The study of parasitic diseases and their controlling agent is vital for the understanding of effectiveness and usage of the drug to fish. A parasite not only affects the fish organs, but also, it can alter the behavior of host and become resistant to drugs that contribute to its survival within the host (Zander, 2013). The increased prevalence of parasitic disease in aquaculture is exacerbated by greater globalization of the trade in aquatic animals and their products (Bondad-Reantaso et al., 2005) and the other risks of translocation and establishment of parasite into new areas. The primary infection of fish ectoparasites (Argulus spp., sea lice, Ichthyophtherius sp) may further open new doors for the secondary bacterial or viral infections, which can be more lethal to affect fish populations (Tully and Nolan, 2002). These parasitic diseases have caused tremendous economic loss in European aquaculture including fish population of both farmed and wild region. There was a loss due to parasitic infection of about US$ 480 million per year in the world salmon industry in North America (Shinn et al., 2015) and about US$ 615.0 per hectare for 1 year in carp farms in India (Sahoo et al., 2013). The economic loss assessment data showed that reduced growth rate, carp mortality and drug expense for controlling argulosis contributed to 8%–10% of total economic loss in Indian carp farming.

To control parasitic diseases such as fish louse (Argulus spp.), gill flukes (Dactylogyrus sp.), Myxobolllus sp., ich (Ichthyophtherius sp.) and gill maggot (Ergasillus sp.), a wide variety of drugs are available in the market and are frequently used by fish farmers. But there are very less approved drugs for parasitic diseases in many countries including India. Due to lack of licensed antiparasitic drugs and their unregulated use in aquaculture has led to the situation of drug resistant parasitic populations (Shinn and Bron, 2012). As per the record of globally used chemotherapeutic agents, which are being used in salmon farming, the eleven active ingredients can be divided into five types of pesticides, which include two urea derivatives (benzoylphenylureas): teflubenzuron and diflubenzuron; two organophosphates: dichlorvos and azamethiphos; three avermectins: ivermectin, emamectin benzoate and doramectin; an oxidizing agent: oxygen peroxide and three pyrethrine: pyrethrum, cypermethrin and deltamethrin (Roth, 2000). In India, EB, Nuvan, Butox Vet, Cliner, Ectodel (2.8%), Hitek Powder and Paracure-IV are commonly used drugs to control parasitic diseases in aquaculture (Mishra et al., 2017). Among different antiparasitic formulations, emamectin benzoate is considered as safe and an effective in-feed anti-parasitic agent for controlling parasitic infestations both in marine and freshwater-reared fish and has been approved by the United States Food and Drug Administration (USFDA) and European Medicines Agency (EMA). It is a semi-synthetic derivative of a chemical produced by the bacterium, Streptomyces avermitilis. It belongs to a class of compounds called avermectins, which are commonly used as an anti-parasitic agent against internal and external parasites in a variety of host species, particularly mammals. It consists of two chemicals with a similar structure (Supplementary Data S1: Figure 1: Chemical structure of emamectin benzoate designed by ACD/ChemSketch) mixed with minimum of 90% 4″ -epimethylamino-4″-deoxyavermectin B_1a and a maximum of 10% 4″-epi-methylamino-4″-deoxyaverrnectin B_1b benzoate (Leibee et al., 1995). The molecular formula of EB is stated in the form of Emamectin B1a: C49H75NO13 and Emamectin B1b: C48H73NO13. The mechanism of action of emamectin is similar to other available avermectins. The binding of emamectin with gamma-aminobutyric acid (GABA) receptors and glutamate-gated chloride channels leads to its blockage which leads to neuromuscular paralysis of parasites (Roberts and Hutson, 1999; CAHS, 2007). In 2000, for the first time, EB was registered as an antiparasitic veterinary drug in the United Kingdom for its application in temperate aquaculture and used as a feed additive in a product formulation under the trade name Slice (Schering-Plough Animal Health) (Singha et al., 2022). The European Medicines Evaluation Committee (EMEC) has set the maximum residue limit (MRL) of EB 100 μg/kg in muscle and fillet and that is considered safe for the human consumption. In the past few years, EB has been used as medicated feed in all jurisdictions. In fact, EB is the mostly used product in Canada (under Emergency Drug Release) and the US (Investigational New Animal Drugs-INAD) for control of parasitic infestations in fish (Chowdhury et al., 2012). Considering, the enhanced attention towards use of EB by global aquaculture practitioners, the safety study is need to be carried out in different fish species and climates in order to combat the parasite resistance towards this drug, toxicity and unregulated use in fish farms. The biosafety and residue depletion studies have been carried out in fish of different climatic region using recommended dose i.e. 50 μg/kg fish per day fed for 7 consecutive days. The toxicity of EB on rainbow trout and Atlantic salmon has been studied by various researchers in different regions and habitats (Roy et al., 2000; Stone et al., 2000; Stone et al., 2002). There are reports of EB toxicity and biosafety in common carp (Braun et al., 2008), Asian Seabass (Raja et al., 2020), Labeo rohita (Choudhary et al., 2022; Kumar et al., 2022) and Nile tilapia (Julinta et al., 2020) from Indian freshwater and brackishwater aquaculture system.

Golden mahseer (Tor putitora) is widely distributed in South and South-East Asia and regarded as an important food, game and sport fish in Afghanistan, Bangladesh, Bhutan, India, Iran, Myanmar, Nepal, Pakistan, Thailand and Sri Lanka (Islam and Tanaka 2007). The prevalence of fish ectoparasites such as Argulus sp. And Trichodina sp. Are reported in mahseer from Indonesia and India (Mallik et al., 2010; Muchlisin et al., 2014), where infection rate was found to be around 50% of the affected fish populations. Myxozoan infection by parasite Myxobolus tambroides, a species novel, was reported in Malaysian mahseer, Tor tambroides from Tasik Kenyir Reservoir, Malaysia (Székely et al., 2012). But, there is hardly any information available on application of EB for the control of parasitic infection, biosafety and tissue depletion in golden mahseer. Even, information is scanty on drug tolerance of fish to the higher concentrations of EB administration for an extended medication period of 21 days (three times the recommended duration of EB application). Considering the above information, the present experimental trial is aimed to determine the effect of EB on biosafety, toxicity, residue depletion and drug tolerance in juvenile golden mahseer as a model candidate coldwater fish species for sport fishery and conservation. The experiment is conducted through an extended medicated feeding of graded doses of EB to golden mahseer for 21 consecutive days at 1 × (50 μg/kg fish/day), 2 × (100 μg/kg fish/day), 5 × (250 μg/kg fish/day) and 10 × (500 μg/kg fish/day) concentrations. The study is also intended to improve and fills the knowledge gap on the correct usage of this drug in coldwater aquaculture system and consumption of fish meat after the drug withdrawal.

Prophylactic use of antibiotics is rare in aquaculture, while the metaphylactic use includes the practice of treating an entire population of fish orally in toto even if only a small percentage of the animals are affected. It is considered as a common practice in aquatic farms. In commercially produced fish species, drugs are often added to the feed to reduce handling stress, and to protect the fish health. The current model of integrated feed therapy is based on the assumption that the distribution of medicated feed is uniform to ensure that all the fish are receiving the appropriate dose of the drug (Whyte et al., 2011). The application of EB has always been preferred over the other chemicals and drugs as an antiparasitic drug. It is reported that hydrogen peroxide (H₂O₂), the only chemical used for the treatment of sea lice infestation, found poor in its efficacy against sea lice juvenile in British Colombia (Riccardo et al., 2007) as compared to EB (Lam et al., 2020). Among other veterinary drugs, though dichlorvos, azamethiphos, pyrethrum, cypermethrin, deltamethrin, doramectin, diflubenzuron and diflubenzuron were in application to treat crustacean ectoparasites infestation in marine fish (Horsberg, 2012; Hannisdal et al., 2020), the EB was registered as an antiparasitic veterinary drug for temperate water aquaculture in the United Kingdom in 2000 (Singha et al., 2022) best on its efficacy over other chemicals and veterinary drugs.

The present study is the first report on EB toxicity and residue depletion from muscle in golden mahseer juveniles fed with graded doses of EB viz., 1× (50 μg/kg fish/day), 2× (100 μg/kg fish/day), 5× (250 μg/kg fish/day) and 10× (500 μg/kg fish/day) for 21 consecutive days. In this study, the EB was administered through medicated feed and the toxicity was assessed by survivability, feeding, behavioral characteristics and histopathological changes in the intestine, liver, kidney and muscle tissues of golden mahseer.

The ad libitum feeding was followed to ensure the maximum feed uptake by the golden mahseer juveniles and avoiding leaching into water. In an EB medicated trial on L. rohita, fishes appeared to have consumed the feed within 15–30 min of receiving the medicated diet which indicates low or undetectable amount of EB leaching into the water (Choudhary et al., 2022). However, it was observed that the feed intake by golden mahseer was not influenced by 1× and 2× EB treatments, but there was a considerable decrease in the 5× and 10× EB treated fish, even after the medication period was over. The EB medication has no impact on the palatability of the feed since there was no difference in feed consumption between the non-medicated control group and fish medicated at recommended dose as reported in Salmon (Armstrong et al., 2000) and Asian seabass (Raja et al., 2020). The nile tilapia fed with EB diet at the prescribed dose of 50 μg/kg fish/day for 7 consecutive days, appeared to consume the majority of the available feed despite an apparent trend indicating a decrease in feeding with increasing EB dose (Julinta et al., 2020). Another study on juvenile nile tilapia found that, daily feed consumption by fish ranged between 70% and 100% based on visual observation on EB treatment days (Neto et al., 2019). The feed consumption by rainbow trout was studied at two different temperatures during EB medication at dose of 50 μg/kg fish/day for 7 consecutive days, where the total feed intake was 96.4% and 99.9% by fish maintained at 6ºC and 15ºC, respectively (Roy et al., 2006). The unconsumed feed within 1 h was leftover by 5× and 10× EB treated fish, therefore rated accordingly for their feeding behavior. The decrease in the feed intake by fish is a sign of stress and intoxication (Bowker et al., 2013). The similar kind of findings were also previously reported in nile tilapia, where EB treatment at dose of 500 μg/kg fish/day showed significant decrease in feed uptake (Julinta et al., 2020). A biosafety study in Asian seabass reported 50% decrease in the feed uptake after 16th day of medication period during EB treatment at dose of 500 μg/kg fish/day for 21 consecutive days (Raja et al., 2020). Even a slight appetite decrease in fish treated with 5× and 10× EB doses did not affect the growth of the golden mahseer juveniles. This finding is supported by similar studies, where the growth of fish were not affected when administered with higher EB doses through medicated feed (Julinta et al., 2020; Raja et al., 2020; Choudhary et al., 2022). Also, the toxicity study in Atlantic salmon and rainbow trout corroborate our finding, where the growth of EB treated fish were not affected as compared to control one fed with EB-free diet (Roy et al., 2000; Bowker et al., 2013). There was no mortality recorded in golden mahseer juveniles fed with 1×, 2×, 5×, and 10× doses of EB for consecutive 21 days. The fish were able to tolerate the 10× dose of EB during and after the medication period, which is supported by EB tolerance study in Asian seabass (Raja et al., 2020) and rainbow trout (Siavash et al., 2014), where no adverse effect was observed on the survivability of EB treated fish. In contrary to above findings, 4.43% mortality was observed in nile tilapia fed with 10× EB-diet for 7 consecutive days (Julinta et al., 2020), that might be due to the toxicity imparted by EB. Various factors may be involved in determining the toxicity of a drug such as treatment duration, age, sex of the organism, species, concentration of drug, water quality, metabolism, excretion and formulation type of drug (Kumar et al., 2018; Bharti and Rasool, 2021). In the present study, no external body surface lesions were observed in control and EB-fed juveniles of golden mahseer. The behavioral characteristics of control, 1× and 2× treatment group fish were normal throughout the experimental period. The fishes of 5× and 10× treatment group showed change in behavioral characteristics as compared to control group such as lethargy, gasping of air and crowding near the inlet of water. The fish treated with 10× EB dose started showing abnormal pigmentation on their body surface at 12th day of medication period. All abnormal behavior by 5× and 10× treatment group fish were reverted back to normal stage after 9–10 days of post-medication period. Similarly, abnormal behavior was reported in Atlantic salmon, rainbow trout, nile tilapia and Asian seabass after administration of high doses of EB through medicated-feed (Roy et al., 2000; Stone et al., 2002; Julinta et al., 2020; Raja et al., 2020). Thus, treating golden mahseer juveniles with the recommended EB treatment regimen of 50 μg/kg fish/day for 7 consecutive days carried a significant safety margin of the drug.

Histopathological studies have long been recognized as reliable biomarkers of stress in fish for several reasons (van der Oost et al., 2003). In present study, the histopathological changes were observed in intestine, liver, kidney and muscle tissues of the golden mahseer juveniles fed with EB-medicated diet for 21 consecutive days. The histopathological changes were distinct at 11th and 21st day of EB medication (Figures 1–6). The observed severe degenerative changes in the organs of EB fed fish included disarrangement of mucosa, abundant goblet cells and dilated lamina propria in intestine (Figure 1); pyknotic nuclei, melanomacrophage centre, vacuolation and necrosis in liver (Figures 2, 3); degenerated renal tubules and Bowman’s capsule dilation in kidney (Figure 4) and necrosis, myofibril disintegration, muscle oedema, migration of inflammatory cells and splitting of muscle fibres in muscle (Figures 5, 6). Similar histological findings were also observed in nile tilapia fed with 1× and 10× doses of EB for 7 consecutive days, which included degenerated renal tubules in kidney, vacuolation in liver, inflammation, epithelial layer disintegration and loss of absorptive vacuoles in intestine (Julinta et al., 2020).

The MRL and acceptable daily intake (ADI) of EB is listed in the Codex Alimentarius, an international food standards published jointly by Food and Agriculture Organization (FAO) and World Health Organization (WHO) (Codex Alimentarius, 2021 (Codex)) and the records are maintained by Codex Alimentarius Commission (CAC). A meeting held by Codex Committee for pesticide residues (CCPR) suggested a MRL of 100 μg/kg fish in fillet/muscle tissues of trout and salmon for EB, which was evaluated by Joint Expert Committee on Food Additives (JECFA) from FAO/WHO. The recommended ADI of 0–0.5 μg/kg bw for a person was established by Joint FAO/WHO Expert Meeting on Pesticide Residues (JMPR), which was based on an overall No-Observed-Adverse-Effect Level (NOAEL) of 0.25 mg/kg bw per day for neurotoxicity from 14- and 53-week studies in dogs. This is supported by an NOAEL of 0.25 mg/kg bw per day from 1- and 2-year studies in rats. The MRL of 100 μg/kg fish for Emamectin B_1a in edible tissues of salmonid also supports the MRL established by European Union (EMEA, 2003). The Emamectin B_1a is considered as prominent marker for EB residue analysis from muscle tissues of the organisms in which this drug is administered (EMEA, 2003; EFSA, 2012; Codex Alimentarius, 2021). Despite the slightly reduced feeding response in 5× and 10× EB treated golden mahseer juveniles, the higher concentration of Emamectin B_1a was detected in muscle during medication and post medication period. The traces of Emamectin B_1a in muscle peaked between 11th and 21st day of medication period followed by gradual depletion in post-medication period. The results indicated that a decrease in total Emamectin B_1a residue concentration level was observed when EB-medicated diet was discontinued followed by feeding with EB-free diet. The muscle residue concentration of Emamectin B_1a was within the MRL of 100 μg/kg fish at the end of the 30 days post-medication. Here, we concluded that EB residue in muscle is within the MRL, even if it is administered for three times the recommended duration of 7 consecutive days at dose of 50 μg/kg fish/day. Similar research has been conducted in different fishes from different geographical locations of the world, where adequate margin of safety of EB was reported at recommended dose (Kim-Kang et al., 2004; Whyte et al., 2011; Collymore et al., 2014; Neto et al., 2019; Julinta et al., 2020; Raja et al., 2020; Choudhary et al., 2022). In our study, the traces of Emamectin B_1a were still detected in muscle tissues of EB treated golden mahseer after 30 days post-medication, which suggests the complete withdrawal of EB require more than 30 days in golden mahseer. However, the detected range falls under the MRL established by European Union and Codex Alimentarius. In Indian tropical climate, the EB withdrawal from muscle tissue of Asian seabass and nile tilapia requires 28 days and 45 days, respectively (Julinta et al., 2020; Raja et al., 2020). The withdrawal period is different in different fish species and climatic regions. A temperature dependent EB withdrawal was reported in rainbow trout (Roy et al., 2006) where, mean Emamectin B_1a residue concentration of 13.7 ± 10.5 ng g⁻¹ at 77 days post-treatment was detected from the fillet in coldwater study. But in case of warm water study the mean Emamectin B_1a residue concentration was 1.6 ± 1.6 ng g⁻¹ at 49 days post-treatment indicating the slow residue depletion in cold water environment. LOD is an important test that indicates presence or absence of the drug, whereas LOQ measures presence of low level of the drug. In the study, the sample concentrations of EB were observed higher as compared to LOD and LOQ values. The negative values of LOD and LOQ state negligible presence of the drug in the samples due to variability of the process, demonstrating no bias in the method.

This study is the first report of the toxicity and residue depletion of EB on golden mahseer in Indian coldwater region. The feed administered EB graded levels up to 500 μg/kg fish/day for consecutive 21 days was not sufficient to cause any mortality in golden mahseer, but considerable change in feeding and behavioral characteristics were observed. The fish treated with 50 μg/kg fish/day of EB for consecutive 21 days showed no behavioral change and the muscle residue of EB was within the MRL (100 μg/kg fish) as per the guidelines of Codex Alimentarius and European Union even during medication period. This ensures the safety of recommended EB dose of 50 μg/kg fish/day for consecutive 7 days for usage as an anti-parasitic agent in aquaculture. The higher EB doses cause cellular changes in various organs such as intestine, liver, kidney and muscle of fish treated for longer duration than the recommended period, which may interfere with the detoxification and metabolism of the drug. More detailed investigation is required to elucidate the molecular mechanism by which prolonged higher EB doses lead to immunogenic and metabolic abnormalities. The consumption of meats from fish treated by recommended nominal dose of EB is safe for a person as the recommended ADI for EB is 0–0.5 μg/kg bw of person as established by JMPR. The study supports the safety of EB in golden mahseer and cold climate aquaculture of India at approved daily dose (ADD_{farm fish}) of 50 μg/kg fish/day for 7 consecutive days to control parasitic infections. As the EB residue for nominal dose is within the MRL, no withdrawal period recommendation is required in case of golden mahseer.