Mutual Effects Of African Giant Snails Achatina Fulica And(Micro) Plastics In Soil

The widespread use of plastic products has caused global plastic pollution.The soil is one of major storages of plastic waste.Plastic litters in the soil could break or degrade into smaller plastic fragments,and plastic particles with size of smaller than 5mm are called microplastics?MPs?,which are considered as a new threat to terrestrial ecosystems.Previous studies of microplastics mostly focus on microplastics in oceans,but little is known about the environmental fate and ecological risks of microplastics in the terrestrial soil environments.Soil animals have high possibility to expose to plastic fragments in the soil.However,few studies have investigated the fate of microplastics in the soil after plastic feeding and digestion,and potential toxic effects on animals.In addition,the biodegradation of plastics by animals has attracted wide attention,but it remains unclear whether soil animals have the ability of biodegradation of plastics.In this study,white jade snail,i.e.Achatina fulica was selected as the test organism,which is one of main species of animals in soil.Polyethyl terephthalate?PET?microfiber?MFs??mean length and diameter are 1257.8?m and 76.3?m,respectively?and polystyrene?PS?foam were used as the representatives of?micro?plastics.The interaction and mutual effects between soil animals and?micro?plastics were fully investigated.The effects of?micro?plastics on the growth and development,histopathology and oxidative stress-related enzyme activity of A.fulicas were analyzed through systematically observed feeding,metabolism and excretion of?micro?plastics by A.fulica.Compared with the original?micro?plastic,changes of surface morphology,particle size distributions,molecular weight and molecular structures of PS in feaces residue was analyzed through stered microscopes observation,scanning electron microscope?SEM?,Gel permeation chromatograph?GPC?,Fourier transform infrared spectroscopy?FTIR?and Proton nuclear magnetic resonance?¹H NMR?.The main results are as follows:?1?After A.fulica were fed with the Nile Red-stained PET MFs for 5 h,MFs were observed to accumulate crop,stomach,anterior intestine,and posterior intestine with high concentrations at 5,10,12,24 h after exposure,respectively.All of MFs were excreted within 48 h.The SEM imagines show the appearance of cracks and deterioration on the surface of MFs after depuration by sanils'digestion.The diameters of the MFs discharged by the snail were significantly decreased 10.81±6.26%compared with the raw PS materials?p<0.001?.?2?After 28 d exposure to PET MFs at concentrations of 0.01,0.14,0.71 g kg^-1,no animals died.0.14-0.71 g kg^-11 MFs exposure caused an average reduction of 24.7-34.9%in food intake,and 46.6-69.7%in excretion rates.There were no significant changes in body weight,shell length and shell diameter in the exposure group in comparison with the control group.Based on the analyzation of the histopathological sections of major digestive organs,0.71 g kg^-1 MFs MFs induced significant villi damages in the gastrointestinal walls of 40%snails,including shortening or breakage of intestinal villi,but did not influence the histology of the liver and kidney.Moreover,0.71 g kg^-1 MFs exposure reduced glutathione peroxidase level?59.3±13.8%?and total antioxidant capacity?36.7±8.5%?,but elevated malondialdehyde level?58.0±6.4%?in the liver,which indicates oxidative stress is involved in the toxic mechanism.?3?After fed with PS foam pre-brushed with lettuce juice and co-diet lettuce for4 w,A.fulica consumed 18.5±2.9 mg PS per animal during the test.Through 30%H₂O₂digestion and filtration,12.8±1.1 mg PS was found in feaces per snail.According to mass balance,30.7%of intaked PS was digested by A.fulica.Morphology and size statistics of PS in feces were carried out,and the particle size distribution was between0.146-3.1 mm.PS ingestion had no significant effects on the growth and development of A.fulicas,and the body weight,shell length and shell diameter of snails showed no obvious changes compared with the control group.GPC analysis indicated an increase in PS molecular weights?Mw from 234,500 Da to 250,300 Da and Mn from 92,000 Da to 106,000 Da?in feces,confirming limited PS depolymerization.FTIR and ¹H NMR confirmed the formation of functional groups of oxidized intermediates and chemical modification.For example,new groups such as Carbon-carbon double bond?-CH=CH-?,carbonyl group?H₂C=O?and hydroxyl group?-OH?appeared.?4?After preliminary treatment of oxytetracycline for 5 d,the amount of gut bacteria in snails decreased by two orders of magnitude.And then these A.fulicas were fed with PS foam for 4 weeks,as similarly as the normal snails without oxytetracycline treatment.The molecular weights of PS were also proved to changed of Mw increasing to 245,900 Da,and Mn increasing to 100,860 Da,which were consistent with that of normal snails.This indicated that suppression of gut microbes with oxytetracycline had no effect on the depolymerization of PS.While High-throughput sequencing analysis revealed significant shifts in the gut microbiome after the snails ingested PS,with increase of family Enterobacteriaceae and Sphingobacteriaceae,suggesting that the gut microorganisms were associated with PS biodegradation.The above results indicate:?1?A.fulica can ingest and excrete MFs,and 5h-exposure could not cause the accumulation of MFs.The digestion of A.fulica can cause the surface of MFs wear and aging mainly through the action of the intestine.?2?The exposure to MFs influences feeding and excretion of snails,and further induces pathological changes in the gastrointestinal tract.Oxidative stress is involved in the toxicity mechanism of microfibers.?3?A.fulica can be induced to ingest PS foam,and break PS foam into microplastics by chewing and digestion.A.fulica can depolymerize PS with low molecular weight by their digestion,which leads to the limited biodegradation on PS.?4?PS feeding and digestion have impacts on the abundance of intestinal microbiota in snails,which indicate that gut microbial communities are involved in the biodegradation of PS by A.fulica.This study,for the first time,has explored the interaction of PET/PS?micro?plastic and A.fulica,which suggests the ecological risks of?micro?plastics on soil animals.In addtion,we have discovered a series of biological effects such as abrasion,fragmentation,and biodegradation of?micro?plastics by soil animals.These novel findings provide scientific basis for fate analysis of microplastics in terrestrial soil environments,and also provide new evidence for eco-environmental risks of?micro?plastic pollution.