Adverse Effects Of Atrazine On The Growth And Development Of Eriocheir Sinensis

Atrazine (2-chloro-4-ethylamino-6-isopropyl-amino-s-triazine, ATR) is a widely used triazine herbicide. Despite limited solubility, ATR is detected in ground and surface water in certain areas. Recent studies have shown that it can induce neurological, immunological, developmental, and biochemical alterations in several model organisms.The Chinese mitten crab Eriocheir sinensis is an important economically aquatic species in China. Because of the habitat and intricate life cycle, the crab is easy to be affected by its environmental factors. Moreover, the precocity and the degeneration of germ plasm are puzzles to the aquaculture. In this study, we tried to detect the potential hazard of ATR on the growth and development of E. sinensis, in order to provid aid to solve those puzzles, and also wish to supply the basic data for toxicology.The acute toxicity of ATR to the juvenile E. sinensis (6.9– 13.2 g in wet weight) was investigated. The results showed that the 96 h half lethal concentration (LC50) exceeded 181 mg/L. ATR showed a low lethal toxicity for the studied species.Juvenile crabs of both sexes were exposed to ATR at doses 0.001, 0.01, 0.1, 1 mg/L for a period of 90 days. Effects on gonad histomorphology were assessed. 0.001 mg/L induced elevated levels of atresic ootyces in ovaries. 0.01 and 1 mg/L induced development ahead of schedule in the testis. Meanwhile, all concentrations of ATR could disadvantage ultrastructure of the mitochondrion in the optic ganglia cells of eyestalks.ELISA was introduced to analysis the levels of serum sex steroids (testosterone (T), 17β-estradiol (E2) and progesterone (PROG)) after ATR exposure for 60 days. ATR induced a decrease of T and a concomitant increase of E2 in male crab. Further, these suppressive effects on serum androgen and the induction in estrogen were dose- and time-related. At relatively higher concentration (0.1, 1 mg/L), ATR could significantly increase concentration in serum PROG of the male crabs. In the female, however, the levels of serum T and E2 were relatively constant; 0.01 mg/L induced a significant decrease of PROG.Molt-inhibiting hormone (MIH) is a kind of neuropeptides hormone secreted from X-organ sinus gland complex which located in the optic ganglia of eyestalks. MIH acts on Y-organ to inhibit the produce and secretion of ecdysone. In this study, we used RT-PCR to detect the expression of MIH in the optic ganglia of eyestalks of E. sinensis which exposed with several concentrations of ATR (0.001, 0.01, 0.1, 1 mg/L) for 30 days. At the same time, serum ecdysone was analyzed by ELISA. The results showed that 0.1mg/L resulted in the up-regulation of MIH, but suppressed the secretion of ecdyson.Separate groups of spermatozoa which obtained from mature male crab were exposed to ATR at 0.001, 0.01, 0.1, 1 mg/L respectively. At 0 h, 0.5 h, 1 h, 2 h, spermatozoa were observed by optical microscope and transmission electron microscope (TEM) to detect the adverse effects of ATR on the mortality, percentage of acrosomal reaction and the alteration of ultrastructure. The results showed that toxic effects on above-mentioned indexes were dose- and time-related. ATR enhanced the mortality, inhibited acrosomal reaction, and resulted in the absence of the chromatin dot in spermatozoa nucleus significantly. However, spermatozoa nucleus DNA was not damaged by any concentration of ATR.In this work, we also evaluated the effects of ATR on DNA damage in hemocytes of the crab. Single-cell gel electrophoresis (comet assay) was used to investigate the status of damage. After being observed by fluorescence microscope, the images were analyzed with CASP, so we could make sure whether DNA was influenced by ATR. At all concentrations, ATR induced DNA damage. With the increase of concentration of ATR, the value of TM and the percent of TailDNA increased gradually. And the most significant effect was happened when the concentration of ATR was 1 mg/L. It was proved that ATR can damage DNA chain and structure of the crab hemocytes.In conclusion, in our research, the juvenile crabs and mature spermatozoa were used as subjects to study the adverse effects of ATR on the growth and develoment of E. sinensis. According to the results from in vivo and in vitro assay, we can conclude that ATR is a low lethal toxicity to this species, but it can induce endocrine-disrupting effects on its growth and develoment. The results of this study may be beneficial to a further understanding of the influence of ATR on the endocrine system of the aquatic animal, and provide a basis for protecting of E. sinensis and other commercially important aquatic crustaceans.