Effects of different fish sizes, temperatures and concentration levels of sodium bicarbonate on anaesthesia in Mozambique tilapia (Oreochromis mossambicus)

Effects of different fish sizes, temperatures and concentration levels of sodium bicarbonate on anaesthesia in Mozambique tilapia (Oreochromis mossambicus)

Aquaculture 529 (2020) 735716 Contents lists available at ScienceDirect Aquaculture journal homepage: www.elsevier.com/locate/aquaculture Effects of...

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Aquaculture 529 (2020) 735716

Contents lists available at ScienceDirect

Aquaculture journal homepage: www.elsevier.com/locate/aquaculture

Effects of different fish sizes, temperatures and concentration levels of sodium bicarbonate on anaesthesia in Mozambique tilapia (Oreochromis mossambicus)

T



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

Corresponding author. E-mail addresses: [email protected], [email protected] (N.N. Gabriel).

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.

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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)

24

28

32

Sodium bicarbonate concentration (g L-1)

30 45 60 30 45 60 30 45 60

Dissolved oxygen (mg L-1)

pH

Before

After

Before

After

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

2

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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