甲醛对中华绒螯蟹不同阶段幼体的毒性研究

Agricultural Science ＆ Technology, 2019, 20(5): 46-50
Copyright ⓒ 2019, Information Institute of HAAS. All rights reserved
1. Introduction
Eriocheir sinensis , also known as Chinese mitten crab or river crab, is a higher crustacean belonging to Grapsidae family and Eriocheir genera. Eriocheir sinensis is a famous Chinese local food characterized by its special taste and high nutrition. The high-grade aquatic species is welcomed by numerous consumers and is also one of China’s key export freshwater aquaculture products. Much progress has been made in the artificial breeding of Eriocheir sinensis with the promotion of outdoor earthen pond ecological breeding [1]. In river crab breeding or indoor breeding studies, the growing of aufwuch such as Carchesium and Zoothamnium would influence the transformation or even lead to deaths of crab larvae. Nystatin and malachite green have good effect in eliminating such infusorians, but they would leave toxic residual or cause resistance to drugs, which explains why many of such chemical products were forbidden [2].
Formaldehyde (HCHO) is the simplest aldehydes which is usually colorless, transparent, combustible and with pungent odour.
Supported by the Guiding Program of Liaoning Key Research & Development Project (2018203002).
E-mail: sunna0911@
A
甲醛对中华绒螯蟹不同阶段幼体的
毒性研究
孙 娜 1
, 王雨浓 2
, 刘 胥 1, 雷衍之 2 （1. 盘锦光合蟹业有限公司，辽宁省中华绒螯蟹育种重点实验室，辽宁 盘锦 124200；2. 大连海洋大学，辽宁 大连 116023）
摘　要　采用半流水实验法，对中华绒螯蟹各期溞状幼体进行急性毒性试验，以确定甲醛对中华绒螯蟹幼体的毒性效应。

结果表明：甲醛对中华绒螯蟹各期溞状幼体毒性作用明显，甲醛浓度越大，幼体死亡率越高。

其中 Z1的24和48 h 半致死浓度分别为 52.029和 29.300 mg/L ，安全浓度为2.79 mg/L 。

Z2的 24 和 48 h 半致死浓度分别为76.323和50.892 mg/L ，安全浓度为6.79 mg/L 。

Z3的24和48 h 半致死浓度分别为108.947 和67.693 mg/L ，安全浓度为7.84 mg/L 。

Z4 的 24 和 48 h 半致死浓度分别为121.414和75.480 mg/L ，安全浓度为8.75 mg/L 。

Z5的24和48 h 半致死浓度分别为128.546和89.312 mg/L ，安全浓度为12.934 mg/L 。

关键词　中华绒螯蟹；急性毒性；甲醛；半致死浓度；安全浓度
A cute Toxicity of Formaldehyde to Eriocheir sinensis Zoea
SUN Na 1, WANG Yu-nong 2, LIU Xu 1, LEI Yan-zhi 2
1. Key Laboratory of Eriocheir sinensis Breeding, Liaoning Province, Panjin Photosynthetic Crab Industry Co. Ltd., Panjing 124200, PRC;
2. Dalian Ocean University, Dalian 116023, PRC
基金项目　辽宁省重点研发计划指导计划项目（2018203002）作者简介　孙　娜 （1983—），女，山东滨洲人，工程师，主要从事水产养殖水质调控工作。

Abstract The acute toxicities of formaldehyde to Chinese mitten crab (Eriocheir sinensis ) zoea at various stages in semi-static system were studied. Each stage larvae was set with 5 concentration gradients and a contrast, and each concentration gradient had three parallels. The LC 50 of formaldehyde to Z1 was found to be 52.029 mg/L for 24 h and 29.300 mg/L for 48 h, with safety concentration of 2.79 mg/L; LC 50 to Z2 was 76.323 mg/L for 24 h, 50.892 mg/L for 48 h, with safety concentration of 6.79 mg/L; LC 50 to Z3 was 108.947 mg/L for 24 h, 67.693 mg/ L for 48 h, with safety concentration of 7.84 mg/L; LC 50 to Z4 was 121.414 mg/L for 24 h, 75.480 mg/L for 48 h, with safety concentration of 8.75 mg/L; LC 50 to Z5 was 128.546 mg/L for 24 h, 89.312 mg/L for 48 h, with safety concentration of 12.934 mg/L.
Key words Chinese mitten crab; Acute toxicity; Formaldehyde; Median lethal concentration; Safety concentration DOI:10.16175/ki.1009-4229.2019.05.008
Formaldehyde can dissolve lipids by combining with the amino group in protein and cause the coagulation and denaturation of the protein. Both gas and liquid formaldehyde present powerful sterilization effect. Similar to the forbidden drugs, formaldehyde also has certain toxic and side effects. But compared with those forbidden drugs, formaldehyde has better effects and smaller side effects. Therefore, formaldehyde has become an ideal drug to eliminate Zoothamnium in small-scale breeding. The dosage of using formaldehyde is critical. Inadequate dosage of formaldehyde is not effective, while overdose may influence the growth of larvae or even lead to deaths. Previous researches have studied the toxic effects of formaldehyde on fish [3], shrimps [4], shellfish [5] and other aquatic animals. For Eriocheir sinensis, only acute toxicities of formaldehyde to the Eriocheir sinensis [6] zoea at various stages in semi-static system were studied. But no general rule for Z5 zoea has been explored. In this paper, the research group studied the tolerances of Eriocheir sinensis zoea at various stages and the megalopa on formaldehyde in Panjin Photosynthetic Crab Industry Co. Ltd. The research aims to find out the most appropriate dosage of formaldehyde used for Eriocheir sinensis zoea at various stages, and provide theoretical basis and practical references for disease control and improvement of larval survival in artificial breeding and family breeding of Eriocheir sinensis.
2. Materials and Methods
2.1. Materials
Tests were conducted in April—July 2017. Erio- cheir sinensis zoea larvae at various stages were provided by Panjin Photosynthetic Crab Industry Co. Ltd. We selected those larvae with neat transformation and good activity and fed them with chlorella and rotifera every day. As the newly transformed larvae are usually weak and the transformation usually takes 2~3 d under test condition, we selected larvae that had transformed after 12 h and tried to keep the samples accurate [6]. The formaldehyde solution was purchased from Shenyang Rongxing Chemicals. The solution is analytical grade and had a content of 36%~40%. The test seawater was obtained from the original parent crab pool processed with aeration treatment. The salinity was 25.0~26.0, temperature 20~25℃ and pH value 8.2~8.4. A 4 L plastic square container (35 cm×50 cm×15 cm) and an 1 L beaker were prepared for the breeding test.
2.2. Methods
Preliminary test is conducted using single factor test approach and acute toxicity test method. According to the principle of equal logarithmic spacing, each stage larvae were set with five concentration gradients and a contrast; each concentration gradient had three parallels; ten larvae were put in each concentration group. The water was replaced completely after 24 h of the test; the formaldehyde solution was prepared each time when needed during the water replacement. In the test, we fed Z1~Z4 zoea larvae with chlorella and rotifer and Z5 larvae with fresh artemia nauplii; continuously observe the larvae for the first 8 h, then observe the activity of larvae every 4 h; record the number of dead larvae at 24 and 48 h respectively, and take the bodies out in time. The standard for judging their deaths is that they sink to the bottom, turn white and have no responding to the outside stimuli.
2.3. Data analysis
The natural mortality correction is conducted using the equation of Abbot [7]: P=P'-C/1-C, where P is the natural mortality (%), P' is the test group mortality (%) and C is the control mortality (%). All the data are based on the average of three parallels.
Median lethal concentration is calculated using linear regression method, i.e. the linear regression of corrected mortality and concentration logarithm. The LC50 at 24 and 28 h and 95% confidence interval are calculated using statistical method. A significance analysis on the linear regression of drug concentration logarithm and mortality of testing samples is conducted through F-test. Then chi-square test is performed to identify the goodness of fit between drug concentration logarithm and the linear regression of
sample mortality. The data processing is performed by SPSS 16.0 software. For specific operations, please refer to the data processing method by AN S L et al. [8] and JIA C S [9].
Safety concentration can be calculated by the following equation, where SC is the safety concentration, 24 h LC50 stands for the median lethal concentration at 24 h, and 48 h LC50 stands for the median lethal concentration at 48 h.
3. Results and Analysis
Table 1 shows that the mortality of Eriocheir sinensis larvae at each stage gradually rises with the increase of the concentration of formaldehyde solution and the test time. The mortality at 24 h is similar to that at 48 h when the concentration of formaldehyde solution is low. When the concentration increases, the difference between the mortality at 24 and 48 h becomes significant, until the mortality at 24 and 48 h both reach 100%. For instance, when the concentration is 5 mg/L, the mortality of Z1 larva at 24 and 48 h is both 4%; when the concentration increases to 23 mg/L, the mortality will increase to 32% at 24 h and 40% at 48 h. When the concentration reaches 110 mg/L, the mortality at 24 and 48 h are both 100%. This rule is also applicable to other stages of larvae.
Table 2 shows the chi-square test results and the “corrected mortality - quality concentration logarithm” regression equation calculated by acute toxicity test. According to the toxicity test of formaldehyde to Eriocheir sinensis zoea, the LC50 and the safety
concentration at 24 and 48 h gradually increase with the growing of zoea. The LC50 of formaldehyde to Z1 was found to be 52.029 mg/L for 24 h and 29.300 mg/L for 48 h, with safety concentration of 2.79 mg/L; LC50 to Z2 was 76.323 mg/L for 24 h, 50.892 mg/L for 48 h, with safety concentration of 6.79 mg/L; LC50 to Z3 was 108.947 mg/L for 24 h, 67.693 mg/ L for 48 h, safety concentration 7.84 mg/L; LC50 to Z4 was 121.414 mg/L for 24 h, 75.480 mg/L for 48 h, safety concentration 8.75 mg/L; LC50 to Z5 was 128.546 mg/L for 24 h, 89.312 mg/L for 48 h, safety concentration 12.934 mg/L. This indicates that the resistance of Eriocheir sinensis zoea to formaldehyde toxicity is strengthening as they grow up.
4. Discussion
According to the test results, as the exposure continues, the Eriocheir sinensis zoea larvae treated with different concentrations of formaldehyde presented different levels of toxic reaction. The toxic reaction of the low concentration group is not significant. The larvae of this group are less active and react slowly. The high concentration group had Table 1 Toxicity of formaldehyde to Eriocheir sinensis zoea
(Z1~Z5)
Stage of
larva
Formaldehyde
concentration
//mg/L
24 h48 h
Mortality
//%
Corrected
mortality
//%
Mor-
tality
//%
Corrected
mortality
//% Z1
016.7016.70
5204204
11301633.3320
2343.33325040
5156.674866.6760
110100100100100 Z2
000 3.330
16 3.33 3.33 6.67 3.45
34202026.6724.14
7243.3343.3376.6775.86
151808093.3393.1
320100100100100 Z3
000 3.330
30 3.33 3.332017.24
5816.6716.6736.6734.48
112404073.3372.41
21793.3393.33100100
420100100100100 Z4
00000
30001010
6116.6716.6726.6726.67
12446.6746.676066.67
2519090100100
510100100100100 Z5
000 3.330
40 6.67 6.671010
76202043.3343.33
14343.3343.3373.3373.33
27093.3393.33100100
510100100100100
apparent toxic reaction at 3 h. The larvae of this group became white and stiff in appendage. Most of them would sink to the bottom of beaker, turn around sometimes, have weak response to the outside stimuli, and start to die at 6 h. The dead larvae would sink to the bottom, turn white and become stiff, obviously larger than normal larvae. Comparatively, the larvae of the control group are active. They move fast, swim at the middle and lower part of the water, or stick to the wall of the beaker. These larvae have strong abdominal contraction and react quickly to the outside stimulus. This result is similar to the observation results of the larvae intoxicating by SHEN H D et al. [6] and LI A et al. [10].
By combining the amino group of protein, formaldehyde can coagulate protein and interfere with the physiological activities of cells. For this reason, formaldehyde can kill most of pathogenic microorganisms and is widely used for disease control in breeding practice. The mechanism is to transform the protein through alkylation reaction, change the functions of enzymes and nucleic acids to realize the disinfection and sterilization. Meanwhile, formaldehyde can dissolve lipids, damage the permeability of cell membrane, and disequilibrate the inner and outer state of river crab larva. That’s why the body of river crab becomes bigger after death [6]. Formaldehyde is featured with broad-spectrum bactericidal properties and has strong killing power on sessile ciliates parasitized on Eriocheir sinensis zoea or surface of prawn, or dactylogyrus, gyrodactylus and trichodina parasitized on fish [2]. Formaldehyde also has certain effect on killing virus [11]. Studies have shown that the safety concentration of formaldehyde on the stage II larva of Orithyia sinica and the Litopenaeus vannamei is 23.4 μL/L and 2.62 mg/L, respectively [4]. Therefore, the safety concentration on crustacean larva is similar, but the sensitivity in descending order is: Litopenaeus vannamei>Eriocheir sinensis>Orithyia sinica. In addition, the safety concent- ration of formaldehyde on juvenile sea cucumber, Babylonia areolata and Megalobrama pellegrini is 3.18×10-6, 0.283 and 7.50 mg/L[12], respectively. To sum up, different species or one species at different stages present different degrees of sensitivity to formaldehyde, and their living conditions have significant effects on these differences.
Compared with other drugs, povidone-iodine has better effect than potassium permanganate and oxytetracycline in terms of the improvement of larval survival and molt rate [13]. The toxicity of the three drugs to Litopenaeus vannamei in descending order is: potassium permanganate>povidone-iodine>formaldehyde [4]. DING C S [14] reported that the safety concentrations of dipterex on the larve of Eriocheir sinensis are 0.001 01, 0.002 62, 0.000 63 and 0.002 02 mg/L, obviously lower than the concentration of this study. In conclusion, the toxicity of the above four drugs to shellfish in descending order is: dipterex>potassium permanganate>povidone-iodine>formaldehyde. The breeding of Eriocheir sinensis is a continual process and the transformation of their larvae may vary due to various factors. Besides, frequent changing of water poses large stress
Table 2 Median lethal concentration, 95% confidence interval and safety concentration of formaldehyde to
Eriocheir sinensis zoea at each stage
Stage of larva Item Median lethal concentration//mg/L95% confidence interval Safety concentration//mg/L
Z124 h52.02930.164~471.647
2.79 48 h29.30019.894~39.176
Z224 h76.32360.443~101.065
6.79 48 h50.89240.694~62.426
Z324 h108.94791.403~132.494
7.84 48 h67.69354.374~82.421
Z424 h121.414101.357~147.340
8.75 48 h75.48061.138~99.839
Z524 h128.546108.066~153.429
12.934 48 h89.31275.413~105.110
on the larvae. Therefore, the author proposes to use the safety concentration at Z1 stage as the maximal dosage in actual production. Specifically, when combined with the studies by SHEN H D et al., the dosage of formaldehyde used in the disease control for Eriocheir sinensis zoea shall not exceed 3 mg/L. References
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Responsible editor: LI Wen
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