Feed additives are nonnutritive products added to the basic feed mix to enhance productive function and growth, preserve feeds, increase the efficiency of feed utilization, or benefit metabolism and animal health ^[1][2][1,2]. Numerous studies and individual experiences gained by livestock owners have shown that the comprehensive feeding of animals, especially for high-yielding cattle, is impossible without highly effective feed additives such as antibiotics ^[3][4][3,4]. The beneficial effect of antibiotics as a growth stimulant was discovered in the 1940s ^[1][2][1,2]. Their uses in aquaculture for disease control, prevention, and growth promoters have been practiced for a long time. However, the unrestricted and widespread use of prophylactic antibiotics in aquaculture has caused a series of developments harmful to human health and the environment [2]. The consumption of antibiotics as feed additives in large amounts increases the tendency of their residual products to settle in aquaculture ecosystems, accumulate in fish meat that compromises their immunity, and cause the emergence of antibiotic-resistant bacteria in aquatic environments ^[5][6][5,6].

RAC is a popular growth promoter extensively used as a feed additive for muscle leanness in cattle. RAC stimulates lipolysis, redirects nutrients from adipose tissue, and increases protein synthesis ^[7][8][9][7,8,9]. RAC and other β-adrenergic agonists were traditionally used to treat respiratory disorders and premature birth in human beings [10]. Before RAC was approved for use as an additive in some countries, it already passed an extensive approval process through the FDA to determine the no-observed-adverse-effect level (NOEL or NOAEL; 0.125 mg/kg/day) and the acceptable daily intake (ADI; 1.25 mg/kg/day) was completed in December of 1999 (FDA, 1999). Although RAC use as a growth stimulant was allowed in 2000, its use has always remained controversial [11]. RAC passed the approval processes from the FDA that are similarly applied for the approval of anabolic implants. This process was administered by FDA veterinarians, animal scientists, biologists, and toxicologists. This strict process leaves a convincing impression that RAC is safe for the health of the aquatic environment.

2. Applications of RAC in Livestock and Poultry Sectors

RAC, a phenethanolamine β–adrenergic agonist, is used as a feed additive to develop leanness, enhance growth performance, and increase feed conversion efficiency in different farm animals, including poultry in some countries ^[12][13][14][12,13,14]. Presently, RAC is being used as a feed additive in livestock rations to decrease the fat contents in carcasses without changing their quality [15]. Consumers nowadays have a high demand for quality carcasses with minimal fat content, leanness, and tenderness, which can be achieved by adding RAC to the animal ration diet [16]. Numerous studies revealed improved feedlot performance and carcass composition when RAC is fed to poultry animals. Previous studies suggest that adding RAC to an animal diet can lower feed intakes, but it improves the feed efficiency in swine when 10–20 ppm (parts per million) of RAC is added ^[16][17][18][16,17,18]. The introduction of RAC in rations also improves carcass protein and decreases the fat percentage in swine ^[18][19][20][18,19,20]. The addition of RAC in the finishing diet of pigs can regulate animal metabolism by optimal carcass muscle production and decrease the lipid concentration ^[21][22][21,22]. Adding RAC in the finishing ration of swine also enhances growth, feed conversion ratio, and carcass dressing percentage [23]. The significant improvement in carcass quality due to RAC feeding is considered by meat producers as a looming economic benefit of hundreds of dollars, potentially overshadowing the perceived environmental problems of RAC uses. Despite the great potential of RAC as a growth stimulant, conflicting research results began to question the efficiency of RAC for the meat industry. Previous studies showed that growth performance declines as the duration of RAC feeding is prolonged, but an improvement in muscular growth continues with the increasing duration of RAC use ^{[24][25][26][27][28]}[26,35,36,37,38]. Outputs from various studies also showed that including RAC in the finishing ration for swine improved ADG (p < 0.001) compared to untreated groups. Experiments from 1990 to 2005 showed an almost similar ADG improvement pattern when swine were given rations containing 5, 10, and 20 mg/kg RAC in the finishing diet ^{[25][29][30][31][32]}[35,39,40,41,42]. However, two studies showed that pigs fed with RAC in the finishing ration had reduced ADG by about 0.9% ^[33][43] and 1.9%, respectively ^[34][44]. Feed intake of swine improved when the ration inclusion level was about 5 mg/kg for 34 days ^[25][35]. Carcass weight was increased by 2.3% when the finishing ration of pigs contained 5 mg/kg of RAC compared to 0 mg/kg RAC ^[25][32][35,42]. In similar studies, the addition of 10 mg/kg RAC in the dietary ration resulted in a decrease in hot carcass weight ^[17][35][17,45]. The addition of 20 mg/kg RAC can either reduce ^[36][37][27,46] or increase the carcass weight from 0.3% to 5.1% ^[32][38][42,47]. An increase from 4.4% to 10.7% in carcass weight was also reported when the pig was fed with 20 mg/kg RAC in the finishing ration before slaughter ^[25][35]. Other studies also reported 1.9% to 5.2% and 5.5% increases in carcass weight when the finishing ration included about 20 mg/kg RAC, respectively ^[29][31][39,41].

3. Biological Basis of the RAC Response in Animal Tissues

RAC is the first βAR ligand to be cleared for use in pigs in the United States. RAC is structurally similar to the natural catecholamines epinephrine and norepinephrine and binds with high affinity to βAR in pig adipose and muscle tissue. Primary attention has been given to understanding how βAR might mediate increased growth and protein accretion ^[39][48]. Catecholamines (CAs) are a group of organic compounds consisting of a hydroxyl group and an amine side chain. Most CAs are used by dopamine, norepinephrine, and epinephrine receptors in the nervous system ^[40][41][49,50]. CAs have received the attention of researchers because of their early recognized involvement in different neurological disorders ^[42][51]. The biological effect of CAs is mediated by two receptors, namely α- and β-adrenergic receptors, and sometimes they are collectively called adrenoreceptors. Alpha- and beta-adrenergic receptors have different locations in animal tissues and respond differently and often oppositely to catecholamines ^[43][52]. An adrenoreceptor stimulated by an α-agonist leads to intracellular effects mediated by adenylate cyclase inhibition. This stimulates smooth muscle contraction in blood vessels that supply peripheral organs such as skin and kidneys, smooth muscle relaxation in the gastrointestinal tract, and blood platelet aggregation. However, stimulating β-adrenergic receptors by β-agonists activates adenylate cyclase, leading to increased glycogenolysis and gluconeogenesis in the liver and skeletal muscle and increased lipolysis in adipose tissue ^[44][53]. Increased blood flow to the brain activates the catecholamine, making animals cope with stressful conditions such as reducing feed during the dry period, mixing animals in unfamiliar groups, transportation, and rough handling ^[45][54]. However, the measurement of CAs in urine and the brain cannot provide a clear picture of stress, fear, and temperament. Some scientists consider CAs a coping hormone as they provide energy to the brain and help reduce deficiency, leading to “energy deficiency syndrome” of the brain ^[46][55].

It is reported that RAC can steer the fat accumulation in the body of cattle and pigs via a prime metabolic channel through adipocyte accumulation and liberation by energizing β-adrenergic receptors because of similarities with catecholamine. As a result, minimal fat is deposited in the carcass. Animals fed with varying levels of RAC have improved carcass development and a lower percentage of adipose tissue compared to control animals ^[32][47][42,56]. Accumulating a lower percentage of fat in carcasses resulted as RAC directly affected adipocytes by increasing the rate of lipolysis and inhibiting the transformation of glucose to triglyceride ^{[47][48][49][50]}[56,57,58,59]. Beta-adrenergic agents such as RAC orchestrate the cellular reaction via β-adrenergic receptors and trigger adenylate cyclase and protein kinase A, affecting the lipogenic activity by two mechanisms. The first one is protein kinase A, which directs the phosphorylation of existing proteins, decreasing their functional activity ^[51][52][53][60,61,62]. The second one is increased protein kinase A activity that reduces the rate of gene transcription and cell content of the main protein ^[54][55][63,64]. However, previous experiments revealed that swine fed with a RAC diet had decreased lipogenic enzyme activity in fat tissues ^[48][49][50][57,58,59]. The β-adrenergic agents affect the metabolism of adipose tissue through activation of β-receptors of protein kinase A. Increased protein kinase A activity results in increased lipolysis through activation of hormone-sensitive lipase ^[56][65]. Moreover, increased protein kinase A activity inhibits glucose conversion to triglycerides, which is considered increased serine phosphorylation. Lipoprotein lipase is an important enzyme that controls the triacylglycerol between the muscle and adipocyte tissue, improves lipid storage, and provides energy or muscle growth. Therefore, including RAC in diet changes lipid metabolism, inducing lipolysis rather than inhibiting lipogenesis in the animal. In summary, strong science supports the use of RAC as feed additives. RAC is an energy repartitioning agent that diverts nutrients by increasing protein synthesis ratio and decreasing protein degradation, promoting muscle growth by inducing muscle hypertrophy, reducing fat deposition, improving feed conversion, and increasing average daily weight gain that improves carcass yield and meat quality.

4. Potential Benefits of RAC Feeding on Fishes

The fast development in aquaculture systems is managed by different factors, including the growing consumption of formulated aqua-feeds and a massive improvement in the culture systems ^[57][58][66,67]. Phenethanolamines (PEOHs) act as a nutrient dissemination agent in intermediary metabolism by transferring nutrients from adipocytes to muscle protein unification ^[47][59][60][56,68,69]. The rationing of PEOH in fishes is not a common practice as fishes have lower energy demand and thus respond to diets with a higher protein–energy ratio than birds and mammals ^[61][70]. Previous data showed that RAC supplementation in channel catfish (Ictalurus punctatus) promoted weight gain and reduced fat deposition ^[62][71]. RAC feeding at 20 mg/kg or lower resulted in a 17% increase in weight gain and a 24% reduction in muscle fat in catfish ^[63][72]. In combination with dietary protein supplements in catfish, RAC yielded higher weight gain than when RAC was only combined with restricted protein supplementation ^[64][73]. In varying concentrations, RAC has been found to increase the feed efficiency in rainbow trout (Oncorhynchus mykiss) (walbaum) while maintaining normal hepatosomatic and viscerosomatic indices throughout the feeding weeks ^[65][74]. In another study, the combined effect of RAC at 0 and 10 mg/kg and l-carnitine at three levels (0, 1, and 2 g/kg) showed that 1 g/kg l-carnitine and 10 mg/kg RAC improved growth performance, feed efficiency, and protein efficiency ratio in juvenile rainbow trout. The combined use of l-carnitine and RAC in trout diets increased body protein, reduced body fat, and altered the fatty acid profile of muscle tissue ^[66][76]. Increasing levels of RAC in the diet of juvenile pacu (Piaractus mesopotamicus) for 60 days did not improve body growth or composition but only altered the hematological and biochemical parameters ^[67][77]. Pacu (Piaractus mesopotamicus) fed with 33.75 ppm of RAC in the finishing phase developed less fat content in its meat and improved antioxidant status inside the freezer ^[68][78].

5. RAC Level Detection in Wastewater Systems

Veterinary drugs in water bodies have become an alarming issue for nontargeted species and human populations ^[69][70][71][81,82,83]. The excessive use of veterinary drugs and steroidal hormones in animal feeding systems is viewed as one factor that negatively affects the health of aquatic species. RAC could be introduced into aquatic systems from water overflow areas rich in toxic waste from ruminants and is the most commonly detected drug (130–500 ng/L) in pond wastewater at pig farms ^[72][84]. RAC was also seen at 50 ng/L in groundwater due to run-offs from ruminants’ waste control facilities ^[72][84]. In China, another β-agonist, clenbuterol, used in treating respiratory illness and premature birth in humans, was identified at a concentration of 11 ng/L in abattoir wastewater ^[73][85]. Hospital sewage is one of the aquatic system’s major sources of β-agonists. In Taiwan rivers, the presence of four β-agonists from hospital discharge was reported, and RAC, at 70% prevalence, is the most common antibiotic present in the collected samples ^[74][86]. Due to the frequent detection of β-agonists in environmental water samples, researchers from various fields have initiated investigations to determine toxic compounds such as RAC. The occurrences of β-agonists in theour environment are at a lower concentration under parts per million or parts per billion. Their intractable chemical behaviors made their detection very difficult, and their detection always poses analytical challenges. Due to the diversity of their physio-chemical features and continuing occurrence of β-agonists in theour water, devastating consequences on both human and aquatic lives are expected in the near future.

6. RAC Poses Physiological and Toxicological Effects on Fishes

The utility of the zebrafish model for evaluating RAC toxicity is very well recognized because the manifestations of toxicity are widely demonstrated in this model ^[75][92]. For instance, RAC exposure of zebrafish at different concentrations for seven days exhibited altered behavior and imbalanced oxidative status ^[76][88]. Zebrafish larvae acutely exposed to RAC displayed altered heart rate and locomotory and exploratory behavior but maintained their survival rate ^[77][89]. In addition, adult male zebrafish exposed to RAC for 21 days revealed poor reproduction and breeding capability and altered behavior ^[78][90]. In the same study, the mating of RAC-fed male adult zebrafish with non-RAC-fed female adult zebrafish resulted in delayed hatching (72 hpf) and a significant number of abnormal embryos with stunted development, edema of the heart, granule formation, degenerated yolk, and yolk deformities.

7. RAC Levels Detected in Poultry Animals and Products

Amendola et al. utilized a GC-ion trap to select precursor and product ions in monitoring the clenbuterol spike in human urine ^[79][93]. Nanoparticles were also utilized to extract and detect RAC and salbutamol by an electrochemical process. Rajkumar et al. used glassy carbon electrode-modified poly taurine/zirconia nanoparticles (ZrO₂) to identify RAC and salbutamol in swine muscle and human urinary samples ^[80][94]. With an additional derivatization procedure, HPLC-UV was a popular method to detect RAC in porcine muscle and urine samples ^[81][82][83][95,96,97]. Nowadays, RAC levels can be estimated by high-performance liquid chromatography (HPLC) coupled with MS detection or HPLC coupled with tandem MS (MS/MS) ^[84][101]. Ultra-pressure liquid-phase chromatography (UPLC) coupled with triple-quadrupole MS was utilized to detect RAC within 5 min ^[81][85][95,102]. 

8. RAC Regulations and Feed Fights

Different regulatory organizations such as the United States Food and Drug Administration (US FDA) and the European Medicinal Agency (EMA) and independent organizations such as the Joint World Health Organization/Food and Agricultural Organization Expert Committee on Feed Additives (JECFA) have determined tolerances and maximum residue limits (MRLs) of antimicrobials and growth stimulants such as RAC, respectively, for muscle, liver, kidney, and fat contents ^[86][106]. The MRLs derived by JECFA are recommended to the Codex Alimentarius Commission (Codex), which determines whether to establish international standards for residues of veterinary drugs in terms of MRLs. The US FDA uses the term tolerance, while other countries and organizations use MRLs. Other developed countries that are not part of the EU develop their own MRLs, while most developing countries adopt EU or Codex MRLs ^[87][107]. At present, RAC is the most controversial food additive in the world. It is well accepted that RAC has been authorized as a feed additive in many countries for the pig and cattle industry. The acceptability of RAC as a feed additive was based on the joint FAO/WHO Expert Committee on Food Additives and Scientific Evidence recommendations. However, it did not achieve a satisfactory judgment result between the European Union (EU) and the United States by the World Trade Organization (WTO) about the residual dosage of RAC, which could cause personal injury. Most jurisdictions, including the European Union (EU), China, Taiwan, Korea, and Russia, have disallowed its use for safety reasons ^[88][108]. This legal division reflects the long-standing disagreement between countries supporting the establishment of maximum residue levels and those who oppose it within the Codex. The Codex, an intergovernmental food standard-setting body with more than 180 members, ratified an MRL for RAC at 10 parts per billion (ppb) in pork and beef muscle meat ^[89][109].  At present, the RAC controversy persists, and countries worldwide are divided on whether to allow or ban the use of RAC in meat production. In the United States, RAC is approved for swine, turkeys, and cattle ^[11][90][11,114]. RAC is also approved for Brazil, Canada, South Korea, and Mexico, but RAC has been banned in China, Taiwan, and the European Union. Despite an international CAC standard, there have been occasions in which exports from the United States to countries with zero-tolerance policies were rejected due to RAC levels under the global MRL ^[32][42]. One of the major arguments is the lack of consistency in how countries set their tissue RAC residue limits and which residue limits are applied to various tissues, specifically edible noncarcass ^[91][115]. The testing results from such countries can be contentious because they employ varying sample handling and testing methods that may impact the results. Diversities in technologies used to detect RAC reflect the inconsistency around how residue limits are established and which residue limits are applied to various tissues in different countries ^[91][115]. The CDC has not yet decided on the standards for the residues of RAC. It means that controversy still exists on the residual dosages of RAC meat, especially of the side effects of the cumulative doses of long-term intake of RAC meat in the human body.

9. Conclusions

Despite the controversy surrounding the use of beta-agonists as growth stimulants, the benefits to sustainability and animal production are apparent. However, the importance of production technologies such as RAC to meet the demands for quality meat of the growing global population cannot be overstated. RAC becomes a source of public concern and triggers endless transatlantic trade disputes. The threatening trade issues and accompanying abilities to detect extremely low concentrations of residues in tissues, variations employed by each country, and unscientific import policies could impact future RAC use. All these issues remain significant as RAC is still usually fed to livestock, and the potential toxicity of this substance is continuously being reported. Despite the looming economic benefits of RAC use, the potentially harmful and adverse effects on the environment and human health should not be overlooked and must be given attention. There is a great need for medical, scientific, pharmacological, and toxicological evidence from animal experiments to prove the causal relationship between RAC and its toxic effects on humans and animals.