A Study On Toxicity Of Typical Organic Additives In Biodegradable Plastics To Caenorhabditis Elegans

Conventional plastics can hardly be degraded naturally,and cause wide environmental pollution.The popularization of biodegradable plastics(BDP)as alternative products is a key to control plastic pollution from the source.Compared with conventional plastics,BDP can break down and degrade more rapidly,thus releasing more additive components into the natural environment and posing potential environmental risks.However,there is little data on the ecotoxicity of typical organic additives in BDP,such as preliminary toxicity assessment studies based on BDP,which is disadvantageous to the popularization of BDP application.Therefore,ecotoxicity assessment of the main typical organic additives in BDP products based on in vivo toxicological evaluation system is significant to the safety evaluation and promotion application of BDP products.In this study,the nematode Caenorhabditis elegans(C.elegans)was used as the model organism.12 types of BDP organic additives were selected,specifically five chain extenders:hexamethylene diisocyanate(HDI),toluene diisocyanate(TDI)and 4-fluorophenyl isocyanate(FIS),diallyl maleate(DIM)and diethylhexyl maleate(DOM);four antioxidants:2,4-di-tert-butylphenol(2,4-DTBP),2,6-di-tert-butyl-4-methylphenol(BHT),2,4-Dimethyl-6-sec-hexadecylphenol(AO1076)and tris(2,4-di-tert-butyl)phosphite phenyl ester(AO168);and three light stabilizers:2,2,6,6-tetramethylpiperidinyl sebacate(T770),2,2,6,6-tetramethylpiperidinooxy(TEMPO)and 2-(2'-hydroxy-3'-tert-butyl-5'-methylphenyl)-5-chlorobenzotriazole(UV326).The lethality and developmental toxicity of these additives to nematodes were studied after48 h exposure from L1 phase.Based on the LD₅₀ values,six types of highly-toxic additives including HDI,DIM,2,4-DTBP,BHT,TEMPO and T770 were further selected for chronic toxicology at sublethal concentrations.Subsequently,the mechanisms associated with the toxic effects were investigated at the genetic level using a variety of transgenic nematodes.The main results are as follows:(1)Exposure to typical organic additive of BDP resulted in a reduction of survival rates in concentration-dependent manners.Among 12 types of organic additives,isocyanate and maleic anhydride chain extender,HDI and DIM,had the smallest LD₅₀values,i.e.12.77 and 12.91?g L^-1;whereas,light stabilizer UV326 had the largest LD₅₀values according to the fit analysis.The descending order of LD₅₀ of these additives was HDI,DIM,BHT,T770,2,4-DTBP,AO1076,TDI,DOM,TEMPO,AO168,FIS and following UV326.Exposure to these additives also inhibited nematodes'body length and body width in concentration-dependent manners,and the growth inhibition rates were analyzed in terms of changes of animal volumes.The results showed that chain extenders-induced growth inhibition ranged from 28.36 to 96.23%and varied greatly between species,with maximum of HDI.Growth inhibition rates induced by the antioxidants and light stabilizers were 41.60-96.67%and 6.15-67.45%,respectively.(2)Exposure to the chain extenders HDI,DIM and the antioxidants BHT and 2,4-DTBP of sublethal concentrations resulted in chronic effects such as considerable reductions in nematodes'lifespan,however,similar effects were not shown in groups of light stabilizers T770 and TEMPO.In addition,HDI,DIM,BHT and 2,4-DTBP induced a significant expression upregulation of gst-4 and ges-1 gene in nematodes,reflecting elevation of glutathione S-transferase levels and changes in mitochondrial function in intestinal cells.In contrast,the light stabilizers T770 and TEMPO had no effect on nematodes'glutathione S-transferase expression,and presented slight damage of intestinal mitochondria.(3)Exposure to sublethal levels of BDP typical organic additives inhibited nematodes'locomotor behavior with various degrees.HDI,DIM,BHT,and 2,4-DTBP exposure depressed frequency of head thrashes,body bends,and pharynx pumping in nematodes,indicating locomotion impairment and bradykinesia.Furthermore,exposure to 6 types of BDP organic additives altered dietary and excretory behavior of nematodes,and showed a decrease of food intake but an increase of excretory cycles.In addition,exposure to the additives HDI,DIM and T770 suppressed the expression of eat-4 and unc-47 in nematodes significantly.The related neurons,glutamatergic and GABAergic neurons,showed morphology characteristics including cytosolic damage or partial cytosolic loss,dendritic reduction and cord breakage.BHT and 2,4-DTBP inhibited the expression of unc-47,but had no significant effects on gene eat-4;otherwise,exposure to TEMPO had no significant effects on these neurons,either.The above results indicate that:(1)typical organic additives of BDP possess lethality and development toxicity to nematodes.The toxicity is related to the type and chemical structure of additives,and some isocyanates and maleic anhydride additives have relatively strong toxicity.(2)HDI,DIM,BHT and 2,4-DTBP have chronic toxic effects such as inhibition of animal longevity at a sublethal concentration.Oxidative stress and damage of intestinal mitochondria are involved with the mechanisms of toxic action.(3)Typical organic additives of BDP have different-degree neurotoxicity to nematodes,triggering behavioral changes such as sluggish movement,reduced food intake and longer excretory cycles,neurotoxic action is related to damage of glutamatergic and GABAergic neurons.This work systematically investigated the toxic effects and related mechanisms of typical organic additives of BDP on C.elegans.For the first time,ecotoxicity of common types of BDP additives were compared and analyzed.Our results revealed that isocyanates and maleic anhydrides had multiple toxic effects;and their ecological risks should be of concern.These findings provide a scientific basis for the toxicity risk assessment of BDP additives and provide an important reference for the environmental safety and promotion of BDP applications.