Toxicity Of Ammonium Salt And Nitrate To Embryonic And Larval Development Of Bufo Bufo Gargarizans

Nitrogen pollution from anthropogenic sources enters aquatic ecosystems through agricultural runoff associated with nitrogenous based fertilizers, livestock, and effluents from human and industrial waste. Therefore, nitrogen (N) has been suggested as a potential factor causing declines in amphibian populations, especially in agricultural landscapes. Nitrogen in the aquatic environment occurs in four forms (ammonium ion, ammonia, nitrite, and nitrate). The most toxic nitrogen to aquatic animals is ammonia followed by ammonium, nitrite, and nitrate. Ammonium and nitrite are quickly oxidized to nitrate by bacteria in the aquatic ecosystems. Furthermore, nitrate can be converted to nitrite in the gut environment.Thyroid hormone (TH) is pleiotropic factor for many morphological and physiological functions in amphibian metamorphosis. TH activity is regulated in a tissue-specific manner by three deiodinases, types I, II and III. Thyroxine (T4) is the major form of thyroid hormone in amphibians, and it is converted to the more active hormone tri-iodothyronine (T3) in peripheral tissues. The type II deiodinases (Dio2) synthesized T3form T4, while type III deiodinases (Dio3) inactivates the hormone by removing iodine from the inner ring of the hormone. Therefore, Dio2and Dio3make vital contributions to this process by controlling levels of the thyroid hormones T4and T3available to different tissues.In this study, we examined the embryonic and larval development of Chinese toad, Bufo gargarizans exposed to ammonium chloride (NH4Cl) and sodium nitrate (NaNO3). The effects of NH4Cl and NaNO3on the development of embryos and larval were investigated using statistical, morphological, histopathological and molecular biology methodology. Based on our observations, we reached six main conclusions:1. To assess the sensitivity of Bufo gargarizans embryo, the LC50values (i.e. the concentration at which50%of embryos die) was determined from24to96h. Our results showed that768.430mg/L NH4-N caused50%mortality in embryos after96h NH4Cl-exposure. The LC50values from24to96h of exposure to NaNO3were978.823mg/L, by means of a4day toxicity test with Bufo gargarizans embryos. Base on our acute toxicity results, the safe concentration of NH4Cl and NaNO3were determined to be7.684mg/L NH4-N,9.788mg/L NO3-N, respectively. 2. Chronic toxicity tests were also conducted to evaluate the sublethal effects of NH4-N and NO3-N; embryos were exposed to different ecologically relevant concentrations (10,50and100mg/L) in static renewal tests from mid gastrula stage to operculum completion stage. NH4Cl inhibited developmental stages of embryos, which was arrested at stage18-19in100mg/L NH4-N-treated group. No significant retardation was observed in developmental stage in the high dose (100mg/L NO3-N) group. The results indicated that NH4Cl is much more toxic to embryos than the NaNO3. In addition, compared to the control treatment, the reduced total length and body weight can be observed in the medium and high dose (50,100mg/L) groups. Total length and body weight of embryos were not significantly affected by NaNO3concentration of10mg/L NO3-N relative to untreated controls.3. Both NH4Cl and NaNO3were highly teratogenic, resulting in reduced body size, body-axis curvature, and abdominal edema as the main adverse effects. At the histopathological examinations, abnormal alteration in the eye, olfactory and adhesive organ of embryos following100mg/L NO3-N was observed. The phenotypes of abnormal eyes included the degenerated lens and retinal layer. The embryos exposed to100mg/L NO3-N showed many melanocytes appeared in the olfactory epithelium and adhesive organ. Additionally, morphological malformations of gill and skin were also observed with scanning electron microscopy. Under magnification, the surface of skin and gill had an irregular corrugated appearance with lifting of the superficial laer.4. The damage of Dio2and Dio3expression exposed to100mg/L NH4-N were analyzed by semiquantitative RT-PCR. Compared to the control group,100mg/L NO3-N did not inhibit Dio2and Dio3gene expression severely. We have isolated Dio2and Dio3from Bufo gargarizans embryos and analyzed its spatial and temporal expression pattern at stage G20in100mg/L NH4-N and100mg/L NO3-N groups. Dio2mRNA expression was more concentrated at the axial structures and head region in100mg/L NH4-N groups. Compared the control, Dio2expression was also found to be in the eye, nose, oral apparatus, branchial arch areas, somites, and tailbud as expected in100mg/L NO3-N groups. The expression of Dio3is getting more restricted to the branchial arches and oral apparatus in100mg/L NH4-N. No Dio3expression was detectable in head region in100mg/L NO3-N groups.5. We examined the effects of chronic NaNO3exposure on metamorphosis of B. gargarizans larvae with various NaNO3concentrations at0,10,50, and100mg/L NO3-N at stage G42. Compared with the control, the100mg/L NO3-N-treated larval had a significant increase in body weight and total length at stage G42. While, no significant changes were found in the remaining treatments. NaNO3at concentration of10mg/L NO3-N significantly inhibited metamorphosis and retarded T0.5. However, NaNO3at concentrations of100mg/L NO3-N significantly increased metamorphosis rates and shortened T0.5of the larvae to complete metamorphosis.6. Larval at stage G42from each group were examined for histological alterations in thyroid glands. Histological changes observed in thyroid gland from the10mg/L NO3-N treatment group includes marked increases in peripheral colloid vacuolation and decreased colloid area in follicle cell. No obviously different changes were observed in the remaining treatments. This result may be explained by the fact that NaNO3can inhibit the formation TH through the inhibition iodine binding to the sodium-iodide symporter on the surface of thyroid follicles. This reduces the levels of the T3and T4, which increases thyroid stimulating hormone (TSH). Chronic stimulation of the thyroid gland by TSH can lead to proliferative changes in follicular cells.In conclusion, NH4-N and NO3-N have toxic effects on the early embryonic development and larval development of the adult toad. The present study indicated chronic toxicity tests may provide more accurate way in formulating the "safe levels" of nitrogenous based fertilizers to amphibian.