| Increased concerns have been raised on the safety of individual and combined plasticizer exposure to aquatic organisms due to their universal distribution and well-known endocrine disruptor activities. In this study, we examined the toxic effects of three plasticizers di (2-ethylhexyl) phthalate (DEHP), dibutyl phthalate (DBP) and acetyl tributyl citrate (ATBC) at an environmental relevant concentrations on the development and reproduction of zebrafish both by individual exposure for twelve months and combined exposure for three months.In the individual exposure experiment, we first observed an interruption on both body growth and gonadal development of exposed embryos and larvae of zebrafish by DEHP, DBP and ATBC individually at concentrations of 0.5 and 0.05 μg mL-1. In detail, (1) compared with the control fish, the body length, weight and gonado-somatic index (GSI) of both exposed male and female zebrafish to DEHP at the concentration of 0.5 μg mL-1 were significantly inhibited by 34%, 32.6% and 43% in males,30%,40% and 32% in females, whereas at 0.05 μg mL-1,69%,30.5% and 56% in male and 24%,31% and 36% in female zebrafish, respectively. (2) However, in 0.5 μg mL-1 DBP,26%,28% and 33% of males and 23%,24% and 51% of females were suppressed, whereas at 0.05 μg mL-1 DBP,28%,33% and 27% of males and 22%,25% and 31% of females were affected as compared with the control group, respectively. (3) In contrast to DEHP and DBP, low toxic effects were observed in 0.5 and 0.05 μg mL-1 ATBC showing an eco-friendly nature. The body length, weight and GSI in 0.5 μg mL-1 ATBC compared to control was about 10%,19% and 20% in males, and 14%,17% and 22% in females. Whereas, in 0.05 μg mL-1 ATBC, the suppression was mainly observed in about 7%,6% and 11% of male and 5%,9% and 17% of female zebrafish, respectively. Moreover, there was also an indication of more decrease in the body weight of male zebrafish exposed to 0.5 and 0.05 μg mL-1 DEHP with-32.6% and-30.4%, whereas-40% and -31% in females, respectively. The similar phenomena were also observed in 0.5 and 0.05 μg mL-1 DBP which was about -28% and -33% in males and -24% and 25% in females, respectively. In contrast, these effects at 0.5 and 0.05 μg mL-1 ATBC were about -19% and -6% in males, whereas -17% and -9% in females.In the individual exposure experiment, toxic effects were also found in the 72 h hatching rates and hatchling survival after 1 month exposure. In DEHP 0.5 and 0.05 μg/mL-1 the hatching rate observed was 63% and 77%, respectively. In DBP the hatching rate was slightly high or somewhat similar compared with DEHP observed. In the DBP 0.5 and 0.05μg/mL-1 was 73% and 83%, whereas the hatching rate in ATBC found to be more than the two phthalates and slightly less than the control group with a hatching percentage of 83% and 89% in 0.5 and 0.05μg/mL-1 respectively. The hatching survival rate observed after 1 month exposure in three plasticizers DEHP, DBP and ATBC were as follows; DEHP 0.5 and 0.05 μg mL-1 34.92% and 38.96%, in DBP 0.5 and 0.05 μg/mL-1 45.21% and 53.25% and in ATBC 0.5 and 0.05 μg/mL-177.11% and 80.90% and where compared with 86.96%, respectively.In the individual exposure experiment, the toxic effects on the fecundity of zebrafish exposed to DEHP, DBP and ATBC (0.5 and 0.05 μg/mL-1) were also observed.In DEHP 0.5μg/mL-1 significant toxicological effect was observed in the first group of exposed female to exposed male, which produced 121 eggs in total, of which, only 37 were fertilized (-69.9% non-fertilization rate). In the second group, exposed female to control male, a total number of 167 eggs were accounted showing 79 fertilized eggs (-52.7% non-fertilization rate).The third group of exposed male to control female produced 155 eggs in total, from which 65 fertilized eggs were obtained (-58.1% non-fertilization rate).In the DEHP 0.05 μg/mL-1 significant toxicological effect was also observed in the three groups of exposed female to exposed male, which produced 188 eggs in total, of which, only 65 were fertilized (-65.6% non-fertilization rate). In the second group, exposed female to control male, a total number of 220 eggs were accounted showing 89 fertilized eggs (-59.7% non-fertilization rate). The third group of exposed male to control female produced 228 eggs in total, from which 110 fertilized eggs were obtained (-51.6% non-fertilization rate).In the DBP 0.5 μg/mL-1 was found to be significantly toxic in the three groups of exposed female to exposed male, which produced 224 eggs in total, of which, only 112 were fertilized (-53.9% non-fertilization rate). In the second group, exposed female to control male, a total number of 220 eggs were accounted showing 89 fertilized eggs (-59.7% non-fertilization rate), the third group of exposed male to control female produced 243 eggs in total, from which 84 fertilized eggs were obtained (-50.2% non-fertilization rate).In the DBP 0.05 μg/mL-1 exposed female to exposed male, which produced 195 eggs in total, of which, only 80 were fertilized (-59.1% non-fertilization rate). In the second group, exposed female to control male, a total number of 224 eggs were accounted showing 99 fertilized eggs (-55.8% non-fertilization rate) and the third group of exposed male to control female produced 227 eggs in total, from which 126 fertilized eggs were obtained (-43.5% non-fertilization rate).In the ATBC 0.5 μg/mL-1 exposed female to exposed male, which produced 334 eggs in total, of which, only 260 were fertilized (-24.4% non-fertilization rate). In the second group, exposed female to control male, a total number of 290 eggs were accounted showing 189 fertilized eggs (-33.7% non-fertilization rate) and the third group of exposed male to control female produced 301 eggs in total, from which 199 fertilized eggs were obtained (34.0% non-fertilization rate). In the ATBC 0.05 μg/mL-1 showed very less effect on the fecundity of zebrafish male and female with more than 70% of fertilization success in the three exposed groups compared to the control group, where the first exposed female to exposed male group produced 387 eggs in total, of which, only 317 were fertilized (-18.08% non-fertilization rate). In the second group, exposed female to control male, a total number of 372 eggs were accounted showing 279 fertilized eggs (-24.6% non-fertilization rate) and the third group of exposed male to control female produced 359 eggs in total, from which 279 fertilized eggs were obtained (22.2% non-fertilization rate).In the individual exposure experiment, the results for histological analysis of the ovaries in the exposed adult zebrafish showed that, the development of exposed ovaries was obviously inhibited after twelve-month exposure of 0.5 and 0.05μg/mL-1 DEHP, characteristics of increased numbers of perinucleolar oocytes, less numbers of early and late cortical alveolar oocytes, less amount of vitellogenic oocytes and mature oocytes. In regard to the results observed from 0.5 μg mL-1 DBP concentration in exposed female ovary, increased number of perinucleolar oocytes of variable sizes and dark blue staining as well as early cortical alveolar oocytes were observed in the exposed ovaries. Whereas in the female ovary exposed to 0.05 μg/mL-1 DBP increased early cortical alveolar oocytes and vacuolation of surrounding granular were observed. In the ovaries exposed to 0.5 and 0.05μg/mL-1 ATBC, perinucleolar oocyte, early cortical alveolar oocyte, late cortical alveolar oocyte, vitellogenic oocyte, post-ovulatory follicles were all observed in the exposed ovaries, but disruption of yolk vesicles, hyalinization, disintegration of zona radiata, and hypertrophy of perifollicular cells occurred.In the combined exposure experiment, the obtained data demonstrated significant adverse effects on the reproductive system of adult zebrafish after three-month exposure, as evidenced by significant decrease in body length, weight, GSI, impairment of fecundity, interruption of oogenesis and spermatogenesis, etc. In detail, in comparison with the control fish, the body lengths, weight and GSI of both exposed male and female zebrafish were significantly inhibited by 37%, 40% and 39% for males,10%,53% and 33% for females, respectively, indicating an interruption of both body growth and gonad development by the combined exposure of the three plasticizers tested, too. Significant toxicological effect was observed in the first group of exposed female to exposed male, which produced 338 eggs in total, of which, only 80 were fertilized (-76.3% non-fertilization rate). In the second group, exposed female to control male, a total number of 202 eggs were accounted showing 56 fertilized eggs (-72.2% non-fertilization rate). And the third group of exposed male to control female produced 253 eggs in total, from which 82 fertilized eggs were obtained (-67.6% non-fertilization rate). In contrast, only 12.5% non-fertilization rate was observed for the normal breeding in the group of control male to control female (488 eggs in total). The above results revealed that combined exposure of the three plasticizers mentioned-above imposed a significant toxic effect on the fecundity of the adult zebrafish after three-month exposure. Of interest, male zebrafish were more sensitive to the combined plasticizer exposure with respect to relatively lower fecundity and more heavily damaged testis than female zebrafish. The maturation of oocytes was inhibited as evidenced by occurrence of more underdeveloped perinucleolar oocytes and early cortical alveolar oocytes as well as atretic oocytes in the exposed ovary. In contrast, most of the spermatocysts were broken and almost all of the spermatogenetic cells of permatogonium, spermatocyte and spermatids were lost in the exposed testis, inferring that Sertoli cells might be the main target cells of the combined plasticizer mixture and subsequent infertility of the exposed male fish.Taken together, each of DEHP, DBP and ATBC contributed to the combined toxic effects and synergestic effects may occur in the combined exposure. Thus great concern should be raised on the safety of individual and combined plasticizer exposure to the environment and organisms. |
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