Occurrence Of Typical Nanoparticles And Their Effects On Antibiotic Resistance Genes In Marine Shellfish Along The Coast Of China

Due to human activities,more and more nanoparticles（NPs）are released into the environment,and the accumulation of NPs in organisms and their ecological risks have attracted more attention,especially for marine shellfish.Based on the simulation of high concentrations of NPs in the laboratory,qualitative and quantitative studies of NPs in marine shellfish in the natural environment are very scarce.At the same time,there are few methods for the extraction and separation of NPs from marine shellfish.The efficient extraction of NPs from marine shellfish and the preparation of a stable sample system are the primary problems to be solved in this study.Filter-feeding marine shellfish represented by mussels are often suggested as indicator organisms for monitoring various toxic pollutants（metals and organic pollutants）due to their wide distribution,poor individual mobility and significant bioaccumulation effect.The coastal environment is an important repository of antibiotic resistance genes（ARGs）.However,in the natural environment,the research on the occurrence characteristics of ARGs mediated by NPs in marine shellfish gut is more scarce.In the shellfish intestines with high concentrations of heavy metals,the occurrence and influencing factors of metal resistance genes（MRGs）and ARGs are also insufficient.Therefore,understanding the occurrence characteristics and influencing factors of ARGs in marine shellfish in the natural environment is essential for maintaining ecological stability and human health.In this study,typical marine shellfish samples were collected along the coastline of China,and a set of standard methods for the extraction and separation of metalcontaining NPs in marine shellfish were established based on single particle inductively coupled plasma mass spectrometry（sp ICP-MS）and electron microscopy techniques.On this basis,the typical NPs in Chinese coastal mussels were characterized qualitatively and quantitatively,and the species and tissue differences of their occurrence were discussed,and a linear fitting was carried out between the total amount of metals and the concentration of NPs.At the same time,based on the Illumina PE150 metagenomic sequencing platform and PCR（polymerase chain reaction）technology,the composition and functional genes of the intestinal microbial community of the coastal mussel were analyzed in depth,and the abundance of resistance genes was discussed and compared.Based on structural equation modeling（SEM）,the effects of typical metal NPs and other natural and human factors on the occurrence of ARGs in the mussel gut were analyzed,and the changes in the occurrence and structure of ARGs in marine shellfish were clarified by metal-containing NPs,microbial communities and MRGs.The following main conclusions are drawn:（1）Five enzyme and two alkaline extraction methods were comprehensively compared and analyzed.A standardized protocol was developed to extract Ag,Au,and indigenous Ti-containing NPs from mussels.The optimal parameters are: dry sample extraction with tetramethylammonium hydroxide（TMAH）,5%（v/v）final concentration of TMAH,extraction at 25 ℃ for 12 h,and separation by centrifugation（3000 rpm for 5 min）.The particle number recoveries of spiked Ag and Au NPs were88 ± 0.9% and 95 ± 1.1%,respectively,while Ti-containing NPs had a particle number concentration of 8.2 × 106 particles/mg and an average size of 70 nm in tested mussels.The applicability of the method was evaluated in 10 marine shellfish using the optimized method.The particle number recoveries of spiked Ag-and Au-NPs were 72-98% and 72-97%,respectively,and the particle number concentrations and average sizes of the Ti-containing NPs were 2.1 × 106 – 8.4 × 106 particles/mg and 70–80 nm,respectively.These Ti-containing NPs,such as Ti O2,accounted for about half of the Ti mass in shellfish,indicating that marine shellfish may be a significant sink for Ticontaining NPs.（2）In this study,a qualitative and quantitative analysis of typical NPs in various tissues of 3 marine shellfish was conducted in 23 sampling sites in 9 coastal cities in China.sp ICP-MS analysis found that the average size and particle number concentration of Ti-NPs in musses were in the range of 84-102 nm and 1.02×106-5.86×106 particles/mg,respectively.The number concentration of particles containing Ti-NPs has obvious latitudinal zonality,generally showing the trend of Bohai Sea >Yellow Sea > East China Sea > South China Sea,and the highest and lowest levels were measured at the two sampling points of Lianyungang and Guangxi Beihai.The particle number concentration of Ti-NPs also has obvious species specificity: M.edulis > M.crassitesta > M.viridis;the average particle size of Ti-NPs also has the geographical difference of Yellow Sea > East China Sea,South China Sea and Bohai Sea.The species distribution difference of M.crassitesta > M.viridis >M.edulis.Generally speaking,the average particle size of the southeastern Bohai Sea and Guangdong is larger;the proportion of Ti-NPs and total Ti is also the same in the East China Sea and the South China Sea> The latitudinal zonality of the Bohai Sea and the Yellow Sea,and the species specificity of M.crassitesta ≈ M.viridis >M.edulis.On the whole,the proportion of the intersections of each sea area is relatively high.The tissue specificity of the number concentration of Ti-NPs-containing particles,Generally,the tissues with the highest concentration and the smallest particle size of containing Ti-NPs were the digestive gland and adductor muscle,respectively.The mass concentrations of Fe-,Znand Pb-NPs in mussel ranged from 129.4 to 15319.3 μg/kg,16.1 to 2787.7 μg/kg,and2.3 to 903.9 μg/kg,respectively.Based on electron microscopy technology,a large number of metal NPs with different shapes and elemental compositions were found in mussels,including rutile,anatase,Tix O2x-1,goethite,hematite,magnetite and other NPs containing Zn,Pb,Cr,Mg.（3）The relative abundances and species of ARGs in marine shellfish at each sampling point along the coast in China are generally latitudinal（South China Sea >Bohai Sea,Yellow Sea and East China Sea）.Moreover,there were significant differences in the occurrence of ARGs in different species.A relatively high resistance gene（lnu B）of lincosamides was found in each sampling point of the M.edulis,and its relative abundance gradually increased with the change of the latitude of the sampling point.The relative abundance of aminoglycoside antibiotic resistance gene（ANT3-IIc）in M.crassitesta was 76.6-154.2 ppm,accounting for 14.2%-20.1%.As for M.viridis,most of the ARGs were multi-drug resistant,and the ade C gene,as a representative of resistance to glycylcycline and tetracycline antibiotics,was much more abundant than other types of ARGs,accounting for about 56% of the total ARGs abundance.And the relative abundance of MRGs compared with each species of mussels generally showed the general trend of M.edulis > M.viridis > M.crassitesta.According to the analysis of the microbial community structure of the mussel intestine,we found the microbial communities with higher abundance between different species have obvious differences.The dominant phyla of the microbial community were Cyanobacteria,Verrucomicrobia and Proteobacteria in M.edulis,Proteobacteria,Mucoromycota,Cyanobacteria and Bacteroidetes in M.crassitesta,Proteobacteria,Planctomycetes and Bacteroidetes in M.viridis,respectively.The composition of the top 35 genera in relative abundance were also significantly different in these species.（4）The direct and indirect effects of anthropic/natural factors,metals and typical metal NPs in mussels on the relative abundance of ARGs were analyzed based on structural equation modeling（SEM）.The analysis showed that climate,agriculture and population all showed a direct negative correlation with the relative abundance of ARGs.Although Ti-NPs and trace metals（TTMs）had no significant direct effect on ARGs,Ti-NPs could directly affect the dominant bacterial phyla.To achieve indirect regulation of the relative abundance of ARGs.In addition,Fe-NPs and TTMs had a direct effect on the relative abundance of ARGs,but had no direct effect on the dominant phyla.Although Pb-NPs and trace metals（UTMs）had no significant direct effect on the relative abundance of ARGs,they could indirectly regulate ARGs by directly affecting Verrucomicrobia and Cyanobacteria.Similar to the influence path of Fe-NPs,Zn-NPs has a direct effect on the relative abundance of ARGs,and the correlation coefficient of its interpretation path shows the law of economy and industry>Zn-NPs>climate and agricultural activities.Fe-NPs and Zn-NPs had a direct effect on the relative abundance of ARGs,while Ti-NPs and Pb-NPs indirectly regulated the relative abundance of ARGs by affecting the bacterial community structure.