| Neonicotinoid insecticides have been registered in more than 120 countries since their initial arrival on the market in the early 1990 s,and currently they have accounted for over 25% of global insecticide market.China is one of the countries with the largest use of neonicotinoids,as well as the main producer and exporter of these compounds.Neonicotinoids act as nicotinic acetylcholine receptor agonists,which have been widely used because they are believed to have little effect on vertebrate species and have little cross-resistance with other insecticides.Nevertheless,neonicotinoids have high water solubility and are highly mobile.As a consequence,neonicotinoids are easily transported into aquatic ecosystems,posing potential risks to non-target aquatic organisms.Meanwhile,previous studies have shown that neonicotinoids have endocrine disrupting effects on birds,mammals,reptiles and invertebrates,such as silkworm.In most developed countries in North America and Europe,imidacloprid and clothianidin are the most extensively used neonicotinoids.To date,therefore,most toxicity data regarding neonicotinoids are limited to imidacloprid and clothianidin,which hindered assessing ecological risk associated with other neonicotinoids,such as acetamiprid.Beside imidacloprid,acetamiprid has been one of the most extensively used neonicotinoid in China,with its annual output being nearly 8,000 tons.Previous studies indicated that detection frequencies and concentrations of acetamiprid residues in surface water in China were comparable to imidacloprid.Therefore,we chose zebrafish as aquatic model organisms to evaluate the toxicity of acetamiprid on the embryonic development of zebrafish and its effect on the reproductive endocrine system.Firstly,the acute toxicity of acetamiprid to zebrafish embryos was evaluated.During the period of 6–120 hours post fertilization(hpf),zebrafish embryos were exposure to acetamiprid with concentrations of 53.8 ± 1.0–974 ± 20 mg/L to evaluate the developmental toxicity of acetamiprid on zebrafish embryos by choosing sublethal(malformation,hatchability,heart rate,body length,spontaneous movement and touch response)and lethal effects.In addition,a toxicity spectrum of this compound was established.The results showed that the 120 hpf LC50 and EC50 for malformations of acetamiprid were 518(469–572)mg/L and 323(303–344)mg/L,respectively,and LC5 and EC5 for malformations were 272(228–333)mg/L and 195(171–229)mg/L,respectively.The calculated EC5 and EC50 for hatchability at 72 hpf were 36(7–140)mg/L and 554(485–633)mg/L,respectively.The EC50 s for tail touch response at 27,36 and 48 hpf were 896(545–1474)mg/L,923(829–1027)mg/L and 888(797–990)mg/L,respectively.The EC50 s for head touch response at 27,36 and 48 hpf were 855(717–1019)mg/L,695(512–943)mg/L and 754(592–960)mg/L,respectively.The toxicity spectrum of this compound showed that spontaneous movement was the most sensitive endpoint with the EC5 and EC50 at 23 hpf being 51(28–104)and 296(167–523)mg/L,respectively.Overall,acetamiprid has obvious developmental toxicity on zebrafish embryos,and has influenced their behavior,growth,morphology,hatchability and survival after acute exposure.Spontaneous movement is the most sensitive endpoint in all test endpoints.Secondly,on the basis of the results from the acute exposure test,juvenile zebrafish at 20 days post fertilization(dpf)were exposed to acetamiprid at concentrations of 0,0.19,1.19,17.2,155 and 1637 ?g/L for 22 weeks to investigate the distribution,metabolism and bioaccumulation of acetamiprid in the brain,gill,gonad,intestine and muscle tissues of the zebrafish and the F1 generation.An ultra performance liquid chromatography-tandem mass spectrometry method was established for the analysis of acetamiprid and its metabolites in various tissues and the F1 generation embryos of the zebrafish.Both acetamiprid and its metabolites were detected and the detected concentrations of the three compounds increased with the increase of the exposure concentrations,and there were obvious characteristics of tissue and gender distribution.The detected concentrations in brain and intestine tissues were significantly higher than those in other tissues.At the same time,acetamiprid concentrations in female fish were higher than those in male fish.The main metabolite of acetamiprid in zebrafish is acetamiprid-N-demethyl,with the ratio of the highest molar concentration to the parent compound being 18.8%.The ratios of the molar concentrations of metabolites to the parent compounds in the offspring generation were higher than those in the F0 generation.Among them,the ratios of the molar concentrations of acetamiprid-N-demethyl in the embryos with parental exposure concentrations of 155 and 1637 ?g/L to the parent compounds were 31.5%and 93.1%,respectively.Bioconcentration factor of acetamiprid in zebrafish decreased with the increase of the exposure concentrations.When the exposure concentration increased to a certain extent,the bioconcentration factor reached a constant value,and most of the calculated values were less than 1,suggesting that acetamiprid was not to be significantly concentrated in zebrafish.Thirdly,the reproductive endocrine epigenetic effects of 20 dpf juvenile zebrafish after chronic exposure to acetamiprid for 22 weeks were studied by evaluating the survival rate,sex ratio,measurement of body constants,spawning of F0 generation and embryo development in F1 generation.Compared with the control group,the survival rates of the exposure groups decreased significantly and the ratios of female to male fish increased significantly.Body weight and body length of the fish in most exposure groups increased significantly,but for the growth condition factor(K),there was no significant difference between the treatments and the control group,except for the females being exposed to 1637 ?g/L,which was significantly increased compared with the control group.Moreover,the hepatic-somatic and brain-somatic indexes showed a downward trend and the female gonadal-somatic index values were not significantly affected,but the gonadal-somatic index values of males significantly decreased in the 17.2 and 155 ?g/L exposure groups.Chronic exposure to acetamiprid did not affect the spawning capacity of the zebrafish in the F0 generation.For the development of the F1 generation embryos,embryo malformation rates increased significantly under the conditions of no further exposure and continuing exposure to acetamiprid with the same concentration as the parental generation.Finally,the mechanism of endocrine disruption effect on the hormone levels,transcriptional expression of genes related to hypothalamic-pituitary-gonadal(HPG)axis and the vtg gene in the liver were determined after 22 weeks of chronic exposure to acetamiprid by 20 dpf zebrafish.The results showed that chronic exposure to acetamiprid led to an increase of estradiol(E2)concentration and a decrease of androstenedione concentration in the zebrafish ovary and testis,and also led to the concentration of estrone increased in testis,which reflected an estrogen effect.The transcriptional expression of genes related to HPG axis and the vtg gene in the liver of the zebrafish changed and showed gender-related characteristics.Chronic exposure to acetamiprid induced the expressions of ar,cyp19 b,fsh?,gnrh2,gnrh3 and lh? genes in the brain of the female fish but inhibited the expressions of these genes in the brain of the male fish.The expression of 3?hsd,cyp11 a,cyp17,cyp19 a,fshr and lhr genes in gonads implied that chronic exposure to acetamiprid more strongly affected females than males.The expression of vtg1 and vtg2 genes in the female liver were significantly down-regulated(p < 0.05)except for the fish in the exposed groups of0.19 and 155 ?g/L,which showed no significant difference compared with the control group.However,the expression of vtg1 in the male liver showed an up-regulated trend under the exposure concentrations of 0.19–1637 ?g/L,and the vtg2 gene expression was significantly down-regulated compared with the control group(p <0.05),with the down-regulated folds ranging from 0.04 ± 0.03 to 0.22 ± 0.16.In summary,acute exposure to acetamiprid affected the embryonic development and chronic exposure disturbed the reproductive endocrine system of the zebrafish.Acetamiprid has different degrees of influence on their behavior,growth,morphology,hatching ability and survival after acute exposure.After chronic exposure of acetamiprid at environment-related concentrations to juvenile zebrafish,acetamiprid and its metabolites can be detected in the tissues of the zebrafish and the main metabolite is acetamiprid-N-demethyl and the concentrations of acetamiprid and its metabolites in the brain and intestine tissues are higher than those in other tissues.At the same time,acetamiprid and its metabolites can be transferred to the offspring embryos after the parents were exposed to acetamiprid.At the same time,chronic exposure to acetamiprid led to a significant decrease in the survival rate of the zebrafish,and the ratios of female to male fish significantly increased,the body mass constant changed and an increase in estradiol concentration and a decrease in androstenedione concentration in ovary and testis and the transcriptional expression of genes related to HPG axis and the vtg gene in the liver of the zebrafish were also changed. |
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