Acute toxicity test of Zn , on Java medaka ( Oryzias javanicus ) fish as an indicator of estuary pollution

The acute toxicity of Zn on Java medaka (Oryzias javanicus) fish was studied. To obtain the results, the experiments were carried out in 3-liter aquariums ( 15 to 20 fish for each aquarium) with static condit ion based on O.E.C.D method with 5 treatments, and 1 bl ank in two repetitions. During the experiment, wate r physico-chemical factors were pH = 7.7 to 7.9, sali nity = 19.3 to 19.7 p.p.t, temperature = 29.1 to 30 .8°C and conductivity = 13.54 to 13.94 ms. On the basis of obtained results, the LC 50-96 h for Zn were determined at 9.75 (9.65 9.85) mg/l for juveniles and 14.32 (13.94 14.70) mg/l for adults, respect ively. Also, the maximum allowable concentration (MAC) val ues for Zn on Oryzias javanicus juveniles and adults were determined at 0.97 mg/l for juveniles a nd 1.43 mg/l for adults. These results will be help ful for future ecotoxicology study based on heavy metal s pollution in estuary areas.


INTRODUCTION
Ecotoxicology deals with adverse effects of toxicants on the ecosystems using two different approaches: acute and chronic toxicity testing.An acute toxicity test determines the concentration of pollutants, which has detrimental effects (change in behavior, death, etc.), on a group of test organisms during a short-term exposure.96 h median lethal concentration(96 h LC 50 ) resulting in 50% mortality of test animals after 96 h of exposure is most commonly used (Mayor et al., 2008).
The term 'heavy metal' has been used extensively to describe metals that are environmental pollutants (Risso de Faverney et al., 2001).According to Chen et al. (2005), even though some metals are essential when taken up by organisms, their excessive presence will reverse the effect and they become toxic and therefore harmful.Heavy metals have been recognized as one of the major factors posing a serious threat to marine biota and its contamination in the marine environment has been reported to cause toxicity to aquatic organisms (Rafael et al., 2008).According to Batzias and Siontorou (2008), heavy metals may enter marine environments in a variety of natural and anthropogenic sources.Heavy metals can *Corresponding author.E-mail: aismail@science.upm.edu.my.be critically important to the life processes of marine organisms.Zinc is an essential heavy metal for one or more organisms and it is necessary in trace amounts for the functioning of biological systems (Huerta-Diaz et al., 2008).Using fish as a biologic bioindicator for pollution monitoring in the aquatic ecosystems is an approach that has been accepted and is gaining both popularity and credibility (Alam and Maughan, 1995).The main goal of pollution monitoring is collecting of continual reliable information about the aquatic environment in the context of the relationship between humans and the ecosystem, organisms and the environment (Giardina et al., 2009).In the bigger picture, monitoring the marine environment is about studying the health of the ecosystem and understanding the dangers it is now facing from pollution and particularly from the presence of heavy metals among other pollutants.Fish may easily absorb pollutants from the ambient water and through their food chain and these absorbed pollutants are then deposited in their tissues through the effects of bio-concentration, bioaccumulation (Chen et al., 2005).The mechanisms of the transport of metals across the membrane barriers have yet to be fully examined.Although it is essential to fish, high concentrations may be toxic.
The availability of heavy metals can be influenced by many environmental factors and by composition of diet; as a consequence, the physiological requirements of the essential elements are difficult to determine (Rafael et al., 2008).In this regard, heavy metals have long been recognized as an important pollutant due to their toxicity and ability to accumulate in marine organisms (Gorski and Nugegoda, 2006).There is therefore a need to have toxicity test using suitable marine organisms such as small fish for purposes of acute and chronic toxicity tests of these pollutants on the live organisms.Laboratory toxicity bioassays have become a convenient test for screening chemical and physical parameters for potency, but predictive results and relevance to aquatic ecosystems in the "real world" have been inadequate in hazard assessment.Direct toxic action of pollutants may occur in limited areas but after the material in question has spread out and become diluted, vastly larger and more diverse habitats and their residents will be exposed to long term sub lethal exposure (Kim and Oh, 2012).
In this study, we have attempted to assess acute toxicity test of Zn in 48 and 96 h on the Java medaka fish collected from Linggi Estuary of Peninsular Malaysia as a new research organism in the field of Ecotoxicology studies in the estuarine and coastal areas.

Solution preparation
Stock solution was prepared by dissolving zinc sulphate (99%, Sigma-Aldrich, St.Louis, Mo, USA) in double distilled water to a final volume of 1.0 l.Each treatment solution was prepared by diluting the stock solution (with calibrated pipette) with double distilled water to achieve the appropriate exposure concentrations.

Toxicity tests
To determine the effects of Zn on survival of juvenile and adult Oryzias javanicus, a series of static-renewal 96 h bioassays was performed (renewed every 24 h).A test series contained different concentrations of the test chemicals with 10 to 15 organisms per concentration.A control was assigned for each test series consisting of 100% only water.During the tests, the water physico-chemical parameters were as described in Table 1 and the 12:12 light/dark cycle was also maintained.
This study was done at the Ecotoxicology Laboratory of Universiti Putra Malaysia.Experiments were performed according to the standard method of Organization of Europe Economic Cooperation and Development (OECD, 1992, 1997) and TRC (1984).In the first step, the weight and dimensions of the fish in both the adult and juvenile groups were measured.After installation of the aquariums, fish that were gathered from the Linggi Estuary of Peninsular Malaysia were kept inside the aquariums, each containing 3 L of Linggi River filtered water for one week to adapt to the new environment, with aeration and filtration provided using sponge filters.Fish were not fed for 24-h before or during acute testing and physico-chemical parameters such as temperature, pH and dissolved O2 were also measured (Table 1).
The toxicity for the 48 and 96 h static-renewal tests was determined by mortality range of the fish.At 24, 48 and 72 h, 90% of test water was replaced with fresh test solutions and the number of dead was noted.The organisms were considered dead if no physical response to stimuli, such as prodding with a glass rod, was observed.Two replicate 96-h tests were performed in two sets.Mean of weight and length of fish and metal concentrations are shown in Table 2.

Record and analysis of results
According to the above method, acute toxicity test (LC50) Zn in 48 and 96 h was done and after that the fish mortality details were recorded in every concentration in the different times (every 24 h).Meanwhile, for greater accuracy, the test was done in two repeats and the results of mortality were almost similar in both sets (Tables 3 and 4).In the end, results were analyzed using SPSS package and Probit analysis method (Finney, 1990) (Tables 5 and 6).
The LC50 values were determined from maximum likelihood estimates of linear functions relating to log metal concentrations to probit transformations of percent mortality (Finney, 1990).The LC50 values were determined using mean assayed Zn concentrations and cumulative mortality.Statistical comparisons between LC50 values were based on the standard error of the difference.When it became apparent that no statistically significant differences in LC50 values had occurred between bioassay replicates (p>0.05),Chisquare tests were performed to test the homogeneity of mortality between replicates.
Fish behavior in the different hours of the experiment were observed and recorded such as excitement, unusual stir, unnatural discharge on the skin and body, and slow movement to relatively complete stop (Dying in the continuo's position).

RESULTS
The results of the toxicity test show that mean of LC 50 for 96 h of Zn were 14.32 mg/l for adult fish and 9.7 mg/l for juveniles respectively and on other hand, mean of LC 50 for 48 h of Zn were 26.43 mg/l for adult fish and 18.80 mg/l for juveniles respectively.According to the results of the acute toxicity test of Zn on the adults and juveniles of Java medaka fish, MATC amounts of Zn were 1.43 mg/l for adults and 0.97 mg/l for juveniles respectively (Table 6).Maximum allowable toxicant concentration (MATC) was determined with attention to LC 50 amounts also (TRC, 1984).Maximum allowable toxicant concentration is the concentration that has no negative effect on fish in the experiment period.According T.R.C guidelines, maximum allowable toxicant concentration is obtained by the division of LC 50 -96 h per 10 and it is called NOEC or No Observed Effect Concentration that in a toxicity experiment is the maximum concentration of substance that will not have any statistical effect on the population of samples compared with the control group.

DISCUSSION
There have been no previous toxicity studies conducted on wild Java medaka fish and few studies have been conducted on comparable species similar assays.However we can gain some insight by comparing this study with studies conducted on Java medaka fish, a closely related marine fish medaka species that has proven to be an excellent candidate for toxicological analysis (Koyama et al., 2008).Despite the high concentration of Zn inside natural aquatic ecosystems especially in marine sediments, it has no specific effect on the aquatic organisms, and the important reason is the compound of Zn with elements containing manganese and exit of water column.On the other hand, this ratio was different in the juveniles (Batzias and Siontorou, 2008).In comparing results between adults and juveniles it was observed that the amount of acute toxicity of Zn in 96 h was almost double the amounts in 48 h in both groups of adults and juveniles.Symptoms of Zn effects show that in low concentrations, fishes had unusual swimming behavior and in the high concentrations they showed heightened activity and irritability, imbalance, changed skin color, exhibited formation of mucus on skin, began swimming vertically, had slipped eye, showed formation of blood spots around the eyes and under the stomach, gill hyperemia, and curvature of the spine (above 12.5 mg/l Zn both in adult and juvenile aquariums).Results of research about acute toxicity test effects on the fish indicated that some fresh water fish are comparable with Java medaka in acute toxicity test results.Research about acute toxicity test of Zinc shows LC 50 of this metal on the adults of Carp, Trout and Salmon were 7.8, 1.19 and 2 mg/l respectively (Mance, 1990), also it was 12 mg/l for Chanos chanos (Herrera and Santos, 1995) and 1.5 mg/l for Flag fish (Jadanella floridae) (Woodling et al., 2002).
Small fishes like Killi fish and Medaka fish have been as suitable models for ecotoxicology studies in the different researches (Inoue and Takei, 2002).
In another study it was reported that LC 50 -96 h, 4chlorophenol, Zinc, hexavalent chromium and residue chlorine were from 3.0 to 4.1, from 12.4 to 14.7, 7.4 and 0.05 mg/l, respectively (Koyama et al., 2008).Java Medaka did not show higher sensitivity to chemicals than fresh water fish (Koyama et al., 2008).It has also been reported that LC 50 -48 h one type of Killi fish named Fundulus heteroclitus for Zn, Cu and Cd were determined as 96.5, 44.4 and 19.0 mg/l, respectively (Dennis et al., 1990).

Conclusion
Environment pollution is a common issue in the world; it is especially important as part of this pollution depended on heavy metals.Industrial developments are important cause of pollutants effluence in ecosystems.In this study, we showed that different concentrations of Zinc exhibited varying degrees of toxicity by using Java Medaka fish and two ages of fish, including juvenile and adult mortality.To obtain information quickly, short-term tests are used.These tests are used to determine the total toxicity of solutions but there is no information about their toxicity levels.Moreover for the assessment of different organisms' responses to toxicity tests, knowing the different positions of some parameters such as temperature and pH is necessary.An amount of LC 50 is a useful criterion for acute toxicity but it does not represent the amount of pollutants in aquatic habitats.The content of waste materials that are not toxic in 96 h could be harmful in the longer period in a water habitat, and thus LC 96 h may be seen only in some parts of long-term toxicity.According to the results of this study, it is inferred that Java Medaka fish is a suitable species for ecotoxicological study and should be introduced as an important fish in the toxicity tests because it is easily collected from natural wild conditions in Malaysia and around several countries.This fish can be useful for testing the endocrine effect of chemicals in the long-term and life-long as well.Medaka is readily available throughout the year from a culture unit and in the wild and they are very hardy during handling and testing.No disease problems or unexplained control mortalities occurred while any of the acute tests was in progress.Therefore, Java Medaka appears to be a good species that can be used for general toxicity testing.

Table 1 .
Range of physicochemical parameters in aquariums.

Table 1 .
Mean of weight and lenght and metal concentrations in juveniles and adults aquariums.

Table 3 .
Mean number of dead adults in the 4 days for Zn different concentrations.
I = First repeat, II = Second repeat.

Table 4 .
Mean number of dead juveniles in the 4 days for every concentrations of Zn.
I = First repeat, II = second repeat.

Table 5 .
LC50 amount for Zn toxicity at 48 and 96 h in adult and juvenile of Oryzias javanicus fish.

Table 6 .
Comparison of acute toxicity estimate(LC50) for Java medaka fish and other aquatic species for 48 and 96 h exposer to Zn.