Long-term exposure of three plasticizers of di-(2-ethylhexyl)phthalate, dibutyl phthalate and acetyl tributyl citrate-individua.pdf

1、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 ex

2、amined 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

3、 of zebrafish both byindividual exposure for twelve months and combined exposure for three months.
　　In the individual exposure experiment，we first observed an interruption on both bodygrowth and gonadal development o

4、f exposed embryos and larvae of zebrafish by DEHP，DBP andATBC individually at concentrations of 0.5 and 0.05 μg mL-1.In detail,(1)compared with thecontrol fish，the body length，weight and gonado-somatic index (GSI) of bot

5、h exposed male andfemale 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

6、％，31％ and 36％ in female zebrafish，respectively.(2)However，in 0.5μg 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％

7、 of femaleswere 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 a

8、nd 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，thesuppression was mainly observed in about 7％，6％ and 11％ of male and 5％，9％ and

9、 17％ offemale zebrafish，respectively.Moreover，there was also an indication of more decrease in thebody 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

10、，respectively.The similar phenomena were also observed in0.5 and 0.05 μg mL-1 DBP which was about ～28％ and ～33％ in males and ～24％ and 25％ infemales，respectively.In contrast，these effects at 0.5 and 0.05 μg mL-1 ATBC were

11、 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 hatchingrates and hatchling survival after 1 month exposure.In DEHP 0.5 an

12、d 0.05 μg/mL-1 the hatchingrate observed was 63％ and 77％，respectively.In DBP the hatching rate was slightly high orsomewhat similar compared with DEHP observed.In the DBP 0.5 and 0.05μg/mL-1 was 73％ and83％，whereas the ha

13、tching rate in ATBC found to be more than the two phthalates and slightlyless 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

14、 1 month exposure in three plasticizersDEHP，DBP and ATBC were as follows; DEHP 0.5 and 0.05 μg mL-1 34.92％ and 38.96％，inDBP 0.5 and 0.05 μg/mL-1 45.21％ and 53.25％ and in ATBC 0.5 and 0.05 μg/mL-1 77.11％ and80.90％ and whe

15、re compared with 86.96 ％，respectively.
　　In the individual exposure experiment，the toxic effects on the fecundity of zebrafishexposed to DEHP，DBP and ATBC (0.5 and 0.05 μg/mL-1) were also observed.
　　In the DEHP 0.

16、05 μ g/mL-1 significant toxicological effect was also observed in the threegroups of exposed female to exposed male，which produced 188 eggs in total，of which，only 65were fertilized (～ 65.6％ non-fertilization rate).In the

17、 second group，exposed female to controlmale，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 tota

18、l,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 exposedfemale to exposed male，which produced 224 eggs

19、 in total，of which，only 112 were fertilized (～53.9％ non-fertilization rate).In the second group，exposed female to control male，a total numberof 220 eggs were accounted showing 89 fertilized eggs (～59.7％ non-fertilization

20、 rate)，the thirdgroup of exposed male to control female produced 243 eggs in total，from which 84 fertilized eggswere obtained (～50.2％ non-fertilization rate).
　　In the DBP 0.05 μg/mL-1 exposed female to exposed male，w

21、hich produced 195 eggs in total,of which，only 80 were fertilized (～ 59.1％ non-fertilization rate).In the second group，exposedfemale to control male，a total number of 224 eggs were accounted showing 99 fertilized eggs(～55

22、.8％ non-fertilization rate) and the third group of exposed male to control female produced227 eggs in total，from which 126 fertilized eggs were obtained (～43.5％ non-fertilization rate).
　　In the ATBC 0.5 tg/mL-1 expos

23、ed 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，exposedfemale to control male，a total number of 290 eggs were accounted sh

24、owing 189 fertilized eggs(～33.7％ non-fertilization rate) and the third group of exposed male to control female produced301 eggs in total，from which 199 fertilized eggs were obtained (34.0％ non-fertilization rate).In the

25、ATBC 0.05 μg/mL-1 showed very less effect on the fecundity ofzebrafish male and femalewith more than 70％ of fertilization success in the three exposed groups compared to the controlgroup，where the first exposed female to

26、 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 tocontrol male，a total number of 372 eggs were accounted showing 279 f

27、ertilized eggs (～24.6％non-fertilization rate) and the third group of exposed male to control female produced 359 eggs intotal，from which 279 fertilized eggs were obtained (22.2％ non-fertilization rate).
　　In the indiv

28、idual exposure experiment，the results for histological analysis of the ovaries in theexposed adult zebrafish showed that，the development of exposed ovaries was obviously inhibitedafter twelve-month exposure of 0.5 and 0.

29、05μg / mL-1 DEHP，characteristics of increased numbersof perinucleolar oocytes，less numbers of early and late cortical alveolar oocytes，less amount ofvitellogenic oocytes and mature oocytes.In regard to the results observ

30、ed from 0.5 μg mL-1 DBPconcentration in exposed female ovary，increased number ofperinucleolar oocytes of variable sizesand dark blue staining as well as early cortical alveolar oocytes were observed in the exposedovaries

31、.Whereas in the female ovary exposed to 0.05 μg/mL-1 DBP increased early corticalalveolar oocytes and vacuolation of surrounding granular were observed.In the ovaries exposedto 0.5 and 0.05μg/mL-1 ATBC，perinucleolar oocy

32、te，early cortical alveolar oocyte，late corticalalveolar oocyte，vitellogenic oocyte，post-ovulatory follicles were all observed in the exposedovaries，but disruption of yolk vesicles，hyalinization，disintegration of zona rad

33、iata，andhypertrophy ofperifollicular cells occurred.
　　In the combined exposure experiment，the obtained data demonstrated significant adverseeffects on the reproductive system of adult zebrafish after three-month expo

34、sure，as evidenced bysignificant decrease in body length，weight，GSI，impairment of fecundity，interruption ofoogenesis and spermatogenesis，etc.In detail，in comparison with the control fish，the body lengths,weight and GSI of

35、 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 interruptionof both body growth and gonad development by the combin

36、ed exposure of the three plasticizerstested，too.Significant toxicological effect was observed in the first group of exposed female toexposed male，which produced 338 eggs in total，of which，only 80 were fertilized (～76.3％

37、non-fertilization rate).In the second group，exposed female to control male，a total number of 202 eggswere accounted showing 56 fertilized eggs (～72.2％ non-fertilization rate).And the third group ofexposed male to control

38、 female produced 253 eggs in total，from which 82 fertilized eggs wereobtained (～67.6％ non-fertilization rate).In contrast，only 12.5％ non-fertilization rate wasobserved for the normal breeding in the group of control male

39、 to control female (488 eggs in total).The above results revealed that combined exposure of the three plasticizers mentioned-aboveimposed a significant toxic effect on the fecundity of the adult zebrafish after three-mon

40、thexposure.Of interest，male zebrafish were more sensitive to the combined plasticizer exposurewith respect to relatively lower fecundity and more heavily damaged testis than female zebrafish.The maturation of oocytes was

41、 inhibited as evidenced by occurrence of more underdevelopedperinucleolar oocytes and early cortical alveolar oocytes as well as atretic oocytes in the exposedovary.In contrast，most of the spermatocysts were broken and a

42、lmost all of the spermatogeneticcells ofpermatogonium，spermatocyte and spermatids were lost in the exposed testis，inferring thatSertoli cells might be the main target cells of the combined plasticizer mixture and subsequ

43、entinfertility of the exposed male fish.
　　Taken together，each of DEHP，DBP and ATBC contributed to the combined toxic effects andsynergestic effects may occur in the combined exposure.Thus great concern should be rais