1 Introduction

Artemia,belong to theArthropoda,Branchiata,Cr-ustacea,Branchiopoda,Anostraca,Branchinectid-ae,Artemia,living in saltwater lakes and salt fields with high salinity[1].Distribution in salt fields and inland high salt waters of China's coastal provinces[2].Because of its wide distribution,strong environmental adaptability,fast reproduction,nutrient rich,convenient source,resting eggs can be stored for a long time and other advantages,Artemiabecome the most widely used live bait in aquatic animal breeding.

Artemia,because of its resting eggs and nauplii,is easy to obtain and to be cultivated.It has become an important material for the toxicity test.At present,there are many experimental study on toxicity of heavy metal ions on the domestic section on Artemia,for example:Guo[3],Xiu[4],Han[5],Xu[6]and others have done Cu2+,Hg2+,Se2+,Zn2+,Pb2+,Ni2+,Cd2+toxicity and other heavy metals onArtemianauplii.However,because of the environmental conditions used in the experiment and the difference in the origin of the Artemia,the result of the test is very different.Zhang[7]have shown the antagonistic effect of Pb2+-Mn2+on Russian Artemia by uniform design table method;Li[8]concluded that the Pb2+-Mn2+showed synergistic effect by using the same toxicity solution method for the Artemiaspecies of Ebinur Lake in Xinjiang.This may be related to the strain,hatching condition and design ofArtemia.

TheArtemianauplite and adult are very important biological bait for fish,shrimp,and crabs.This paper selects the fish breeding common resting eggs and more suitable for the water environment ofArtemiagrowth,studied the acute toxicity of copper,manganese and zinc onArtemianauplii and adults and larvae to three kinds of heavy metal ions on the joint toxicity of nauplii.Through the study of the acute toxicity effect of Cu2+,Mn2+and Zn2+onArtemia,improve the survival rate ofArtemianauplii and adults as well as quality,can reduce the cost in the process of aquaculture,so as to improve the economic benefit;on the other hand,using the sensitivity ofArtemiato heavy metals to monitor water is contaminated,especially the polluted water does not necessarily contain a heavy metal ions,has very important practical significance for the joint toxicity of various heavy metals onArtemiaas test,can put the environment biological indicators.

2 Materials and Methods

2.1 Material

2.1.1 Artemia

The resting eggs produced by a company in Tianjin were incubated in a full temperature oscillation incubator at 28.5℃,1 500 lx,hatching for 36 h.Nauplii at 15±3℃,salinity 30,and feeding on algae feed for 16 days in an aerobic environment.

2.1.2 Test drugs and equipment

CuSO4·5H2O(AR)，ZnCl2(AR)，MnCl4·4H2O(AR)，Full temperature oscillating incubator(HZQ-F160)

2.2 experiment design

The joint toxicity test of nauplii was carried out by the method of equal toxicity solution[8].The toxicity of each group was one toxic unit(48 h LC50of a certain test heavy metal ion to Artemia nauplius).The 48 h LC50of A and B heavy metal ions were multiplied by 0.8,0.6,0.5,0.4 and 0.2,respectively,and then the two combinations,as shown in Table 2.The test conditions were the same as the single factor toxicity test,and the mortality of 48 h was recorded in each group.

3.1.1 Toxicity of Cu2+to nauplii and adult ofArtemia

Tab.1 The concentration of metal ions in a single acute toxicity test

Note:Ⅰ-nauplius,Ⅱ-imago

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Take 100ml small beaker astest container,adding proper amount of water for each beaker,then according to the data of heavy metals solution pre-test results were added,each beaker into 40Artemianauplii or 20 adults,set a total of 7 concentration gradient and 1 control groups,each with three parallel,concentration setup is shown in Table 1.The test temperature was about 15±3℃.During the experiment,no bait was used and pH was kept in the optimum range.The death rate of artemia parasites in 24 hours and 48 hours was recorded.The 48 h LC50of each heavy metal onArtemiawas obtained by using EXCEL software to analyze and process the test data.

After the data were recorded,the experimental data were analyzed and processed by EXCEL software.According to the experimental results,the concentration-mortality curves were made,and the types of interaction between heavy metal ions were analyzed and determined.

2.2.2 Experimental design of combined toxicity of heavy metals

2.2.1 Design of single factor test for heavy metals

从“伙伴”到“对手”：《美国国家安全战略报告》的话语空间分析 ……………………… 刘文宇 徐博书（6.8）

Tab.2 Combined toxicity test concentration combination

Test combination A+B 1 2 3 4 5 0.8+0.2 0.6+0.4 0.5+0.5 0.4+0.6 0.2+0.8 Cu-Mn 0.8Cu+0.2Mn 0.6Cu+0.4Mn 0.5Cu+0.5Mn 0.4Cu+0.6Mn 0.2Cu+0.8Mn Cu-Zn 0.8Cu+0.2Zn 0.6Cu+0.4Zn 0.5Cu+0.5Zn 0.4Cu+0.6Zn 0.2Cu+0.8Zn Mn-Zn 0.8Mn+0.2Zn 0.6Mn+0.4Zn 0.5Mn+0.5Zn 0.4Mn+0.6Zn 0.2Mn+0.8Zn

2.3 Manipulation data

平扫下的肝局灶性结节性增生表现出了等密度或低密度肿块,界限清晰,钙化的情况比较少见。有20%的患者会有低密度瘢痕。使用增强扫描时,早期的肿块有快速明显的均匀强化,瘢痕在平扫时表现出低密度,瘢痕内供血动脉清晰,门静脉期的瘢痕逐渐强化或高密度表现。此次研究中,约有80%的肝脏局灶性结节性增生患者都没有明显的症状,一般都是在体检的时候发现典型CT表现,如平扫密度稍低,使用增强扫描动脉有中央瘢痕,病灶均匀强化,门脉期的病灶密度下降,延迟期的病灶为等密度表现,使用动态CT增强扫描在13个病灶中发现了7个中央瘢痕,肝脏局灶性结节性增生的主要特征就是在延迟期有中央瘢痕强化。

3 Results and Analysis

3.1 Single factor toxicity test of three kinds of heavy metal ions onArtemia

The preparation of three kinds of heavy metals Cu2+,Mn2+and Zn2+in solution with distilled water solution on the basis of the test results:copper sulfate concentration(as Cu2+)in the seawater salinity is 30 to 10-15 mg/L began to appear cloudy,after static precipitation.Testpreparation ofcopper,manganese and zinc in motherliquor concentration (with heavy metal ionsmeter)are 1 g/L,5 g/L and 20 g/L.The concentration of heavy metals is determined by pre-test,which is shown in Table 1.

As can be seen from Fig.1 the toxicity of Cu2+toArtemianauplius and adults increased with the increase of Cu2+concentration.The mortality of adultArtemiawas 90mg/L when the concentration of Cu2+was 4.0 mg/L.The mortality rate of adultArtemia increased more.The results of above showed that there was no significant difference between them(P>0.05).But Figure 1 shows that when the concentration of Cu2+reaches 4 mg/L,the mortality of nauplii is only about 60%,while the adult mortality is higher than 90%.

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Fig.1 Acute toxicity of Cu2+to Artemia nauplii and adults

3.1.2 Toxicity of manganese toArtemianaupli and adults

As can be seen from Fig.2,with the increase of the concentration of Mn2+,the toxicity of theArtemianaupli and the adult increased,and the death rate of theArtemiain the same concentration was higher than that of the adult.When the concentration of Mn2+is higher than 20 mg/L,which changes each unit,Artemiaadult mortality changes more.The variance analysis of the test results found that the difference ofArtemiatoxic effects of Mn2+on the two period was significant(0.012+).The results showed that Mn2+had a certain toxic effect on the growth and development ofArtemia.The toxicity of Mn2+to the adult ofArtemiawas greater,and the toxicity of Mn2+to the adult ofArtemia was linear.

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Fig.2 Acute toxicity of Mn2+toArtemianauplii and adults

As can be seen from Fig.3,with the increase of Zn2+concentration,the toxicity to Artemiain two periods increased.When Zn2+>20 mg/L,its toxicity toArtemiaadults was greater than that of nauplii.When the concentration of Zn2+increased by one unit,the adultArtemiavariance are more toxic.Analysis of the two stages ofArtemiamortality were not significant different in mortality in two cases (P>0.05),but can be seen from Figure 3 when the concentration of Zn2+reached 50 mg/L,nauplius mortality rate was only about 40%,and the adult mortality rate was as high as 90%.

Fig.3 Acute toxicity of Zn2+to Artemianaupli and adults

3.1.4 Comparison of Acute toxicity of three heavy MetalIons toArtemisnaupliand adults

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As can be seen from Fig.4,theArtemianauplii of Cu2+,Mn2+and Zn2+tolerance of the three kinds of heavy metals in 48 h LC50were 3.03,20 and 125 mg/L.The 48 h LC50of three heavy metals of Cu2+,Mn2+and Zn2+ofArtemiaadults were 1.5,6.63 and 32.5 mg/L,respectively.From these two groups of data,it can be seen that the toxicity of the three heavy metal ions to adultArtemiais higher,and the toxicity of Cu2+is greater than that of Zn2+.The order of toxicity was Cu2+>Mn2+>Zn2+.

AD9833可将DAC数据的最高有效位输出。通过对OPBITEN（D5）控制位置1，可将VOUT引脚输出DAC数据的最高有效位。这可以作为粗调时钟源。这个方波还可在输出之前进行2分频。控制寄存器的DIV2（D3）bit控制VOUT引脚提供的此输出的频率。

3.1.3 Toxicity of Zinc toArtemisnauplii and adults

Fig.4 Comparison of the 48 h semi lethal concentration of three heavy metals to the two stages ofArtemia

3.1.5 The safe concentration of heavy metals to nauplii and adults ofArtemia

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The safety concentration of various heavy metal ions onArtemianauplii and adults is shown in table 3.

Tab.3 Safe concentrations of various heavy metal ions for Artemia nauplii and adults Various heavy metal ions

Various heavy metal ions Safety concentration ofArtemianauplii and adult Artemianaupli Adult Cu 00.40 0.13 Mn 02.72 0.68 Zn 11.60 3.42

3.2 Combined toxicity test of heavy metals to nauplii ofArtemia

Fig.5 Combined toxicity test of three kinds of heavy metal ions

As shown in Fig.5,Cu-Mn,Cu-Zn and Mn-Zn ofArtemianauplii showed antagonistic effect.Three cases were mortality and theoretical analysis of variance results:There was a significant difference between the mortality rate and the theoretical mortality rate in the three conditions(P<0.01).The combination of Cu-Mn,Cu-Zn and Mn-Zn reduced the toxicity ofArtemianaupli.The mortality analysis of the three cases showed that:mortality difference was not significant(P>0.05),that the antagonistic between each combination has no significant difference.

4 Discussion

Copper,zinc and manganese are essential trace elements in animal body.Their toxicity to organisms is not only dependent on the chemical nature and form of metal,but also affected by the environmental factors and the physiological conditions of the individual animals.There are certain limits of essential elements in organisms,which can promote the growth and development of organisms in the range of physiological concentration.Too much will have a toxic effect on biology[8].

4.1 Toxicity of single heavy metals to Artemis nauplii and adults

The acute toxicity test of the three kinds of heavy metal ions onArtemianauplii and adults was tested in this experiment.The toxicity of different heavy metal ions is different from that ofArtemiaduring the same period.ForArtemianauplii,Cu2+showed greater toxicity,about 40 times of Zn2+,while for adults,the toxicity of Cu2+was about 20 times of Zn2+.The differences of the three kinds of heavy metals on the acute toxicity test on Artemia,may be due to the chemical structure of three kinds of heavy metal ions of different toxicological mechanism of different causes;may also be due to various heavy metal ions invade the body in different ways,together with the different absorption rate of Artemiaon different causes;there may be three kinds of trace elements in the body of the function.Copper is the active center of enzyme composition and variety of enzymes,content of medium can promote the growth of body[8];manganese activator of many enzymes,excess into the body it will produce toxic effects on the nervous system,and also can inhibit the activity of related enzymes[8];zinc is involved in the composition of more than 300 enzymes and functional proteins in animals,affecting many metabolic functions of the body.When the content is moderate,it can promote the growth of organism,and excessive will do harm to the organism[9].

The metal tolerance ofArtemiawas different in different developmental stages.The tolerance concentration of theArtemianauplii to heavy metals was higher,and the acute toxicity of heavy metal ions on the adultArtemiawas enhanced.At present,the results of acute toxicity test of heavy metal ions toArtemiaare still different.Xu[6]experiments showed that the 48 h LC50of Cu2+and Mn2+for the naupli ofArtemiawere 2.10 mg/L and 12.15 mg/L.Xiu[4]experiments showed that the 48 h LC50of acute toxicity of Zn2+to stageⅡ-Ⅲ Artemia was 19.1 mg/L.It is different from this test,which may be related to the strain,hatching and experimentalconditions of theArtemia(salinity,temperature,DO,light,pH and so on).

4.2 Combined toxicity test of a variety of heavy metals onArtemianauplii

The joint toxicity test of the three heavy metal ions toArtemiawas carried out in this experiment.The results showed that Cu-Mn,Cu-Zn and Mn-Zn showed antagonistic effects onArtemianauplii,but the antagonism between each group was not significant.The joint toxicity of Cu-Mn onArtemianauplii showed antagonistic action,the reason may be thatcopper is the active center of the composition and enzymes in organism,manganese is the activator of many enzymes in aquatic organisms,when the mass enters the body,may cause certain effects on the nervous system,while suppressing the activity of the enzyme.Therefore,the combined action of the two metal ions may prevent each other,thus reducing the toxicity of the twoArtemianauplii[8].Cu-Zn showed the reason of antagonism.It may be because copper thiophil belongs to heavy metals,and some biological tissue enzyme thiol has a great affinity[8].Zinc is part of many biological active site cofactor,zinc ion and copper ion compete in living organisms,because the active site metal into the cells of the body is limited,when zinc ion is far greater than the concentration of copper ion at the active site of these cells which mostly occupied by Zn2+,the toxicity Cu2+cannot enter the cell[10],so the two are showing antagonism.Mn-Zn showed antagonistic effect,may be due to zinc is part of many coenzyme,manganese activator of many enzymes in aquatic organisms,may increaseArtemianauplii activity of two kinds of metal ions when combined,thereby reducing its toxicity,so the two are showing antagonism.

Chen[10]and others studied the combined action of Zn2+,Cu2+,Hg2+,Cd2+and Pb2+on Hydra.The experiment showed that the four cases showed an antagonistic effect on the mortality of Hydra,and the mortality of Hydra in four cases was not significant.The results of this paper are similar.On one hand,it is possible that one of the metal ions is more easily absorbed by the body and occupies the active sites of relatively more biological cells in advance because of the effect of heavy metals.Because of the limited activity site of cells,the damage of other heavy metals to the body is very small.There may be no significant difference in the number of active sites occupied by several kinds of heavy metals,so the antagonistic effect is also not significant.On the other hand,it may be that the experimental conditions are limited,and the toxicity test method is not perfect,so the difference between the test results is not significant.Therefore,we need to constantly combine the knowledge of biometrics and constantly improve the test methods.It will be seen from this that the toxicity of two kinds of heavy metal ions onArtemiamay be weakened or enhanced.After the study of toxicity to organisms,we can take into account the possible interactions between different compounds.

It can be seen from this experiment that the toxicity test of single heavy metal to Artemiacan only depend on its physical and chemical properties and absorption speed.The joint toxicity of many heavy metal ions may also depend on the interaction between two heavy metal ions.In this experiment,the effect of the death ofArtemiawas used as a standard to detect the effect of heavy metals on theArtemia.Only a combination of different heavy metal ions between the joint toxicity was tested.Result analysis is just a list of the line graph,and no equation was written because of the complex effects of heavy metals on Artemiacan not rely on a simple equation to express its influence.Using mathematical statistics related knowledge to increase the number of treatment,improve the analysis of design and test software,joint toxicity analysis of multi factors is an important research direction in the future.

陈东成在《大易翻译学》中指出，翻译应坚持两条基本原则：求同存异，守经达权。文无定诠，译无定法，变文之数无方，唯有灵活运用，通权达变，方可译出使人知之，乐知，好知的译文。翻译时译者应遵循“适旨、适性、适变、适度”的调整原则，译文便可产生翻译之美。翻译是一个经纬交织的过程，亦是一个经权结合的过程，有坚持，也有变，将体相用融会贯通，指导具体的翻译实践。

5 Conclusion

The acute toxic effects of Cu2+,Mn2+and Zn2+on the nauplii and adult ofArtemiawere studied in this experiment.Based on these,the joint toxicity tests of three heavy metal ions againstArtemianauplii were carried out.The test results showed thatArtemianauplii tolerance of the three kinds of metal ions were higher than that of adults,Cu2+,Mn2+and Zn2+48 h LC50were 4.0、27.19 and 115.95 mg/L,Cu2+,Mn2+and Zn2+and the adult 48 h LC50 is only 1.31,6.86 and 34.23 mg/L.The results of combined toxicity test showed that Cu-Mn,Cu-Zn and Mn-Zn were all antagonistic to theArtemianauplii.

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