Safety of emamectin benzoate administered in feed to Nile tilapia Oreochromis niloticus (L.)

Journal Pre-proof Safety of emamectin benzoate administered in feed to Nile tilapia Oreochromis niloticus (L.) Roy Beryl Julinta, Thangapalam Jawahar Abraham, Anwesha Roy, Jasmine Singha, Avishek Bardhan, Tapas Kumar Sar, Prasanna Kumar Patil, K. Ashok Kumar

PII:

S1382-6689(20)30024-7

DOI:

https://doi.org/10.1016/j.etap.2020.103348

Reference:

ENVTOX 103348

To appear in:

Environmental Toxicology and Pharmacology

Received Date:

16 October 2019

Revised Date:

25 January 2020

Accepted Date:

29 January 2020

Please cite this article as: Julinta RB, Abraham TJ, Roy A, Singha J, Bardhan A, Sar TK, Patil PK, Kumar KA, Safety of emamectin benzoate administered in feed to Nile tilapia Oreochromis niloticus (L.), Environmental Toxicology and Pharmacology (2020), doi: https://doi.org/10.1016/j.etap.2020.103348

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Safety of emamectin benzoate administered in feed to Nile tilapia Oreochromis niloticus (L.)

Roy Beryl Julintaa, Thangapalam Jawahar Abrahama,*, Anwesha Roya, Jasmine Singhaa, Avishek Bardhana, Tapas Kumar Sarb, Prasanna Kumar Patilc, and K. Ashok

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Kumard

Department of Aquatic Animal Health, Faculty of Fishery Sciences, West Bengal University

of Animal and Fishery Sciences, Chakgaria, Kolkata-700 094, West Bengal, India

Department of Veterinary Pharmacology, Faculty of Veterinary and Animal Sciences, West

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Bengal University of Animal and Fishery Sciences, Belgachia, Kolkata-700 037, West

Animal Health and Environment Division, ICAR-Central Institute of Brackishwater

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cAquatic

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Bengal, India

Aquaculture, Raja Annamalai Puram, Chennai-600 028, Tamil Nadu, India d

Fish Processing Division, ICAR-Central Institute of Fisheries Technology, Willington

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Island, Cochin-682 029, Kerala, India

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Running title: Safety of emamectin benzoate in Nile tilapia

* Corresponding author. E-mail: [email protected] Orcid ID orcid.org/0000-0003-0581-1307

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Graphical abstract

Oral emamectin benzoate (EB)-dosing impaired the growth of Oreochromis

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

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Highlights

niloticus

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EB-residue levels were well within the permissible limit during the dosing period

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The edible tissues had traces of residues even on day 45 post-EB-dosing

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EB-dosing effected mild histopathological lesions in the kidney, liver and intestine

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Fish reverted back to near normalcy with time upon termination of oral-EB-dosing

ABSTRACT

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Emamectin benzoate (EB) premix top-coated onto feed is extensively used to treat

ectoparasitic crustacean infestations in aquaculture. This study evaluated the safety of EBdosing in Nile tilapia Oreochromis niloticus at the recommended dose and dosage of 50

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µg/kg biomass/day for 7 consecutive days (1X) and compared with control and 10 times the

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recommended dose (10X). Depletion of EB-residues in the edible muscle of 1X-dosed Nile tilapia was also studied. Mortality, behavioural changes, feed consumption, biomass, EB-

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residue depletion, and histopathological alterations in the kidney, liver and intestine were determined at slated intervals. Significant dose-dependent reduction in feed intake and

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biomass and insignificant mortalities were noted in 1X and 10X EB-dosed fish. In 1X EBdosed fish muscle, the residues peaked on day 7 EB-dosing (9.72 ng/g) and decreased

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subsequently. Nevertheless, the residue levels were within the acceptable limit of the European Commission and the Canadian Food Inspection Agency even during the EB-dosing

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period. Histologically, tubule degeneration in the kidney, mild glycogen vacuolation in the liver, and loss of absorptive vacuoles, inflammation and disintegration of the epithelial layer in the intestine of Nile tilapia fed the 1X EB-diet were observed. The fish reverted back to their normal functions with time upon termination of oral-EB-dosing. This work contributed scientific data on the safety of EB particularly on the feed intake, growth reduction, mortality, 3

histopathological alterations, and EB-residue levels in the edible tissues of Nile tilapia fed at the recommended dose and dosage, which suggested that EB-therapy might be reasonably risky in a tropical climate.

Keywords: Oreochromis niloticus, Biosafety, Emamectin benzoate-dosing, Residue depletion, Histopathology

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1. Introduction Tilapias (Oreochromis spp.) are intensively reared farmed fish worldwide with production ranging from backyard ponds to large commercial operations (Jansen et al., 2019). Nile tilapia

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Oreochromis niloticus is considered as the world's major cultured species of tilapia with

production increased from 603,034 tons in 1996 (FAO, 1998) to 1.6 million tons in 2016

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(FAO, 2018). The overcrowded situation of fish farming enhances the risk of spreading

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diseases worldwide, causing huge economic losses (Jansen et al., 2019; Julinta et al., 2019). Amid several issues that affect the production of Nile tilapia, one important factor, which is often overlooked is parasitic infestations or diseases. Argulus is one of the most damaging

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fish ectoparasites distressing the aquaculture industry from production to marketing (Poly, 2008; Kumar et al., 2016). They may parasitize both marine and freshwater fish species at

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variable temperature ranges and its intensity is higher in culture conditions than in natural

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environment (Natarajan, 1982; Poly, 2008; Woo and Buchmann, 2012). Argulus japonicus infestation on the skin of Oreochromis spp. has been well documented (Sara et al., 2013; Trujillo-González et al., 2018). Epidemics of argulosis have been reported in carp with direct effects such as dermal ulceration, osmotic imbalance, physiological stress, and immunosuppression. The total loss due to argulosis in carp culture has been assessed to the magnitude of Rs. 29,524.40 (US$ 425)/ha/year in India (Sahoo et al., 2013). The disease 4

control in aquaculture relies on an amalgamation of good management practices, use of few approved and commercially available drugs and vaccines, and prevention of infection. SLICE® (0.2% emamectin benzoate [EB]) is a novel semi-synthetic avermectin derivative, produced by the fermentation of soil bacterium Streptomyces avermitilis (MSD Animal Health, 2012), that was developed initially as an insecticide for use with food crops. Several studies have examined the efficacy of EB in USA, Canada and Europe (Roy et al., 2000; Stone et al., 2000a,b; 2002; Gustafson et al., 2006, Lees et al., 2008a,b, Saksida et al., 2010).

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Like other avermectins, emamectin interferes with neural transmissions by binding or blocking gamma-aminobutyric acid (GABA) receptors and glutamate-gated chloride

channels, causing neuromuscular paralysis (CAHS, 2007; Kumar et al., 2016; Sahoo et al.,

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2019). This product became the treatment of choice for its effectiveness against all life stages, prolonged effect, and ease of administration in the feed (Stone et al., 2000a, b). The improved

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productivity from the use of veterinary medicinal products (VMPs) in food-producing

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animals is associated with drug residues that remain in the tissues of treated animals and poses a health hazard to the consumer (Health Canada, 2013). Sensitive and selective methods are required to monitor residue levels in aquaculture species for routine regulatory

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analysis. The LC-MS/MS has become the method of choice for drug residue analysis in foodproducing animals due to its robustness, high sensitivity, selectivity, and structural

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elucidation capabilities (Sichilongo et al., 2015).

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Very limited drugs are permitted for use in aquaculture only in countries where the regulation subsists and no such regulations are prevailing in most of the developing countries including India. Except on the registration of antibiotic-free aquaculture inputs, the information on the quantities of aquadrugs used and the standard approaches to be followed for their application in aquaculture is not available in India (http://caa.gov.in). Such regulatory guidelines are necessary for successful aquaculture. It is assumed that in a tropical 5

climate the oral administration of EB may affect the safety of fish by altering the functions of the vital organs and impact the depletion of residues in the edible muscle tissues at the recommended dose and dosage. Lack of these studies and scientific data on the safety of aquadrugs particularly on EB for use with commercial freshwater finfish species in tropical conditions necessitated the exploration of such information through this study.

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2. Materials and methods 2.1. Experimental fish and design

The healthy juvenile Nile tilapia O. niloticus (11.80±1.55 cm and 14.40±1.50 g) were

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procured from Sonarpur (Lat 22°27′50.2158″ N; Long 88°23′7.4004″ E), South 24 Parganas district, West Bengal, India and transported to the laboratory in oxygen-filled bags. The fish

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were acclimated in five circular 500-L fibreglass reinforced plastic tanks for 15 days and fed

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twice daily at 2% body weight (BW) with commercial pellet feed (CP Private Limited, India). The fish, without any gross abnormality, from the reference population, were then collected, individually counted and randomly allocated among 12 rectangular study tanks (L58 cm ×

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H45 cm × W45 cm; Volume: 80L) with 30 fish each. The treatments were allotted into 3 groups, viz., group 1: 0X control feed, group 2: 1X EB-diet (50 µg/kg fish/day), and group 3:

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10X EB-diet (500 µg/kg fish/day) in quadruplicate. About 50% of the water was replaced

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weekly thrice to avoid the accumulation of excretory products. The water quality parameters were maintained optimally during the experimentation period (water temperature: 23.0-31.0 °C; pH: 7.6-8.3; dissolved oxygen: 4.1-5.6 mg/L; nitrite: 0.12-0.33 mg/L and nitrate: 0.270.51 mg/L). 2.2. Emamectin benzoate-diets preparation

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New medicated EB-diets were freshly prepared one week prior to the dosing period, based on the estimated average weight for all fish per tank. The inclusion rate for the emamectin benzoate (Sigma-Aldrich, India: Cat no 31733-250MG) was calculated to deliver an approximate dosage of 50 µg active ingredient/kg biomass/day for 7 consecutive days. Initially, EB stock solution was prepared by the addition of 50 mg EB powder in 5 mL ethyl acetate and 50 µL tween 80 and vortexed for 5 min (Feng et al., 2016). The top coated 1X and 10X EB-diets were prepared by mixing the required volume of EB suspension/kg feed

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with 5.0 mL vegetable oil as the binder. Medicated diets were prepared in order of increasing EB concentration. The feeds were air-dried overnight before being placed into plastic sealable containers. The control diet did not contain any medication but was coated with vegetable oil

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to minimize variation between the feeds. Feeding rates and methods were identical for control and dosing groups during the trial. The experimental protocols were approved by the Indian

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Council of Agricultural Research, Government of India, New Delhi under the All India

legal requirements of India.

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2.3. Dose administration

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Network Project on Fish Health and fulfilled the ethical guidelines including adherence to the

The 59-day study included 7-days acclimation (pre-dosing), 7-days EB-dosing, and 45-days post-EB-dosing periods. During the pre-dosing period, the fish groups were fed with

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a control diet. During the dosing period, EB-diets were administered to the respective groups

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and the control diet was administered to the control group. During the post-dosing period, the control diet was administered to all the tanks. The feed ration, 2.0% of mean fish BW, was divided into three equal portions/day and the feed consumption was determined daily. 2.4. Necropsy and histopathology The fish were euthanized using clove oil (100 µL/L) and examined for gross lesions. After necropsy, kidney, liver and intestine samples of EB-fed fish on day 0 and day 7 EB7

dosing were immediately fixed in Bouin's solution for a minimum of 24 h. The fixed samples were dehydrated by a series of increasing concentrations of ethanol solutions, followed by paraffin wax impregnation. Paraffin blocks were sectioned 5μm thick on a microtome and resultant sections were stained with hematoxylin and eosin (Roberts, 2012). The histopathology scores of Bowker et al. (2013) was followed to quantify the extent of tissue damage, i.e., 0 as no change, 1 as normal (<5% tissues affected), 2 as mild (5-15% tissues affected), 3 as moderate (15-25% tissues affected), 4 as marked (25-50% tissues affected) and

2.5. Emamectin benzoate-residue analysis by LC-MS/MS

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5 as severe (>50% of tissues affected).

2.5.1. Collection of edible muscle, and extraction and purification of emamectin benzoate

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The samples for EB-residue analysis were collected only from the control and 1X EBfed groups. Two fish were randomly picked from each experimental tank at each sampling

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time (day 0, day 3 EB-dosing, day 7 EB-dosing, day 3, 7, 14, 28 and 45 post-EB-dosing). The

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selected fish from the test tanks were euthanized using clove oil (100 µL/L), dissected, beheaded, degutted, washed thoroughly, packed in polythene bags separately and stored at 20°C for further analysis by LC-MS/MS. The extraction and purification of EB from the fish

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homogenates were as per Dong et al. (2015) that was amended slightly by the ICAR-Central Institute of Fishery Technology, Cochin, India. In brief, 5 g of thawed fish muscle tissue was

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spiked with 12.5, 15 and 30 µg/kg EB and transferred to a 50-mL polypropylene centrifuge

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tube. Then 10 mL of 1% acetic acid (CH3COOH) in acetonitrile (ACN) was added to the homogenized portion and vortexed. To which added was 1.5 g of sodium acetate (C2H3NaO2) and 4 g of magnesium sulphate (MgSO4) and vortexed. The supernatant was then collected and passed through a 2 mL DISQUE tube, which was then vortexed and centrifuged at 540 x g for 4 min. The supernatant fluid was collected and evaporated to dryness using N2 evaporator at 45ºC. To which added was 2 mL of 50% ACN filtered through 0.2 PTFE 8

syringe filter and then transferred to LC-MS/MS vials. The extracts, approximately 450 μL in volume, were received into an autosampler vial for LC-MS/MS analysis. All blanks reagent and sample blanks were spiked with a known amount of internal standard before extraction. 2.5.2. Liquid chromatography Five micro-litre of the concentrated extract was analysed using LC-MS/MS system (ABSCIE 4000 QTRAP®; MS-Applied Biosystem/MDS/Sciex, API-3000), with data acquisition and processing performed by the data analyst software (version 1.4.2). The mass

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spectrometry (MS) detector was configured as positive electric spray ionization (ESI+) with the multiple reaction monitoring (MRM) modes. Chromatographic separation was achieved on a Kinetex® 2.6 µm C18 100 Å (LC Column 1500 × 2.1 mm) analytical column

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(Phenomenex) at room temperature on a reversed-phase column with an organic mobile

phase. The different gradient mobile phase compositions of 5 mM ammonium formate in

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water containing 0.1% formic acid (A) and 5 mM ammonium formate in methanol/

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acetonitrile containing 0.1% formic acid (B). The gradient programme consisted of 90A:10B to 90A:10B (0.1–0.5 min), 90A:10B to 40A:60B (0.6–3.0 min), 40A:60B to 10A:90B (3.1–

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10.0 min), 10A:90B to 10A:90B (10.1–12.0 min), 10A:90B to 90A:10B (12.1–13.0 min), 90A:10B to 90A:10B (13.1–15.0 min). The flow rate was 0.8 mL/min with a column temperature of 30 ºC. The mass spectrometer MASSLYNX 4.0 software calculated the

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results automatically. The calibration curves were constructed using the best fit of two

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replicated determinations per concentration level for the calculation of the residue values when the correlation coefficient was at least 0.9. 2.5.3. Validation

The entire procedure was validated for selectivity, sensitivity (limits of detection and quantification), accuracy, precision, recovery and robustness according to 2002/657/EC Decision (European Commission, 2002). 9

2.6. Statistical analyses The data were expressed as a mean±standard deviation. The feeding behaviour data were analyzed by non-parametric Kruskal–Wallis test with pair-wise comparisons. The data on mortalities and biomass were analyzed by one-way ANOVA and Tukey HSD post-hoc for the comparison of means. All the statistical analyses were done using Statistical Package for Social Sciences (IBM-SPSS) Version: 22.0, considering a probability level of P < 0.05 for the significance of the collected data. Linear regression analysis was done to estimate the

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elimination period in Microsoft Excel 2010 package. 3. Results

3.1. General and feeding behaviour of emamectin benzoate-fed Nile tilapia

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The Nile tilapia juveniles were distributed throughout the water column of the tanks

during the study period. Abnormal behaviour was not observed during the acclimation period.

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No gasping, loss of equilibrium, and unusual behaviour were observed during the dosing and

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post-EB-dosing periods. Increased rate of subdued behavioural responses including lying at the tank bottom, no curiosity in feeding during the EB-dosing and early part of post-EBdosing periods were noted in the 10X EB-dosing groups. A few fish of this group appeared

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dark in colour with discoloured intestine, inflamed kidney and spleen during the dosing regimen, which abated during the post-EB-dosing period with time. The results of the feeding

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behaviour score are tabulated in Table 1. There were no observations of inactive feeding in

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the control group all through the experimentation. The belligerent feeding was lacking and the feed intake rating of the 1X group was 3.57±0.53 (mean±standard deviation of 7 days of EB-dosing), a reduction of 10.75% during the dosing period. During the pre-dosing and postEB-dosing periods, the feed consumption was normal (score: 4.00). The feed intake of 10X group slashed with the mean score of 2.86±0.69 (mean±standard deviation of 7 days of EBdosing), a reduction of 28.50% during the dosing period. The decreased feed intake was also 10

noted during the early post-EB-dosing period, which subsequently increased to 3.96±0.21 (mean±standard deviation of 45 days of post-EB-dosing). The reduction in the biomass of 1X and 10X EB-fed fish groups compared to the control group were 6.91 and 12.18%, respectively. 3.2. Mortalities in emamectin benzoate-fed Nile tilapia The mortalities recorded in Nile tilapia juveniles fed EB-feeds for 7 consecutive days are presented in Fig. 1. There were no mortalities during the pre-dosing period. At the

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completion of 7-day EB-dosing regimen, 2.23% and 4.43% mortalities were noted in 1X (50 µg/kg fish/day) and 10X (500 µg/kg fish/day) groups, respectively. No significant differences existed in the mortalities among the three groups.

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3.3. Residue depletion in emamectin benzoate-fed Nile tilapia

The chromatograms of EB-residues in the edible muscle tissues of control and 1X

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EB-diet fed fish are presented in Fig. S1a-f. No residues were found in the control group. The

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EB-residues present in the edible muscle of 1X EB-diet fed Nile tilapia were 0, 3.66, 9.72, 6.57, 4.17, 0.51, 0.20 and 0.022 ng/g, on day 0, 3 and 7 EB-dosing, and day 3, 7, 14, 28 and 45 post-EB-dosing, respectively (Fig. 2). Traces of EB-residues in nanogram levels were

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noted till day 45 post-EB-dosing. However, the linear regression analysis ensued complete elimination of residues on the 41st day of cessation of EB-dosing based on the LC-MS/MS

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residue accrual and depletion data.

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3.4. Histopathology

The kidney of control diet-fed fish had normal tissue architecture with no pathological

abnormalities (Fig. 3a). The 7 days of 1X EB-dosing caused the disintegration of renal tubules, dilated Bowman’s space and regenerating tubules in the kidney (Fig. 4a). Manifestations like shrunken glomerulus with dilated Bowman’s space, fragmented glomerulus, the disintegration of renal tubules and necrotised renal interstitium were noted in 11

the kidney tissues of 10X EB-fed Nile tilapia on day 7 (Fig. 4b). The liver tissue of control fish exhibited regular hepatocytes in cords and plates with the normal portal tract and central vein (Fig. 3b). The histological sections of the liver of 1X (Fig. 5a) and 10X EB-fed (Fig. 5b) Nile tilapia revealed mild to moderate glycogen vacuolation and degeneration of the hepatocytes. The histological sections of the control fish intestine showed normal distal intestine, characterized by the presence of epithelial layer comprising mainly absorptive vacuoles and

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mucus-secreting or goblet cells and lamina propria (Fig. 3c). The 7 days of 1X EB-dosing documented inflammation and disintegration of the epithelial layer, loss of absorptive

vacuoles and deeply stained lamina propria in the intestine (Fig. 6a). Evident changes

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including necrosis in the intestinal villi, mucinous degeneration, deeply stained lamina

propria, inflammation and degenerated epithelial layer and necrotised absorptive region were

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observed in the intestinal tissues of 10X EB-fed Nile tilapia on day 7 (Fig. 6b).

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4. Discussion

In commercial aquaculture, obliteration of Argulus infestations has been challenging even with the use of chemicals, viz., sodium chloride, formaldehyde, potassium

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permanganate, formalin, trichlorfon, EB, powdered quick lime, etc (Singhal et al., 1986; Wildgoose and Lewbart, 2001; Tam, 2005; Kumar et al., 2016). As on date, no approved

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drugs are available for the treatment and control of crustacean ectoparasites including

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Argulus spp. However, in salmon farming two organophosphates: dichlorvos and azamethiphos; three pyrethrine: pyrethrum, cypermethrin and deltamethrin; an oxidizing agent: oxygen peroxide; three avermectins: ivermectin, EB and doramectin; and two urea derivatives (benzoyl phenyl ureas): teflubenzuron and diflubenzuron are used against the salmon louse (Roth, 2000). Also, optimization of the existing treatment methods such as the use of formalin (300 mg/L), sodium chloride (20 g/L), peracetic acid (Perotan®, 40 mg/L), 12

chlorinated lime (20/30/40 mg/L), chloramine T (40 mg/L), hydrogen peroxide (40 mg/L) and Aquahumin (300 mg/L) against A. foliaceus infestations in carp and trout was documented (Weisman et al., 2004). Many of these chemical compounds are unapproved, hazardous to the operator and the fish, or simply ineffective. For use in freshwater tropical fish, data must be engendered to demonstrate that the standard EB-treatment regimen (50 μg/kg fish/day) administered for 7 consecutive days is not only effective but also safe for application to the representative target animals. In an earlier

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study, the licensed oral treatment product, Slice® (0.2% EB) for lice in salmonids was reportedly effective in controlling the experimentally induced Argulus infestation in koi carp (Cyprinus carpio) at 5 μg/kg fish/day and goldfish (Carassius auratus auratus) at 50 μg/kg

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fish/day (Hanson et al., 2011). In the present study, the EB-dosing at the recommended dose (50 µg/kg fish/day) did not cause loss of equilibrium, gasping, flashing and hyperactivity. In

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contrast, the 10X EB-dosed Nile tilapia exhibited abnormal swimming behaviour, darkened

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body colour, unusual feeding behaviour like gulping and expelling the EB-diet once it senses the drug and alterations in the internal organs. Similarly, dark colouration, abnormal swimming behaviour, and inappetence were reported among fish that were exposed to EB-

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doses (Roy et al., 2000; Stone et al., 2002). Stone et al. (2002) noted darker colouration amongst 50% of the smolts that received high EB-dose, and uncoordinated swimming

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behaviour in 1% of the smolts. Likewise, Gaikowski et al. (2013) observed discolouration on

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the external surfaces of Nile tilapia fed antibiotic, florfenicol. Discrete variation in feed intake, during the dosing period, is inevitable in the

voluntary feeding studies. It has been predicted that diminished feed consumption is the first sign of intoxication to avermectins, which confines further toxic effects (Bowker et al., 2013). While there was an apparent trend indicating a decrease in feeding with increasing EB dose in the feed, the Nile tilapia still appeared to consume most of the feed offered. The 13

reduced feed consumption (10.75%) as indicated by the feed intake rating score during the dosing period signified that EB toxicity may minimize the aggressive feeding activities of Nile tilapia. During the latter part of the post-EB-dosing period, no abnormalities were observed and all fish groups behaved almost normal. One concern with administering topcoated, medicated feed to fish is the potential for the drug to leach into the water. As of now, no reports of EB leaching from top-coated feeds are available unlike antibiotics (Yanong et al., 2005).

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The mortalities were observed only during the EB-dosing period in Nile tilapia fed 1X and 10X-doses, with the highest being at 10X-dose (4.43%), possibly due to the EB toxicity. Likewise, in an experiment with Atlantic salmon, Roy et al. (2000) noted meagre mortality in

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high-dose exposure group at day 2 post-exposure. Yet, the mortalities observed in the present study were higher than those observed in temperate fish (Roy et al., 2000), possibly due to

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the increased EB toxicity in a tropical climate. According to Hentschel et al. (2005), any drug

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at a higher concentration than its allowable limit turns out to be toxic to the host organism and results in toxicity reaction. Our results on the increased mortality with the increase in EB concentrations uphold their observations. The observed changes in the internal organs such as

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inflamed kidney and spleen probably indicated the concern of EB toxicity upon oral-dosing at 10X concentration and the probable adaptive changes and tissue alterations in Nile tilapia

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exposed to EB as confirmed by histopathology. During the post-EB-dosing period, all fish

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groups resumed normal feeding and regained growth. The consequences of the dosing trials with EB-diets at different concentrations and the reluctance to feed the EB-diet during the dosing period highlights the critical importance of proper monitoring of fish health and early intervention with oral administration of EB. In the European Union, the maximum residue limit (MRL) of 100 μg/kg emamectin B1a in the edible tissue of farmed salmonids has been established (EMEA, 2003). The 14

maximum residue level for fish intended for human consumption established by the Veterinary Drugs Directorate (VDD) of Health Canada and Canadian Food Inspection Agency (CFIA) is 100 ng/g for muscle and 1000 ng/g for skin (Health Canada, 2017). Even with the reduced feeding response, the experimental Nile tilapia fed the 1X EB-diet for 7 consecutive days recorded residues at levels in the range of 0-9.72 ng/g, well within the permissible limit. The guidelines for MRL were set at 42 ng/g and salmon were not allowed to be harvested for the food chain for 68 days following the last SLICE® treatment at water

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temperatures of 5°C or greater (ACRDP, 2013). The MRL at this level was not detected in our study with Nile tilapia grown at water temperatures in the range of 24.0-31.0°C, thus,

specifying that the actual residue levels of EB did not exceed the set limits (ACRDP, 2013).

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Nonetheless, a negligible level of residues (0.022 ng/g) was noted in Nile tilapia on the 45th day of cessation of EB-dosing, thus demonstrating the long-term persistence of drugs in

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tissues. The observed highest residue level of 9.72 ng/g instantly after the 7th day of EB-

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dosing was much lower than the MRLs set by various organizations (ACRDP, 2013; EMEA, 2003). In contrast, Whyte et al. (2011) recorded the EB-residue concentrations measured in the muscle tissues of Atlantic salmon ranged from 60.5 ng/g at day 1 post-treatment to 7.3

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ng/g at day 45 post-treatment. Interestingly, both in temperate and tropical climates, residues were detected even after 45 days of cessation of EB-dosing. Most international aquaculture

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jurisdictions that have similar restrictions set higher MRLs (e.g., 100 ng/g) and shorter

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withdrawal periods, e.g., 0 day in UK and Chile, and 7 days in Norway (ACRDP, 2013). As revealed by the linear regression analysis, elimination of EB-residues in edible muscle tissue could be achieved approximately on the 41st day of cessation of EB-dosing. Histopathological investigations have long been accepted to be reliable biomarkers of stress in fish for several reasons (van der Oost et al., 2003). Kidneys are quite susceptible to toxic injury as they are exposed directly to the blood plasma via open fenestrae of their 15

glomerular capillaries (Sanchez et al., 2001). Mild histopathological changes (5-15% of the tissues affected) comprising disintegration of renal tubules, dilated Bowman’s space and regenerating tubules were observed on day 7 of 1X EB-dosing. Similarly, inflammation of Bowman's capsule and induced degenerative changes in renal tubules in carbofuran exposed kidney were reported (Sastry and Sharma, 1979). The degenerative process can lead to tissue necrosis in more severe cases (Hibiya, 1982). But, the existence of tubule degeneration together with lack of necrosis in the 1X EB-dosed Nile tilapia kidney indicated that the

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kidney bared damage after exposure to EB, but the diminutive dosage may have prevented the establishment of necrosis in this organ. Contrarily, the degree of kidney damage was

observed mild to marked (5-50% tissues affected) with shrunken and fragmented glomerulus,

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the disintegration of renal tubules with necrotised renal interstitium in 10X EB-dosed Nile

tilapia, which indicated a severe EB-toxicity. Alike, shrinkage in glomerulus was documented

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in Channa striatus (Arora and Kulshrestha, 1984) and Anabas testudineus (Bakthavathsalam

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et al., 1984) when exposed to pesticides. Also, the absence of severe changes in the histological sections of the kidney of 1X EB-dosed Nile tilapia could be as a result of tolerability of the EB-diet to the fish kidneys. Hinton and Laurén (1990) and Cormier et al.

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(1995) reported an increase in the frequency of new nephrons and regenerated tubules, during the process of the retrieval of the damaged kidney in fish. Likewise, the EB-dosed rainbow

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trout fingerling at 150 μg/kg fish/day exhibited posterior kidney regenerating tubules

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(Bowker et al., 2013). The histopathological observations demonstrated that the low EBdosage (50 μg/kg fish/day) initiated the tubule regeneration process in 7 days of EB-dosing. While, the previous studies documented that this phenomenon may take 2-4 weeks after exposure to the stressor (Reimschuessel, 2001), and could even take 2 months to complete (Gernhofer et al., 2001).

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The histopathology of the liver of Nile tilapia fed the 1X EB-diet showed mild glycogen vacuolation. The 10X EB-diet fed Nile tilapia showed moderate glycogen vacuolation and degeneration of the hepatocytes. The presence of glycogen vacuolar degeneration of hepatocytes in Nile tilapia fed the 10X EB-diet may be as a result of excessive exertion required by the fish’s liver to get rid of the EB-toxicant from its body during the process of detoxification. Likewise, liver degeneration and vacuolation were observed in rainbow trout fingerling fed 150 μg EB/kg fish/day (Bowker et al., 2013) and

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Carassius auratus gibelio fed trichlorfon at 1 and 2 g/kg (Lu et al., 2018). Similarly, diffuse hydropic degenerations and vacuolations in the hepatocytes were identified in Nile tilapia

exposed to fipronil, a new broad-spectrum phenylpyrazole insecticide (El-Murr et al., 2015).

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Vacuolar degeneration is a reversible injury, and cells can recover their normal functions

(homeostasis) when the stress is removed (Szende and Suba, 1999). Yet, the reclamation of

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cells will depend on the severity and duration of exposure to the stressors. Conflictingly, no

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pathognomonic signs of EB-toxicity were identified during gross necropsy or histopathological examination in Atlantic salmon and rainbow trout (Roy et al., 2000). The histopathological changes in the intestine may vary depending on the

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experimental species and feeds. The intestine of the control Nile tilapia was characterized by the presence of well-differentiated enterocytes with many absorptive vacuoles and goblet

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cells, which secrete mucus to lubricate and defend the intestinal mucosa against physical,

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chemical and microbial destruction. The presence of distinctive simple columnar epithelium in association with mucus-producing goblet cells in the post-gastric intestinal mucosa has been observed in many fish (Olufemi et al., 2013). The lamina propria helps in contractile activity, liberating the glandular/foveolar secretions into the gastric lumen and indirectly averts the occlusion of the foveolae by ingested food. The connective tissue components of the lamina also contain presumed immune-competent cells (Osman and Caceci, 1991). The 17

present study documented inflammation and disintegration of the epithelial layer, loss of absorptive vacuoles and deeply stained lamina propria on day 7 of 1X EB-dosed Nile tilapia intestine. In contrast, moderate and marked morphological changes including necrosis in the intestinal villi, mucinous degeneration and necrotised absorptive region were observed in 10X EB-dosed Nile tilapia. The intestine of Nile tilapia subjected to 0.014 mg/L of fipronil for 4 days revealed mucinous degeneration (El-Murr et al., 2015) similar to those observed in the present study. Earlier studies indicated epithelial degeneration and inflammatory cells

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infiltration in submucosal oedema in Puntius gonionotus intestine treated with diazinon and sumithion (Hoque et al., 1993) and tilapia exposed to carbofuran (Soufy et al., 2007). The

histopathological alterations observed in the intestinal layers of the present study may be due

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to the direct consequence of EB, thus, revealing the fact that EB even at the recommended

dose can cause apparent pathological changes in the intestine of Nile tilapia. Nevertheless,

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the effects of EB on the vital organs of Nile tilapia are reversible upon discontinuation of EB-

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dosing. 5. Conclusion

The aim of this research was to demonstrate the effect of oral EB-dosing in apparently

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healthy Nile tilapia, that are being cultured in over 135 tropical and sub-tropical countries. The observed mortalities (2.23%), reduction in feed intake during the dosing period

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(10.75%), negligible levels of EB-residues even after 45 days of cessation of EB-dosing and

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mild histopathological alterations in the kidney, liver and intestine at the recommended dose and dosage in Nile tilapia suggested EB might be reasonably risky in a tropical climate and caution must be exercised before its choice for use as a fish anti-parasitic agent. The overall impact of EB on the kidney, liver and intestine may seriously affect the metabolic as well as physiologic activities and could impair the growth and production of Nile tilapia.

18

Statement of relevance  Use of drugs in aquaculture has been consistent with their negative effects on aquaculture species  Studies on the efficacy and safety of antiparasitic drugs particularly emamectin benzoate (EB) on tropical fish are scanty  In this study, drug residues were detected in the edible tilapia tissues even on day 45 post-EB-dosing

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 Compliance on the EC and Canadian regulations even during the dosing period could

Conflict of interest

Declaration of interests

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We declare no conflict of interest.

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be a useful tool for the researchers on aquadrugs and policymakers

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The authors declare that they have no known competing for financial interests or personal relationships that could have appeared to influence the work reported in this paper. Funding

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The work was supported by the Indian Council of Agricultural Research, Government

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of India, New Delhi under the All India Network Project on Fish Health (Grant F. No. CIBA/AINP-FH/2015-16 dated 02.06.2015). Acknowledgements The authors thank the Vice-Chancellor, West Bengal University of Animal and Fishery Sciences, Kolkata for providing necessary facilities to carry out the work.

19

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https://doi.org/10.1577/H03-065.1

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Figure 1. Mortalities in emamectin benzoate (EB)-dosed Oreochromis niloticus at the recommended dose (50 µg/kg fish/day) and ten times the recommended dose (500 µg/kg

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fish/day) for 7 consecutive days.

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Figure 2. Emamectin benzoate (EB) residues depletion in the edible muscle tissues of

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Oreochromis niloticus fed 50 µg/kg fish/day for 7 consecutive days.

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Figure 3. Histology of non-medicated feed fed Oreochromis niloticus showing [a] regular epithelial cells with no pathological abnormalities in the kidney, [b] regular hepatocyte in cords and plates with the normal portal tract and central vein in the liver and, [c] epithelial layer (E), absorptive vacuoles (AV) mucus-secreting or goblet cells (GC) and lamina propria

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(LP) in the intestinal tissue, X200 H&E staining.

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Figure 4. Histological changes in the kidney of EB-dosed Oreochromis niloticus for 7 consecutive days [a] at the recommended dose (50 µg EB/kg fish/day) showing the

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disintegration of renal tubules (DR), dilated Bowman's space (DB) and regenerating tubules (RT), and [b] at ten times the recommended dose (500 µg EB/kg fish/day) showing shrunken

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glomerulus (SG) with dilated Bowman’s space (DB), fragmented glomerulus (FG), the

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staining.

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disintegration of renal tubules (DR) and necrotised renal interstitium (NRI), X200 H&E

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Figure 5. Histological changes in the liver of EB-dosed Oreochromis niloticus for 7 consecutive days [a] at the recommended dose (50 µg EB/kg fish/day) showing mild glycogen vacuolation (V) of the hepatocytes, and [b] at ten times the recommended dose (500 µg EB/kg fish/day) showing moderate glycogen vacuolation (V) and degeneration (D) of the

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hepatocytes, X200 H&E staining.

Figure 6. Histological changes in the intestine of EB-dosed Oreochromis niloticus for 7

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consecutive days [a] at the recommended dose (50 µg EB/kg fish/day) showing inflammation (I) and disintegration of the epithelial layer (DE), loss of absorptive vacuoles (LAV) and

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deeply stained lamina propria (DSL), and [b] at ten times the recommended dose (500 µg

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EB/kg fish/day) showing necrosis in the intestinal villi (NIV), mucinous degeneration (MD), deeply stained lamina propria (DSL), inflammation (I) and degenerated epithelial layer (DE)

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and necrotised absorptive region (NA), X200 H&E staining.

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Table 1 Feeding behaviour rating scores of Oreochromis niloticus juveniles fed with emamectin benzoate (EB) diets at the recommended dose (50 µg/kg fish/day) and 10 times the recommended dose (500 µg/kg fish/day) for 7 consecutive days Period

EB ration: µg/kg fish/day 50 µg (1 X)

500 µg (10 X)

Pre-dosing

4.00

4.00

4.00

(0-7 days)

(4.00±0.00)

(4.00±0.00)

(4.00±0.00)

EB-dosing

4.00

3.00 - 4.00

2.00 - 4.00

(8-14 days)

(4.00±0.00)

(3.57±0.53)*#

Post-EB dosing

4.00

4.00

(15-59 days)

(4.00±0.00)

(4.00±0.00)

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0 µg (0 X)

(2.86±0.69)*# 3.00 - 4.00

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(3.96±0.21)

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* P < 0.05 comparing the pre-dosing and post-EB-dosing periods of a particular treatment. # : P < 0.05 comparing exposure to control (0 X). Data are expressed as Mean ± standard

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deviation. •

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