotransformation in the liver and potential renal excretion.4
Although commonly used, MS-222 is reported to have retinotoxic effects in fish, frogs, and humans.2,5–8 A commonly cited clinical report7 describes a 62-year-old ichthyologist who developed vision loss with decreased ERG wave amplitudes after chronic low-level exposure to MS-222 over approximately 30 years. The authors of that report7 based the diagnosis of retinopathy associated with chronic exposure to MS-222 partially on information found in a series of studies2,5,8 that indicated the in vitro exposure of eye cups (ie, all tissues remaining after removal of the cornea, lens, and vitreous from the enucleated eye) to high concentrations of MS-222 resulted in altered ERG waves in retinas of eyes isolated from frogs. Interestingly, 7 months after cessation of exposure to MS-222, the vision of the patient described in that report7 had clinical improvement and his ERG waves had doubled in amplitude.
Electroretinography is considered the gold standard to assess retinal function, and b-wave amplitudes provide evidence of the function of inner retinal components.9 Results of ERG are useful in differentiating retinal from postretinal blindness because a positive ERG wave is indicative only of retinal function, providing no information regarding the remainder of the visual pathway (eg, optic nerve or visual cortex).
Considering that fish are routinely anesthetized with MS-222, it is logical to question whether repeated or prolonged exposure to the drug results in vision loss or retinal damage in these species. In our experience, fish anesthetized repeatedly with MS-222 do not have apparent behavioral changes associated with vision loss, such as difficulty locating food or objects in their environment or avoiding capture. Additionally, to the authors' knowledge, there have not been reports of vision loss or retinotoxic effects attributed to MS-222 exposure in fish.
The purpose of the study reported here was to determine whether repeated exposure to clinically relevant concentrations of MS-222 would alter retinal function or induce histologically detectable retinal lesions in koi carp (Cyprinus carpio). We hypothesized that koi carp anesthetized with MS-222 for a total exposure time of 20 minutes daily on 1 to 13 consecutive days would not have changes in retinal function (as assessed via ERG b-wave amplitudes) or histopathologic retinal changes indicative of toxic effects.
Materials and Methods
Fish—Eighteen 12- to 15-month-old koi carp measuring approximately 30 cm in length (approx 11.8 in, measured from snout to tail) and weighing 227 to 420 g (0.5 to 0.92 lb) at the start of the study were acquired from a commercial fish hatchery. The fish were determined to be healthy on the basis of visual examination. After 1 week of acclimation in the tank at our facility, 2 arbitrarily selected fish were netted and restrained for scale and skin scrape and a gill biopsy (removing < 2 mm of the gill edge) for histologic examination to further assess their overall health. No lesions or infectious agents were detected.
Fish were acclimated for 2 weeks and housed throughout the study in a 1,900-L fiberglass tank. The tank contained 5 sponge filters and 5 air stones and was filled with 1,100 L of water and covered with mesh netting. Water for the tank originated from municipal water, was stored in an 11,350-L storage tank, and was treated with sodium thiosulfate and allowed to neutralize for 24 hours before use. Tank water was treated with a commercially available water conditionera (0.5 mL/L) and aquarium-grade salt (1 g/L) once weekly during acclimation and twice weekly throughout the study period. Throughout the acclimation and study periods, daily water quality testing for pH and concentrations of ammonia, nitrate, and nitrite was performed. Water was changed as necessary to maintain appropriate concentrations of ammonia (range, 0 to 0.5 mg/L), nitrite (0 to 0.004 mg/L), nitrate (0 to 40 mg/L), and pH (6.8 to 7.6). The fish were fed a commercially available koi dietb 3 times weekly (Monday, Wednesday, and Friday). Once the study began, food was provided between 2 and 4 hours after all fish had recovered from anesthesia. The study protocols were approved by the Institutional Animal Care and Use Committee of the North Carolina State University College of Veterinary Medicine.
Procedures—Tricaine methanesulfonatec stock solution and dilutions were prepared daily. Ten grams of MS-222 were measured on a gram scale and added to 1 L of sodium thiosulfate–treated water to achieve a stock solution of 10 g/L buffered with 10 g of sodium bicarbonate. Subsequent dilutions were prepared from this stock solution. Temperature in fish tanks, induction buckets, and maintenance tanks was measured with a waterproof, calibrated digital thermometer and maintained at 24° to 25°C (75° to 77°F). Each piece of equipment was cleaned daily with a biosafe cleansing agent.d The fish and all study products used on a per-weight basis were weighed with a digital gram scale.
On day 1, 2 fish were arbitrarily selected, netted, and injected with 390 mg of sodium pentobarbital/fish (approx 930 to 1,800 mg/kg [423 to 818 mg/lb]) intracoelomically, and immediately placed in a holding bucket with tank water until deceased. Following euthanasia, both eyes were enucleated and immediately fixed in Davidson solution with glacial acetic acid (11% [vol/vol]) added directly prior to sample collection. The fixed samples were processed for paraffin embedding, serially sectioned at 3- to 5-μm thicknesses dorsoventrally, and stained with H&E according to standard techniques.10 Two sections of each globe were evaluated histologically (JRT and LBB) for the presence, thickness, and cellularity of each retinal layer and for evidence of inflammation, degeneration, or necrosis.
The remaining fish (n = 16) were anesthetized with MS-222 (200 mg/L) in an anesthetic induction bucket, with loss of righting reflex, decreased opercular movement rate (subjective assessment), and lack of response to stimuli (tactile stimulation by hand) used as indicators of appropriate anesthetic induction. Anesthesia was maintained with a motorized water pump used to recirculate water containing MS-222 (initial concentration, 150 mg/L) at a rate of 2.0 L/min into the mouth and over the gills of the anesthetized fish via plastic tubing. The pump was immersed in a 10-L maintenance tank, over which the fish were placed on a wedged sponge platform.11 Anesthetic depth was assessed by observation of opercular movement and purposeful fin and tail movement. Anesthetic concentration was altered by adding stock solution (in increments of 25 mg/L) or treated MS-222–free water as necessary to maintain an acceptable anesthetic depth. Electroretinography was performed for both eyes of all fish, and fish were weighed while anesthetized. Two fish were arbitrarily selected for euthanasia while anesthetized and ocular histologic evaluation, as described. The remaining fish (n = 14) were placed in individual recovery buckets and observed until opercular movements returned to subjectively normal frequency and righting ability was regained. Fish that did not have obvious opercular movements in the recovery bucket were manually advanced in the water until spontaneous opercular movement returned. Following recovery, fish were returned to their shared housing tank.
The described procedures were repeated for remaining fish on days 2 through 7 (n = 14) and 8 through 13 (12), with ERG performed for both eyes of all fish on days 7 and 13. The ERG was followed by euthanasia of 2 fish as described, with subsequent collection and submittal of the eyes for histologic evaluation. In total, 8 fish were euthanized, with ocular histologic evaluation performed.
Photopic and scotopic ERG was performed via an ERG systeme with a software programf and a recording electrode, which contacts the cornea and has a built-in, high luminance diode to generate a white light stimulus (Kooijman active electrode).g Following a 10- to 15-minute dark-adaptation period and achievement of an adequate plane of anesthesia for all patients, hydroxypropyl methylcellulose (0.3%) was applied to the cornea to temporarily adhere the integrated flash Kooijman electrode. Ground electrodesh were placed lateral to midline on the right side of the caudal peduncle, 1 to 2 mm dorsal to the lateral line. Reference electrodes were placed caudal to the globe and dorsal to the operculum. Evaluation of impedance was conducted prior to all measurements, and a baseline requirement for both the active and reference electrodes was an impedance ≤ 2.5 kΩ. Right eyes were routinely evaluated prior to the left. A scotopic standard intensity response was elicited at 3.5 candelas/m2 (mean for 20 cycles), followed immediately by photopic evaluation with a background illuminance of 30 candelas/m2 and concurrent pulses of standard intensity (LED flash, 3.5 candelas/m2). Time required for the entire protocol was approximately 60 s/eye.
The motorized water pump delivering anesthetized water was turned off during ERG measurements because it caused electrical interference. The water pump was resumed for 2 minutes in between ERG of the right and left eyes.
Statistical analysis—Analysis was performed with a statistical software package.i Normal distribution of the data was confirmed via visual inspection and evaluation of skewness and kurtosis. Potential differences in b-wave amplitudes were compared among study days 1, 7, and 13 via repeated-measures ANOVA. The amplitudes were tested for the right eye and left eye separately, and a Bonferroni correction for multiple comparisons was performed. Corrected values of P ≤ 0.05 were considered significant.
Results
Median body weight of all fish on days 1, 7, and 13 was 320.5 g (0.71 lb), 303.5 g (0.67 lb), and 318 g (0.70 lb), respectively. Minimal physical changes in the form of scale loss and minor bruising from daily handling were detected in a few fish during the study. No fish lost the ability to avoid capture or find food or had any other behaviors suggesting vision loss during the study period. Except for the 8 fish euthanized for histologic evaluations, no fish died during the study.
The concentration of MS-222 required for anesthetic induction was 200 mg/L of water for all fish. A concentration of 125 mg/L was required for maintenance of anesthesia on days when ERG was not performed, whereas a concentration of 150 mg/L was required on days when ERG was performed, for all fish.
Median b-wave amplitudes for right eyes on days 1, 7, and 13 were 17.7 μV (range, 2.2 to 31.2 μV), 20.9 μV (11.0 to 64.0 μV), and 17.6 μV (7.1 to 23.6 μV), respectively. Median b-wave amplitudes for left eyes on days 1, 7, and 13 were 15.1 μV (range, 5.0 to 39.3 μV), 16.9 μV (3.6 to 40.7 μV), and 14.3 μV (5.8 to 23.0 μV), respectively. No significant (P = 0.08 and P = 0.92 for right and left eyes, respectively) differences were detected among different study days. A representative ERG response tracing from one of the study fish is shown (Figure 1). No histologic abnormalities were detected in the 4 eyes evaluated from 2 untreated control fish or the 12 eyes evaluated from 6 fish that underwent anesthesia with MS-222 for 1 to 13 consecutive days.
Discussion
In the study reported here, we evaluated whether repeated exposure to clinically relevant concentrations of MS-222 would alter retinal function or induce histologically detectable retinal lesions in koi carp as evidenced by decreases in ERG b-wave amplitude over time or development of histologically detectable retinal lesions. For the koi carp assessed in this study, anesthesia with MS-222 at concentrations of 125 to 200 mg/L for a total exposure time of 20 minutes daily for up to 13 consecutive days did not result in significant changes in ERG b-wave amplitudes or histologic changes indicative of retinal damage, indicating that short-term, repeated exposures did not affect retinal structure or function.
To the authors' knowledge, there have been no in vivo studies completed to investigate the effects of MS-222 on the retina, nor have any studies been performed to detect the presence of histologic lesions after exposure to the drug in any species. Electroretinography is used in human and veterinary ophthalmology as the diagnostic method of choice to assess retinal function12 and differentiate between retinal and postretinal causes of blindness,9 such as optic nerve or visual cortex dysfunction. The ERG waves are created via light-induced extracellular currents, which cause a retinal response. Recordings of ERG responses most commonly contain a negative a wave followed by a positive b wave. The a wave is created by closure of sodium channels within photoreceptor cells due to light perception. Sodium channel closure causes hyperpolarization of photoreceptor cells, and this leads to deflection of the ERG wave below the 0-μV line. The b wave results from an electrical signal that is conducted from photoreceptor cells to bipolar cells within the adjacent layer of the retina. Amplitude of the b wave is determined by measuring the difference between the trough of the a wave and the peak of the b wave. Electroretinography was chosen for use in the present study because it is the most specific test of retinal function; therefore, a change in the ERG would indicate a change in retinal function only (rather than other aspects of the visual pathway). The lack of significant changes in ERG b-wave amplitudes for the duration of the study period and the fact that the fish in this study did not have any behaviors during or after the study period that would indicate retinal dysfunction, such as difficulty finding their food or avoiding capture, supported the hypothesis that repeated exposure to MS-222 would not result in changes in retinal function.
Histologic samples were evaluated from 8 fish (including 2 untreated controls at the start of the study) throughout the 13-day study period. Following each ERG assessment, samples from 2 fish were evaluated for the presence of degeneration, inflammation, necrosis, and atrophy. Degeneration and necrosis generally cause nuclear swelling, nuclear bursting, and debris within affected cells. No atrophy of the retinal layers (detectable as loss of retinal layers or an apparent decrease in thickness of ≥ 1 retinal layer)13 or structural lesions were identified, and no other histologic changes were noted. Although the retinas of fish are fundamentally structurally similar to those of other vertebrates, teleost fish can regenerate the neural portion of the eye, and warm-blooded vertebrates cannot.14 This ability to regenerate retinal tissue could have diminished our ability to detect structural changes in the retinas of koi carp in the present study.
Results of the study reported here conflict with previous studies,2,5,8 which showed evidence of MS-222– associated ERG changes in frogs. The general conclusions of those investigators indicated that MS-222 interferes with rhodopsin regeneration. Rhodopsin is a photosensitive chemical that is integral in light detection. It is intimately associated with the photoreceptor cells. Once exposed to light, rhodopsin splits into 2 components: scotopsin and all-trans-retinal. After decomposition, rhodopsin is regenerated by the conversion of all-trans-retinal to 11-cis-retinal, which can bind to scotopsin to form rhodopsin, allowing the process to recur.9 Tricaine methanesulfonate interferes with rhodopsin regeneration within the retinal cells via Schiff base formation between MS-222 and 11-cis-retinal.2,5,8 Thus, MS-222 could potentially delay rhodopsin regeneration, resulting in abnormal photoreceptor function and retinal dysfunction.2 However, this effect on the rhodopsin cycle is transient.2,8 Aromatic amines have also been found to interfere with the rhodopsin cycle in the eye cups of frogs.5 Although MS-222 is an aromatic amine, the study5 in which this effect was demonstrated did not evaluate MS-222 specifically. The previous studies2,5,8 in frogs were performed on eye cups in vitro following decapitation, and it is unclear how decapitation or death may have affected ERG wave characteristics.
The route of MS-222 exposure may also have contributed to the different effects reported in different species. Route of exposure in the reported7 human case was most likely via skin absorption (ungloved hands in water that contained MS-222). Conversely, for studies2,5,8 of the effects on frog eyes, investigators placed the eyes directly into solutions containing MS-222, and the route of exposure was likely transretinal because anterior portions of the globe were removed prior to placement in the solution. Interestingly, the fish in the present study likely absorbed the drug through 2 routes. Given that anesthesia was necessarily induced via an immersion technique, followed by delivery across the gills with a pump, most of the drug was thought to be absorbed via the gills, but we cannot preclude some corneal absorption because the eyes were directly exposed to the solution in the anesthetic induction container and to a lesser degree during anesthesia with the anesthetic recirculation system.
The rhodopsin regeneration cycle remains intact in dark-adapted retinas,2,8 and dark adaptation was performed in fish of the present study prior to ERG. Dark adaptation is a necessary condition for ERG interpretation.9 Rhodopsin does not split unless light is perceived, so dark adaptation could have prevented the observation of changes in ERG wave amplitude if this process was affected by MS-222. However, if the changes in ERG wave amplitude were due to delayed rhodopsin regeneration, the effects should have been transient.
Because of problems inherent in restraint of conscious fish, all ERG testing was performed on anesthetized fish. Although ERG wave formation and amplitude are decreased in anesthetized patients, it is common practice to heavily sedate or anesthetize veterinary patients to avoid artifact caused by movement.15–17 Although ERG amplitudes of the anesthetized fish may have been lower than if the fish were conscious, they did not change over the 13-day period. The b-wave amplitudes for fish in the present study were also low, compared with those typically detected in many mammalian species. Although electrical interference and water movement could have contributed to this apparent difference, it is also likely that b waves of fish are typically smaller than those of mammals. Frogs, turtles, and skates have been reported to have b-wave amplitudes as low as 2 to 10 μV,18 and although there are no reported reference ranges for these species or for fish, it is likely that the amplitudes for fish would be more similar to those for these species than to mammals.
Tricaine methanesulfonate is commonly used worldwide for anesthesia in fish, and appropriate withdrawal times are required in fish used for human consumption.19 The possibility of retinotoxic effects in such a frequently used drug is a concern for humans and for nonhuman animals. However, results of our study in koi carp did not reveal evidence of functional or histopathologic retinal changes following repeated short-term exposure to MS-222.
ABBREVIATIONS
ERG | Electroretinography |
MS-222 | Tricaine methanesulfonate |
API Stress Coat Aquarium Water Conditioner, Mars Fishcare Inc, Chalfont, Pa.
Blackwater Creek Pelletted Koi Food, Blackwater Creek Koi Farms Inc, Eustis, Fla.
MS-222, Tricaine S, Western Chemical Inc, Ferndale, Wash.
Versa-Clean, Fisher Scientific, Waltham, Mass.
Retiport system, model 32, Acri Tec Inc, Salt Lake City, Utah.
ERG-LED program, Roland Consult, Brandenburg, Germany.
Kooijman active electrode, Roland Consult, Brandenburg, Germany.
Ground electrodes, 27-gauge, stainless steel, subdermal needle electrodes, CareFusion, Middleton, Wis.
Stata, version 10, StataCorp LP, College Station, Tex.
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