The Reproductive And Developmental Toxicity And Mechanism Of Polybrominated Diphenyl Ethers In Rotifer Branchionus Plicatilis

Polybrominated diphenyl ethers (PBDEs), a class of persistent organic pollutant,are widespread in marine ecosystem. Rotifers, Branchionus plicatilis, a class ofcommon seawater invertebrate zooplankter, have played a basal role in enrichmentand transfer of pollutants in marine ecosystems. In addition, their populationdynamics might directly or indirectly affect marine food chains. A study ofdevelopmental and reproductive toxicity of PBDEs in B. plicatilis is important inassessing the risk of persistent organic pollutants of emerging concern on aquaticbiota and food webs. The current knowledge concerning effects of PBDEs on B.plicatilis is very limited, so the present study initiated to determine the potential forPBDEs (tetrabrominated diphenyl ether-47, BDE-47; and decabrominated diphenylether-209, BDE-209) to cause developmental and reproductive toxicity in B. plicatilis.The underlying mechanisms of PBDEs on B. plicatilis, with a focus on oxidativestress, were investigated.The main methods and results as follows:1. The results of Gas Chromatography-Mass Spectrometer (GC-MS) showed thatdesigned concentrations of BDE-47and BDE-209in our study were above thedissolved concentrations, and the water solubility of BDE-209is lower than thatof BDE-47.2.24h-EC50was estimated by log-probit method, and the value of BDE-47was7.923mg/L (95%confidence intervals,3.626-13.634mg/L) in B. plicatilis.3. The structure of B. plicatilis ovaries were examined by light microscopy,results suggesting that the structure of ovaries were damaged by both0.2mg/LBDE-47and BDE-209. 4. The indexes of reproduction (first reproduction time, last reproduction time,number of eggs laid, number of production larvae and production rate) wereexamined by anatomical lens throughout the course of the experiment. Exposureto the low and moderate concentration of BDE-47(0.05mg/L,0.1mg/L) causeda significant decreases (P<0.05) in time of first reproduction relative to thatobserved in the control, and highly significant decreases (P<0.001) wereobserved in0.2mg/L BDE-47and all three BDE-209groups. Significantdecrease (P<0.05or P<0.001) in time of last reproduction was detected in eachtreatment except for0.05mg/L BDE-47and BDE-209groups compared to thecontrol. Compared to the control group, highly significant decreases (P<0.001) innumber of egg laid and number of production larvae were observed in all ofBDE-47groups. The significant decreases of production rate (P<0.001) occurredin0.1mg/L,0.2mg/L BDE-47groups. Remarkably, these three indicatorsincreased slightly in all BDE-209groups.5. The indexes of development (pre-reproductive phase, reproductive phase andpost-reproductive phase) were examined five times daily by anatomical lensthroughout the course of the experiment. Results showed that both BDE-47andBDE-209caused the pre-reproductive phase significant decreases (P<0.01orP<0.001) in each treatment compared to the control. Reproductive phasedeclined significantly (P<0.001) at0.2mg/L concentration of BDE-47andBDE-209groups compared to the control, even at0.1mg/L BDE-209. Highlysignificant decreases (P<0.01or P<0.001) in post-reproductive phase wereobserved in0.1mg/L,0.2mg/L BDE-47groups, and0.2mg/L BDE-209treatment compared to the control.6. Factor Analysis (FA) analysis showed that reproduction phase and lastreproduction time were more susceptible than other indexes to BDE-47orBDE-209. The results of separate analysis of BDE-47and BDE-209demonstrated that the toxicity of BDE-47to the B. plicatilis would be given more emphasis on the reproductive toxicity in the stage of adult, and the toxicity ofBDE-209inclined slightly to affect the developmental toxicity of larvae.7. Reactive oxygen species (ROS) production was labeled with DCFH-DA, and thechange of intracellular calcium level ([Ca2+]in) was determined using Fluo-3/AM.The fluorescence is detected with fluorescence microscope, and then the valuesare calculated by Image-Pro Plus system. It has been demonstrated that exposureto0.05mg/L BDE-47and0.05mg/L,0.1mg/L BDE-209did not induce anysignificant increase in intracellular ROS levels as compared to controls.However, higher concentrations of BDE-47(0.1mg/L,0.2mg/L) and ofBDE-209(0.2mg/L) significantly elevated (P<0.01or P<0.001) ROS levels.Similarly, the two congeners were able to induce an increase in calcium levels.Treatment of0.1mg/L,0.2mg/L of BDE-47, and0.2mg/L of BDE-209causesignificant effect (P<0.01or P<0.001) on [Ca2+]inas compared to control.8. Real-Time quantitative PCR (qRT-PCR) analysis showed that vasa and nanosmRNA were activated by BDE-209, but inhibited by BDE-47.The expressions ofvasa mRNA had significantly decrease in0.2mg/L BDE-47group (P<0.05).Lower BDE-209(0.05mg/L) significantly increased (P<0.05) the expressions ofvasa mRNA. The results of nanos showed that it is distinctly up-regulatedmRNA levels in0.05mg/L BDE-209exposure group compared with control(P<0.001), and then the induction decreased with increased BDE-209concentration. In addition, vasa mRNA was expressed at lower levels than nanosmRNA in both congeners. The expressions of sod mRNA significantly increased(P<0.01) at the BDE-47concentration of0.2mg/L. The expression of cat had nosignificant difference between the two congeners. Treatment of0.2mg/L of thetwo congeners showed significant increase on cam as compared to control(P<0.05or P<0.01).Major conclusions are that：1. The results of GC-MS proved that PBDEs has poor water solubility and the water solubility of BDE-209is lower than that of BDE-47.2. The24h-EC50data suggested BDE-47could cause higher acute toxicity thanBDE-209.3. Based on studies included morphological criteria, physiological indexes, andconserved germ cell-specific markers, the vasa and nanos mRNA expression, wesuggested that the ovary of B. plicatilis is the target organ affected by BDE-47and BDE-209.4. Whether BDE-47or BDE-209had capacity to cause damage and to restraindevelopment and reproduction in B. plicatilis. It also confirmed that BDE-47wasmore noxious than BDE-209in B. plicatilis. Furthermore, low level BDE-209might have a potential estrogenic effects in B. plicatilis.5. The results of FA indicated that reproduction phase and time of last reproductioncould be considered as the promising candidates for biomarkers.6. Combining of the experimental results, it is probable that PBDEs are able tocause an increase intracellular ROS levels in ovaries, which induce oxidativestress, regulate the activities of sod mRNA and cat mRNA, disrupt calciumhomeostasis, and elevate the expression of cam mRNA, events that finallyprotected B. plicatilis against PBDEs-induced toxicity. Our study presented thatantioxidant system played an important role in PBDEs-induced toxicity in B.plicatilis. Antioxidant enzymes (sod and cat) and non-enzymatic compounds(Ca2+and cam) were involved in the mechanism of PBDEs toxicity.In summary, our researches have provided relatively complete assessment on thereproductive and developmental toxicity of BDE-47and BDE-209in B. plicatilisthrough the investigation both of physiological function and oxidative stress processes.The current study may be of use for evaluating the toxicity of environmental PBDEsexposures.