Acute and early life stage toxicity of industrial effluent on Japanese medaka (Oryzias latipes)

Abstract

To develop the whole effluent toxicity testing methods (WET), embryo larval stage toxicity test using Japanese medaka (Oryzias latipes) was conducted to evaluate an effluent from a banknote printing plant (BPP). The method is based on acute toxicity using endpoint of 96-h larval morality and on chronic toxicity using endpoints such as the time to hatch, hatching success, deformity, growth rate, swim-up failure, accumulative mortality and sexual ratio. In test for 96-h larval mortality, LC50 (the concentration was lethal to 50% of newly hatching medaka larvae) was 72.9%. In chronic toxicity test, newly fertilized embryos (< 5-h old) were exposed to 1%, 6.25%, 12.5%, 25%, 50% effluent concentrations and to 200 μg/l BPA in a 24-h static renewal system at 25 ± 1 °C until 15 day post-hatch. The results showed that all chronic endpoints were significantly different from the control at 50% dilution (p < 0.01). Embryos began to show lesions on 4th day at higher concentrations (12.5%, 25%, 50% BPP effluent concentrations). Treatment group of 25% dilution showed delayed time to hatch. A reduction in body weight was observed at 25% dilutions for males and females, respectively. Deformities were observed in newly hatched larvae at 25% and 50% BPP effluent concentrations. At 25% dilution, sex ratio of larvae was alternated and there was feminization phenomenon. We conclude that embryo larval stage test using medaka is feasible to evaluate both acute and chronic toxicities and potential endocrine disrupting activity of industrial effluents.

Introduction

Industrial effluent discharges directly to surface waters or municipal sewage treatment plants. The current standards for effluent discharges are not based on risk or impacts upon receiving waters. Many effluents are of a complex nature (Vyryan et al., 1999) and discharge a vast number and diversity of chemical substances to the receiving water, many of which are unknown (Trevizo and Nirmalak, 1999). In addition to chemical measurements, whole effluent toxicity (WET) methods generate the aggregate toxic effect of an effluent or its potential impact on receiving (ambient) waters. In recent WET tests, short-term chronic adverse effects were included and embryo-larval survival and teratogenicity of fathead minnow (Pimephales promelas) were used as the test methods (USEPA, 2002). However, the test method is based on the total frequency of mortalities and gross morphological deformities. Many field studies suggest estrogenic effects on fish in polluted habitats, including reduced reproduction success (Petersen et al., 1997), reduced the levels of plasma estrogens in males (Johnson et al., 1988), and alternation in sex ratios (Lang et al., 1995). However, some reports showed anti-estrogenic activity on fish, for example masculination of fish (Leblac et al., 1997), reduced gonad growth (Sanstrom et al., 1997), disrupted steroid synthesis by the ovary and altered peripheral metabolism of steroids (Van der Kraak et al., 1992). In addition, M74 syndrome (environmentally related disease 1974) in Baltic salmon and the equivalent Early Mortality Syndrome (EMS) in salmonids of North American Great Lakes were observed in fry and embryo (AFS, 1996). Some studies have reported that effluent from sewage treatment plants have both teratogenicity and estrogenic activity (Fuerchker et al., 2001, Montsrrat et al., 2001), while estrogenic activity has not been included in recent WET.

Medaka (Oryzias latipes) serves as an excellent fish model to determine acute and chronic toxicities, including endocrine disrupting activity of chemicals. Many reports have suggested that medaka is sensitive to toxicants and endocrine disrupting chemicals (Scholz et al., 2000, Metcalfe et al., 2001, Islinger et al., 2002), especially in early life stage.

The characteristics and effects of industrial effluents vary and the effluent guidelines need to be identified independently. For example, previous studies have demonstrated that effluent from sewage treatment plants (STP) were estrogenic to juvenile fish (McArdle et al., 2000, Julia et al., 2002). Kovacs et al. (1995) reported lower egg production and delayed reproduction in fathead minnow exposed to bleached kraft pulp mill effluents (BKME). However, no significant effects were observed in maturing rainbow trout exposed to effluent of a pulp and paper mill (Heuvel et al., 2002). USEPA is announcing a draft strategy that describes a process to identify industries for which effluent guidelines need to be revised or developed (USEPA, 2002). There is little work being carried out on different types of industrial effluents in China.

In the present study, acute and chronic toxicity of effluent from a banknote printing plant (BPP) in medaka (Oryzias latipes) were assessed based on a variety of endpoints. The aims of the study are to determine the toxicity of effluents from BPP, the sensitivity of different endpoints, including lethal, sub-lethal, growth, deformities and sexual ratio, as well as the feasibility of using medaka as model animal in whole effluent toxicity testing methods.