Mechanistic Insights Into Sensitivity Difference Of Two Earthworm Species To Tetrabromobisphenol A And Cadmium In Soil

The quality of soil,especially agricultural soil,is directly related to food safety,human healthy and sustainable development of economy and society.A nationwide soil survey in China in 2014 showed that cadmium?Cd?is the most serious pollutant in soil and its effect on ago-ecological system has threatened to the agricultural product safety,therefore Cd has attracted extensive concerns of government and public.Tetrabromobisphenol A?TBBPA?is the world's most widely used brominated flame retardant?BFR?,and has been ubiquitously detected in environmental media.However due to its tendency to forming bound residues and to biotransformation,the environmental behavior of TBBPA is difficult to be studied using conventional methods,and the study on biological process and effect of TBBPA is less reported.E-waste is one of the fastest growing wastes,containing high concentrations of Cd and TBBPA.Therefore,unlegal processing leads to the release of the two coexisting pollutants into soil.Most of the studies on the combined pollution of organic contaminants and heavy metals have focused on traditional organic compounds?such as pesticides and polycyclic aromatic hydrocarbons?,and few literatures on BFRs are available,and mainly on polybrominated diphenyl ethers at present.Additionally,the reference earthworm species,Eisenia fetida,is commonly applied as bioindicator in soil ecotoxicological tests.However,this species is not a typical soil organism and has high tolerance to many contaminants,which can not indicate the real soil risk of pollutants.In the present study,using epigeic E.fetida and anecic Metaphire guillelmi as target organisms,the bioavailability in soil,internal behavior?including accumulation,distribution and/or metabolism?and biological effects?such as lethality,growth,and oxidative stress response?of Cd and TBBPA in dividual and co-existed form were investigated.The species dependent sensitivity and the mechanism were analyzed.The major results are as follows:?1?Results from the experiment of single Cd pollution indicated that among the battery of biochemical indexes,reactive oxygen species?ROS?and protein carbonyl groups served as sensitive biomarkers.According to their respective response thresholds,M.guillelmi was more sensitive to Cd than E.fetida and they differed in their dose-response relationships.In E.fetida,the activities of three antioxidant enzymes?superoxide dismutase?SOD?,catalase?CAT?and glutathione S-transferase?GST??exhibited hormesis-like U-shaped dose-response relationships,while in M.guillelmi SOD,glutathione peroxidase?GPx?and GST showed inverted U-shaped relationships.The concentrations of Cd in the subcellular fractions and whole body of the earthworms well fit?R2>0.9?a saturation model versus bioavailable Cd concentrations determined by the diffusive gradients in thin films technique.Despite the lower accumulation capacity of M.guillelmi,the Cd-binding capacity of its subcellular heat-stable protein fraction,the so-called biologically detoxified metal pool,was 2.7 times lower than that of E.fetida,whereas the Cd binding affinity of its heat-denatured protein fraction,i.e.the metal-sensitive fraction,was 3.0 times higher,which accounted for the low detoxification ability and the high susceptibility of M.guillelmi to soil Cd.?2?¹⁴C labeled TBBPA was used to study its internal behavior in earthworms.M.guillelmi accumulated more ¹⁴C than E.fetida,evidenced by a 1.3-fold higher biota-soil accumulation factor and more bound residues were formed in the former.¹⁴C accumulated mostly in the gut of M.guillemi and in the skin of E.fetida,suggesting that its uptake by M.guillelmi was mainly via gut processes whereas in E.fetida epidermal adsorption predominated.TBBPA was rapidly metabolized in earthworms.Besides polar metabolites,the major metabolites in both earthworms were O-methylation products of TBBPA,i.e.its mono-and dimethyl ethers?MeO-TBBPA and diMeO-TBBPA?.The TBBPA transformation potential was greater in M.guillelmi than in E.fetida?To distinguish the toxicity of TBBPA parent and metabolites,MeO-TBBPA and diMeO-TBBPA were synthesized and used in toxicity tests.Lethal and sub-lethal toxicity assessments showed that the toxicity of three compounds ranked as:TBBPA>MeO-TBBPA>diMeO-TBBPA.It indicated that earthworms used O-methylation to detoxify TBBPA,and M.guillelmi exhibited the higher detoxification ability than E.fetida.By soil experiment,synthetic toxicity of TBBPA to earthworms was further examined.It indicted that the sensitivity to TBBPA at low doses??10 mg kg-1?resembled each other for the two earthworm species,while high doses??50 mg kg-1?induced severer oxidative stress,and higher doses??250 mg kg-1?even led to lethality for M.guillelmi.By contrast,E.fetida sustained higher tolerance.According to the TBBPA metabolism in earthworms,M.guillelmi had great ability to O-methylate TBBPA,which decreased at high doses.It demonstrated that the species-specific sensitivity to TBBPA was related to earthworms' metabolic ability.?3?In the presence of Cd?<10 mg kg-1?,the internal behavior of TBBPA in earthworms almost did not significantly?P>0.05?change,and TBBPA??10 mg kg-1?also did not affect the behavior of Cd in body.However,the two chemicals had interaction on their fates in soil.In the presence of earthworms,the interaction became more complicated.Generally,because of the stronger biodisturbance of M.guillelmi than that of E.fetida,the effects of M.guillelmi on chemicals'environmental behavior were more obvious,showing that the mineralization and bound fraction?especially the humin-bound fraction?of TBBPA in the M.guillelmi processed soils reduced significantly,radioactivity residues?mainly the extractable fraction?reduced,being attributed to the earthworm uptake,and bioavailability of Cd in soil great decreased in the presence of the worm species.The co-toxicity of TBBPA and Cd was assessed.Results indicated that high doses??50 mg kg-1?of TBBPA inhibited the Cd uptake in E.fetida,while stimulated the uptake in M.guillelmi,which may result from the reduction of Cd bioavailability in soil and the different exposure routes of the two earthworms.Besides,the subcellular distribution of Cd also changed in the presence of TBBPA at high concentrations.Combined pollution induced synergistic acute toxicity,inhibited earthworm growth,exacerbated histopathological toxicity to body wall,and led to severer oxidative stress,including ROS accumulation,the change of antioxidant defense system indices?the inhibition of activity and/or gene expression level of SOD and CAT/GPx,as well as up-regulation of heat stress protein HSP 70?,and severe oxidative damage?the increase of malonaldehyde?.In contrast,M.guillelmi was more sensitive to the combined pollution than E.fetida.In summary,M.guillelmi had higher sensitivity to both single and combined pollution of TBBPA and Cd compared to the standard species E.fetida.The comprehensive analysis revealed the mechanism underlying the species-specific sensitivity based on the inherent relationship of environmental process,biological process,and biological effect.The application of M.guillelmi in ecotoxicological study will facilitate the accurate assessment of soil pollution risk.The present study provides scientific basis for screening the earthworm sensitive to TBBPA and Cd.