Studies On Metabolic Kinetics And Residues Of Phthalic Acid Esters In Fish

Phthalic Acid Esters (PAEs) are generally represented a class of compounds, which was widely used as plasticizers in a variety of consumer products and household industries such as paints, coatings, printing, dyeing, pesticides, cosmetics and asspices. But phthalate esters are not chemically bound to the plastics, so they can be released easily from products and migrate into the aquaculture water that comes into direct contact. And some researches have found that PAEs exhibited estrogen-like activities, which have been classified into environmental endocrine disruptors. PAEs chemicals could influence the hormone metabolism in animals’ reproduction and development process, thus affect the normal behavior of them. At the same time, organism lived in aquaculture water including fishes would absorbed and accumulated them. Further more, the bioaccumulation of PAEs in organisms will bring potential threats to aquatic and human beings through food chain transfer. PAEs chemicals were determined by RP-HPLC, GC and LC-MS/MS in this study, which included DMP, DEP, DBP, BBP and DEHP. Moreover, the studies of PAEs chemicals on metabolic kinetics and residues were also developed and clarified. Then all the studies will provide theory basis for aquatic toxicology, fishing sources protection and aquatic products safety. And the research was following below.At first, the methods for simultaneous determination of PAEs have been established by HPLC and GC in fish’s plasma and tissues.DMP and DEP were detected in plasma after liquid-liquid extraction. The mobile phase was composed of acetonitrile-water (40:60, v/v) at a flow-rate of1ml/min. DMP and DEP separation was achieved on a SHIMADZU ODS Cis reversed-phase column (5μm,150mm×4.6mm) which had an ODS C18pre-column (5μm,12.5mm×4.6mm). And the results showed that there was a good linearity over the range0.05-10.0μg/mL (r>0.99). The recoveries were more than85.0%in plasma, and the intra-and inter-day coefficients of variation were less than12.0%in all cases. The limit of detection (LOD) was0.01μg/mL and the lower limit of quantification (LLOQ) was0.05μg/mL. The analysis of tissue samples (muscle, skin, brain and myocardium) was carried out based on the method of plasma analysis, with some modification to suit different tissues. Some of the tissue samples were transferred to a10mL tapered glass-tube. The following steps were similar to the plasma analysis procedure, except that the extraction of tissues for analysis was pretreated with solid-phase extraction column (SPE).Secondly, the comparative studies on the metabolic kinetics and residues of DMP were developed in fish. The Dimethyl phthalate (DMP) concentrations in plasma and muscle tissues were determined by the reversed-phase high performance liquid chromatography (RP-HPLC) after a single oral administration at20mg/kg for Tilapia, Oreochromis niloticus×O.aureus and Snakehead fish, Ophiocephalus argus. The results indicate that DMP absorption, distribution and excretion in Snakehead fish were more slowly than in Tilapia. And the differences of metabolic kinetics and residues of DMP were significant in Tilapia and Snakehead fish. Besides, the comparative studies metabolic kinetics and residues of DMP and DEP was also performed. The results showed that DMP absorption, distribution and excretion in were more slowly than DEP in Tilapia. And the different chemical structures of PAEs significantly affected the metabolic kinetics and residues of DMP and DEP in Tilapia. Moreover, the study was conducted to evaluate the toxicokinetic disposition and residue of Dimethyl phthalate (DMP) in tilapia at different water temperatures (18±2℃and28±2℃). After oral administration of DMP with a single does of20mg/kg body weight, the tilapia’s plasma, muscle, brain, skin and myocardium were sampled separately at different time to determine the concentration of DMP. Then the data were analyzed using3p97Pharmacokinetics software written by the Chinese Society of Mathematical Pharmacology, which indicated that the DMP absorption rate and elimination rate all were faster in tilapia at28℃than that at18℃. No matter how, the DMP residue was detected after oral administration for360hrs at both water temperatures, but the research had revealed that water temperature had great impact on the DMP absorption, distribution and elimination in tilapia.Lastly, the in vivo and in vitro metabolism was investigated in this study. The article developed a method for simultaneous determination of diethyl phthalate (DEP), dibutyl phthalate (DBP), butyl benzyl phthalate (BBP) and di-(2-ethylhexyl) phthalate (DEHP) by using GC-FID. Meanwhile, the metabolism of four PAEs chemicals was also studied in Ctenopharyngodon idellus organs and hepatocyte through LC-MS/MS. The results showed that PAEs chemicals were not metabolized significantly in hepatocyte, but they were metabolized quickly in blood serum, intestinal tract and hepato-pancreas homogenate, especially in the serum. And the main metabolites were mono-phthalates, including monoethyl phthalate (mEP), monobutyl phthalate (mBP), monobenzyl phthalate (mBzP) and monoethylhexyl phthalate (mEHP). Therefore, it was concluded that the PAEs chemicals were mainly hydrolyzed by means of the proteolytic enzymes in Ctenopharyngodon idellus organs, and the blood and intestine were the first metabolized location of PAEs chemicals absorbed by the fish gill and intestine. Besides, Ctenopharyngodon idellus were anesthetized, then the catheter was inserted and ligated into their ureter, after that, they were put into clean water. DBP and BBP were administered by gavage when they woke up. Urine samples were thus collected for12h after administration. After extraction, the samples were injected directly into the LC-MS/MS system. The results showed that the DBP and BBP were not firstly metabolized by P450oxidases in liver, but some of the hydrolytic enzymes in the tissues. As the intestine, blood and other organs were important ways of exogenous compounds absorbed into the body by mouth or gill, biological transformation has occurred before they got into the liver.In summary, PAEs chemicals were quantitatively determined by HPLC and GC chromatography. Then the metabolic kinetics and residues were studied after oral administration of PAEs by the detection methods mentioned above. Moreover, the metabolites and metabolic ways of PAEs were also investigated. Therefore, the elimination characters of PAEs chemicals were clarified in the article.