The Efficacy of Clove Oil for Anaesthesia of Eight Species of Australian Tropical Freshwater Teleosts

LIMNOLOGY and Limnol. Oceanogr.: Methods 13, 2015, 463–475 VC 2015 The Authors Limnology and Oceanography: Methods published by Wiley Periodicals, Inc. on behalf of Association for the OCEANOGRAPHY: METHODS Sciences of Limnology and Oceanography doi: 10.1002/lom3.10040

The efficacy of clove oil for anaesthesia of eight species of Australian tropical freshwater teleosts

Frederieke J. Kroon†* 1CSIRO Division of Ecosystem Sciences, Atherton, Queensland, Australia

Abstract The efficacy of clove oil for anaesthesia was examined on eight species of Australian tropical freshwater fishes: the native Mulgrave goby (Glossogobius bellendenensis, Gobiidae), empire gudgeon (Hypseleotris compressa) and sleepy cod (Oxyeleotris lineolatus) (both Eleotrididae), Eastern rainbowfish (Melanotaenia splendida, Melano- taeniidae), Pacific blue-eye (Pseudomugil signifer, Pseudomugilidae), and eel-tailed catfish (Tandanus tandanus, Plotosidae), and the non-native species spotted tilapia (Pelmatolapia mariae, Cichlidae) and guppy (Poecilia reticulata, Poecilidae). Induction to anaesthesia, recovery from anaesthesia and survival of fish were determined for five concentrations (20 mg/L, 30 mg/L, 40 mg/L, 80 mg/L, and 160 mg/L). A 20 mg/L clove oil concentration was not sufficient to induce a loss of reflex reactivity within 10 min for most individuals (> 85%) in the eight species examined. Effective clove oil concentrations for anaesthesia induction and recovery (40–80 mg/L) for seven out of the eight species examined overlap with those reported for other tropical freshwater fish species. Differences in induction and recovery times among treatments were not always independent of fish size for G. bellendenensis, M. splendida, O. lineolatus, and P. mariae, emphasizing the importance of taking biological and environmental factors into account when determining and applying effective clove oil concentrations for anaesthesia in fish. In contrast, long recovery times and mortality for T. tandanus in even relatively low concentrations suggests that clove oil may not be an appropriate anaesthetic for at least some fish species.

In the last two decades, clove-oil-derived eugenol has been freshwater fish species (Keene et al. 1998; Waterstrat 1999; recognized as an effective anaesthetic for fish in aquaculture Sladky et al. 2001; Jayathilake et al. 2003; Small 2003; Cooke and aquatic research (Keene et al. 1998; Ross and Ross 2008). et al. 2004; Hoskonen and Pirhonen 2004; Roubach et al. Clove oil has a variety of biological effects, including anaes- 2005; Kaiser et al. 2006; Simoes~ et al. 2011; Morato-Fernandes thetic properties, with eugenol (4-allyl-2-methoxyphenol) et al. 2013), as well as for freshwater life history stages of eels being the primary component (Chaieb et al. 2007). Clove oil (Walsh and Pease 2002) and salmonids (Woody et al. 2002; is derived from the flower buds, i.e., cloves, of the clove tree Iversen et al. 2003; Hoskonen and Pirhonen 2004). Syzygium aromaticum (L.) Merrill and Perry (Myrtaceae) The freshwater fish fauna of Australia is unique in the (Chaieb et al. 2007). Effective concentrations of clove oil for world (Allen et al. 2003), with a high incidence of endemism anaesthetics have been determined for a variety of marine in the southern, central, and western provinces, as well as (Soto and Burhanuddin 1995; Munday and Wilson 1997; Grif- the North East Queensland region (Unmack 2001). This fiths 2000; King et al. 2005; Mylonas et al. 2005; Cunha and uniqueness attracts freshwater biologists to conduct both Rosa 2006; Park et al. 2008; Gullian and Villanueva 2009) and field and laboratory research on this fauna, with new fish species being discovered on a regular basis (Pusey et al. 2004; Thuesen et al. 2011; Welsh et al. 2014). However, effective † Present address: Australian Institute of Marine Science, Townsville, clove oil concentrations for anaesthetics have been pub- Queensland, Australia *Correspondence: [email protected] lished for only two Australian freshwater fish species, namely This is an open access article under the terms of the Creative Commons the tropical Western rainbowfish (Melanotaenia australis, Mel- Attribution-NonCommercial-NoDerivs License, which permits use and dis- anotaeniidae) (Castelnau 1875) (Young 2009) and the sub- tribution in any medium, provided the original work is properly cited, the tropical longfinned eel (Anguilla reinhardtii, Anguillidae) use is non-commercial and no modifications or adaptations are made. Steindachner 1867 (Walsh and Pease 2002). Interestingly, The copyright line for this article was changed on 23 September 2015 after the effective concentrations for these two species are higher original online publication. compared to non-Australian freshwater fish species from

463 Kroon Clove oil anaesthesia in Australian ﬁsh

Table 1. Australian tropical freshwater fish species submitted to anaesthesia with clove oil. For each fish species, the mean length (total length, TL) 6 standard error (SE) and range (minimum–maximum), the number of individuals tested for each clove oil concentration, the total number of fish tested, and the mean water temperature of acclimation aquaria 6 SE and range (minimum–maximum), are given.

Number of fish tested for each clove TL (mm) oil concentration Temperature (8C) Range 20 30 40 80 160 Total # Range Fish species Mean 6 SE (min–max) mg/L mg/L mg/L mg/L mg/L of fish Mean 6 SE (min–max) Glossogobius 45.8 6 1.5 34.8–72.5 76977 3625.3 6 0.1 24.5–26.0 bellendenensis Hypseleotris compressa 44.4 6 1.4 23.0–73.5 15 19 19 20 16 89 25.4 6 0.2 25.0–27.5 Oxyeleotris lineolatus 69.5 6 21.5 34.0–583.0 55555 2525.1 6 0.1 25.0–26.0 Melanotaenia splendida 84.5 6 1.6 49.0–125.0 24 16 24 24 14 102 25.0 6 0.2 22.0–26.5 Pseudomugil signifer 36.4 6 0.8 25.8–49.6 10 10 11 10 12 53 24.5 6 0.1 23.5–25.0 Tandanus tandanus 93.5 6 3.8 67.9–127.1 30645 1825.1 6 0.1 25.0–26.0 Pelmatolapia mariae* 66.1 6 4.2 28.9–128.8 12 19 13 14 12 70 24.8 6 0.0 24.0–26.0 Poecilia reticulata* 24.5 6 0.7 16.7–40.7 4 10 10 10 12 46 24.7 6 0.1 24.0–26.0 *Denotes species non-native to Australia. temperate (Keene et al. 1998; Hoskonen and Pirhonen 2004), belonging to families Gobiidae (Mulgrave goby, Glossogobius subtropical (Waterstrat 1999; Small 2003; Cooke et al. 2004; bellendenensis Hoese and Allen 2009), Eleotridae (empire Mylonas et al. 2005; Morato-Fernandes et al. 2013), and gudgeon, Hypseleotris compressa (Krefft 1864), sleepy cod, tropical regions (Sladky et al. 2001; Jayathilake et al. 2003; Oxyeleotris lineolatus (Steindacher 1867)), Melanotaeniidae Roubach et al. 2005; Kaiser et al. 2006; Simoes~ et al. 2011). (Eastern rainbowfish, Melanotaenia splendida (Peters 1866)), This highlights that freshwater biologists studying Australian Pseudomugilidae (Pacific blue-eye, Pseudomugil signifer Kner fish cannot necessarily rely on clove oil concentrations 1865), Plotosidae (eel-tailed catfish, Tandanus tandanus determined for non-Australian fish, and that further work is Mitchell 1838), Cichlidae (spotted tilapia, Pelmatolapia needed to facilitate the collection, handling and transporta- mariae Boulenger 1899), and Poecilidae (guppy, Poecilia retic- tion of Australian freshwater fish. This is particularly perti- ulata Peters 1859) were examined (Table 1). All species exam- nent for Australia’s tropical north where research on the ined are native to Australia, except for P. mariae and P. freshwater fish fauna is urgently required to minimise the reticulata. potential impacts of proposed agricultural expansion (Slezak The following criteria were used to select the eight spe- 2014). cies: importance to recreational fisheries (e.g., T. tandanus, O. In this study, induction to anaesthesia, recovery from lineolatus), importance to aquarium trade and hobby (e.g., H. anaesthesia and survival rates were determined for eight Aus- compressa, M. splendida, P. signifer), and importance to con- tralian tropical freshwater fish species exposed to five differ- servation, such as endemic (e.g., G. bellendenensis) and non- ent concentrations of clove oil (20 mg/L, 30 mg/L, 40 mg/L, native (e.g., P. mariae, P. reticulata) species. In Queensland, 80 mg/L, and 160 mg/L). The induction and recovery times the six native species are not listed as threatened or endan- were compared for six native species (Pusey et al. 2004), and gered under the Nature Conservation (Wildlife) Regulation two non-native species with established populations in tropi- 2006, while Tilapia spp. are declared noxious fish under the cal Australia (Kroon et al. 2015). Moreover, the effect of fish Fisheries Regulation 2008. Both P. mariae and P. reticulata size on induction and recovery times was examined for all have established populations in Queensland (Department of eight species. The results will contribute to the use of effec- Primary Industries 2001), including in the Wet Tropics tive clove oil concentrations for anaesthesia in both field region (Kroon et al. 2015). and laboratory research on tropical freshwater fish. Fish were collected using seine nets, funnel traps, and backpack electrofishing, in the Barron and Mulgrave river basins in the Wet Tropics region in 2011 and 2012. Since Materials and procedures our collections, a new species of Tandanus has been identi- Description and collection of study species fied to co-occur with T. tandanus in the Mulgrave-Russell This study was conducted at the CSIRO laboratory in river drainage, namely Tandanus tropicanus (Plotosidae) Atherton (latitude 178150, longitude 1458290) in the Wet (Welsh et al. 2014). Our study occurred before the identifica- Tropics region of North Queensland, Australia. Eight species tion of T. tropicanus; hence we use the name T. tandanus.

464 Kroon Clove oil anaesthesia in Australian ﬁsh

Fish were transported to the laboratory the same day, and 1985; Ross and Ross 2008). All trials were conducted during sorted by species and acclimated in 60-L glass aquaria filled daylight hours. Following recovery, fish were gently netted with rainwater for at least 1 week. The average temperature, and transferred to a holding aquarium and held for 2 days pH, and dissolved oxygen of the rainwater was 25.6 6 0.058C, noting any mortality. 7.7 6 0.038C, and 6.7 6 0.088C, respectively. Aquaria were Statistical analyses static with power filters and kept in an ambient room tem- For each individual fish species, normality and homoge- perature of 25.0 6 0.98C and a 12: 12-h light: dark cycle. Fish neity of variance were examined for each of the four criteria were fed ad-lib twice daily with most fish taking the food of anaesthesia for both nontransformed and log within 1 week. Eating was taken as a sign that fish has accli- (x 1 1) transformed data. In general, the assumptions were met for mated to their holding conditions; only fish that were eating log transformed but not for nontransformed data. Sub- were subsequently used in the clove oil experiments. (x 1 1) sequent analyses were conducted on log(x 1 1) transformed Clove oil concentrations data which also enabled the display of linear relationships. Five different clove oil dosages were examined, namely First, for each individual fish species the relationships 20 mg/L, 30 mg/L, 40 mg/L, 80 mg/L, and 160 mg/L. Clove between each of the four criteria of anaesthesia and clove oil oil (85.0% min Eugenol, Chem-Supply Pty Ltd) was first dis- concentrations was examined using linear regression. Specifi- solved in 0.05% (vol) ethanol (EtOH, 100%) to obtain a cally, for each species the time of induction (Stage 4 or 5) or 100 mg/mL stock solution for further use. At this low con- recovery (Stage 3 or 5) was included as a dependent variable, centration EtOH has no anaesthetizing effects on fishes with the different clove oil concentrations included as an (Munday and Wilson 1997; Cunha and Rosa 2006; Kaiser independent variable. Second, for each individual fish spe- et al. 2006). Fresh stock solutions were made daily. For each cies differences in each of the four criteria of anaesthesia test concentration, the required volume of stock solution among the different clove oil concentrations were examined was mixed thoroughly into a 2-L glass treatment aquarium separately, using analysis of covariance (ANCOVA) and filled with rainwater. Tukey’s honest significant difference (HSD) test (Whitlock and Schluter 2009). Specifically, for each species the time of Induction of, and recovery from anaesthesia induction (Stage 4 or 5) or recovery (Stage 3 or 5) was Four criteria for efficacy were chosen, namely total loss of included as a dependent variable, with the different clove oil equilibrium (Stage 4) and loss of reflex reactivity (Stage 5) concentrations included as a categorical variable and TL as a for anaesthesia induction, and total recovery of equilibrium numerical variable. In the first round of model fitting, the (Stage 3) and total behavioural recovery with normal swim- interaction between the treatment (i.e., clove oil concentra- ming (Stage 5) for anaesthesia recovery (Keene et al. 1998). tions) and the covariate (i.e., TL) was tested for each species Keene et al. (1998) describe the behaviour exhibited during and each of the four criteria of anaesthesia (Whitlock and Stage 4 and 5 induction as “Total loss of muscle tone and Schluter 2009). If an interaction was detected, differences in equilibrium; slow but regular opercular rate; loss of spinal the time of induction (Stage 4 or 5) or recovery (Stage 3 or reflexes,” and “Total loss of reactivity; opercular movements 5) among treatments were considered to be not independent slow and irregular; heart rate very slow; loss of all reflexes,” of TL. If no interaction was detected, the interaction term respectively. was dropped from the model and the treatment effect was Clove oil experiments were conducted in September, tested in the second round of model fitting (Whitlock and November, and December 2011, and May, June, July, August, Schluter 2009). STATISTICA 12 was used for all analyses and November 2012. To start an exposure, individual fish (StatSoft 2013). Fish that did not recover from anaesthesia or were gently netted and transferred from their respective died in the subsequent 48 h were excluded from the statisti- acclimation aquarium to the 2-L treatment aquarium. Fol- cal analyses, but were considered in the overall assessment lowing Stage 5 induction fish were measured (total length, of effective clove oil concentrations for anaesthesia. TL, 0.1 mm). Subsequently, fish were transferred to another 2-L recovery aquarium filled with continuously aerated rainwater until Stage 5 recovery. The times from (1) entry into Results the treatment aquarium to Stage 4 and 5 induction, (2) exit Survival from the treatment aquarium to entry in the recovery aquar- No mortality occurred during the induction or recovery ium (i.e., maintenance stage, Ross and Ross 2008), and (3) of anaesthesia, and during the 2 days post exposure, in five exit from the treatment aquarium to Stage 3 and 5 recovery of the eight species (Table 2). For the 18 T. tandanus, nine was recorded using a stopwatch. Maintenance stage was kept did not recover to Stage 5 within 10 min, and four did not constant to 1 min for all fish. A clove oil concentration was recover from anaesthesia at all (two in 40 mg/L, two in considered in-effective if either loss of reflex reactivity (Stage 160 mg/L). The exposure experiments were subsequently ter- 5) or total behavioural recovery with normal swimming minated for this species, with no 30 mg/L exposures con- (Stage 5) did not occur within 10 min (Marking and Meyer ducted. For P. signifer, 7 out of 53 individuals did not recover

465 Kroon Clove oil anaesthesia in Australian ﬁsh

Table 2. Number of individuals of eight Australian tropical ferences in induction times were not independent of fish freshwater fish species that did not recover from anaesthesia, or size for G. bellendenensis and M. splendida (Table 3). At died in the subsequent 48 h (in between brackets), after submis- 30 mg/L, the mean time for Stage 5 ranged from c. 226 s for sion to anaesthesia with clove oil. G. bellendenensis to c. 718 s for O. lineolatus. Differences between mean time for Stage 5 among the eight species were Clove oil concentration less pronounced at 160 mg/L, ranging from c. 45 s for G. bel- (mg/L) lendenensis to c. 115 s for O. lineolatus. Species 20 30 40 80 160 Total Recovery from anaesthesia Glossogobius bellendenensis 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) The time for total recovery of equilibrium (Stage 3) Hypseleotris compressa 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) increased significantly with increasing clove oil concentra- Oxyeleotris lineolatus 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) tion for six species, showed an increasing trend for T. tanda- Melanotaenia splendida 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) nus, with no significant effect detected for H. compressa (Fig. Pseudomugil signifer 0 (0) 0 (1) 1 (3) 0 (0) 6 (0) 7 (4) 2A). For all species, except M. splendida and T. tandanus, the Tandanus tandanus 0 (0) 0 (0) 2 (0) 0 (0) 2 (0) 4 (0) mean time for Stage 3 was significantly longer at 160 mg/L Pelmatolapia mariae* 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) compared to 30 mg/L and 40 mg/L (Fig. 2A; Table 3). These Poecilia reticulata* 0 (0) 0 (0) 0 (1) 1 (0) 1 (2) 2 (3) differences in recovery times were not independent of fish *Denotes species non-native to Australia. size for O. lineolatus and P. mariae. Post hoc comparison revealed no significant differences among the four clove oil M. splendida p > from anaesthesia (one in 40 mg/L, six in 160 mg/L), and concentrations for (Tukey’s HSD, 0.05). At four fish did not survive during the 48 h post exposure 30 mg/L, the mean time for Stage 3 ranged from c. 67 s for M. splendida O. lineolatus period; the 160 mg/L exposures were subsequently termi- to c. 222 s for . Differences between nated for this species. For P. reticulata, one out of 46 individ- mean time for Stage 3 among the eight species were more H. com- uals did not recover from anaesthesia (160 mg/L), and four pronounced at 160 mg/L, ranging from c. 77 s for pressa O. lineolatus fish did not survive during the 48 h post exposure period. to c. 639 s for . The time for effective total behavioural recovery with normal swimming (Stage 5), i.e., 10 min (Marking and Meyer Induction of anaesthesia 1985; Ross and Ross 2008), was exceeded by 34 individuals For all eight species, loss of reflex reactivity (Stage 5) at (10% of total) comprising all but one (M. splendida) species 20 mg/L was more than 10 min for most (> 85%) individu- (Fig. 2B). Most of these individuals (28) were from three spe- als. Hence, this clove oil concentration was considered in- cies (O. lineolatus, T. tandanus and P. mariae), and most of effective, and not included in the statistical analyses. these exceedances (25) occurred in two clove oil concentra- The time for total loss of equilibrium (Stage 4) decreased tions (30 mg/L and 160 mg/L). The time to reach Stage 5 significantly with increasing clove oil concentration for all increased significantly with increasing clove oil concentra- eight species (Fig. 1A). For all species, except O. lineolatus tions for four species, showed a decreasing trend for H. com- and T. tandanus, the mean time for Stage 4 was significantly pressa and P. mariae, with no significant effect detected for shorter at 40 mg/L, 80 mg/L, and 160 mg/L compared to M. splendida and P. reticulata (Fig. 2B). Significant differences 30 mg/L (Fig. 1A; Table 3). These differences in induction in the mean times for Stage 5 were not as pronounced as for times were not independent of fish size for M. splendida Stage 4, with recovery times being significantly longer at (Table 3). At 30 mg/L, the mean time for Stage 4 ranged 160 mg/L compared to 30 mg/L and/or 40 mg/L for only from c. 69 s for H. compressa to c. 236 s for P. reticulata. Dif- three species (G. bellendenensis, O. lineolatus, P. signifer) (Fig. ferences between mean time for Stage 4 among the eight 2B; Table 3). The differences in induction times were not species were less pronounced at 160 mg/L, ranging from c. independent of fish size for M. splendida and P. mariae. Post 18 s for P. reticulata to c. 49 s for O. lineolatus. hoc comparison revealed no significant differences among The time for effective loss of reflex activity (Stage 5), i.e., the four clove oil concentrations for M. splendida (Tukey’s 10 min (Marking and Meyer 1985; Ross and Ross 2008), was HSD, p > 0.05). At 30 mg/L, the mean time for Stage 5 exceeded by 19 individuals (6% of total) of five species, ranged from c. 230 s for P. reticulata to c. 631 s for O. lineola- namely H. compressa, O. lineolatus, M. splendida, P. mariae, tus. Differences between mean time for Stage 5 among the and P. reticulata (Fig. 1B). All exceedances occurred in the eight species were more pronounced at 160 mg/L, ranging 30 mg/L clove oil concentration. Similarly to Stage 4, the from c. 233 s for M. splendida to c. 1288 s for T. tandanus. time to reach Stage 5 decreased significantly with increasing clove oil concentration for all eight species (Fig. 1B). For all species, except P. signifer and T. tandanus, the mean time for Discussion Stage 5 was significantly shorter at 40 mg/L, 80 mg/L, and The general findings of this study show that induction to 160 mg/L compared to 30 mg/L (Fig. 1B; Table 3). These dif- anaesthesia decreased with increased clove oil

466 Kroon Clove oil anaesthesia in Australian ﬁsh

Fig. 1. Relationship between induction time of anaesthesia, namely total loss of equilibrium (Stage 4) (A) and loss of reflex reactivity (Stage 5) (B), and clove oil concentration for eight Australian tropical freshwater fish species. Linear regression lines and 95% confidence intervals, together with regression statistics, are presented. Different letters indicate statistically significant differences in mean induction times following ANCOVA and Tukey HSD. Please see Table 1 for the number of individuals tested for each species and each clove oil concentration, and Table 2 for detailed ANCOVA results. concentrations, while recovery from anaesthesia increased, Ross and Ross 2008). These results corroborate previous stud- albeit not always, with increased clove oil concentrations. In ies (Iversen et al. 2003; Jayathilake et al. 2003; King et al. the eight species of tropical freshwater fish examined, a con- 2005; Gullian and Villanueva 2009; Simoes~ et al. 2011) that centration of 20 mg/L clove oil was ineffective in inducing this concentration is generally, but not always (Keene et al. loss of reflex reactivity (Stage 5) for anaesthesia within a rea- 1998; Cho and Heath 2000), insufficient to achieve loss of sonable timeframe (i.e., 10 min) (Marking and Meyer 1985; reflex reactivity in fish. Several studies, however, have

467 Kroon Clove oil anaesthesia in Australian ﬁsh

Fig. 1. (Continued) demonstrated that clove oil concentrations of 20 mg/L can to clove oil exposure, to the extent that the exposure experi- be sufficient for achieving lighter stages of anaesthesia useful ment was terminated for T. tandanus, and exposure to high for collecting (Cunha and Rosa 2006), handling (Woody concentrations (160 mg/L) was ceased for P. signifer. Mortal- et al. 2002; Iversen et al. 2003; King et al. 2005) and trans- ity in fish exposed to clove oil has been documented previ- porting fish (Cooke et al. 2004; Kaiser et al. 2006; Iversen ously, and generally involves either relatively high et al. 2009; Simoes~ et al. 2011). concentrations or prolonged exposures. For example, in fin- Three of the eight fish species examined (T. tandanus, gerling channel catfish Ictalurus punctatus (Rafinesque 1818), P. signifer, P. reticulata) appeared to be particularly susceptible 50% died following a 10 min exposure to 300 mg/L clove oil

468 Kroon Clove oil anaesthesia in Australian ﬁsh

(Waterstrat 1999). Some mortality occurred in perch Perca min. In juvenile cobia Rachycentron canadum (Linnaeus ﬂuviatilis Linnaeus, 1758, exposed to 80 mg/L and 120 mg/L 1766), a 10 min exposure to 60 mg/L resulted in approxi- (Hoskonen and Pirhonen 2004). In juvenile Nile tilapia Oreo- mately 25% mortality, increasing to 100% with exposure to chromis niloticus (Linnaeus 1758), exposure to 100 mg/L clove 100 mg/L (Gullian and Villanueva 2009). In female P. reticu- oil for 10 min resulted in 20% mortality (Simoes~ et al. 2011). lata 75% mortality occurred after 1 h exposure to 35 lg/L Prolonged exposures to 90 mg/L clove oil resulted in 50% and 40 lg/L each (Jayathilake et al. 2003). Exposure experi- mortality after 20 min, increasing to 100% mortality after 30 ments with the Lake Victoria cichlid Haplochromis

Table 3. Results of analysis of covariance (ANCOVA) to examine the efficacy of clove oil for anaesthesia in eight Australian tropical freshwater fish species. For each fish species the time of induction (Stage 4 or 5) or recovery (Stage 3 or 5) was included as a dependent variable, with the different clove oil concentrations included as a categorical variable and total length (TL) as a numerical variable. The first round of model fitting included the interaction between the clove oil concentrations and the TL. If no interaction was detected, the interaction term was dropped and the treatment effect was tested in the second round of model fitting. Significant p values are in bold. The results of post-hoc comparisons of mean induction (Stage 4 or 5) and mean recovery (Stage 3 or 5) times are presented in Figs. 1 and 2, respectively. Induction Stage 4 Clove oil TL Clove oil x TL Species F df pFdf pFdf p Glossogobius bellendenensis 107.13 3.24 <0.01 5.89 1.24 0.02 — — n.s Hypseleotris compressa 41.63 3.69 <0.01 0.50 1.69 0.48 — — n.s Oxyeleotris lineolatus 15.39 3.15 <0.01 0.05 1.15 0.83 — — n.s Melanotaenia splendida 3.67 3.70 0.02 0.01 1.70 0.91 3.14 3.70 0.03 Pseudomugil signifer 45.60 3.26 <0.01 1.50 1.26 0.23 — — n.s Tandanus tandanus 7.72 2.7 0.02 1.08 1.7 0.33 — — n.s Pelmatolapia mariae* 108.68 3.53 <0.01 0.16 1.53 0.69 — — n.s Poecilia reticulata* 45.75 3.32 <0.01 9.60 1.32 <0.01 — — n.s

Induction Stage 5 Clove oil TL Clove oil x TL Species F df pFdf pFdf p Glossogobius bellendenensis 5.69 3.21 <0.01 23.58 1.21 <0.01 4.17 3.21 0.02 Hypseleotris compressa 79.90 3.69 <0.01 2.82 1.69 0.10 — — n.s Oxyeleotris lineolatus 82.98 3.15 <0.01 1.19 1.15 0.29 — — n.s Melanotaenia splendida 14.09 3.70 <0.01 2.98 1.70 0.09 10.42 3.70 <0.01 Pseudomugil signifer 73.20 3.26 <0.01 1.55 1.26 0.23 — — n.s Tandanus tandanus 7.26 2.7 0.02 1.87 1.7 0.21 — — n.s Pelmatolapia mariae* 179.32 3.53 <0.01 60.17 1.53 <0.01 — — n.s Poecilia reticulata* 87.04 3.32 <0.01 3.69 1.32 0.06 — — n.s

Recovery Stage 3 Clove oil TL Clove oil x TL Species F df pFdf pFdf p Glossogobius bellendenensis 15.57 3.24 <0.01 0.60 1.24 0.45 — — n.s Hypseleotris compressa 4.86 3.69 <0.01 3.62 1.69 0.06 — — n.s Oxyeleotris lineolatus 3.64 3.12 0.05 2.89 1.12 0.12 3.75 3.12 0.04 Melanotaenia splendida 2.81 3.73 0.05 3.77 1.73 0.06 — — n.s Pseudomugil signifer 20.16 3.26 <0.01 1.79 1.26 0.19 — — n.s Tandanus tandanus 1.99 2.7 0.21 0.40 1.7 0.55 — — n.s Pelmatolapia mariae* 10.18 3.50 <0.01 4.43 1.50 0.04 11.80 3.50 <0.01 Poecilia reticulata* 5.16 3.32 <0.01 2.88 1.32 0.10 — — n.s

469 Kroon Clove oil anaesthesia in Australian ﬁsh

Recovery Stage 5 Clove oil TL Clove oil x TL Species F df pFdf pFdf p Glossogobius bellendenensis 9.44 3.24 <0.01 1.37 1.24 0.25 — — n.s Hypseleotris compressa 10.99 3.69 <0.01 3.73 1.69 0.06 — — n.s Oxyeleotris lineolatus 9.89 3.15 <0.01 0.05 1.15 0.83 — — n.s Melanotaenia splendida 4.45 3.70 <0.01 2.40 1.70 0.13 4.16 3.70 <0.01 Pseudomugil signifer 10.09 3.26 <0.01 0.28 1.26 0.60 — — n.s Tandanus tandanus 2.96 2.7 0.12 0.00 1.7 0.99 — — n.s Pelmatolapia mariae* 5.46 3.50 <0.01 2.96 1.50 0.09 4.52 3.50 <0.01 Poecilia reticulata* 3.85 3.32 0.02 8.74 1.32 <0.01 — — n.s

obliquidens (Hilgendorf 1888) were terminated following and > 10 min for O. lineolatus. At 40 mg/L and 80 mg/L, time mortality during a 24 h exposure to 30 lL/L clove oil (Kai- to total behavioural recovery was < 10 min for most individu- ser et al. 2006). The results of these and our study demon- als of all seven species. In other tropical freshwater fish spe- strate a narrow margin of safety for clove oil concentrations cies, recovery of equilibrium without mortalities occurred and exposure times (Sladky et al. 2001; Kaiser et al. 2006), within 10 min following exposure to 50–100 mg/L for P. bra- further emphasising the need to determine species-specific chypomus (Sladky et al. 2001), 35–100 mg/L for juvenile and clove oil concentrations for anaesthesia. Moreover, the long 35–135 mg/L for subadult C. macropomum (Roubach et al. recovery time (> 10 min) and some mortality of T. tandanus 2005), and 90 mg/L for juvenile O. niloticus (Simoes~ et al. following exposure to even relatively low concentrations 2011). Hence, the effective clove oil concentrations of 40– suggests that clove oil may not be an appropriate ana- 80 mg/L for anaesthesia for seven out of the eight species esthetic for at least some fish species (Hoskonen and documented in this study are within the same range as (Jaya- Pirhonen 2004). thilake et al. 2003; Roubach et al. 2005), or even slightly lower For seven out of the eight species examined, a range of than (Sladky et al. 2001; Simoes~ et al. 2011) those reported for clove oil concentrations were found to be effective, i.e., induc- other tropical freshwater fish species. ing loss of reflex reactivity (Stage 5) within 10 min or less In this study, differences in induction and recovery times (Marking and Meyer 1985; Ross and Ross 2008). Effective con- among treatments were not always independent of fish size centrations for anaesthesia induction started at 30 mg/L for for some species, namely G. bellendenensis, M. splendida, O. some species (G. bellendenensis, P. signifer), while slightly lineolatus, and P. mariae. Effect of fish size on anaesthesia has higher concentration of 40 mg/L resulted in shorter induction been documented in other fish species, including female P. times for all seven species. For P. reticulata, this corresponds reticulata (Jayathilake et al. 2003) and juvenile and subadult with effective clove oil concentrations for anaesthesia C. macropomum (Roubach et al. 2005). While the lack of size reported by Jayathilake et al. (2003). For all seven species effect in the other four species may in part be an artefact of examined here, mean induction times further decreased with the smaller size ranges tested, induction and recovery times increasing clove oil concentration, however, concomitant during clove oil anaesthesia are not always affected by varia- recovery times increased significantly with complete lack of tions in fish length (Walsh and Pease 2002; Cunha and Rosa recovery for some P. signifer and P. reticulata at 160 mg/L. 2006; Young 2009). Moreover, various studies have also These concentrations effective for induction of anaesthesia demonstrated an effect of water temperature on induction overlap with those documented for other tropical freshwater and recovery times during clove oil anaesthesia, including fish species, namely 50–200 mg/L for red pacu Piaractus bra- for temperate (Hoskonen and Pirhonen 2004; Woolsey et al. chypomus (Cuvier 1818) (Sladky et al. 2001), 35–135 mg/L for 2004), subtropical (Walsh and Pease 2002; Mylonas et al. juvenile and subadult C. macropomum (Roubach et al. 2005), 2005), and tropical (Park et al. 2008) fish species. The lack of and 80–90 mg/L for juvenile O. niloticus (Simoes~ et al. 2011). temperature effect on induction and recovery times docu- Similarly, total behavioural recovery with normal swimming mented for seven tropical reef fish is likely a result of the (Stage 5) was found to occur within 10 min (Marking and very small temperature range examined (Cunha and Rosa Meyer 1985) across a range of clove oil concentrations. At 2006). The influence of fish size and water temperature on 30 mg/L recovery to Stage 5 occurred effectively for four spe- anaesthesia is thought to be related to the uptake and meta- cies (G. bellendenensis, M. splendida, P. signifer, P. reticulata), but bolic rate of clove oil in fish (Hoskonen and Pirhonen 2004; was surprisingly long for two species (H. compressa, P. mariae), Guenette et al. 2007). These effects highlight the

470 Kroon Clove oil anaesthesia in Australian ﬁsh

Fig. 2. Relationship between recovery time of anaesthesia, namely total recovery of equilibrium (Stage 3) (A) and total behavioural recovery with normal swimming (Stage 5) (B), and clove oil concentration for eight Australian tropical freshwater fish species. Linear regression lines and 95% confidence intervals, together with regression statistics, are presented. Different letters indicate statistically significant differences in mean recovery times following ANCOVA and Tukey HSD. Please see Table 1 for the number of individuals tested for each species and each clove oil concentration, and Table 2 for detailed ANCOVA results. importance of taking both biological and environmental Recent studies have documented physiological effects in factors into account when determining and applying fish following exposure to clove oil, highlighting the impor- effective concentrations of anaesthetics for fish (Ross and tance of considering potential side-effects of this anaesthetic Ross 2008). when designing aquatic research. For example, largemouth

471 Kroon Clove oil anaesthesia in Australian ﬁsh

Fig. 2. (Continued)

black bass Micropterus salmoides (Lacepe`de 1802), exposed to steelhead trout Oncorhynchus mykiss (Walbaum 1792), was sig- clove oil concentrations that induced Stage 4 or 5 anaesthesia nificantly lower than in nonexposed fish (Pirhonen and exhibited cardiovascular recovery of up to 2 h (Cooke et al. Schreck 2003). A reduced stress response (e.g., plasma cortisol 2004). In European eel (Anguillia anguilla) (Linnaeus 1758), concentrations) following clove oil anaesthetisation has been clove oil anaesthesia resulted in the transcriptional induction reported in at least some fish species (Iversen et al. 2003, 2009, of antioxidant genes in the brain (Renault et al. 2011). During 2013; Pirhonen and Schreck 2003; Small 2003; Wagner et al. recovery from clove oil anaesthesia, feed intake in juvenile 2003; Renault et al. 2011). Similarly, studies comparing

472 Kroon Clove oil anaesthesia in Australian fish different anaesthetics have demonstrated different physiologi- Cunha, F. E. A., and I. L. Rosa. 2006. Anaesthetic effects of cal effects in fish during anaesthesia induction and recovery. clove oil on seven species of tropical reef teleosts. J. Fish For example, opercular ventilation rates differed significantly Biol. 69: 1504–1512. doi:10.1111/j.1095-8649.2006.01213.x during anaesthesia induction by clove oil and 2- Department of Primary Industries. 2001. Control of exotic pest pheonxyethanol, in both European sea bass Dicentrarchus lab- fishes. An operational strategy for Queensland freshwaters- rax (Linnaeus 1758) and gilthead sea bream Sparus aurata Lin- 2000–2005, p. 45. Department of Primary Industries. naeus 1758 (Mylonas et al. 2005). Some significant differences Griffiths, S. P. 2000. The use of clove oil as an anaesthetic and were documented during recovery following clove oil or tri- method for sampling intertidal rockpool fishes. J. Fish Biol. caine methanesulphonate (MS222) anaesthesia in haematocrit 57: 1453–1464. doi:10.1111/j.1095-8649.2000.tb02224.x and total leucocyte in juvenile chinook salmon Onchorynchus Guenette, S. A., F. C. Uhland, P. Helie, F. Beaudry, and P. tshawytscha (Walbaum 1792) (Cho and Heath 2000), and in Vachon. 2007. Pharmacokinetics of eugenol in rainbow plasma concentrations of blood glucose, cortisol, lactate and trout (Oncorhynchus mykiss). Aquaculture 266: 262–265. tri-iodothyronin in rainbow trout O. mykiss (Wagner et al. doi:10.1016/j.aquaculture.2007.02.046 2003; Holloway et al. 2004). Hence, the choice of anaesthetic Gullian, M., and J. Villanueva. 2009. Efficacy of tricaine is dependent on the sensitivity of the study species, as well as methanesulphonate and clove oil as anaesthetics for juve- on the procedures to be performed and the physiological nile cobia Rachycentron canadum. Aquacult. Res. 40: 852– parameters to be examined. 860. doi:10.1111/j.1365-2109.2009.02180.x In summary, exposure of eight tropical freshwater fish Holloway, A. C., J. L. Keene, D. G. Noakes, and R. D. Moccia. species to five different concentrations of clove oil (20– 2004. Effects of clove oil and MS-222 on blood hormone 160 mg/L) showed that 40–80 mg/L is most effective for profiles in rainbow trout Oncorhynchus mykiss, Walbaum. induction of and recovery from anaesthesia. In contrast, a Aquacult. Res. 35: 1025–1030. doi:10.1111/j.1365-2109. 20 mg/L clove oil concentration did not induce a loss of 2004.01108.x reflex reactivity within ten min for most individuals (> 85%) Hoskonen, P., and J. Pirhonen. 2004. Temperature effects on in the eight species examined. Long recovery times and anaesthesia with clove oil in six temperate-zone fishes. J. some mortality for T. tandanus at even relatively low concen- Fish Biol. 64: 1136–1142. doi:10.1111/j.1095-8649.2004. trations suggest that clove oil may not be an appropriate 00359.x anaesthetic for at least some fish species. Differences in Iversen, M., R. A. Eliassen, and B. Finstad. 2009. Potential bene- induction and recovery times were not always independent fit of clove oil sedation on animal welfare during salmon of fish size for four species. Combined, these results high- smolt, Salmo salar L. transport and transfer to sea. Aquacult. light the importance of examining the appropriateness of Res. 40:233–241.doi:10.1111/j.1365-2109.2008.02091.x certain anaesthetics for individual species and considering Iversen, M., B. Finstad, R. S. Mckinley, and R. A. Eliassen. biological factors such as size for effective anaesthesia in 2003. 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Acknowledgments Submitted 10 December 2014 I would like to thank Dean Jones, Yves Le Bras, and Alice Gouzerh for Revised 5 May 2015 ﬁeld collections, ﬁsh husbandry, clove oil exposures, and data entry, and Accepted 6 May 2015 two anonymous reviewers for comments on a previous version of the manuscript. This work was funded by CSIRO’s Water for a Healthy Coun- Associate editor: George Waldbusser

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