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Abstract ［Objectives］ This study was conducted to investigate the toxic effects of 2,2′,4,4′,5,5′-hexabromobiphenyl ether on Chlorella vulgaris and provide basic data for protecting aquatic ecosystems.
［Methods］ The acute toxicity effects of 2,2′,4,4′,5,5′-hexabromodiphenyl ether on C. vulgaris was investigated by the semi-static water contacting acute toxicity method.
［Results］ The 48, 72 and 96 h- EC 50 of 2,2′,4,4′,5,5′-hexabromodiphenyl ether to C. vulgaris were 23.58 , 18.71 and 14.75 μg/L, respectively, and the safe concentration of 2,2′,4,4′,5,5′-hexabromodiphenyl ether to C. vulgaris was 1.475μg/L. For the water solubility of 2,2′,4,4′,5,5′-hexabromodiphenyl ether is extremely low (1 μg/L), it could not cause the acute poisoning death of C. vulgaris . According to the grading standards for the assessment of the toxicity on algae, 2,2′,4,4′,5,5′-hexabromodiphenyl ether was extremely highly toxic to C. vulgaris .
［Conclusions］The extremely high toxicity of 2,2′,4,4′,5,5′-hexabromodiphenyl ether to C. vulgaris shows that it has heavy potential harm to aquatic ecosystems and the maximum residue limit standards of 2,2′,4,4′,5,5′-hexabromodiphenyl ether in water should be formulated to better protect aquatic ecosystems.
Key words 2,2′,4,4′,5,5′-Hexabromodiphenyl ether; Acute toxicity; Chlorella vulgaris ; 96 h EC 50
Received: January 29, 2020Accepted: March 23, 2020
Supported by Central Public-interest Scientific Institution Basal Research Fund, Chinese Academy of Fishery Science (2017HY-ZD0208); China Agricultural Research System-Freshwater Fish (CARS-46).
Shunlong MENG (1982-), male, P. R. China, associate researcher, PhD, devoted to research about environmental toxicology, fisheries environmental protection, and aquatic product quality and safety risk assessment.
*Corresponding author. E-mail: chenjz@ffrc.cn; xup@ffrc.cn.
Polybrominated diphenyl ethers (PBDEs) are brominated aromatic hydrocarbons with the general formula C12 H(0-9) Br(1-10) O. According to the number of bromine atoms in polybrominated diphenyl ether molecules, they are divided into 10 homologous groups, including 209 homologues. PBDEs are highly flame-retardant and are often added to high molecular synthetic materials such as polyurethane, resin, rubber and polystyrene to produce fireproof materials. They are also widely used in the fields of textiles, processing, coatings, home decoration, furniture, electrical equipment and building materials［1］. In the 1940s and 1950s, PBDEs were industrially produced and added to various industrial products. In the 1960s and 1970s, PBDEs and their products were widely used due to their excellent flame retardant effects［2］. However, as PBDEs are continuously detected in environmental samples, the environmental problems caused by them are getting more and more attention from the society. Currently PBDEs have been restricted to production and use as new POPs［3］. However, since PBDEs are already present in industrial products and have strong stability, they are released into the environment with the use of industrial products and incineration of waste industrial products. Existing research has confirmed that PBDEs have become an important type of environmental pollutants, which is widely detected in air, water, sediment, soil, plants, wildlife and human body［4］. The concentration of PBDEs in the sediments of the Great Lakes［5-7］ in North America, Tokyo Bay［8］ in Japan, and the Pearl River Delta［9］ in China showed a trend of increasing with time. The concentration of PBDEs in airborne dust around an electronic waste treatment plant is as high as 1.96-340.71 μg/g［10］; the concentration of PBDEs in river sediments near an incineration point in Guangdong is up to 63 300 ng/g［11］; the content of PBDEs in the sediment of the Pearl River Estuary reaches 0.04-94.70 ng/g［12］; and the content of PBDEs in the water body of the Pearl River estuary reaches 26.1-94.6 pg/L［13］. Moreover, since PBDEs can enter organisms through the food chain, PBDEs are also detected in organisms in some areas and exhibit biomagnification［14］. At present, there are many studies on the toxic effects of PBDEs on mammals, but there are few studies on aquatic organisms. Since PBDEs in the environment are mainly tetrabromodiphenyl ether, pentabromodiphenyl ether, hexabromodiphenyl ether and decabromodiphenyl ether［14］, in this study, 2,2′,4,4′,5,5′-hexabromobiphenyl ether was selected as a representative substance, the acute toxicity of which on the key organism Chlorella vulgaris in the aquatic ecosystem and aquatic food chain serving as a representative organism was investigated, aiming to provide basic data for the protection of aquatic ecosystems.
Materials and Methods
Test organism
In order to improve the representativeness and comparability of the test results, the alga used in this test was common C. vulgaris . The test was carried out by the standardized acute toxicity test method.
Common C. vulgaris is a single-cell freshwater alga of Chlorophyta. C. vulgaris grows and reproduces through photosynthetic autotrophy, and is widely distributed. It is also one of the main algae common in aquaculture waters. The C. vulgaris used in the test was purchased from the Institute of Hydrobiology, Chinese Academy of Sciences. It was cultured in BG11 medium at a temperature of (25±1) ℃ under a light-dark ratio at 12 h∶12 h with a light intensity of 4 000 lx and a pH value of 7.0-7.5. The conical flasks were shaken once every 2 h during the light period, and stood in the dark period, and meanwhile, the positions of the conical flasks were randomly changed, so that the algal liquids were evenly illuminated.
Test water
The test water was dechlorinated tap water aerated for 7 d, which had a temperature of (20±1) ℃ and a pH value of 7.0-7.5, and contained dissolved oxygen 6.5-7.0 mg/L. The test water met the fishery water quality standard (GB 11607-89).
Test drug
The used 2,2′,4,4′,5,5′-hexabromobiphenyl ether (BDE-153) was produced by Wuhan Kaymke Chemical Technology Co., Ltd., and had a purity equal to or higher than 98%. Because BDE-153 is slightly soluble in water, according to the sensitivity of the test organisms to cosolvents in relevant literatures, N,N-dimethylformamide (DMF) was selected as a cosolvent for acute toxicity test of the alga. A BDE-153 mother liquor was prepared and stored in a refrigerator at 4 ℃ for use. During the test, it was diluted with water to a test solution with a desired mass concentration. Test methods
The test was carried out with 250 ml flasks. Before the test, the flasks were immersed in dilute nitric acid for 48 h, rinsed with purified water, and air-dried for use. Two control groups were set up in the test, which were the blank control group and the cosolvent control group, and the concentration of the cosolvent in the cosolvent control group was the same as that in the highest-dose group. The concentrations of BDE-153 in the test were: 0.1 , 1.0 , 5.0, 10.0 and 50.0 μg/L. C. vulgaris in the exponential growth phase was inoculated into a triangular flask containing 150 ml of BG11 medium, and the initial algal density was 1×105 cells/L. Each of the concentration gradients was done in three parallels. The algal fluid was collected at 24, 48, 72, and 96 h of the test, and microscopically examined to calculate the cell density of C. vulgaris .
Data processing
The 96 h-median effect concentration ( EC 50 ) of BDE-153 against C. vulgaris was calculated according to the method specified by the OECD. According to the experimental data, the growth curves of C. vulgaris under the various treatment concentrations of BDE-153 at 96 h were constructed. The areas under the growth curves were calculated according to following formula:
A=N1-N02×t1+N1+N2-2N02×(t2-t1)+…Nn-1 +Nn-2N02×(tn-tn-1 ),
where A is the area under the growth curve; N 0 is the initial cell number at t 0 (cells/ml); N 1 is the algal number determined at t 1 (cells/ml); Nn is the algal number determined at tn (cells/ml); t 1 is the time for the first determination at the beginning of the test; and tn is the n th determination after the test is started. The cell growth inhibition percentage (IA) of C. vulgaris was calculated according to following formula:
IA=Ac-AtAc×100% ,
where IA is the cell growth inhibition percentage of C. vulgaris in the treatment group; Ac is the area under the growth curve of C. vulgaris of the control group; and At is the area under the growth curve of C. vulgaris of the treatment group. The concentration-effect equation was obtained by the probability unit-concentration logarithm method; and when the probability unit was 5, the 96 h EC 50 was calculated. Statistical Analysis
The significance of the correlation coefficient of the regression equation was tested by t test. The significance level was α=0.05.
Results and Analysis
The trends of C. vulgaris density with culture time at different treatment concentrations of BDE-153 are shown in Fig. 1. The test results (Fig. 1) showed that the C. vulgaris of the control groups showed a rapid growth trend, and good exponential growth characteristics as well. With the extension of treatment time, the growth trend of C. vulgaris in each BDE-153 group was significantly changed compared with the control groups. The growth of C. vulgaris in each treatment group was significantly inhibited, and the inhibition of BDE-153 to C. vulgaris was gradually aggravated with the increase of the treatment concentration and the extension of the treatment time. It indicated that the inhibitory effect of BDE-153 on C. vulgaris increased with the increase of the treatment time and treatment concentration, and a good dose-response relationship was shown between the inhibitory effect and BDE-153 exposure concentration.
The regression equation, correlation coefficient and EC 50 calculated by the linear regression method are listed in Table 1. The correlation coefficients at 48, 72 and 96 h were 0.989, 0.982 and 0.983, respectively. The t test was used to test the correlation coefficient under each treatment time, and the results reached a significant level ( P <0.05). The 48, 72 and 96 h EC 50 of BDE-153 on C. vulgaris were 23.58, 18.71 and 14.75 μg/L, respectively, and the safe concentration was 1.475 μg/L.
Shunlong MENG et al. Acute Toxicity Effects of 2,2′,4,4′,5,5′-Hexabromodiphenyl Ether on Chlorella vulgaris
Conclusions and Discussion
Acute toxicity test is the fundamental basis for mastering the acute toxicity of exogenous chemicals to organisms, and also provides the first-hand information for toxicological safety evaluation. It is generally required in the regulatory procedures of different types of chemicals［15］. This study showed that the 96 h- EC 50 of BDE-153 on C. vulgaris was 14.75 μg/L. According to the grading standards for the assessment of the toxicity on algae［16］, 96 h- EC 50 <1 mg/L, 1 mg/L<96 h- EC 50 <10 mg/L, 10 mg/L<96 h- EC 50 <100 mg/L and 96 h- EC 50 >100 mg/L stand for very high toxicity, high toxicity, medium toxicity and low toxicity, respectively. Therefore, BDE-153 was very highly toxic to C. vulgaris . From the perspective of the food chain and ecosystem composed of phytoplankton, zooplankton and fishes, algae are an important producer in aquatic ecosystems, a fundamental link in the food chain, which can synthesize nutrients in water through photosynthesis into substances necessary for ecosystems and store them in the form of energy to maintain the normal functioning of aquatic ecosystems, playing an important role in the material cycle and energy conversion of aquatic ecosystems. Therefore, the extremely high toxicity of BDE-153 to C. vulgaris shows its great harm to aquatic ecosystems. It is necessary to establish the maximum residue limit standards of PBDEs in water to better protect aquatic ecosystems. Algae are sensitive to PBDEs, and the results of the acute toxicity tests of2,2′,4,4′-tetrabromodiphenyl ether (BDE-47) on algae showed that the 48 h- EC 50 for Skeletonema costatum was 70 μg/L［17］ and the 96 h- EC 50 was 1.99 μg/L［18］; the 96 h- EC 50 for C. vulgaris, Chaetoceros mulleri and Heterosigma akashiwo were 0.79 , 1.52 and 2.25 μg/L, respectively［18］; the 96 h- EC 50 for Dunaliella salina and Tetraselmis subcordiformis were 119.93 and 113.66 μg/L, respectively［19］; and the 96 h- EC 50 for Chrysophyceae, H. akashiwo, Karenia mikimotoi and Platymonas helgolandica were 0.016, 0.046, 0.250 and 2.000 mg/L, respectively［20］. The 96 h- EC 50 of BDE-153 on T. subcordiformis and D. salina were 996 and 1 249 μg/L, respectively［19］. The substitution number of bromine atoms affects the toxicity of PBDE homologues to algae. It is generally believed that the toxicity of PBDE homologues to food algae decreases with the increase of the substitution number of bromine atoms［19］. This study also found the same trend. The previous experimental research on BDE-47 showed that its 96 h- EC 50 to C. vulgaris was 3.97 μg/L, which was less than the 96 h- EC 50 of BDE-153 to C. vulgaris (14.75 μg/L). The acute toxic effects of PBDEs homologues on microalgae depend on the difficulty (or bioavailability) of such chemicals into algal cells. The biological enrichment factor (BAF) of hydrophobic chemicals has a parabolic relationship with its n-octanol/water partition coefficient (Kow). When log Kow>6.5, the extent of the absorption of chemicals by organisms is reduced due to the steric hindrance of superhydrophobic compounds［21］. The log Kow value of BDE-47 is 6.81, while the log Kow value of BDE-153 is 7.90, which is higher than BDE-47, and its corresponding steric hindrance is also the larger. At the same concentration, the microalgaes absorption of BDE153 is less than the low-bromine homologue BDE-47. Large steric hindrance hinders the uptake, enrichment, metabolism and transmission of PBDEs［22-24］, resulting in decreased toxicity. In this study, the 96 h- EC 50 value of BDE-153 is an order of magnitude higher than that of low-bromine BDE-47. The reason may be that BDE-153 has a large number of substituents and large molecules, which makes the intake by microalgae difficult［19］. The 96 h- EC 50 of BDE-153 on T. subcordiformis and D. salina were 996 and 1 249 μg/L, respectively［19］, which was far higher than the 96 h- EC 50 of BDE-153 on C. vulgaris (14.75 μg/L). BDE-153 has different toxic effects on different types of microalgae, which may be related to the morphology and structure of different algae, such as the cell wall and extracellular coatings［11］. The thickness and presence or absence of cell walls and extracellular coatings may affect the acute toxicity of pollutants to algae. Meanwhile, these differences in acute tests may also be related to the purity of the drug used, the health condition of the test organisms and the physical and chemical characteristics of the test water［25］.
The 48, 72 and 96 h- EC 50 of 2,2′,4,4′,5,5′-hexabromobiphenyl ether on C. vulgaris were 23.58, 18.71 and 14.75 μg/L, respectively, and the safe concentration was 1.475 μg/L. Since 2,2′,4,4′,5,5′-hexabromobiphenyl ether has extremely low solubility in water (1 μg/L), 2,2′,4,4′,5,5′-hexabromobiphenyl ether is unlikely to cause acute poisoning death of C. vulgaris in the natural environment.
According to the grading standards for toxicity assessment for algae, 2,2′,4,4′,5,5′-hexabromobiphenyl ether is an extremely highly toxic chemical to C. vulgaris . The extremely high toxicity of 2,2′,4,4′, 5,5′-hexabromobiphenyl ether to C. vulgaris shows that it has great harm to aquatic ecosystems, and it is necessary to establish the maximum residue limit standards of PBDEs in water to better protect water ecosystems.
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Editor: Yingzhi GUANGProofreader: Xinxiu ZHU
［Methods］ The acute toxicity effects of 2,2′,4,4′,5,5′-hexabromodiphenyl ether on C. vulgaris was investigated by the semi-static water contacting acute toxicity method.
［Results］ The 48, 72 and 96 h- EC 50 of 2,2′,4,4′,5,5′-hexabromodiphenyl ether to C. vulgaris were 23.58 , 18.71 and 14.75 μg/L, respectively, and the safe concentration of 2,2′,4,4′,5,5′-hexabromodiphenyl ether to C. vulgaris was 1.475μg/L. For the water solubility of 2,2′,4,4′,5,5′-hexabromodiphenyl ether is extremely low (1 μg/L), it could not cause the acute poisoning death of C. vulgaris . According to the grading standards for the assessment of the toxicity on algae, 2,2′,4,4′,5,5′-hexabromodiphenyl ether was extremely highly toxic to C. vulgaris .
［Conclusions］The extremely high toxicity of 2,2′,4,4′,5,5′-hexabromodiphenyl ether to C. vulgaris shows that it has heavy potential harm to aquatic ecosystems and the maximum residue limit standards of 2,2′,4,4′,5,5′-hexabromodiphenyl ether in water should be formulated to better protect aquatic ecosystems.
Key words 2,2′,4,4′,5,5′-Hexabromodiphenyl ether; Acute toxicity; Chlorella vulgaris ; 96 h EC 50
Received: January 29, 2020Accepted: March 23, 2020
Supported by Central Public-interest Scientific Institution Basal Research Fund, Chinese Academy of Fishery Science (2017HY-ZD0208); China Agricultural Research System-Freshwater Fish (CARS-46).
Shunlong MENG (1982-), male, P. R. China, associate researcher, PhD, devoted to research about environmental toxicology, fisheries environmental protection, and aquatic product quality and safety risk assessment.
*Corresponding author. E-mail: chenjz@ffrc.cn; xup@ffrc.cn.
Polybrominated diphenyl ethers (PBDEs) are brominated aromatic hydrocarbons with the general formula C12 H(0-9) Br(1-10) O. According to the number of bromine atoms in polybrominated diphenyl ether molecules, they are divided into 10 homologous groups, including 209 homologues. PBDEs are highly flame-retardant and are often added to high molecular synthetic materials such as polyurethane, resin, rubber and polystyrene to produce fireproof materials. They are also widely used in the fields of textiles, processing, coatings, home decoration, furniture, electrical equipment and building materials［1］. In the 1940s and 1950s, PBDEs were industrially produced and added to various industrial products. In the 1960s and 1970s, PBDEs and their products were widely used due to their excellent flame retardant effects［2］. However, as PBDEs are continuously detected in environmental samples, the environmental problems caused by them are getting more and more attention from the society. Currently PBDEs have been restricted to production and use as new POPs［3］. However, since PBDEs are already present in industrial products and have strong stability, they are released into the environment with the use of industrial products and incineration of waste industrial products. Existing research has confirmed that PBDEs have become an important type of environmental pollutants, which is widely detected in air, water, sediment, soil, plants, wildlife and human body［4］. The concentration of PBDEs in the sediments of the Great Lakes［5-7］ in North America, Tokyo Bay［8］ in Japan, and the Pearl River Delta［9］ in China showed a trend of increasing with time. The concentration of PBDEs in airborne dust around an electronic waste treatment plant is as high as 1.96-340.71 μg/g［10］; the concentration of PBDEs in river sediments near an incineration point in Guangdong is up to 63 300 ng/g［11］; the content of PBDEs in the sediment of the Pearl River Estuary reaches 0.04-94.70 ng/g［12］; and the content of PBDEs in the water body of the Pearl River estuary reaches 26.1-94.6 pg/L［13］. Moreover, since PBDEs can enter organisms through the food chain, PBDEs are also detected in organisms in some areas and exhibit biomagnification［14］. At present, there are many studies on the toxic effects of PBDEs on mammals, but there are few studies on aquatic organisms. Since PBDEs in the environment are mainly tetrabromodiphenyl ether, pentabromodiphenyl ether, hexabromodiphenyl ether and decabromodiphenyl ether［14］, in this study, 2,2′,4,4′,5,5′-hexabromobiphenyl ether was selected as a representative substance, the acute toxicity of which on the key organism Chlorella vulgaris in the aquatic ecosystem and aquatic food chain serving as a representative organism was investigated, aiming to provide basic data for the protection of aquatic ecosystems.
Materials and Methods
Test organism
In order to improve the representativeness and comparability of the test results, the alga used in this test was common C. vulgaris . The test was carried out by the standardized acute toxicity test method.
Common C. vulgaris is a single-cell freshwater alga of Chlorophyta. C. vulgaris grows and reproduces through photosynthetic autotrophy, and is widely distributed. It is also one of the main algae common in aquaculture waters. The C. vulgaris used in the test was purchased from the Institute of Hydrobiology, Chinese Academy of Sciences. It was cultured in BG11 medium at a temperature of (25±1) ℃ under a light-dark ratio at 12 h∶12 h with a light intensity of 4 000 lx and a pH value of 7.0-7.5. The conical flasks were shaken once every 2 h during the light period, and stood in the dark period, and meanwhile, the positions of the conical flasks were randomly changed, so that the algal liquids were evenly illuminated.
Test water
The test water was dechlorinated tap water aerated for 7 d, which had a temperature of (20±1) ℃ and a pH value of 7.0-7.5, and contained dissolved oxygen 6.5-7.0 mg/L. The test water met the fishery water quality standard (GB 11607-89).
Test drug
The used 2,2′,4,4′,5,5′-hexabromobiphenyl ether (BDE-153) was produced by Wuhan Kaymke Chemical Technology Co., Ltd., and had a purity equal to or higher than 98%. Because BDE-153 is slightly soluble in water, according to the sensitivity of the test organisms to cosolvents in relevant literatures, N,N-dimethylformamide (DMF) was selected as a cosolvent for acute toxicity test of the alga. A BDE-153 mother liquor was prepared and stored in a refrigerator at 4 ℃ for use. During the test, it was diluted with water to a test solution with a desired mass concentration. Test methods
The test was carried out with 250 ml flasks. Before the test, the flasks were immersed in dilute nitric acid for 48 h, rinsed with purified water, and air-dried for use. Two control groups were set up in the test, which were the blank control group and the cosolvent control group, and the concentration of the cosolvent in the cosolvent control group was the same as that in the highest-dose group. The concentrations of BDE-153 in the test were: 0.1 , 1.0 , 5.0, 10.0 and 50.0 μg/L. C. vulgaris in the exponential growth phase was inoculated into a triangular flask containing 150 ml of BG11 medium, and the initial algal density was 1×105 cells/L. Each of the concentration gradients was done in three parallels. The algal fluid was collected at 24, 48, 72, and 96 h of the test, and microscopically examined to calculate the cell density of C. vulgaris .
Data processing
The 96 h-median effect concentration ( EC 50 ) of BDE-153 against C. vulgaris was calculated according to the method specified by the OECD. According to the experimental data, the growth curves of C. vulgaris under the various treatment concentrations of BDE-153 at 96 h were constructed. The areas under the growth curves were calculated according to following formula:
A=N1-N02×t1+N1+N2-2N02×(t2-t1)+…Nn-1 +Nn-2N02×(tn-tn-1 ),
where A is the area under the growth curve; N 0 is the initial cell number at t 0 (cells/ml); N 1 is the algal number determined at t 1 (cells/ml); Nn is the algal number determined at tn (cells/ml); t 1 is the time for the first determination at the beginning of the test; and tn is the n th determination after the test is started. The cell growth inhibition percentage (IA) of C. vulgaris was calculated according to following formula:
IA=Ac-AtAc×100% ,
where IA is the cell growth inhibition percentage of C. vulgaris in the treatment group; Ac is the area under the growth curve of C. vulgaris of the control group; and At is the area under the growth curve of C. vulgaris of the treatment group. The concentration-effect equation was obtained by the probability unit-concentration logarithm method; and when the probability unit was 5, the 96 h EC 50 was calculated. Statistical Analysis
The significance of the correlation coefficient of the regression equation was tested by t test. The significance level was α=0.05.
Results and Analysis
The trends of C. vulgaris density with culture time at different treatment concentrations of BDE-153 are shown in Fig. 1. The test results (Fig. 1) showed that the C. vulgaris of the control groups showed a rapid growth trend, and good exponential growth characteristics as well. With the extension of treatment time, the growth trend of C. vulgaris in each BDE-153 group was significantly changed compared with the control groups. The growth of C. vulgaris in each treatment group was significantly inhibited, and the inhibition of BDE-153 to C. vulgaris was gradually aggravated with the increase of the treatment concentration and the extension of the treatment time. It indicated that the inhibitory effect of BDE-153 on C. vulgaris increased with the increase of the treatment time and treatment concentration, and a good dose-response relationship was shown between the inhibitory effect and BDE-153 exposure concentration.
The regression equation, correlation coefficient and EC 50 calculated by the linear regression method are listed in Table 1. The correlation coefficients at 48, 72 and 96 h were 0.989, 0.982 and 0.983, respectively. The t test was used to test the correlation coefficient under each treatment time, and the results reached a significant level ( P <0.05). The 48, 72 and 96 h EC 50 of BDE-153 on C. vulgaris were 23.58, 18.71 and 14.75 μg/L, respectively, and the safe concentration was 1.475 μg/L.
Shunlong MENG et al. Acute Toxicity Effects of 2,2′,4,4′,5,5′-Hexabromodiphenyl Ether on Chlorella vulgaris
Conclusions and Discussion
Acute toxicity test is the fundamental basis for mastering the acute toxicity of exogenous chemicals to organisms, and also provides the first-hand information for toxicological safety evaluation. It is generally required in the regulatory procedures of different types of chemicals［15］. This study showed that the 96 h- EC 50 of BDE-153 on C. vulgaris was 14.75 μg/L. According to the grading standards for the assessment of the toxicity on algae［16］, 96 h- EC 50 <1 mg/L, 1 mg/L<96 h- EC 50 <10 mg/L, 10 mg/L<96 h- EC 50 <100 mg/L and 96 h- EC 50 >100 mg/L stand for very high toxicity, high toxicity, medium toxicity and low toxicity, respectively. Therefore, BDE-153 was very highly toxic to C. vulgaris . From the perspective of the food chain and ecosystem composed of phytoplankton, zooplankton and fishes, algae are an important producer in aquatic ecosystems, a fundamental link in the food chain, which can synthesize nutrients in water through photosynthesis into substances necessary for ecosystems and store them in the form of energy to maintain the normal functioning of aquatic ecosystems, playing an important role in the material cycle and energy conversion of aquatic ecosystems. Therefore, the extremely high toxicity of BDE-153 to C. vulgaris shows its great harm to aquatic ecosystems. It is necessary to establish the maximum residue limit standards of PBDEs in water to better protect aquatic ecosystems. Algae are sensitive to PBDEs, and the results of the acute toxicity tests of2,2′,4,4′-tetrabromodiphenyl ether (BDE-47) on algae showed that the 48 h- EC 50 for Skeletonema costatum was 70 μg/L［17］ and the 96 h- EC 50 was 1.99 μg/L［18］; the 96 h- EC 50 for C. vulgaris, Chaetoceros mulleri and Heterosigma akashiwo were 0.79 , 1.52 and 2.25 μg/L, respectively［18］; the 96 h- EC 50 for Dunaliella salina and Tetraselmis subcordiformis were 119.93 and 113.66 μg/L, respectively［19］; and the 96 h- EC 50 for Chrysophyceae, H. akashiwo, Karenia mikimotoi and Platymonas helgolandica were 0.016, 0.046, 0.250 and 2.000 mg/L, respectively［20］. The 96 h- EC 50 of BDE-153 on T. subcordiformis and D. salina were 996 and 1 249 μg/L, respectively［19］. The substitution number of bromine atoms affects the toxicity of PBDE homologues to algae. It is generally believed that the toxicity of PBDE homologues to food algae decreases with the increase of the substitution number of bromine atoms［19］. This study also found the same trend. The previous experimental research on BDE-47 showed that its 96 h- EC 50 to C. vulgaris was 3.97 μg/L, which was less than the 96 h- EC 50 of BDE-153 to C. vulgaris (14.75 μg/L). The acute toxic effects of PBDEs homologues on microalgae depend on the difficulty (or bioavailability) of such chemicals into algal cells. The biological enrichment factor (BAF) of hydrophobic chemicals has a parabolic relationship with its n-octanol/water partition coefficient (Kow). When log Kow>6.5, the extent of the absorption of chemicals by organisms is reduced due to the steric hindrance of superhydrophobic compounds［21］. The log Kow value of BDE-47 is 6.81, while the log Kow value of BDE-153 is 7.90, which is higher than BDE-47, and its corresponding steric hindrance is also the larger. At the same concentration, the microalgaes absorption of BDE153 is less than the low-bromine homologue BDE-47. Large steric hindrance hinders the uptake, enrichment, metabolism and transmission of PBDEs［22-24］, resulting in decreased toxicity. In this study, the 96 h- EC 50 value of BDE-153 is an order of magnitude higher than that of low-bromine BDE-47. The reason may be that BDE-153 has a large number of substituents and large molecules, which makes the intake by microalgae difficult［19］. The 96 h- EC 50 of BDE-153 on T. subcordiformis and D. salina were 996 and 1 249 μg/L, respectively［19］, which was far higher than the 96 h- EC 50 of BDE-153 on C. vulgaris (14.75 μg/L). BDE-153 has different toxic effects on different types of microalgae, which may be related to the morphology and structure of different algae, such as the cell wall and extracellular coatings［11］. The thickness and presence or absence of cell walls and extracellular coatings may affect the acute toxicity of pollutants to algae. Meanwhile, these differences in acute tests may also be related to the purity of the drug used, the health condition of the test organisms and the physical and chemical characteristics of the test water［25］.
The 48, 72 and 96 h- EC 50 of 2,2′,4,4′,5,5′-hexabromobiphenyl ether on C. vulgaris were 23.58, 18.71 and 14.75 μg/L, respectively, and the safe concentration was 1.475 μg/L. Since 2,2′,4,4′,5,5′-hexabromobiphenyl ether has extremely low solubility in water (1 μg/L), 2,2′,4,4′,5,5′-hexabromobiphenyl ether is unlikely to cause acute poisoning death of C. vulgaris in the natural environment.
According to the grading standards for toxicity assessment for algae, 2,2′,4,4′,5,5′-hexabromobiphenyl ether is an extremely highly toxic chemical to C. vulgaris . The extremely high toxicity of 2,2′,4,4′, 5,5′-hexabromobiphenyl ether to C. vulgaris shows that it has great harm to aquatic ecosystems, and it is necessary to establish the maximum residue limit standards of PBDEs in water to better protect water ecosystems.
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