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Abstract The large plastic produced by human production and life is degraded into micro??plastics in the environment. Micro??plastics are harmful to organisms and the environment. In order to determine the hazards of micro??plastic pollution， zebrafish embryos were used as the tested organism， and 0.6-1.0 ??m polystyrene micro??plastic was used to carry out embryo exposure experiment. The toxic effects of polystyrene plastic particles on zebrafish embryos were investigated comprehensively， and the toxicity was evaluated. The results of this study showed that 0.6-1.0 ??m polystyrene micro??plastic had certain toxicity on zebrafish embryos， and it had a lethal effect when the concentration reached 250 mg/L； 25 mg/L 0.6-1.0 ??m polystyrene particles can cause cyrtosis and pericardial edema of zebrafish embryos and other non??lethal toxic effects； and 250 and 1 000 mg/L exposure concentration can slow down heart rate. A hazard evaluation index system of polystyrene particles to zebrafish embryos was established， and it is determined that the exposure to 250 mg/L or lower concentration of polystyrene particles has slight hazard.
Key words Micro??plastics； Polystyrene； Zebrafish； Embryos
Plastic particles with a diameter within 5 mm are known as micro??plastics[1]. In recent years， studies on micro??plastics have increased sharply[2]. These studies are mostly concentrated on ecological effect and investigation of source， distribution and abundance. The studies on the ecological effect of micro??plastics are to investigate the accumulation of micro??plastics in living body and corresponding effect through laboratory simulation， and are mostly conducted at the coastal regions and oceans?? surface layer[1-4]. Harmful pollutants also could adhere to the surface of micro??plastics， forming a combination which negatively influences environment and organisms[5].
Lu et al.[5] studied polystyrene micro??plastic with zebrafish embryos as experiment materials， which were exposed to microplastic environment for 7 d. The results showed that 5 ??m micro??plastic accumulated in the gill， liver and digestive tract （viscera） of zebrafish embryos， while 20 ??m microplastic only accumulated in fish gill and digestive tract （viscera）. There may be two reasons for such phenomenon. Firstly， 5 ??m microplastic particles could enter circulatory system and be transported to liver. Secondly， the difference observed between large particles and small particles might be due to concentration and the probability of one particle to contact tissue. For instance， the quantity of 5 ??m polystyrene microplastic particles per unit area is higher than 20 ??m polystyrene micro??plastic particles by two magnitudes. In this study， on the basis of zebrafish embryo model， an in??vitro exposure experiment was carried out using commercial micro??plastic particles （0.6-1.0 ??m polystyrene micro??plastic）， with an attempt to provide basic data for the evaluation of the toxicity of micro??plastics through the detection of a series of indices.
Materials and Methods
Materials
The tested organisms in this study were AB wild type zebrafish. The eggs were cultured in a glass aquarium with non??interrupted water recycling and filtration and aeration at （26??1）?? to fish， which was fed twice daily. The used water was aerated tapped water with a pH value of 7.5-8.0 and saturation percentage of dissolved oxygen higher than 80%. The fish reaching sexual maturity could be induced to naturally mate and spawn according to OECD236 laboratory manual. Fish eggs were collected 1 h later and flushed for 3-5 times. Normally??developed fertilized eggs were selected under a dissecting microscope and used for the experiment 2 h after fertilization. The used micro??plastic was 0.6-1.0 ??m polystyrene microspheres （CAS 9003??53??6， with a purity o 2.5%） produced by Shanghai Jingchun Biochemical Technology Co.， Ltd.
Instruments
Vacuum instrument： Olympus BX51 vertical fluorescence microscope； OlympusIX71 inverted fluorescence microscope； Olympus DP71 digital microscopic camera （Olympus， Japan）； LRH??250??GSB1 manual climatic box （Taihong Medical Equipment Co.， Ltd.）.
Experimental methods
Preliminary experiment The fertilized zebrafish eggs were placed in 48??well culture plates. Five groups were set， each of which had four parallel samples. Into each well， 3 ml of blank control solution and 10 fertilized eggs were added sequentially. The original polystyrene micro??plastic liquid was diluted to 10 times， 100 times， 1 000 times and 10 000 times groups， each of which （2 ml） was added into corresponding experimental group， to carry out preliminary exposure experiment. The blank group （CK） was free of treatment. The eggs were cultured in an illumination constant??temperature incubator to 96 h. Every 24 h， the exposure liquids and blank liquid were replaced， and the death and hatching conditions of the embryos were recorded. The development condition of zebrafish embryos was observed under a microscope and recorded according to different stages. The formal experimental doses were determined according to the results of the preliminary experiment. Exposure experiment of zebrafish embryos 0.6-1.0 ??m polystyrene particles were used for the exposure experiment. The four experiment concentrations designed according to the preliminary experiment were 1 000， 250， 25 and 2.5 mg/L， respectively. Each group included five parallel samples. The specific operation was the same as the preliminary experiment. In the experimental process， the mortality and hatchability of the embryos were observed by semi??static method. When replacing the toxic liquid， coagulated eggs were also removed， and the development status of zebrafish embryos was observed under a microscope. The lethal indicators were egg coagulation， no beginning of gastrulae differentiation， no termination of gastrulae differentiation， no somite， no tail extension， no heartbeat and no hatching. The teratogenic indicators included changed body length， cyrtosis， malformed tail， and changed heart rate. For the computation of heart rate， 9 embryos were randomly selected from the four replicates of each treatment to carry out 1 min of cardiac impulse statistics. The observation indicators of zebrafish embryos at various exposure time were shown in Table 1.
Statistic analysis Mortality， hatchability， malformation rate and heart rate were recorded. Significance of differences in various indices was analyzed between the control group and experimental groups by one??way analysis of variance （ANOVA）， in which P??0.05 indicates a significant difference （*）， and P??0.01 indicates a very significant difference （**）.
The obtained toxicological indicators were classified （according to the study of Nagel et al.[6]）， into lethal indicators and nonlethal indicators. The sum of weights of lethal indicators （summarized as mortality） and nonlethal indicators was hazard index P， the formula of which is shown as below：
P=??fI?¤DI+??nfIIn?¤DIIn
Wherein fI is the weight of lethal indicator； DI is mortality； n is the number of nonlethal indicators； fIIn is the weight of nonlethal indicator； and DIIn is the occurring rate of nonlethal indicator.
The establishment of the hazard evaluation index system of polystyrene to zebrafish embryos and the determination of the weight of hazard evaluation index were performed by analytic hierarchy process[7]. Each indicator was subjected to pairwise comparison on degree of importance， so as to evaluate stepwise. During the analysis， judgment matrix was established， followed by the determination of the eigenvector corresponding to the largest eigenvalue by square root methods and the acquisition of approximate values， and finally， the importance weight of single indicator to the general objective was obtained. The hazard evaluation index system and weight of polystyrene to zebrafish embryos are shown in Table 2. The hazard grade of polystyrene microplastic to zebrafish embryos was divided using its hazard index. The hazard grades corresponding to hazard index are shown in Table 3.
Results and Discussion
Effects of polystyrene to zebrafish embryos
As shown in Fig. 1， zebrafish embryos developed normally at 24 （Fig. 1a） and 48 h （Fig. 1b） in the CK， while in higher micro??plastic concentrations， some zebrafish embryos died （Fig. 1c） or developed slowly with malformation （Fig. 1d）.
Tetratogenic situation of zebrafish embryos exposed to polystyrene
Zebrafish embryos were exposed to 2.5 to 1 000 mg/L poly??
styrene toxic liquid and observed for 72 h. The mortality and hatchability of zebrafish embryos are shown in Fig. 2.
The mortality of zebrafish embryos increased significantly with the concentration increasing. The mortality was 7% averagely at the polystyrene concentration of 250 mg/L， and was 98% averagely at the polystyrene concentration of 1 000 mg/L， with a very significant difference from the CK （P??0.01）. Furthermore， the mortality of zebrafish embryos exhibited dose??effect relationship.
The 72 h hatchability of zebrafish embryos decreased with the polystyrene concentration increasing. The eggs in the CK were all hatched， with an average hatchability of 99%， and the 2.5， 25， 250 and 1 000 mg/L groups showed the average hatchability of 97%， 89%， 59% and 0%， respectively. Among the various concentrations， the 25 mg/L group was significantly different from the CK group （P??0.05）， and the 250 and 1 000 mg/L groups were very significantly different from the CK （P??0.01）.
The teratogenic effect of polystyrene microplastic on zebrafish embryos is shown in Fig. 3. The microscopic observation at 48 h showed that zebrafish embryos suffered from cyrtosis， and when the concentration reached 25， 250 and 1 000 mg/L， the average cyrtosis rate increased largely， to 32%??65% and 100%， respectively. When the concentration reached 25 mg/L， very significant cyrtosis was observed （P??0.01）.
Variation in heart rate of zebrafish embryos exposed to polystyrene
In the 48 and 72 h observation， the pericardial edema rate of zebrafish embryos increased greatly with the concentration increasing. The 48 h experimental groups had the average pericardial edema rates of 3%， 32%， 62% and 95%， respectively， and when the concentration reached 25 mg/L， the difference from the CK became very significant. The 72 h experimental groups showed the 72 h average pericardial edema rates of 5%， 30%， 65% and 86%， respectively， and also， when the concentration reached 25 mg/L， the difference from the CK became very significant. The heart rate variation of zebrafish embryos caused by the exposure to polystyrene microplastic is shown in Fig. 4. In the 24 and 48 h heart rate observation， little differences were observed between the CK and the experimental groups， and only the highest??dose group （1 000 mg/L） showed remarkably decreased heart rate. In the 24 h observation， the average heart rates of the CK and the experimental groups were 53， 52， 54， 46 and 35 times/min， respectively， and the 1 000 mg/L group was very significantly from the CK （P??0.01）. During the 48 h heart rate observation， the average heart rates of the CK and the experimental groups were 105， 103， 98， 91 and 57 times/min， respectively and a very significant difference was observed at the concentration of 1 000 mg/L.
Table 4 shows the hazard grades of various polystyrene concentrations. The polystyrene at the concentrations of 2.5， 25 and 250 mg/L had a hazard of grade I， which was light. When the concentration increased to 1 000 mg/L， the hazard was grade IV， which was strong.
Discussion
The results of this study showed that polystyrene did not cause death of zebrafish embryos at the concentration lower than 25 mg/L， and had an observed lethal concentration of 250 mg/L， and when the concentration increased to 1 000 mg/L， the mortality of zebrafish embryos nearly reached 100%. During the 72 h experimental observation， various experimental groups all exhibited delayed hatching， and the hatchability decreased sharply with the increase of polystyrene concentration.
Zebrafish embryos suffered from malformation at 48 h， which was mainly reflected by cyrtosis and pericardial edema， and cyrtosis rate and pericardial edema rate both increased with the polystyrene concentration increasing. The highest??dose group showed a cyrtosis rate of 100% at 48 h. There was no big difference in pericardial edema rate between 48 and 72 h. These results also reflected the dose??effect relationship of polystyrene liquid， and the observed little difference was related to selection of samples.
In this study， heartbeat of zebrafish embryos was observed at 24 h， and heart rate was measured at 24 and 48 h， respectively. The 48 h heart rates were all higher than the 24 h heart rates. In the 24 h observation， there were no big differences between the experimental groups and the CK， only the highest??dose group had a very significant difference from the CK. At 48 h， the 2.5 and 25 mg/L groups still had no big differences from the CK， but when the experimental concentration reached 250 mg/L， the heart rate began to decrease remarkably， and had a sharp drop at 1 000 mg/L. The effect of polystyrene microplastic on zebrafish embryos was firstly reflected by heart rate， which might be because in the development process of zebrafish embryos， heart is the organ developing the earliest[7-8]. Yang et al.[8] studied the effect of BPAF on zebrafish embryos and juvenile fish. The research results showed that BPAF exposure very significantly inhibited the heart rates of zebrafish embryos and juvenile fish， which both had a trend of increasing at first and decreasing then， indicating that heart is closely related to the survival of living organisms， and unsound heart development affected blood circulation of zebrafish embryos which thus suffered from delayed hatching and malformation. The variation in 24 h heart rates of various experimental groups was not as remarkable as that in 48 h heart rates of various experimental groups， and the 48 h heart rates of various experimental groups were all higher than the 24 h ones， suggesting that the effect of polystyrene micro??plastic on the heart of zebrafish embryos needs to accumulate to certain time. The heart rates of high??dose groups were remarkably lower than those of low??dose groups， suggesting that polystyrene micro??plastic only could affects zebrafish embryos when reaching certain concentration， while 25 mg/L and lower concentrations are safe exposure concentrations.
Conclusions
0.6-1.0 ??m polystyrene micro??plasctic has certain toxicity on zebrafish embryos， and can cause death of zebrafish embryos at the concentration of 250 mg/L.
25 mg/L 0.6-1.0 ??m polystyrene particles would cause nonlethal toxic effects such as cyrtosis and pericardial edema of zebrafish embryos， and 250 and 1000 mg/L can slow down their heart rate.
A hazard evaluation index system of polystyrene particles to zebrafish embryos was established， and it is determined that the exposure to 250 mg/L or lower concentration of polystyrene particles has slight hazard.
References
[1] LIAO Q， QU JS， WANG JP， et al. Pollution analysis of plastic and improving proposal in marine environment[J]. World Sci??Tech R & D， 2015（2）： 206-211， 217.
[2] ZHOU Q， ZHANG HB， LI Y， et al. Progress on microplastics pollution and its ecological effects in the coastal environment[J]. Chinese Science Bulletin， 2015（33）： 3210-3220.
[3] ZHOU Q. Micro??plastic pollution characteristics and ecological risk in typical coastal tidal flat and paralic environment[D]. Shandong： Yantai Institute of Coastal Zone Research， Chinese Academy of Sciences， 2016： 1-106.
[4] SUN XX. Progress and Prospect on the Study of the ecological risk of microplastics in the ocean[J]. Advances in Earth Science， 2016（6）： 560-566.
[5] LU Y， ZHANG Y， DENG Y， et al. Uptake and accumulation of polystyrene microplastics in zebrafish （Danio rerio） and toxic effects in liver[J]. Environ. Sci. Technol.， 2016（50）： 4054-4060.
[6] NAGEL R， DAR T. The embryo test with the zebrafish （Danio rerio）： A general model in ecotoxicology and toxicology[J]. Altex， 2002， 19（suppl1）： 38-48.
[7] YU FF， TANG TL， BAI JJ， et al. Toxicities and hazard classification of reclaimed water after disinfection of different approaches by zebrafish embryos bioassay[J]. Asian Journal of Ecotoxicolog， 2015（2）： 313-319.
[8] YANG Y， CHEN YW， TANG TL， et al. Toxic effects of bisphenol AF on zebrafish embryos and larvae[J]. Research of Environmental Sciences， 2015（8）： 1219-1226.
Key words Micro??plastics； Polystyrene； Zebrafish； Embryos
Plastic particles with a diameter within 5 mm are known as micro??plastics[1]. In recent years， studies on micro??plastics have increased sharply[2]. These studies are mostly concentrated on ecological effect and investigation of source， distribution and abundance. The studies on the ecological effect of micro??plastics are to investigate the accumulation of micro??plastics in living body and corresponding effect through laboratory simulation， and are mostly conducted at the coastal regions and oceans?? surface layer[1-4]. Harmful pollutants also could adhere to the surface of micro??plastics， forming a combination which negatively influences environment and organisms[5].
Lu et al.[5] studied polystyrene micro??plastic with zebrafish embryos as experiment materials， which were exposed to microplastic environment for 7 d. The results showed that 5 ??m micro??plastic accumulated in the gill， liver and digestive tract （viscera） of zebrafish embryos， while 20 ??m microplastic only accumulated in fish gill and digestive tract （viscera）. There may be two reasons for such phenomenon. Firstly， 5 ??m microplastic particles could enter circulatory system and be transported to liver. Secondly， the difference observed between large particles and small particles might be due to concentration and the probability of one particle to contact tissue. For instance， the quantity of 5 ??m polystyrene microplastic particles per unit area is higher than 20 ??m polystyrene micro??plastic particles by two magnitudes. In this study， on the basis of zebrafish embryo model， an in??vitro exposure experiment was carried out using commercial micro??plastic particles （0.6-1.0 ??m polystyrene micro??plastic）， with an attempt to provide basic data for the evaluation of the toxicity of micro??plastics through the detection of a series of indices.
Materials and Methods
Materials
The tested organisms in this study were AB wild type zebrafish. The eggs were cultured in a glass aquarium with non??interrupted water recycling and filtration and aeration at （26??1）?? to fish， which was fed twice daily. The used water was aerated tapped water with a pH value of 7.5-8.0 and saturation percentage of dissolved oxygen higher than 80%. The fish reaching sexual maturity could be induced to naturally mate and spawn according to OECD236 laboratory manual. Fish eggs were collected 1 h later and flushed for 3-5 times. Normally??developed fertilized eggs were selected under a dissecting microscope and used for the experiment 2 h after fertilization. The used micro??plastic was 0.6-1.0 ??m polystyrene microspheres （CAS 9003??53??6， with a purity o 2.5%） produced by Shanghai Jingchun Biochemical Technology Co.， Ltd.
Instruments
Vacuum instrument： Olympus BX51 vertical fluorescence microscope； OlympusIX71 inverted fluorescence microscope； Olympus DP71 digital microscopic camera （Olympus， Japan）； LRH??250??GSB1 manual climatic box （Taihong Medical Equipment Co.， Ltd.）.
Experimental methods
Preliminary experiment The fertilized zebrafish eggs were placed in 48??well culture plates. Five groups were set， each of which had four parallel samples. Into each well， 3 ml of blank control solution and 10 fertilized eggs were added sequentially. The original polystyrene micro??plastic liquid was diluted to 10 times， 100 times， 1 000 times and 10 000 times groups， each of which （2 ml） was added into corresponding experimental group， to carry out preliminary exposure experiment. The blank group （CK） was free of treatment. The eggs were cultured in an illumination constant??temperature incubator to 96 h. Every 24 h， the exposure liquids and blank liquid were replaced， and the death and hatching conditions of the embryos were recorded. The development condition of zebrafish embryos was observed under a microscope and recorded according to different stages. The formal experimental doses were determined according to the results of the preliminary experiment. Exposure experiment of zebrafish embryos 0.6-1.0 ??m polystyrene particles were used for the exposure experiment. The four experiment concentrations designed according to the preliminary experiment were 1 000， 250， 25 and 2.5 mg/L， respectively. Each group included five parallel samples. The specific operation was the same as the preliminary experiment. In the experimental process， the mortality and hatchability of the embryos were observed by semi??static method. When replacing the toxic liquid， coagulated eggs were also removed， and the development status of zebrafish embryos was observed under a microscope. The lethal indicators were egg coagulation， no beginning of gastrulae differentiation， no termination of gastrulae differentiation， no somite， no tail extension， no heartbeat and no hatching. The teratogenic indicators included changed body length， cyrtosis， malformed tail， and changed heart rate. For the computation of heart rate， 9 embryos were randomly selected from the four replicates of each treatment to carry out 1 min of cardiac impulse statistics. The observation indicators of zebrafish embryos at various exposure time were shown in Table 1.
Statistic analysis Mortality， hatchability， malformation rate and heart rate were recorded. Significance of differences in various indices was analyzed between the control group and experimental groups by one??way analysis of variance （ANOVA）， in which P??0.05 indicates a significant difference （*）， and P??0.01 indicates a very significant difference （**）.
The obtained toxicological indicators were classified （according to the study of Nagel et al.[6]）， into lethal indicators and nonlethal indicators. The sum of weights of lethal indicators （summarized as mortality） and nonlethal indicators was hazard index P， the formula of which is shown as below：
P=??fI?¤DI+??nfIIn?¤DIIn
Wherein fI is the weight of lethal indicator； DI is mortality； n is the number of nonlethal indicators； fIIn is the weight of nonlethal indicator； and DIIn is the occurring rate of nonlethal indicator.
The establishment of the hazard evaluation index system of polystyrene to zebrafish embryos and the determination of the weight of hazard evaluation index were performed by analytic hierarchy process[7]. Each indicator was subjected to pairwise comparison on degree of importance， so as to evaluate stepwise. During the analysis， judgment matrix was established， followed by the determination of the eigenvector corresponding to the largest eigenvalue by square root methods and the acquisition of approximate values， and finally， the importance weight of single indicator to the general objective was obtained. The hazard evaluation index system and weight of polystyrene to zebrafish embryos are shown in Table 2. The hazard grade of polystyrene microplastic to zebrafish embryos was divided using its hazard index. The hazard grades corresponding to hazard index are shown in Table 3.
Results and Discussion
Effects of polystyrene to zebrafish embryos
As shown in Fig. 1， zebrafish embryos developed normally at 24 （Fig. 1a） and 48 h （Fig. 1b） in the CK， while in higher micro??plastic concentrations， some zebrafish embryos died （Fig. 1c） or developed slowly with malformation （Fig. 1d）.
Tetratogenic situation of zebrafish embryos exposed to polystyrene
Zebrafish embryos were exposed to 2.5 to 1 000 mg/L poly??
styrene toxic liquid and observed for 72 h. The mortality and hatchability of zebrafish embryos are shown in Fig. 2.
The mortality of zebrafish embryos increased significantly with the concentration increasing. The mortality was 7% averagely at the polystyrene concentration of 250 mg/L， and was 98% averagely at the polystyrene concentration of 1 000 mg/L， with a very significant difference from the CK （P??0.01）. Furthermore， the mortality of zebrafish embryos exhibited dose??effect relationship.
The 72 h hatchability of zebrafish embryos decreased with the polystyrene concentration increasing. The eggs in the CK were all hatched， with an average hatchability of 99%， and the 2.5， 25， 250 and 1 000 mg/L groups showed the average hatchability of 97%， 89%， 59% and 0%， respectively. Among the various concentrations， the 25 mg/L group was significantly different from the CK group （P??0.05）， and the 250 and 1 000 mg/L groups were very significantly different from the CK （P??0.01）.
The teratogenic effect of polystyrene microplastic on zebrafish embryos is shown in Fig. 3. The microscopic observation at 48 h showed that zebrafish embryos suffered from cyrtosis， and when the concentration reached 25， 250 and 1 000 mg/L， the average cyrtosis rate increased largely， to 32%??65% and 100%， respectively. When the concentration reached 25 mg/L， very significant cyrtosis was observed （P??0.01）.
Variation in heart rate of zebrafish embryos exposed to polystyrene
In the 48 and 72 h observation， the pericardial edema rate of zebrafish embryos increased greatly with the concentration increasing. The 48 h experimental groups had the average pericardial edema rates of 3%， 32%， 62% and 95%， respectively， and when the concentration reached 25 mg/L， the difference from the CK became very significant. The 72 h experimental groups showed the 72 h average pericardial edema rates of 5%， 30%， 65% and 86%， respectively， and also， when the concentration reached 25 mg/L， the difference from the CK became very significant. The heart rate variation of zebrafish embryos caused by the exposure to polystyrene microplastic is shown in Fig. 4. In the 24 and 48 h heart rate observation， little differences were observed between the CK and the experimental groups， and only the highest??dose group （1 000 mg/L） showed remarkably decreased heart rate. In the 24 h observation， the average heart rates of the CK and the experimental groups were 53， 52， 54， 46 and 35 times/min， respectively， and the 1 000 mg/L group was very significantly from the CK （P??0.01）. During the 48 h heart rate observation， the average heart rates of the CK and the experimental groups were 105， 103， 98， 91 and 57 times/min， respectively and a very significant difference was observed at the concentration of 1 000 mg/L.
Table 4 shows the hazard grades of various polystyrene concentrations. The polystyrene at the concentrations of 2.5， 25 and 250 mg/L had a hazard of grade I， which was light. When the concentration increased to 1 000 mg/L， the hazard was grade IV， which was strong.
Discussion
The results of this study showed that polystyrene did not cause death of zebrafish embryos at the concentration lower than 25 mg/L， and had an observed lethal concentration of 250 mg/L， and when the concentration increased to 1 000 mg/L， the mortality of zebrafish embryos nearly reached 100%. During the 72 h experimental observation， various experimental groups all exhibited delayed hatching， and the hatchability decreased sharply with the increase of polystyrene concentration.
Zebrafish embryos suffered from malformation at 48 h， which was mainly reflected by cyrtosis and pericardial edema， and cyrtosis rate and pericardial edema rate both increased with the polystyrene concentration increasing. The highest??dose group showed a cyrtosis rate of 100% at 48 h. There was no big difference in pericardial edema rate between 48 and 72 h. These results also reflected the dose??effect relationship of polystyrene liquid， and the observed little difference was related to selection of samples.
In this study， heartbeat of zebrafish embryos was observed at 24 h， and heart rate was measured at 24 and 48 h， respectively. The 48 h heart rates were all higher than the 24 h heart rates. In the 24 h observation， there were no big differences between the experimental groups and the CK， only the highest??dose group had a very significant difference from the CK. At 48 h， the 2.5 and 25 mg/L groups still had no big differences from the CK， but when the experimental concentration reached 250 mg/L， the heart rate began to decrease remarkably， and had a sharp drop at 1 000 mg/L. The effect of polystyrene microplastic on zebrafish embryos was firstly reflected by heart rate， which might be because in the development process of zebrafish embryos， heart is the organ developing the earliest[7-8]. Yang et al.[8] studied the effect of BPAF on zebrafish embryos and juvenile fish. The research results showed that BPAF exposure very significantly inhibited the heart rates of zebrafish embryos and juvenile fish， which both had a trend of increasing at first and decreasing then， indicating that heart is closely related to the survival of living organisms， and unsound heart development affected blood circulation of zebrafish embryos which thus suffered from delayed hatching and malformation. The variation in 24 h heart rates of various experimental groups was not as remarkable as that in 48 h heart rates of various experimental groups， and the 48 h heart rates of various experimental groups were all higher than the 24 h ones， suggesting that the effect of polystyrene micro??plastic on the heart of zebrafish embryos needs to accumulate to certain time. The heart rates of high??dose groups were remarkably lower than those of low??dose groups， suggesting that polystyrene micro??plastic only could affects zebrafish embryos when reaching certain concentration， while 25 mg/L and lower concentrations are safe exposure concentrations.
Conclusions
0.6-1.0 ??m polystyrene micro??plasctic has certain toxicity on zebrafish embryos， and can cause death of zebrafish embryos at the concentration of 250 mg/L.
25 mg/L 0.6-1.0 ??m polystyrene particles would cause nonlethal toxic effects such as cyrtosis and pericardial edema of zebrafish embryos， and 250 and 1000 mg/L can slow down their heart rate.
A hazard evaluation index system of polystyrene particles to zebrafish embryos was established， and it is determined that the exposure to 250 mg/L or lower concentration of polystyrene particles has slight hazard.
References
[1] LIAO Q， QU JS， WANG JP， et al. Pollution analysis of plastic and improving proposal in marine environment[J]. World Sci??Tech R & D， 2015（2）： 206-211， 217.
[2] ZHOU Q， ZHANG HB， LI Y， et al. Progress on microplastics pollution and its ecological effects in the coastal environment[J]. Chinese Science Bulletin， 2015（33）： 3210-3220.
[3] ZHOU Q. Micro??plastic pollution characteristics and ecological risk in typical coastal tidal flat and paralic environment[D]. Shandong： Yantai Institute of Coastal Zone Research， Chinese Academy of Sciences， 2016： 1-106.
[4] SUN XX. Progress and Prospect on the Study of the ecological risk of microplastics in the ocean[J]. Advances in Earth Science， 2016（6）： 560-566.
[5] LU Y， ZHANG Y， DENG Y， et al. Uptake and accumulation of polystyrene microplastics in zebrafish （Danio rerio） and toxic effects in liver[J]. Environ. Sci. Technol.， 2016（50）： 4054-4060.
[6] NAGEL R， DAR T. The embryo test with the zebrafish （Danio rerio）： A general model in ecotoxicology and toxicology[J]. Altex， 2002， 19（suppl1）： 38-48.
[7] YU FF， TANG TL， BAI JJ， et al. Toxicities and hazard classification of reclaimed water after disinfection of different approaches by zebrafish embryos bioassay[J]. Asian Journal of Ecotoxicolog， 2015（2）： 313-319.
[8] YANG Y， CHEN YW， TANG TL， et al. Toxic effects of bisphenol AF on zebrafish embryos and larvae[J]. Research of Environmental Sciences， 2015（8）： 1219-1226.