Reprotoxic Effect of Malachite Green on African Catfish Clarias gariepinus (Burchell 1822)

MATERIALS AND METHODS

Sampling and exposure of broodstock to malachite green: This study was carried out in the month of July 2010. Two each, male and female broodstocks weighing 0.93±0.25 kg and total length 19.2±0.87 cm, were purchased from a private fish farm in Ibadan, Nigeria. Fish were acclimatized for two weeks after which they are subjected to treatment. During the period of acclimation, fish were fed commercially prepared pellets at 3% body weight. Stock preparation of Malachite green was done by adding 20 g of malachite green to one litre of distilled water (20 mg of malachite green per mL of stock solution).

One male and female broodstock were each exposed to malachite green preparation at 1 mL of stock solution per 20 L of well aerated water for 45 min. After which fishes were removed and placed in fresh water. This procedure was repeated every other day for three treatments. The other male and female broodstocks were not exposed to any chemical and were regarded as the control for the experiment.

Assessment of water quality: Water quality assessments were carried out for both the treatment and the control (i.e., at the start and towards the end of the experiment). The water quality parameters determined are: alkalinity, ammonia, carbondioxide, chloride, dissolved oxygen, nitrite, pH and hardness. Water quality parameters were determined using Hach® water quality test kits.

Artificial spawning of broodstocks: Spawning was induced in the females (both treatment and control) using Ovupin® according to recommended manufacturer’s dosage rate of 0.5 mL kg^-1. Twenty-four hours later, fish were stripped of egg into a dry sterile petri dish. Egg samples were obtained for histological assessment. The remaining eggs were mixed with the milt from corresponding male broodstock and fertilization was activated with distilled water. Fertilized eggs were spread on carcaban in two separate flow-through hatching system for the treatment and control at a constant flow-rate of 3.5 L per min. The set-up was allowed to run for twenty-four hours to allow for hatching of the fertilized eggs. Newly hatched frys swam into fresh water, while the unhatched and dead eggs were siphoned out. The flow-through system was allowed to run for four days, while regression of yolk sac, growth rate and abnormalities in hatchlings were monitored daily using camera mounted light microscope.

Histological assessment: After sacrificing the male broodstocks to obtain milt; necropsy was done and samples of skin and testes was harvested and preserve in Bouin’s fluid for 24 h, after which tissues were fixed in 10% phosphate-buffered formalin until processing. Processing involves dehydrating tissues, putting them into a xylene phase and impregnating them with paraffin wax under vacuum. Following this process, the tissues were embedded in wax and sectioned on a microtome into 5 μm sections. Selected sections were floated and stretched on a hot-water bath, mounted on clean glass slides and placed on a warming tray to dry and adhere. Following staining with haematoxylin and eosin; sections were covered with a coverslip and mounted on a light-microscope for evaluation by the pathologist (Kiernan, 1990). Abnormalities were documented using a digital camera.

RESULTS AND DISCUSSION

In order to establish aquaculture as a successful and efficient agricultural activity, there is a need to control reproductive processes in fish, in order to obtain high-quality seed and produce juveniles for grow-out without the need to obtain them from the wild. Throughout the exposure of the catfish to malachite green-treated water, the skin of fish was stained lightly. Fish were calm with no observable discomfort. Water quality was monitored in the fish throughout the experimental period and the results are presented as Fig. 1. Water quality assessment indices are also used to identify the relative health of cultured fish specie (Adeyemo and Babalobi, 2008). Malachite green administered at therapeutic level did not significantly affect the quality of water compared with control (Fig. 1). The no-effect of malachite green on water quality agrees with the report of Omitoyin (2007) following exposure of Clarias gariepinus juveniles to 0.15 ppm malachite green. However, malachite green significantly affected reproductive success as observed in Fig. 2-4. When viewed under microscope, the eggs collected from control sample were ovoid (Fig. 2) while that of the malachite treated fish had irregular shape with focal and necrotic lesions (Fig. 3). In the male, the milt collected was watery, brownish in colour and reduced in quantity. Twenty four hours later; normal larvae were hatched in the control (Fig. 4), while the treatments were un-hatched.

Fig. 1:	Comparative values of water quality parameters of malachite green-treated tank and the non-treated tank (control)

Fig. 2:	Normal eggs from the control sample (x 400)

Fig. 3:	Distorted and necrotic eggs collected from malachite treated Broodstock (x 400)

Significant developmental abnormalities in eggs, predominantly chromosome breaks, have been reported in rainbow trout, Oncorhynchus mykiss after long-term exposure to malachite green (Meyer and Jorgenson, 1983).

According to Stentiford et al. (2003) and Zimmerli et al. (2007), histological analysis represents a useful tool to assess the degree of pollution, particularly for sub-lethal and chronic effects. The histological changes observed in this experiment revealed that malachite green causes detrimental effects to the skin and testes of Clarias gariepinus. The pathological lesions in the testes of treatments compared to control (Fig. 5) was necrosis, disruption and depletion of the seminiferous tubules (Fig. 6). Additionally, compared with the skin of the control (Fig. 7); treatment had focal localized degeneration of the skin (Fig. 8). This is in agreement with the degenerative changes, reported to have occurred in the gonads of catfish following acute and chronic exposures to subacute and sublethal concentrations of Malachite green (Srivastava et al., 1998).

Fig. 4:	Larvae within twenty-four hours of hatching in the control group (x 400)

Fig. 5:	Normal histology of the testes, observed in control broodstock (H and E x100)

Fig. 6:	Necrotic, disrupted and depleted seminiferous tubules observed in malachite treated broodstock (H and E x100)

Fig. 7:	Normal histology of skin as observed in the control (H and E, x 400)

Fig. 8:	Focal localized vacuolation of the skin in malachite treated broodstock (H and E, x 400)

As observed in the skin of catfish in the present study, vacular degenerations of the liver was similarly reported in Oreochromis niloticus subsequent to exposure to copper sulphate, another fish paraciticide (Osman et al., 2009) and in the liver of Clarias lazera exposed to untreated dyestuff effluent (Abdel-Moneim et al., 2008). According to Desciens and Bablet (1994), malachite green is a multi-organ toxin. A zero tolerance of 0.01 mg kg^-1 for the sum of malachite green and leucomalachite green in edible fish has been established (Klein et al., 1991). A pharmaceutical alternative to malachite green, ‘Pyceze’ with ‘bronopol’ as its active ingredient, has been developed in UK. It is being used for the treatment of fish and their ova; and appears to be a safe and effective replacement (Kaijser et al., 2001).

CONCLUSION

The present study showed that malachite green has a negative effect on the reproductive outcome of broodstocks. Histological changes were also observed in the testes and skin of the treated fish. Accumulation of malachite green in various tissues of fish was published by Alderman and Clifton-Hadley (1993) and Srivastava et al. (2004). It is, therefore, timely to explore further the potential of alternatives for the eventual complete replacement of malachite green in Nigeria’s aquaculture industry.