Effect of Water Temperature on the Depletion of Eugenol in Sea Bass under the Simulated Settings in Handling and Transport

Featured Application

This could be used in live fish handling and transport, not only to decrease the physical injuries and mortality of the fish, but also to reduce the residual amount of eugenol in fillet.

Abstract

The edible safety of eugenol-treated fish is one of the concerns limiting the anesthetic applied in the aquaculture industry. The depletion of eugenol was investigated at different water temperatures under the simulated settings of sea bass handling and transport. Sea bass was exposed to eugenol at a concentration of 60 mg/L for 3 min, then immediately transferred to clean water to be purged for 24 h and sampled at different time intervals. The left fish were re-exposed to eugenol in a repeat of the first exposure, purged for 48 h in clean water, and sampled for eugenol detection. Under the simulated settings, the sea bass could accumulate more eugenol residue in fillet when exposed to water at a temperature of 20 °C than that at 13 °C. However, eugenol could be depleted much faster when the sea basses were exposed to the higher water temperature. The half-lives of eugenol in sea bass were 0.28 h and 0.29 h for the first and second purging stage at a water temperature of 20 °C, while at 13 °C, the half-lives were 2 and 4.5 h, respectively. Therefore, increasing the water temperature may be an effective way to accelerate eugenol depletion in fish fillet and reduce exposure risk for the consumer.

1. Introduction

Eugenol is a widely used fish anesthetic. It has been reported to have a high efficacy in fish, crustaceans, and other aquatic species [1,2,3,4,5,6,7,8,9]. During handling and transport, eugenol can reduce the stress of fish, decrease the physical injury or mortality of live individuals, and facilitate the operations. Therefore, eugenol has been officially approved as a food fish anesthetic in many countries, such as Japan, New Zealand, South African, and some southeast Asian countries [10,11,12,13].

However, the edible safety of eugenol-treated fish has also aroused great concern, which has become a limiting factor for the application of eugenol in the aquaculture industry. The National Toxicology Program (NTP) determined that eugenol is an equivocal carcinogen [14]. The Food and Drug Administration (FDA) is concerned that the use of eugenol may adversely affect human food safety and animal food safety [15]. Therefore, eugenol is not approved as a food fish anesthetic in the United States. Although eugenol is approved as a food fish anesthetic in Japan, the Maximum Residue Limit (MRL) in fillet is set at 0.05 mg/kg and the withdrawal time is set at 7 days for fish and 10 days for crustaceans, respectively [16]. The WHO/FAO Joint Expert Committee on Food and Additives (JECFA) has proposed an acceptable daily intake (ADI) for eugenol, ranging from 0 to 2.5 mg/kg b.w. [17]. The daily intake of eugenol below the upper limit of the ADI value would not cause an appreciable health risk. This also means that less exposure to the anesthetic would lead to a higher safety margin in human health risk. Therefore, finding a way to reduce the residual amount of eugenol in edible tissues would not only improve the edible safety of eugenol-treated fish, but also benefit the aquaculture industry.

Live fish can remove the residual eugenol in their tissues when purged in clean water. However, the ability to remove the anesthetic by fish is greatly affected by many factors, such as species, dosage, fish size, administration method, and water temperature [18]. As shown in previous reports, the half-life of eugenol in plasma was 12.14 h in rainbow trout in water at 4 °C, 0.3 h in Japanese flounder in water at 24 °C, 19.97 h in grass carp in water at 25 °C, and 21.6 h in mandarin in water at 25 °C water, respectively [19,20,21,22]. In the fillet tissue, the half-life was 26.25 min in rainbow trout [23], 10.27 h in grass carp and 5.1 h in mandarin. It indicated that the influencing factors were very complicated. Therefore, in live fish handling and transport, to ascertain and fully utilize the key influencing factors, it is very important to reduce the residue of eugenol in edible fish tissues in order to minimize the exposure risk of consumers.

Sea bass is one of the most productive seawater species in China, as well as one of the species with the highest detection frequency of eugenol in the fish market [24]. This indicates that eugenol has been used in the handling and transport of sea bass, although the anesthetic has not been approved in the Chinese aquaculture industry. As investigated, from pond to market, sea basses were anesthetized for the first time in the culture ponds before being captured, and then transported to the market for 14–36 h; the second anesthetization was carried out on the transport vehicles before the sea basses were unloaded in the market, and then, the live individuals were temporarily held in a water tank for sale. In both the tanks that the fish were transported in or temporarily held in at the market, the water was free of eugenol. The water temperature was controlled throughout the whole procedure to ensure the survival rate of the sea bass; it was usually set at 17–20 °C during the summer and at 13–15 °C during the winter. By the end of the whole procedure, the sea basses had been quickly anesthetized twice, followed by two stages to purge the eugenol. Although eugenol has been used in this way, it is still unclear how the residual amount of eugenol changes in sea bass fillet and is affected by the water temperature. Therefore, taking sea bass as an example, our aim was to reveal the depletion of eugenol in the fish fillet by simulating the whole procedure of sea bass from pond to market and evaluating the effect of water temperature on the depletion of the fish anesthetic. We hope that our results can provide a way to control the eugenol residues in sea basses and minimize the exposure risk for the consumer through fish consumption. We also hope that our results can provide references for policymaking regarding on the application of eugenol as a food fish anesthetic in the aquaculture industry.

2. Materials and Methods

2.1. Materials for Exposure Experiment

Market-sized sea basses (Lateolabrax japonicas) (body weight 0.5 ± 0.2 kg) were purchased from a farmer’s pond in Zhuhai, China and then transported to the Estuary Fishery Research Center laboratory at the South China Sea Fisheries Research Institute. Before the exposure experiment, the fish were acclimatized in a pool (3 × 4 × 1.5 m) and constantly supplied with aerated brackish water for a week. The brackish water was obtained from the culture pond and pre-filtered with sand prior to introduction. The fish were fed with the same feed as that in farm ponds and then fasted for 24 h prior to the exposure experiments.

Eugenol for the fish exposure experiment was purchased from Shanghai Medical Instrument Co., Ltd. (Shanghai, China). Prior to the preparation, eugenol was dissolved in anhydrous ethanol (analytical grade) at a ratio of 1:10 (v/v), and then prepared into the concentrations needed for the exposure experiment.

2.2. Exposure Experiment

Sea basses were usually anesthetized within 3 min before being captured in the culture pond or unloaded from the transport vehicle. Under an anesthetic state, they were kept immobile with the body turning over, breathing slowly and rhythmically and un-reacting to stimuli. The anesthetic dosage was determined at 60 mg/L for sea basses after the preliminary tests. Bathing at this concentration, sea basses could be anesthetized within 3 min and showed physiological states similar to that observed in practice. The exposure experiment simulated the procedures that are carried out when live sea basses are handled from pond to market.

One hundred and twenty sea basses were randomly selected from the acclimation pool, transferred to a tank (120 × 90 × 87 cm, r_top × r_bottom × h; water volume, 700 L) with a eugenol aqueous solution previously prepared at a concentration of 60 mg/L, and then bathed for 3 min. After the first exposure, all the fish were immediately captured with nets, rinsed with clean water, and then transferred to a depuration pool with eugenol-free and constantly aerated water. Sea basses were held in the pool for 24 h and sampled at 0, 0.5, 1, 2, 4, 8, 12, and 24 h. A total of 6 individuals were randomly caught at each sampling time. The individuals left in the pool were captured, transferred to another tank with eugenol at the same concentration as the first exposure, and then re-bathed for 3 min. After the second exposure, the fish were treated in the same way that they were after the first exposure and were held 48 h in another depuration pool. Sea basses (n = 6) were collected from the pool at the time interval of 0, 0.5, 1, 2, 4, 8, 12, 24, and 48 h. Throughout the whole procedure, the water temperature in the exposure tanks or depuration pools was maintained at 20 °C using a heat pump thermostat (DIYI025E, Diyi Electric Heat Pump Co., Ltd., Foshan, China). In order to evaluate the effect of temperature on the residue of eugenol in the fish, the temperature of the water was maintained at 13 °C in another independent exposure experiment. The procedures were almost the same as that carried out at the water temperature of 20 °C.

The fish caught in the depuration pools were sacrificed with a blow to the head, and individually dried with absorbent paper. The fillet was removed from each fish, sealed in a plastic bag, and frozen at −18 °C in a refrigerator. After collecting the fillet of each fish, the knife and board were cleaned with water and dried with absorbent paper to avoid cross-contamination.

2.3. Eugenol Detection

To detect eugenol in the fish fillet, the method previously developed by us was used [25]. Briefly, 2 g of fillet was weighted into a 50 mL plastic tube, extracted using ultrasonication with 5 mL of hexane over 10 min, and followed by centrifugation at 5000 rpm for 5 min. The supernatant was applied to a phenyl solid phase extraction (SPE) column. Then, the target was eluted with 2 mL of ethyl acetate for quantification after the column rinsed with 5 mL of hexane. Eugenol measurement was conducted on an Agilent 7890N gas chromatograph (GC) coupled to a 7000B tandem mass spectrometer (GC-MS/MS) with a triple quadrupole analyzer (QqQ) that was also equipped with an Agilent J&W Scientific DB-17 MS capillary column (30 m × 0.25 mm, ID 0.25 µm). The method limits of quantification (LOQs) was 1.2 μg/kg for eugenol. Concentrations in the range of 5–500 μg/L performed good linearity with a coefficient of determination (r²) greater than 0.999. The recoveries for the target ranged from 76.4% to 99.9% with relative standard deviations (RSD) ranging from 2.18% to 3.60%.

2.4. Data Analyses

The estimated daily intake (EDI) of eugenol via fish consumption was evaluated using the equation given by EDI = C × CR/BW [26], where C is the eugenol concentration in fish; CR is the upper limit for the daily rate of fish consumption (75 g/day) for an adult, as indicated by dietary guidelines for Chinese residents [27]; and BW is the body weight (kg) of adult people (61.8 kg) based on the Report on the Nutrition and Chronic Disease Status of Chinese Residents [28].

Statistical analyses were performed using SPSS 16.0 for Windows (SPSS Inc., Chicago, IL, USA). Eugenol residue was fitted to a mono-exponential regression model given by Y_t = A*exp (−βt), where Y_t is the concentration in the fillet tissue at time t [23]. Regression model fit was assessed on R square. The half-life (t_1/2) of eugenol in the fillet tissue was calculated using the following formula: t_1/2 = 0.693/β, where β = the rate constant (slope) of the terminal phase of the elimination curve. A non-parametric chi-squared test was used to compare the differences of eugenol residue in the fish fillet between the different treatment groups using a statistical significance level of p < 0.05. The figure was generated using Origin 8.0 software (OriginLab Inc., Northampton, MA, USA).

3. Results

As shown in Figure 1, the maximum residual amount of eugenol in sea bass fillet was different in the two purging stages at a water temperature of 13 °C. The maximum residue was 31,712.0 ± 848.9 μg/kg in the first purging stage and 5846.1 ± 2694.1 μg/kg in the second purging stage. The maximum value of eugenol in the fillet was much higher in the second purging stage than that in the first stage, but it was not significant (p = 0.056). The half-life of eugenol in the fillet was 2.0 h in the first purging stage, and the residual amount in the fish fillet was less than 1.0 mg/kg after 4 h of purging. In the second purging stage, the half-life was 4.5 h, which was more than twice the half-life of the first stage. Additionally, it took 12 h for the residual eugenol to fall below 1.0 mg/kg.

When the water temperature was 20 °C, the change of eugenol residues in the fish was similar to that of 13 °C. The maximum residual amount of eugenol in the fillet in the first purging stage was 5247.0 ± 1021.5 μg/kg, which was significantly lower than the maximum value of 10,025.7 ± 2442.3 μg/kg in the second purging stage (p < 0.05). The time for the residual eugenol fall below 1.0 mg/kg was 1 and 2 h, respectively. The half-lives of eugenol in the fillet in the two purging stages for the sea basses exposed to 20 °C were almost the same; the value of the first purging stage was 0.28 h and the value of the second purging was 0.29 h (shown in

Table 1. Depletion models for eugenol in fillet tissue from sea bass exposed to a concentration of 60 mg/L for 3 min.

Exposure Temperature	Purging Stage	Model Equation	R2	Half-Life (h)
13 °C	First	Yt = 3255 × e−0.34t	0.968	2.0
	Second	Yt = 5667 × e−0.154t	0.978	4.5
20 °C	First	Yt = 5377 × e−2.444t	0.963	0.28
	Second	Yt = 9932 × e−2.405t	0.977	0.29

).

By comparing the maximum residual eugenol in the fillet, the value detected in the first purging stage at the water temperature of 20 °C was significantly higher than that detected in the first purging stage at 13 °C (p < 0.05). The difference in the maximum residual eugenol was also significant in the second purging stage between the water temperature at 20 and 13 °C (p < 0.05). Meanwhile, as shown in Table 1, eugenol depleted more rapidly in the fillet of sea basses bathed at the water temperature of 20 °C. Especially in the purging time from 0 to 0.5 h, the residual amount of eugenol in the fish fillet was significantly deceased, and the p-value was 0.018 in the first purging stage and 0.01 in the second. In contrast, the decreases in the sea basses purged at the water temperature of 13 °C were not significant. After 24 h post- exposure, the residual amount of eugenol in the fish fillet still decreased at 13 °C in the second purging stage, but the residual amount had reached a steady state at 20 °C and the values were much lower.

4. Discussion

Anesthetics used in fish are absorbed into tissues through gills and result in residues [29]. Meanwhile, the gills are also an important pathway for eliminating the anesthetics [30,31]. Gill ventilation has the greatest impact on the accumulation and elimination of fish anesthetics [32]. The frequency of fish ventilation increases significantly at higher temperatures [33]. It has been reported that the residual amount of isoeugenol, a fish anesthetic structurally similar to eugenol in the rainbow trout fillet, increased with exposure to the increasing water temperature [34]. Similar results have also reported in silver perch, in which residual eugenol was detected at a higher level when the fish immersed in water of a higher temperature [35]. Furthermore, the maximum residues of eugenol in the fillet of sea basses were almost determined in the first sampling time (t = 0-h), immediately after the fish were exposed to the anesthetic. The maximum values at the water temperature of 20 °C were significantly higher than that at 13 °C. This indicated that sea bass could accumulate more eugenol in their fillet at a higher water temperature, even if they were exposed to the anesthetic for a very short time (3 min). During the purging stage, the half-lives of eugenol in the fish fillet were also much shorter at the water temperature of 20 °C. As shown in Figure 1, under the simulated settings, when the sea basses were bathed in water with a temperature of 20 °C, the time for the residual eugenol in the fillet to below 1.0 mg/kg was less than 2 h, while at the water temperature of 13 °C, the time was more than 4 h, even as long as 12 h. This indicated that sea bass could deplete eugenol more rapidly when bathing at a higher water temperature. Therefore, water temperature played a very important role in the accumulation or the depletion of eugenol in sea bass.

Under the simulated settings, the sea basses were exposed to eugenol twice throughout the whole procedure, and accordingly, there were two stages to purge the anesthetic in clean water. As shown above, the maximum residual eugenol detected in the second purging stage were much higher than that in the first purging stage, regardless of whether the water was at a temperature of 13 or 20 °C. When exposed to 13 °C, the half-life of eugenol in the fish fillet was much longer in the second purging stage. The same phenomenon has also been presented in silver perch [35]. It is thought that the repeated administrations may reduce the capacity of the fish to clear the eugenol residue from their tissues [20]. However, for sea basses bathed at the water temperature of 20 °C, the capacity to purge eugenol did not seem to decrease, as the half-lives of eugenol in the fillet were almost the same in the two purging stages. Whether the effect of repeated anesthesia on the ability of the fish to deplete eugenol could be overcome by increasing the water temperature, or whether it only occurred in sea bass, should be further studied.

The EDI was assessed at 0.014 mg/kg b.w. for Chinese residents, which was estimated using the maximum residual eugenol detected in sea bass (11.5 mg/kg). It was much lower than the ADI value proposed by JECFA [17]. This showed that the residue amount of eugenol in sea bass fillets could pose very little risk to human health. Therefore, if sea basses were treated with eugenol in the way practiced in their handling and transport, it was unnecessary to set a withdrawal time. Furthermore, sea basses bathing at a higher water temperature could more rapidly deplete eugenol in the fillet. Therefore, for the duration of time the fish are on sale in market, increasing the temperature of the water in which the fish are temporarily held may be an effective method to accelerate the depletion of eugenol and reduce its residue in the edible tissue of the fish, and eventually minimize the exposure risk of humans to eugenol.

5. Conclusions

In this study, the eugenol in the sea bass fillet can be rapidly depleted under the simulated settings and the residue of eugenol in the sea basses posed little risk to human health. The depletion of eugenol in the sea bass fillet could be faster if the fish were bathed at a higher water temperature. Thus, increasing the water temperature during fish handling and transport can accelerate the depletion of eugenol in fish and minimize the exposure risk, especially during the period the fish are on sale in the fish market, as this is the final procedure before the fish are sold to consumers. A huge amount of live fish are transported across the country each day in China. There is an urgent need for fish anesthetic to help reduce the losses suffered during fish handling. Taking sea bass as an example, eugenol, as a fish anesthetic, may be a good choice to solve the problems in the Chinese aquaculture industry.