Aquaculture 529 (2020) 735716
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Eﬀects of diﬀerent ﬁsh sizes, temperatures and concentration levels of sodium bicarbonate on anaesthesia in Mozambique tilapia (Oreochromis mossambicus)
Ndakalimwe Naftal Gabriela, , Victoria Ndinelago Erasmusb, Andreas Namwoondec a
Department of Fisheries and Aquatic Sciences, University of Namibia, Private Bag 462, Hentiesbay, Namibia National Marine Information and Research Centre (NatMIRC), Ministry of Fisheries and Marine Resources, Strand Street, Box 912, Swakopmund, Namibia c Sam Nujoma Marine and Coastal Resources Research Centre, Sam Nujoma Campus, University of Namibia, Henties Bay, Namibia b
A R T I C LE I N FO
A B S T R A C T
Keywords: Anaesthesia Cichlid Fish welfare Sodium bicarbonate
This study was conducted to determine the eﬀects of diﬀerent ﬁsh sizes, temperatures, and concentration levels of sodium bicarbonate (baking soda) in Mozambique tilapia (Oreochromis mossambicus). Three diﬀerent body size classes of Mozambique tilapia; (i) small (0.92 ± 0.00 g, 3.4.21 ± 0.11 cm), (ii) medium (12.35 ± 2.71 g, 9.87 ± 1.23 cm), (iii) large (47.24 ± 3.12 g, 13.92 ± 2.61 cm) were subjected to three concentrations of sodium bicarbonate (30, 45, 60 g L−1) at 24, 28, or 32 °C, each replicated three times. The anaesthetic induction and recovery times were monitored at each treatment combination in ﬁve litres glass aquaria. Sodium bicarbonate proved to possess anaesthetic capacity for Mozambique tilapia, and its anaesthetic induction time was signiﬁcantly aﬀected by the concentration, water temperature, and ﬁsh size (P < .05). The induction time decreased with increase in temperature and sodium bicarbonate concentration, but increased with ﬁsh body size. Moreover, ﬁsh size and sodium bicarbonate concentration had a signiﬁcant eﬀect on the recovery time (P < .05). Inversely, anaesthetic recovery time increased with decrease in sodium bicarbonate concentration and temperature for all ﬁsh sizes; recovery time increased signiﬁcantly with increased ﬁsh size (P < .05). No mortality was recorded during the anaesthetic induction and recovery time or within three days post recovery. This study indicates that, sodium bicarbonate is eﬀective as anaesthetic in Mozambique tilapia at 45 and 60 g L−1 concentrations at all tested ﬁsh sizes and water temperature. Therefore, sodium bicarbonate is recommended as a satisfactory anaesthesia that can be used at all levels of aquaculture to improve ﬁsh welfare.
1. Introduction Namibia has a large ﬁshing sector, which is the 3rd highest contributor to its gross domestic product (GDP). The Namibian ﬁshing sector has two sub-sectors; the marine-based resource exploitation and aquaculture sector, which is still relatively new and under-exploited. The Namibian aquaculture industry has a potential to complement the wild capture based ﬁshing industry, to boost the country's economy, contribute to food security and improve the livelihood of citizens (Villasante et al., 2015; Chiripanhura and Teweldemedhin, 2016). The culture of animals involves regular activities such as sampling, tagging, grading, artiﬁcial breeding and transportation, which at most times, have been identiﬁed as a source of stress to ﬁsh. Handling of live ﬁsh without the use of ﬁsh anaesthetic has been associated with ﬁsh injury and impairment of their commercial value (Bodur et al., 2018). A considerable number of synthetic substances including tricaine ⁎
methanesulphonate (MS-222) (Skår et al., 2017), 2-phenoxyethanol (Ghanawi et al., 2013) and etomidate (Readman et al., 2017) have been used as ﬁsh anaesthetics. Although these substances are eﬀective, they are often unavailable in most parts of the world, expensive, and have demonstrated undesirable side eﬀects such as stress, and haematological disorders in ﬁsh (Gressler et al., 2014). There is thus a need to develop readily available, aﬀordable and easy to administer ﬁsh anaesthetics with limited side eﬀects on cultured ﬁsh. Historically, carbon dioxide (CO2) has been identiﬁed as a potential ﬁsh anaesthetic during ﬁsh handling (Fish, 1943). The gas has been proven to be non-toxic in ﬁsh, human,and environment especially when used in a ventilated environment and is considered economical compared to the other anaesthetics (Summerfelt and Smith, 1990; Iwama and Ackerman, 1994; Prince et al., 1995). The solubility properties of CO2 in water makes it suitable to be introduced and bubbled in the water directly through an airstone or indirectly by addition of sodium
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https://doi.org/10.1016/j.aquaculture.2020.735716 Received 29 May 2020; Received in revised form 9 July 2020; Accepted 12 July 2020 Available online 18 July 2020 0044-8486/ © 2020 Elsevier B.V. All rights reserved.
Aquaculture 529 (2020) 735716
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Table 1 Water quality parameters in diﬀerent treatment combinations recorded before and after the addition of sodium bicarbonate. Preset water temperatures (°C)
Sodium bicarbonate concentration (g L-1)
30 45 60 30 45 60 30 45 60
Dissolved oxygen (mg L-1)
5.50 ± 0.12 5.52 ± 0.22 5.48 ± 0.62 5.48 ± 0.12 5.50 ± 0.12 5.50 ± 0.32 5.37 ± 1.03 5.37 ± 0.16 5.01 ± 0.19
3.75 ± 0.00 3.56 ± 0.02 2.95 ± 0.16 3.55 ± 0.10 3.19 ± 0.07 2.75 ± 0.05 3.15 ± 0.18 2.95 ± 1.12 2.17 ± 0.05
7.20 ± 0.13 7.18 ± 0.11 7.20 ± 0.02 7.17 ± 0.10 7.20 ± 0.31 7.19 ± 0.22 7.10 ± 0.13 7.15 ± 0.16 7.21 ± 0.32
7.71 ± 1.11 7.89 ± 0.10 7.92 ± 0.13 7.52 ± 0.92 7.89 ± 1.01 8.02 ± 1.05 7.57 ± 0.12 7.91 ± 0.08 8.52 ± 1.15
Measured water temperature range (°C)
23.71 – 24.
27.62 – 28.41
31.02 – 32.55
Values are presented as mean ± standard error. Values in the same column are not signiﬁcantly diﬀerent (P > .05).
exchange rate at the time was 1US$ = N$14.98). For the experiment, three diﬀerent concentrations of sodium bicarbonate (30, 45, 60 g L−1) were prepared; a modiﬁcation of concentrations previously tested in tilapia species (Opiyo et al., 2013; Avillanosa and Caipang, 2019). After acclimatization, nine (9) glass aquaria (ﬁve litres) were ﬁlled with water of diﬀerent temperatures (24, 28, or 32 °C), each replicated three times. To maintain the set experimental temperatures, the aquaria were placed in thermostatically controlled water baths. In each of water temperature treatment tank, 30, 45, or 60 g L−1 of baking soda was added. This set of aquaria was designated as anaesthesia aquaria. The water was stirred continuously until complete dissolution was achieved, and the experiment commenced 30 min after complete dissolution of the baking soda as demonstrated by Avillanosa and Caipang (2019). Another set of nine (9) aerated glass aquaria (ﬁve litres) with the same water temperature treatments, replicated three times were used as recovery aquarium for the ﬁsh. A total of 135 ﬁsh were used in this study (45 ﬁsh per size class, and ﬁve ﬁsh in each of treatment combination). Water quality parameters in diﬀerent treatment combinations were recorded before and after the addition of sodium bicarbonate (Table 1). To test the anaesthetic response of Mozambique tilapia at diﬀerent sodium bicarbonate concentrations and water temperatures, one ﬁsh was placed in each anaesthesia aquarium and observed until it reached stage III anaesthesia according to the description by Iwama and Ackerman (1994), and Mirghaed et al. (2016). The ﬁsh at stage III anaesthesia were described to have complete loss of equilibrium and loss of body and irregular opercular movement (Table 2). Once stage III was reached, the ﬁsh was immediately removed from the anaesthesia aquarium and transferred to the recovery aquarium and observed until the ﬁsh attained stage II recovery. Trained laboratory technicians with a stopwatch, one technician per stage, recorded the time taken for each ﬁsh to reach each induction and recovery stage. The survival rate and other post-exposure behaviors in each recovery tank were monitored every 24 h for three days.
bicarbonate (NaHCO3, baking soda), which releases CO2 when dissolving in water (Prince et al., 1995; Altun et al., 2009). The eﬀectiveness of sodium bicarbonate as an anaesthetic has been reported in various ﬁsh species including African catﬁsh (Clarias gariepinus) (Githukia et al., 2016), rainbow trout (Oncorhynchus mykiss), brook trout (Salvelina fontinails) and common carp (Cyprinus carpio) (Booke et al., 1978; Altun et al., 2009). In a previous study by Avillanosa and Caipang (2019), a successful anaesthesia in red hybrid tilapia juveniles, was attained at a concentration of 50 g L−1 sodium bicarbonate at 26 °C - 29 °C water temperature in both freshwater and brackish water. Studies on the use of sodium bicarbonate to anaesthetize ﬁsh, provides evidence that the anaesthetic eﬀects of this chemical is dependent on several factors such as concentration levels, environmental parameters (i.e. temperature, and pH), ﬁsh species, body size and maturation stage of a ﬁsh(Pawar et al., 2011; Oberg et al., 2015; Aydın et al., 2015; Li et al., 2018a, 2018b). Mozambique tilapia (O. mossambicus) is among the freshwater ﬁsh species that have been adopted for aquaculture in Namibia. However, the way they are handled in captivity is not adequately documented. Limited studies exist on the use of sodium bicarbonate as an anaesthetic in ﬁsh, especially on O. mossambicus. There is also limited information on the interacting eﬀects of diﬀerent factors on the eﬃcacy level of sodium bicarbonate in individual ﬁsh species. Hence, there is a need to perform multifactorial studies to best optimize and use sodium bicarbonate as an inexpensive anaesthetic in individual ﬁsh species. For this reason, the present study was designed to investigate the anaesthetic eﬀects of sodium bicarbonate at diﬀerent concentrations and temperatures on O. mossambicus of diﬀerent body sizes. 2. Materials and methods 2.1. Fish and experimental design This study was carried out at Sam Nujoma Marine and Coastal Resources Research Center (SANUMARC), Sam Nujoma Campus, University of Namibia, Henties Bay in February 2020. Mozambique tilapia of diﬀerent sizes were produced and raised at SANUMARC research facilities. Fish were categorized in three size classes by length and weight as follows: small (with 0.92 ± 0.00 g body weight, and 3.4.21 ± 0.11 cm length), medium (with 12.35 ± 2.71 g body weight, and 9.87 ± 1.23 cm length), and large (47.24 ± 3.12 g body weight, and 13.92 ± 2.61 cm length). They were acclimatized for 14 days in 0.18 m3 rectangular glass aquaria (30 ﬁsh /tank) supplied with 150 L dechlorinated and adequately aerated freshwater (dissolved oxygen 5.96 ± 0.18 mg L−1, pH 7.31 ± 1.21, and 29.76 ± 1.51 °C water temperature). The ﬁsh were fed twice daily with commercial sinking pellets as at libitum. Sodium bicarbonate (household brand: Robertsons with 99% purity) used in this study was purchased from Shoprite holding Ltd., a local grocery store in Swakopmund, Namibia, for US$1.27/500 g (the
Table 2 Stages of anaesthesia and recovery in ﬁsh (adapted from Iwama and Ackerman, 1994; Mirghaed et al., 2016). Behaviors/response Stages of anesthesia I II III Stages of recovery I II
Fish exhibits partial loss of equilibrium Complete loss of equilibrium, and response to stimuli loss of body movement, no response external stimuli, and irregular opercular movement. Partial regain of equilibrium, no active swimming. Full equilibrium regained, and normal active swimming.
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Fig. 1. Induction time for anaesthesia (stage III) for small (A), medium (B), and large (C) Oreochromis mossambicus at diﬀerent sodium bicarbonate concentrations and temperatures. Diﬀerent lowercase letters indicate signiﬁcant diﬀerences within a temperature at each size (ANOVA; P < .05). Diﬀerent uppercase letters indicate signiﬁcant diﬀerence within a concentration at each size (ANOVA; P < .05).
Fig. 2. Anaesthetic recovery time (stage II) for small (A), medium (B), and large (C) size Oreochromis mossambicus at diﬀerent sodium bicarbonate concentrations and temperatures. Diﬀerent lowercase letters indicate signiﬁcant diﬀerences within a temperature at each size (ANOVA; P < .05). Diﬀerent uppercase letters indicate signiﬁcant diﬀerence within a concentration at each size (ANOVA; P < .05).
2.2. Statistical analyses
Whereas, the shortest induction time was recorded at the highest concentration (60 gL−1) and highest temperature (32 °C) for all sizes (small, 54.20 ± 10.85; medium, 100.80 ± 14.72; large, 107.40 ± 17.85 s) (P < .05), however, no signiﬁcant diﬀerence was presented when this concentration was compared at other temperatures (P > .05). A signiﬁcant interaction between concentration and ﬁsh size on induction time was presented (P < .05). Recovery time (stage II) for all sizes and concentrations at all temperatures is presented in Fig. 2. Fish size and sodium bicarbonate concentration had signiﬁcant eﬀect on the recovery time (P < .05). Anaesthetic recovery time increased with decrease in concentration and temperature for all sizes (R2 = 0.63), an inverse trend in relation to anaesthetic induction times. Similar to induction time, recovery time increased signiﬁcantly with increase in ﬁsh body size (P < .05). In small ﬁsh, at 60 g L−1 concentration, recovery time was 64.62 ± 3.75 s at 32 °C, which was not signiﬁcantly diﬀerent at other temperatures (P > .05). At the same temperature (32 °C) and concentration (60 g L−1), 104.60 ± 12.19 and 142.22 ± 15.22 s was recorded for medium and large ﬁsh, respectively. The shortest recovery time was recorded at 30 gL−1 and 24 °C for all sizes (small, 101.60 ± 7.20 s; medium, 89.60 ± 1216 s; large size, 55.61 ± 5.48 s). Similar to the induction times, the signiﬁcant synergistic eﬀect on the recovery time was only presented between ﬁsh
All the data were tested for normality by Shapiro-Wilk's test and homogeneity of variance by Levene's test before analysis. The eﬀects of temperature, sodium bicarbonate concentrations, ﬁsh size and their interaction on anaesthesia and recovery time were compared using both one-way and three-way ANOVA, and signiﬁcant diﬀerences among means were determined by Duncan's multiple range test. The signiﬁcance level for all analyses was set at P < .05. Statistical analyses were performed in SPSS (version 21, IBM Corp, Armonk, NY, USA). 3. Results The induction time (stage III) for all ﬁsh sizes and concentrations at all temperatures are presented in Fig. 1. Sodium bicarbonate proved to possess anaesthetic capacity in Mozambique tilapia, and its anaesthetic induction time was signiﬁcantly aﬀected by the water temperature, concentration, and ﬁsh size (P < .05). The induction time had decreased in a linear fashion with increasing temperature and concentration within a ﬁsh size (R2 = 0.92) (Fig. 1). On the other hand, the induction time increased signiﬁcantly with increase in ﬁsh size (P < .05). Additionally, within each temperature level, the induction time was longer at the lowest concentration (30 g L−1), with the longest time recorded at 24 °C for all ﬁsh size classes (small, 295.00 ± 20.65; medium, 270.04 ± 39.43; large, 619.80 ± 43.37 s) (P < .05). 3
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bicarbonate exposure is explained to be a result of high level of carbon dioxide in ﬁsh blood (hypercapnia) and low blood pH, which subsequently reduces oxygen transport to the brain (Post, 1979). During recovery in sodium bicarbonate free water, carbon dioxide in the blood reduces through diﬀusion, blood pH elevates, and ﬁsh gain its equilibrium and normal swimming (Gelwicks et al., 1998). Moreover, carbon dioxide anaesthetization has been reported to aﬀect ﬁsh size diﬀerently. The current study and that by Opiyo et al. (2013) reported induction and recovery time to be shorter in smaller ﬁsh size compared to bigger size. This could be explained by faster ventilation and metabolism in smaller ﬁsh size (Mylonas et al., 2005). In addition, carbon dioxide as an anaesthetic has been reported to successfully induce anaesthesia in ﬁsh at a wide range of temperatures (Gelwicks et al., 1998) which has been demonstrated in the present study. In conclusion, sodium bicarbonate aqueous solution especially at concentrations between 45 and 60 g L−1 can be a safe, inexpensive, readily available, and an eﬀective anaesthetic for Mozambique tilapia across diﬀerent ﬁsh body sizes, and water temperatures for short-time handling which may include minor surgery. In addition to this discovery, knowledge about the physiological eﬀects (i.e. haemato-biochemicals, histology and long-term survival) of sodium bicarbonate anaesthetic in ﬁsh is deemed necessary for better optimization. However, until sustainable ﬁsh anaesthetics become readily accessible, sodium bicarbonate can be adopted for use as a suitable anaesthetic for Mozambique tilapia.
size and concentration (P < .05). Moreover, no mortality was observed during the anaesthetic induction and recovery experiment. Similarly, no mortality or abnormal behaviors were observed within three days post recovery. 4. Discussion Sodium bicarbonate as a potential anaesthetic in ﬁsh has been studied in a numbers of ﬁsh species (Altun et al., 2009; Opiyo et al., 2013; Oberg et al., 2015; Githukia et al., 2016; Avillanosa and Caipang, 2019; Hasimuna et al., 2019). However, to the best of our knowledge, this is the ﬁrst study to assess the eﬀectiveness of this chemical at diﬀerent concentrations and temperatures on diﬀerent sizes of Mozambique tilapia. This study illustrated that sodium bicarbonate, especially at 45 and 60 g L−1, was eﬀective in inducing anaesthesia in Mozambique tilapia at all sizes (between 0.92 and 47 g) and temperatures (24, 28 and 32 °C). These sodium bicarbonate concentrations conform to the recommended criteria of an ideal anaesthetic that can be used during ﬁsh handling; anaesthetic induction and recovery time should be within 180 s and 300 s, respectively (King et al., 2005; Ross and Ross, 2008). Even though, there is no published report on anaesthetic eﬀects of sodium bicarbonate in Mozambique tilapia, our ﬁndings are comparable to other timed sodium bicarbonate anaesthetization on tilapia species reported to date. In accordance to our ﬁndings, Opiyo et al. (2013) reported the eﬃcacy of sodium bicarbonate as an anaesthetic in Nile tilapia (Oreochromis niloticus) juveniles, thereby induction and recovery time increased signiﬁcantly with increasing body weight, and its anaesthetic eﬀectiveness was more apparent at 45 g L−1 concentration and 24 °C water temperature. In red tilapia (O. mossambicus x O. niloticus), a concentration of at least 50 g L−1 was recommended to be eﬀective in anaesthetizing juveniles at water temperature ranging from of 26–29 °C (Avillanosa and Caipang, 2019). Additionally, sodium bicarbonate has been successfully used as an anaesthetic in greenhead tilapia (O. macrochir) broodstock maintained at 25 °C water temperature (Hasimuna et al., 2019). Besides tilapia species, this chemical has also been eﬀectively used in other freshwater ﬁsh species such as common carp (Cyprinus carpio) (Altun et al., 2009), and African catﬁsh (Clarias gariepinus) (Githukia et al., 2016), and in some marine ﬁsh species like red drum (Sciaenops ocellatus), southern ﬂounder (Paralichthys lethostigma), common snook (Centropomus undecimalis), and Florida pompano (Trachinotus carolinus) (Oberg et al., 2015). The anaesthetic response of sodium bicarbonate may vary across ﬁsh species depending on their biological requirements such as habitants and oxygen availability (Oberg et al., 2015). In the current study, 30 g L−1 concentration did not meet the induction time recommended for an ideal anaesthetic for ﬁsh handling; the anaesthetic induction times were greater than 180 s at all temperatures and sizes. The same was reported in O. macrochir broodstocks, thereby anaesthetic induction time was attained after 672 s at 10 g L−1 concentration of sodium bicarbonate (Hasimuna et al., 2019). A study by Altun et al. (2009) indicated that sodium bicarbonate concentrations between 0.2 and 2 g L−1 failed to anaesthetize C. carpio juveniles within the expected time. It seems evident that low sodium bicarbonate concentrations (≤ 30 g L−1), may not qualify to be used for anaesthetization in tilapia species. Optimum sodium bicarbonate anaesthetic concentrations may be species speciﬁc, because diﬀerent ﬁsh species may have diﬀerent biological requirements such as oxygen demands, as demonstrated in a study by Oberg et al. (2015). The study by Avillanosa and Caipang (2019) recommended 50 g L−1 sodium bicarbonate in freshwater as an ideal anaesthetic for red tilapia juveniles, but not the same concentration in brackishwater (20 ppt), this support the premise. Furthermore, the power for sodium bicarbonate to induce anaesthesia in ﬁsh lies in its ability to free carbon dioxide in the water, as aforementioned in this study, and its mechanism of action seems to be fairly understood. The loss of consciousness in ﬁsh following sodium
Author statement Ndakalimwe Naftal Gabriel: was the main principal investigator for this project, who was involved in the design of the experiment, procuring of the research materials, collection of data, data analysis, and manuscript drafting. Victoria Ndinelago Erasmus: was involved in the design of the experiment, and proof reading of the manuscript. Andreas Namwoonde: Was involved in data collection. Declaration of Competing Interest Authors declare that there is no conﬂict of interest. Acknowledgement The authors are appreciative for the experimental facilities provided by the Sam Nujoma Marine and Coastal Resources Research Center, Sam Nujoma Campus, University of Namibia, where the study was conducted (SANUMARC, No. 4515/2901). The authors are grateful for the support from the Sam Nujoma Campus students especially Wilhelm Haihambo (Hons. BSc student) and Lusia Kaavela and Hilma Likius (MSc. students) during data collection. The authors are thankful for the insightful comments and suggestions from the anonymous reviewers. The authors also acknowledge that experimental ﬁsh were handled in agreement with the scientiﬁc research protocols of University of Namibia (Windhoek, Namibia) and conformed to all relevant local and international animal welfare laws, guidelines, and policies. References Altun, T., Bilgin, R., Danabas, D., 2009. Eﬀects of sodium bicarbonate on anaesthesia of common carp (Cyprinus carpio L., 1758) juveniles. Turk. J. Fish. Aquat. Sci. 9, 29–31. Avillanosa, A.L., Caipang, C.M.A., 2019. Use of sodium bicarbonate as an inexpensive general anesthetic for juvenile red tilapia hybrids. Int. J. Aquatic Res. 11, 287–294. https://doi.org/10.1007/s40071-019-00235-1. Aydın, İ., Akbulut, B., Küçük, E., Kumlu, M., 2015. Eﬀects of temperature, ﬁsh size and dosage of clove oil on anaesthesia in turbot (Psetta maxima Linnaeus, 1758). Turk. J. Fish. Aquat. Sci. 15, 899–904. https://doi.org/10.4194/1303-2712-v15_4_13. Bodur, T., Afonso, J.M., Montero, D., Navarro, A., 2018. Assessment of eﬀective dose of new herbal anesthetics in two marine aquaculture species: Dicentrarchus labrax and Argyrosomus regius. Aquaculture 482, 78–82. https://doi.org/10.1016/j.aquaculture.
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