Acute Toxicity of Four Disinfectants to Brine Shrimp, Artemia (Crustacea: Anostraca)

Song Jiang1,2,3, Zhenhua Ma1,2, Falin Zhou1,2, Xu Chen1, Jing Hu1, Rui Yang1, Shengjie Zhou1, Yundong Li2 and Qibin Yang1* 1Tropical Fisheries Research and Development Center, South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Sanya 572018, China 2Key Laboratory of South China Sea Fishery Resources Exploitation and Utilization, Ministry of Agriculture and Rural Affairs, P.R. China; South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou, China 510300 3Shenzhen Base of South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Shenzhen, 518108,China Article Information Received 23 June 2020 Revised 05 September 2022 Accepted 24 September 2022 Available online 14 June 2023 (early access)

T he brine shrimps Artemia (Crustacea, Anostraca) are distributed in salt lakes and ponds worldwide with the exception of Antarctica (Stappen et al., 2001). The Artemia is widely used in laboratory toxicology studies due to its small body size, short lifespan and its availability from dry cysts (Litvinenko et al., 2015). Due to expanding aquaculture, the water quality in offshore areas is aggravating and the breeding of aquatic animals are becoming increasingly unstable. As a result, largescale bacterial diseases are routinely being observed Speer et al., 2018). Therefore, various disinfection and sterilization drugs are being applied to inhibit breeding of pathogens and to ensure the success of aquatic animals breeding and cultivation (Yukihira et al., 2006;Liu et al., 2009;Fan et al., 2014). Consequently, there are emerging evidence that these drugs may influence aquatic animals. Zhao et al. (2014) studied the acute toxicity of benzalkonium bromide, dibromohydantoin, methionine iodian and glutaraldehyde on Babylonia areolate. Liu and Wang (1994) studied the impact of SC on vitality, ingestion rate and daily growth rate of D-larvae and the umbo-larvae of Pinctada martensii. Fan et al. (2014) determined acute toxicity of Hg 2+ and Cd 2+ on juvenile Pinctada maxima (Fan et al., 2014). Here, we aim to determine the acute experimental toxicity, safe concentration, and tolerance of glutaraldehyde, dibromohydantoin, methionine iodian and bleaching powder against Artemia. These findings provide scientific basis for the rational use of disinfectants in the cultivation of Artemia.

Materials and methods
Artemia's cysts (purchased from Aquamaster Company) were hatched in a funnel shaped plastic container filled with synthetic seawater. Newly hatched Artemia were processed following the procedure described by Litvinenko et al. (2015). Four kinds of disinfectants were used including glutaraldehyde (20% effective concentration, Beijing Zhongnong Huazheng Veterinary Drug Limited Liability Company), dibromohydantoin O n l i n e

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(20% effective concentration, Beijing Biological and Fisheries Technology Limited Liability Company), methionine iodine (50% effective concentration, Shanxi Shenlong Tianyi Science and Technology Limited Company) and bleaching powder (50% effective chlorine, Guangxi Nanning Chemical Industry Group Corporation). These disinfectants were prepared into mother liquor before application. The experimental container (glass beakers) were filled with 2 L of sea water was first filtered by sand and then inflated with protein skimmer. During the experiment, the dissolved oxygen contents in the water was kept at more than 5 mg/L and the temperature of water during the experiment was maintained at 29±0.5℃, pH at 8.4±0.2, salinity at 30~33ppt. A total of 100 Artemia were kept in 1 L of seawater containing glass beaker. Different volume of disinfectant mother liquor was supplemented in each glass beaker. Throughout the experiment, the water was kept inflated. The Artemia was kept off-feed, and disinfectant solution was changed every 12 h. The death of the Artemia was recorded to determine the highest survival zero lethal concentrations(LC 0 ) after 48 h of treatment and the LC 100 was determined when all Artemia were dead after 24 h.
Based on the experimental results of pre-test, five treatment groups of different mass concentrations and one control group were set according to the numerical arithmetic interval method. Each treatment was applied in triplicates and each group of 100 Artemia were placed in one experimental unit. The survival of Artemia was observed at 48 h and any dead Artemia was removed swiftly. Artemia were considered dead when the swimming foot stopped moving and the body sang to the bottom of the glass beaker.
The mortality rates after 24 h and 48 h in each group were calculated according to the following formula: The mortality rate (%) = number of death/total number of experimental Artemia × 100 The regression equation of the probability unit of the mortality rate and the usual logarithm of drug mass concentration, the death concentration of the drugs (LC 50 ) was obtained by using the linear interpolation method. The X-axis of the regression equation was the usual logarithm of drug mass concentration, and the Y-axis of the regression equation was the probability unit of the mortality rate. Then the safety quality concentration (SC) was obtained according to the following formula: SC=48 h LC 50 × 0.3/(24 h LC 50 /48 h LC 50 ) 2

Results
Based on the analysis of the pre-test data, the LC 0 and LC 100 for glutaraldehyde, dibromohydantoin, methionine iodine and bleaching powder were 14.00 mg/L and 142.00 mg/L, 4.00 mg/L and 97.00 mg/L, 18.00 mg/L and 105.85 mg/L, 5.60 mg/L and 18.58 mg/L, respectively. Five mass concentration of four chemicals were determined according to two mass concentrations by equal spacing method. The results of the test of the toxicity of four chemicals to the larvae of Artemia are shown in Table I. During the entire analysis period, there was no death recorded in the control group. Analysis revealed that regression curve equations between the unit of mortality after 24 h and the four chemicals concentration were Y=4.74X-3.33, R 2 =0.98, Y=2.66X+0.88, R 2 =0.95, Y=5.17X-4.51, R 2 =0.99, Y=6.28X-2.26, R 2 =0.96, respectively (Fig. 1). It was

Discussion
Artemia was the most sensitive to bleaching powder with concentration of 5.60-18.58 mg/L followed by dibromohydantoin, methionine iodine and glutaraldehyde with the concentration was 4.00-97.00 mg/L, 18.00-105.85 mg/L, 14.00-142.00 mg/L, respectively. These results were comparable to Zhao et al., who have studied juvenile B. areolata (Zhao et al., 2014). The analysis of four kinds of disinfectants to Artemia showed that after 24 h of treatment, the LC 50 value of bleaching powder against Artemia was 14.24 mg/L, however, 48 h post-treatment the LC 50 was 11.26 mg/L. It was also noticed that the difference between two data was the least, which illustrated the toxicity of bleaching powder was stronger than others against larvae. The toxicity of three kinds of disinfectants in descending order was for dibromohydantoin, methionine iodine and glutaraldehyde. By calculating the safe concentration (SC), the sensitiveness of Artemia to four kinds of disinfectants in descending order was concluded to be bleaching powder, dibromohydantoin, methionine iodine and glutaraldehyde.
Chlorine reacts with water to produce atomic oxygen which acts as sterilizing agent. The sterilization efficacy of bleaching powder against the bacteria in aquaculture water has been studied. It was found that the minimum bactericidal concentration (MBC) of bleaching powder to marine vibrio was 8-19 mg/L; the minimum inhibitory concentration (MIC) of vibrio was 4.6 mg/L (Jiang et al., 2009;Yang et al., 1999). According to our results analysis, the SC of bleaching powder to Artemia was inferior to the MIC, which was only 1.93 mg/L. It highlights that the sterilizing effect will not be obvious if safe concentration of bleaching powder is used. Therefore, the use of bleaching powder is not recommending for disinfection of the water of the Artemia.
The reports about the research on bactericidal effect of dibromohydantoin in aquaculture can be easily found in shrimps and crabs, fish, Stichopus japonicas, B. areolata and the SC to the animals above ranged from 0.6 mg/L to90 mg/L, however, there were few reports on the bactericidal effect of dibromohydantoin to Artemia (Zhao et al., 2014;Shi et al., 2008;Zha et al., 2010;Sun et al., 2008). Compared with the aquatic animals reported above, the Artemia were more sensitive to the toxicity of dibromohydantoin and the SC was only 0.41 mg/L. Therefore, in the production, it should not use dibromohydantoin disinfect aquatic water, avoiding the damage to Artemia.
A previous study has found that the five species of fish, such as Megalobrama amblycephala, Carassius auratus, Ctnopharyngodon idellus, Hypophthalmichthys molitrix, Spinibarbus sinensis were sensitive to methionine iodine and the SC of methionine iodine was 2.03-2.38 mg/L in these fish (Ye and Tu, 2009). Our study identified that the SC of methionine iodine was 2.73 mg/L against Artemia, which was slightly higher than the manufacture's recommended SC of 2.0 mg/L. However, during production, farmers are accustomed to use several times higher than the recommended dosage of disinfection drugs. Therefore, the dosage of disinfectant should be strictly controlled when using methionine iodine in the aquatic water for Artemia.
Glutaraldehyde is a broad-spectrum sterilizing agent to kill microorganisms (Thorn et al., 2013;Denyer and Stewart, 1998). Several studies have showed that MBC (1.6-4.2 mg/L) and MIC (0.9-3.2 mg/L) of glutaraldehyde O n l i n e

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varied against the Vibrio and Aeromonas and other bacteria in the aquaculture water (Kaleta, 2013). The results of our study showed that the SC of the glutaraldehyde against Artemia was 7.81 mg/L, which was much higher than the minimum bactericidal concentration against some pathogens. Therefore, glutaraldehyde is safe and effective to prevent and treat bacterial diseases in the water used for the production of Artemia.

Funding
This study was funded by the Youth Fund of Hainan Natural Science Foundation (321QN351) Industrial Technology System of Modern Agriculture (CARS-48), Special fund project for scientific and technological innovation and industrial development in Dapeng New Area (KJYF202101-08), National key research and development plan project (2022YFD2400104), Fangchenggang Science and Technology Plan Project (Fang ke AB22013015), Sanya Science and Technology Project (2018YD11).

IRB approval
The experimental protocol was approved by the animal ethics committee of South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences.

Ethical statement
The whole experiment was conducted according to the guidelines established by the National Institutes of Health.

Statement of conflict of interest
The author have declared no conflict of interest.