| Enrofloxacin(Enrofloxacin,ENR)is the third-generation synthetic quinolone antibacterial agent and special for veterinary and aquatic animals.It is widely used in aquaculture due to its high-efficiency antibacterial ability.The high-density farming model has also led to a sharp increase in the use of enrofloxacin and other antibacterial agents.The use of enrofloxacin has increased sharply too.And at the same time,the use of antibacterial agents in aquaculture has led to the continuous accumulation of residual antibacterial agents in the aquaculture pood,which will eventually enter the natural waters with the aquaculture tail water.The problem of antibacterial agent residues in aquaculture tail water has received more and more attention from society.How to purify the antibacterial agents in the aquaculture tail water has become a major problem for the aquaculture industry.Submerged macrophytes can directly or indirectly remove chemical pollutants in the water environment through direct absorption,root secretion,and root zone microorganisms.Therefore,it is necessary to study the accumulation,metabolism,and elimination of antibacterial agents by submerged macrophytes for establishing removal technology of antimicrobial agents in aquaculture tailwater,which will provide a theoretical basis and support for aquatic aquaculture technology.This paper takes Vallisneria natans,Elodea nuttallii,and Hydrilla verticillata as the research objects,and explores the accumulate,elimination,and metabolism of enrofloxacin in three submerged macrophytes.The main results of the research are as follows.1.Establishment of HPLC method for the detection of enrofloxacin and ciprofloxacinin submerged macrophytesThe Qu Ech ERS pretreatment method was improved.The sample was vortexed with 1% formic acid acidified acetonitrile for 15 minutes,then extracted and dehydrated by sodium chloride,and then PSA(Ethylenediamine-N-Propyl Silanized Silica Gel)and GCB(Graphitized Carbon Black)Remove impurities such as fat and pigment in the sample.After centrifugation,the supernatant was evaporated to dry by the rotary evaporator under reduced pressure,and the residue was dissolved by flow phase and was filtered by a 0.22μm membrane filter before injection.The flow phase was acetonitrile/0.01mol/L tetrabutylammonium bromide solution(Phosphoric Acid adjusted to p H=2.8,10:90(V/V)).The fluorescence detector was performed at Ex=280nm,Em=450nm.The external standard method was used for quantitative analysis.The instrument detection limits of enrofloxacin and ciprofloxacin were 0.001μg/g and 0.001μg/g,respectively,and the RSD values were79.60%-110.15%and 57.26%-102.00%,respectively.The sample detection limits of enrofloxacin and ciprofloxacin were 0.003μg/g,0.005μg/g,respectively,and the RSD values were0.86%-10.90% and 0.80-11.72%,respectively.2.The accumulation,metabolism,and elimination of enrofloxacin by submergedmacrophytesThe accumulation of enrofloxacin in the three submerged macrophytes showed the same accumulation pattern,all of which increased rapidly and then decreased slowly.The highest values were all reached at 24 h after exposure,and were 0.82μg/g,0.86μg/g,and 2.34μg /g,respectively.Then it followed by a slow elimination.At 600 h after exposure,the concentration of enrofloxacin decreased to 0.09μg/g,0.15μg/g,and0.22μg/g,respectively.And the area under the curve was 192.06 μg/g·h,209.92μg,respectively /g·h and 471.44 μg/g·h,respectively.At 24 h,the bioconcentration coefficients of enrofloxacin for the three submerged macrophytes were 9.7,10.2,and27.8,respectively.The concentration of ciprofloxacin in the three submerged macrophytes showed an overall upward trend and then a downward trend.They all reached the highest value at 72 h after exposure,which were 0.027 μg/g,0.029 μg/g,and 0.037 μg/g,respectively.After 300 h,it showed a slow declining trend.The areas under the curve were 8.42 μg/g·h,8.21 μg/g·h and 12.21 μg/g·h,respectively.The percentages of the highest content of metabolite ciprofloxacin and the highest content of enrofloxacin in Vallisneria natans,Elodea nuttallii and Hydrilla verticillata were 3.29%,3.37%,and 1.58%,respectively.The percentages of the area under the curve of the metabolite ciprofloxacin and enrofloxacin were 4.39%,3.87%,and 2.59%,respectively.3.Identification of enrofloxacin metabolites and metabolic The percentages ofmetabolic pathways in submerged macrophytesThe metabolites of enrofloxacin in three submerged macrophytes were identified by Ultra high-performance liquid chromatography-Quadrupole time-of-flight mass spectrometry(UPLC-Q-TOF),A total of 8 metabolites were produced,which were divided into 4 metabolic pathways,namely N4 deethyl(M7,M8),Piperazine ring cleavage(M3,M4,M5),defluorination(M6-1,M6-2)and decarboxylation(M2)pathways,of which M8 is a unique metabolite of H.verticillata,and M5 is a unique metabolite of V.natans.V.natans produced 7 metabolites(M2,M3,M4,M5,M6-1,M6-2,M7)and 4 metabolic pathways.M1(prototype)was deethylated to form M7,or piperazine ring was cleavage to form M3,M3 carbonyl group was replaced by hydroxyl group to form M4,in which C2 and C3 double bonds were reduced to single bonds to form M5.M1 was hydroxylated to M6-1 or M6-2 by removing fluorine C5 or C8;After the decarboxylation of M1,C3 was oxidized to produce carbonyl group,then C2 and C3 break,C2 and C3 obtain carbonyl group and carboxyl group respectively,and then the carboxyl group of C3 was removed,C2 and C4 phase chain form M2.E.nuttallii produces 6 metabolites(M2,M3,M4,M6-1,M6-2,M7)and 4 metabolic pathways.M1(prototype)was deethylated to generate M7,or piperazine ring was cleavaged to generate M3,M3 carbonyl group was replaced by hydroxyl group to generate M4.M1 piperazine ring was cleaved to M3 and regenerated to M4.M1 dehydroxylates fluorine C5 or C8 to form M6-1 or M6-2.After the decarboxylation of M1,C3 was oxidized to produce carbonyl group,then C2 and C3 break,C2 and C3 obtain carbonyl group and carboxyl group respectively,and then the carboxyl group of C3 was removed,C2 and C4 phase chain form M2.H.verticillata produced 7 metabolites(M2,M3,M4,M6-1,M6-2,M7,M8)and 4 metabolic pathways.M1(prototype)was deethylated to generate M7,M7 was defluorinated to generate M8,or piperazine ring was cleavage to generate M3,M3 carbonyl group was replaced by hydroxyl group to generate M4.M1 piperazine ring was cleaved to M3 and regenerated to M4.M1 was hydroxylated to M6-1 or M6-2by removing fluorine C5 or C8;After the decarboxylation of M1,C3 was oxidized to produce carbonyl group,then C2 and C3 break,C2 and C3 obtain carbonyl group and carboxyl group respectively,and then the carboxyl group of C3 was removed,C2 and C4 phase chain form M2. |
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