Lidocaine Hydrochloride Compared with MS222 for the Euthanasia of Zebrafish (Danio rerio)

Abstract

Despite several shortcomings, MS222 is the most commonly used chemical agent for euthanasia of zebrafish. Although lidocaine hydrochloride has some advantages over MS222, its effectiveness as a euthanasia agent for zebrafish is unknown. Larvae at 9 to 16 d postfertilization were exposed to 250 mg/L MS222 or 400, 500, 600, 700, 800, 900, or 1000 mg/L lidocaine and observed for cessation of heartbeat. Adult zebrafish were exposed to 250 mg/L MS222 or 400, 500, or 600 mg/L lidocaine; times to loss of righting reflex, cessation of opercular movement, and complete recovery; body length; aversive behavior; and gross and microscopic evidence of acute toxicity were evaluated. The heartbeat was not lost from any larvae in any group, regardless of drug or dosage. For adults, time to loss of righting reflex was greatest in the 500-mg/L lidocaine group. Opercular movement ceased earlier in all lidocaine groups compared with the MS222 group. Fish in the 500-mg/L lidocaine group were smaller than those in other groups. Fewer fish in the lidocaine groups displayed aversive behavior (erratic swimming and piping) compared with the MS222 group. No fish in the lidocaine hydrochloride groups (n = 30) recovered from euthanasia, whereas one fish in the MS222 group did (n = 10). Neither the MS222 nor lidocaine groups showed any gross or histologic changes suggestive of acute toxicity. Our results suggest that lidocaine hydrochloride may be an effective alternative chemical euthanasia agent for adult zebrafish but should not be used in larval fish.

MS222 is the most common chemical agent for euthanizing zebrafish. It depolarizes sodium ion channels, leading to unconsciousness, immobility, and ultimately vascular collapse and death due to hypoxia.^10,17 Recent studies have linked MS222 to reversible euthanasia,¹⁹ ineffective euthanasia in larval zebrafish,¹⁸ and possibly distress during the euthanasia procedure.^14,20 In addition, MS222 requires special precautions regarding handling, as it is a potent skin, eye, and respiratory irritant in its powder form.¹⁷ Given the concerns associated with MS222 and the large numbers of zebrafish used in research, safe alternatives for the chemical euthanasia of zebrafish are urgently needed.

Lidocaine hydrochloride was recently demonstrated to have a low margin of safety for anesthesia of adult zebrafish: 30% of fish exposed to 350 mg/L died acutely.⁶ Like MS222, lidocaine leads to unconsciousness and eventually death in zebrafish by depolarizing sodium ion channels.¹⁷ Given lidocaine's relatively rapid onset of action as an induction agent,^6,17 its general safety and wide availability, and previous work suggesting that it is less aversive to zebrafish than is MS222,¹⁴ we elected to investigate the use of lidocaine hydrochloride for euthanasia of zebrafish.

To be euthanized effectively, larval zebrafish require higher doses of chemical agents than are recommended for adult fish.^10,16,18 For zebrafish younger than 6 d postfertilization (dpf), doses of MS222 up to 1000 mg/L are not effective for euthanasia.¹⁰ The LC₅₀ for MS222 in embryos at 3 dpf was 1633 mg/L, whereas the LC₅₀ at 9 dpf was 730 mg/L when loss of heartbeat was used as a measure of death.¹⁶ One group recommends the use of 900 mg/L MS222 followed by a secondary method for the euthanasia of larval zebrafish at 14 dpf.¹⁸ Cutaneous gas exchange becomes limited at 12 to 14 dpf; therefore the cause of death from MS222 in larvae is thought to be heart failure rather than hypoxia, as is observed in adults.¹⁶ Previous work demonstrated that lack of heartbeat was more accurate than using opercular movement to determine whether euthanasia occurred for larval zebrafish.^16,18 A secondary method of euthanasia should be used while larvae are anesthetized,¹⁸ given that fish need to remain in solution for longer than 1 h to ensure effectiveness.¹⁶ Few publications discuss the efficacy of chemical agents for euthanizing larval zebrafish.

When exposed to chemical agents, adult zebrafish may display aversive behavior such as rapid opercular movement, piping (gasping for air at the water surface), erratic swimming, and body twitching.^6,14,19,20 Aversive behaviors associated with exposure to MS222 at dose levels used for euthanasia have been described in 40% to 90% of zebrafish.¹⁹ Whether these behaviors might be normal as fish pass through various stages of anesthesia remains unclear;¹⁹ however, such behavior is frequently associated with MS222 compared with other chemical agents.^14,20 Although fish exposed to lidocaine hydrochloride quickly exited a solution containing the compound, no aversive behaviors have been reported in adult zebrafish.^6,14 Whether a rapid exit from a compartment containing anesthetic is evidence of aversive behavior or stress over loss of body control is unknown.¹⁴

The AVMA criteria for performance-based euthanasia indicate that the method must lead to rapid loss of consciousness and death, minimize pain and distress, be reliable and irreversible, and be safe for personnel.^1,10 The goal of the current study was to compare the effectiveness of lidocaine hydrochloride and MS222 for the euthanasia of zebrafish larvae and adults. Although not evaluated in this study, rapid chilling (that is, hypothermal shock) is another method of euthanasia for zebrafish.^1,10,19 We chose to compare 2 chemical agents—one novel and one established—for the euthanasia of zebrafish because rapid cooling is not an approved method of euthanasia in the CCAC Guidelines on the Euthanasia of Animals Used in Science.³ We hypothesized that lidocaine hydrochloride would lead to rapid, less distressing euthanasia of adult fish without recovery and that only high doses of lidocaine would be effective for the euthanasia of larvae. The findings of this study may be useful for improving the wellbeing of laboratory zebrafish undergoing euthanasia.

Materials and Methods

Humane care and use of animals.

Zebrafish were housed and cared for in compliance with guidelines of the Canadian Council on Animal Care and the Animals for Research Act of Ontario.^4,12 All procedures were approved by the University of Toronto Faculties of Medicine and Pharmacy Animal Care Committee.

Animals and housing.

Mixed-sex transgenic human nuclear receptor ligand zebrafish on a wildtype background of various ages (n = 180) that have been bred inhouse for more than 3 generations were used for this study. Fish consisted of animals collected from the sump tanks of 3 racks connected to a single life-support system and maintained in a sentinel tank for health-monitoring purposes. Fish were considered free of Pseudoloma neurophilia, Pseudocapillaria tomentosa, Edwardsiella ictaluri, and pathogenic Mycobacterium spp. (except M. chelonae) as determined by annual sentinel health monitoring. Fish were housed at a density of 10 fish per liter on 3 recirculating systems (Aquaneering, San Diego, CA), each with mechanical, biologic, chemical, and UV filtration. Reverse-osmosis–purified water was balanced to pH 6.5 to 7.5 by using sodium bicarbonate (Sigma–Aldrich Canada, Oakville, ON) and maintained at a conductivity of 480 to 600 µS by using a marine salt mixture (Instant Ocean Sea Salt, United Pet Group, Blacksburg, VA). Ammonia, nitrite, and nitrate levels were maintained below 2 ppm, 1 ppm, and 40 ppm, respectively. Room temperature was maintained at 26 °C on a 12:12-h light:dark cycle. Adult fish were fed twice a day on weekdays by using a commercial flake diet (TetraMin Tropical Flakes, Tetra, Blacksburg, VA).

To produce larvae for the study, 3 female–male pairs of transgenic human nuclear receptor ligand fish were housed overnight in a breeding tank containing system water. Fifteen minutes after lights on, the divider between the fish was removed, and fish were allowed to spawn for 3 h prior to being returned to their home tank. Embryos and larvae were maintained in culture dishes at room temperature (26 °C), with daily water changes for the duration of the experiment. Larvae were fed a diet of Hatch-Fry (grade 1, Argent Laboratories, Redmond, WA) and Spirulina powder (Brine Shrimp Direct, Ogden, UT) twice daily from 5 to 14 dpf. From 14 to 60 dpf, larval food was supplemented with brine shrimp (Platinum Grade Argentemia Cysts, Argent Laboratories, Redmond, VA) once daily.

Solution preparation.

Immediately before use, all solutions were prepared with water from the main recirculating system. MS222 powder (Tricaine-S, Western Chemical, Ferndale, WA) was measured in a fume hood and placed directly into 1 L of system water. Sodium bicarbonate was added until the pH was between 7.0 and 7.4. Lidocaine hydrochloride (Lurocaine, Vetoquinol, Lavaltrie, Quebec, Canada) was instilled beneath the surface of 1 L of system water by using a 25-gauge needle and was stirred into solution to achieve the desired concentration; sodium bicarbonate was added as needed as described earlier. Euthanasia solutions were prepared and tested at 24 to 27 °C.

Experimental design.

Fish were divided into 2 age groups: larvae (9 to 16 dpf) and adults (older than 90 dpf). Adult fish and larvae were randomly allocated into experimental group. Groups of larvae (n = 10 per group) were exposed to 400, 500, 600, 700, 800, 900, and 1000 mg/L lidocaine hydrochloride and to 250 mg/L MS222. Larvae were placed as a group into culture dishes containing 10 mL of solution and observed under a dissecting microscope (Leica Microsystems, Concord, Ontario, Canada) for loss of heartbeat every 2 min for up to one hour. Effective euthanasia was defined as loss of the heartbeat with no recovery after removal from the euthanasia solution once the heart had stopped.¹⁸

Adult fish (n = 10 per group) were exposed to 400, 500, and 600 mg/L lidocaine hydrochloride and to 250mg/L MS222. The fish were placed into 1 L of euthanasia solution and observed continuously for the duration of the procedure for loss of righting reflex (indicating hyporeflexia and muscle relaxation consistent with loss of consciousness), loss of opercular movement (indicating cessation of respiration), and aversive behaviors (specifically, rapid opercular movement, piping, twitching, and erratic swimming).^6,14,17 Once opercular movement was lost, fish remained in the solution for 10 min and then were transferred to an agent-free recovery tank for observation. Body length was measured at the time of transfer. Animals were observed for 30 min for recovery. Effective euthanasia was defined as a treatment that resulted in rapid loss of opercular movement, that provoked minimal aversive behaviors, and that was irreversible. After euthanasia, a subset of animals was fixed in 10% neutral buffered formalin for assessment of potential toxicity through whole-body microscopic analysis.

Assessment of tissues for acute toxicity.

Paraffin-embedded sagittal sections were stained with hematoxylin and eosin (Animal Health Laboratory, University of Guelph, Guelph, Ontario, Canada) and examined by light microscopy by a pathologist blinded to treatment. Tissues examined included skin, gills, kidney, liver, heart, swim bladder, pyloric cecae, spleen, skeletal muscle, gingiva, eyes, and brain.

Statistical analyses.

Data for righting reflex, opercular movement, body length, and recovery were not normally distributed according to the Shapiro–Wilks normality test, and log transformation did not normalize the data. The Kruskal–Wallis test followed by pairwise analysis using the Mann–Whitney–Wilcoxon test with Bonferroni correction was used to identify the groups with significant differences. Data for aversive behavior was analyzed by using the chi-square test. A P value of 0.05 or less was considered significant. Data were analyzed by using statistical analysis software (STATA software, StataCorp, College Station, TX).

Results

Efficacy of euthanasia solution.

Larvae.

The heartbeat was not lost in any zebrafish larvae held in any euthanasia solution, indicating a lack of efficacy for all chemical methods tested. Heartbeat slowed in all but 2 larvae exposed to 900 and 1000 mg/L lidocaine hydrochloride.

Adult zebrafish.

Righting reflex.

Fish in the 500 mg/L lidocaine hydrochloride group took longer to lose their righting reflex than fish in all other groups (P < 0.01). There was no difference in time to loss of righting reflex between any other groups (Figure 1).

Figure 1.

Time to loss of righting reflex (mean ± SEM; n = 10 per group) in adult zebrafish after exposure to 250 mg/L MS222 (MS222) or 400 mg/L (L400), 500 mg/L (L500), or 600 mg/L (L600) lidocaine hydrochloride. *, Value differs significantly (P < 0.01, Kruskal–Wallis test followed by Bonferroni correction) from values for all other groups.

Opercular movement.

Zebrafish in the MS222 group took significantly (P < 0.01) longer to lose opercular movement than did all lidocaine hydrochloride groups. Fish in the 500-mg/L lidocaine group took longer to lose opercular movement than did those in the 400- and 600-mg/L groups (P < 0.01). Time to loss of opercular movement did not differ between the 400- and 600-mg/L lidocaine groups (Figure 2).

Figure 2.

Time to loss of opercular movement (mean ± SEM; n = 10 per group) in adult zebrafish exposed to 250 mg/L MS222 (MS222) or 400 mg/L (L400), 500 mg/L (L500), or 600 mg/L (L600) lidocaine hydrochloride. *, Value differs significantly (P < 0.01, Kruskal–Wallis test followed by Bonferroni correction) from those for all other groups; +, value significantly different from indicated groups.

Body length.

Fish in the 500-mg/L lidocaine group were significantly (P < 0.05) shorter than were those in all other groups. There was no difference in body length between the other groups (Figure 3).

Figure 3.

Body length (mean ± SEM; n = 10 per group) in adult zebrafish exposed to 250 mg/L MS222 (MS222) or 400 mg/L (L400), 500 mg/L (L500), or 600 mg/L (L600) lidocaine hydrochloride. *, Value differs significantly (P < 0.01, Kruskal–Wallis test followed by Bonferroni correction) from those for all other groups.

Recovery.

Recovery time did not differ significantly between the groups; however, one fish in the MS222 group recovered after placement in the recovery tank. No fish from any of the lidocaine hydrochloride groups recovered after exposure.

Aversive behaviors.

The number of fish that displayed distress behavior did not differ between the groups. One fish in each of the 400- and 500-mg/L lidocaine groups displayed body twitching. Three fish in the MS222 group displayed piping, body twitching, and erratic swimming (Figure 4).

Figure 4.

Number of adult fish per group that displayed aversive behaviors when exposed to 250 mg/L MS222 (MS222) or 400 mg/L (L400), 500 mg/L (L500), or 600 mg/L (L600) lidocaine hydrochloride. The number of fish displaying aversive behavior did not differ between any of the groups.

Acute toxicity.

No microscopic changes were found in any of the tissues examined from fish in either the MS222 or lidocaine treatment groups.

Discussion

At the doses evaluated in this study, neither lidocaine hydrochloride nor MS222 were effective euthanasia agents for zebrafish younger than 16 dpf. Larval zebrafish use skin more than gills for respiration and physiologic functions¹⁵ and therefore are expected to differ from adults in their responses to exposure to different chemical euthanasia solutions. In adult fish, gills are required for respiratory gas exchange, ion and water balance, excretion of nitrogenous wastes, and maintenance of acid–base balance.¹⁵ The dominant tissue of oxygen uptake gradually changes from the skin to gills during the first 3 wk of development,^15,18 and this process has implications for how fish react to immersion anesthetics.^15,16 Zebrafish larvae are considerably more tolerant of the lethal effects of MS222 than are adult fish.^10,16,18 Our findings are consistent with previous work demonstrating that significantly higher concentrations of MS222 are required for euthanasia of larval fish than for adult fish.¹⁸ Our results also support findings from other researchers that suggest that zebrafish become more sensitive to the cardiac depressive effects of MS222 as they age.^7,8 During exposure to lidocaine hydrochloride, the heartbeat of most larval fish slowed; however, the time required for this effect to occur was excessive. Whether lidocaine hydrochloride ultimately leads to cardiac arrest and death in zebrafish larvae is unknown. Given that lidocaine hydrochloride and MS222 have a similar mechanism of action, greatly increased doses of lidocaine will likely be required for euthanasia of zebrafish larvae as well. Whether exposure to these more concentrated solutions will result in acute skin irritation or toxicity prior to death is unknown.

Adult zebrafish placed in the 500-mg/L solution of lidocaine took longer to lose their righting reflex than did all other fish. Otherwise, time to loss of righting reflex was similar between the groups, indicating that lidocaine hydrochloride and MS222 lead to loss of righting reflex at a similar rate. The time to loss of consciousness after exposure to 400-mg/L lidocaine in the current study (20 s) was slightly shorter than previously reported (34s).⁶ This finding further confirms that loss of consciousness occurs more rapidly in adult zebrafish with exposure to higher doses of lidocaine hydrochloride. The loss of righting reflex data obtained for MS222 in adult zebrafish was similar to that described by other groups.¹⁹

All lidocaine -exposed groups of adult zebrafish lost opercular movement more rapidly than did the MS222 group. Overall, the time to death was significantly reduced with lidocaine hydrochloride at all doses compared with MS222 in adult fish. All local anesthetics, including MS222 and lidocaine hydrochloride, bind to voltage-gated sodium channels present in the nervous system, heart, and skeletal muscle.^2,5,9,11,13 Concerns have been raised about the possibility that these drugs might act as neuromuscular blocking agents rather than general anesthetics that lead to loss of sensory nerve function. Two recent papers have demonstrated that, in both zebrafish and Xenopus laevis, MS222 blocks sensory nerves and leads to general anesthesia in these species.^2,13 This result indicates that both MS222 and lidocaine hydrochloride cause loss of consciousness and sensation prior to death and do not cause distress associated with neuromuscular blockade alone. The reduction in time to euthanasia may be significant for facilities currently using MS222 for euthanasia of adult zebrafish at a dose of 250 mg/L. A more rapid method of euthanasia can save costs yet still be simple, quick to prepare, and less distressing to the animals. In this study, the time to loss of opercular movement from MS222 was considerably longer than previously reported,¹⁹ and we are unable to explain this difference. The results obtained in the current study are closer to those reported previously for ‘slowing’ of opercular movement.⁶ Possible explanations for the differences noted may include water composition, temperature, genotype, and individual fish responses.

Body size affects the response of zebrafish to different chemical euthanasia agents.^10,16,18 Fish in the 500-mg/L lidocaine group were significantly smaller than their counterparts in other groups. This effect was unintended and was only noted at the time of euthanasia. The shorter body length corresponded with increased time to loss of righting reflex and loss of opercular movement. These findings are similar to previous studies demonstrating that fish size and developmental stage have significant effects on responsiveness to chemical agents.^15,16 In addition, the decreased body size may indicate that these fish were generally of a younger developmental age. Fish age has been demonstrated to influence response to chemical agents in that these fish require exposure to higher doses to chemical agents for longer periods of time to ensure euthanasia.¹⁸ To our knowledge, no work has been performed to investigate whether adult fish of varying body lengths respond differently to anesthetics. Despite this feature, all fish in this group were successfully euthanized with 500 mg/L lidocaine. In this study, lidocaine hydrochloride was determined to cause death more rapidly than MS222 for all groups, regardless of the noted variation in body size. Facilities euthanizing large numbers of zebrafish should consider tailoring their methods to the size and developmental stage of the fish. Exposure times to chemical agents may need to be prolonged or higher doses used for smaller and younger fish.

One fish in the MS222 group recovered whereas none in the lidocaine groups recovered. Euthanasia should be a rapid and irreversible process.¹ A previous study noted that MS222 did not result in a uniform time to death in exposed fish, with some fish recovering.¹⁹ Current guidelines on euthanasia recommend a secondary physical or chemical method to ensure brain death after exposure to a chemical agent.^1,3 This practice is prudent for fish exposed to MS222 at concentrations of 250 mg/L, at which some fish may recover. All evidence suggests that lidocaine hydrochloride at concentrations of 400 mg/L or greater leads to irreversible death. This advantage may be significant when using this agent compared with MS222 for zebrafish euthanasia.

Three adult zebrafish displayed piping, erratic swimming, and body twitching when exposed to MS222. These behaviors have been associated with induction stress in fish.^14,19,20 In the current study, only 2 fish displayed evidence of aversive behaviors in the presence of lidocaine hydrochloride. Recent studies have shown that fish exit anesthetic-containing compartments more quickly than those without anesthetics.^14,20 Whether this behavior is generated by aversion to the anesthetic agent itself or to stress due to loss of control of balance and orientation is unclear.¹⁴ In a previous study, adult zebrafish exited a compartment containing lidocaine hydrochloride less quickly than they did one containing MS222.¹⁴ Lidocaine hydrochloride has been shown to be less aversive than MS222, clove oil, and benzocaine, indicating that the use of lidocaine might lead to improved wellbeing in fish undergoing euthanasia.²⁰ Although not statistically significant, fewer adult zebrafish in this study demonstrated aversion to lidocaine hydrochloride than MS222, as determined by the behavior of the fish. We did not correlate behavioral response with cortisol level during the process of euthanasia. Previous studies have indicated zebrafish display higher cortisol levels when exposed to MS222 compared with other chemical agents.⁹ Future work correlating serum cortisol assays with behavioral assays would be useful to determine whether lidocaine hydrochloride induces physiologic stress or inhibits response detection.

No lesions were noted in adult zebrafish exposed to either MS222 or lidocaine hydrochloride. Previous studies similarly did not find any lesions associated with MS222 in the musculature of exposed zebrafish.¹⁹ These agents may not cause microscopic lesions in fish. Alternatively, the lesions may not have sufficient time to develop prior to placement in buffered formalin. Although lidocaine hydrochloride does not appear to cause acute toxicity to zebrafish at the doses used in this study, no data are available regarding other possible physiologic effects its use might have in adult zebrafish.

International guidelines for euthanasia of zebrafish recommend a number of chemical agents or methods.^1,3 AVMA criteria for performance-based euthanasia recommend that any chemical used be safe for personnel.^1,10 Many currently recommended chemical methods are hazardous to personnel during preparation or use. For example, MS222 requires preparation in a fume hood and the use of personal protective equipment when manipulating the powder form.¹⁷ Clove oil (eugenol) has been described as an equivocal carcinogen by the US Food and Drug Administration.¹⁷ Benzocaine powder, similar to MS222, is a respiratory irritant¹⁷ and requires special handling procedures. Furthermore, unless used as the hydrochloride salt, benzocaine and quinaldine require dissolution in acetone or alcohol prior to placement in water.¹⁷ Lidocaine hydrochloride does not require special handling precautions or storage procedures and poses little hazard to humans unless inadvertently injected in large volumes. Lidocaine is safe to humans at the doses used in this study.¹⁷ The advantages of lidocaine hydrochloride as a euthanasia agent in comparison with MS222 include one-step preparation, reduced health hazards during preparation, and minimal storage security concerns.

In conclusion, lidocaine hydrochloride was effective for euthanasia of adult zebrafish. This compound produced death more rapidly than did MS222. In contrast, neither lidocaine hydrochloride nor MS222 was an effective euthanasia agent for zebrafish younger than 16 dpf. We propose the use of lidocaine hydrochloride at 400 mg/L as an effective and humane alternative chemical euthanasia agent to MS222 in adult zebrafish. We recommend that zebrafish facilities develop euthanasia procedures for different sizes and developmental stages of fish to ensure adequate euthanasia. It is possible that fish of different genotypes may react differently to exposure to chemical euthanasia agents, and this area requires further study. Small groups of fish should be exposed prior to using a chemical for euthanasia of fish on a wider scale. Finally, current euthanasia guidelines do not take into account the effect of body size and stage of development of fish on the efficacy of chemical methods of euthanasia.^1,3 Facilities and assessment panels should consider these factors when developing or reviewing institutional standard operating procedures for euthanasia of zebrafish.