Acute Toxicity Bioassay of a Pyrethroid Pesticide Bifenthrin to the Asian Stinging Catfish, Heteropneustes Fossilis (Bloch)

Bifenthrin is a type-I synthetic neopyrethroid pesticide having eight different stereoisomers.The current study aims to assess the short-term toxic effect of Bifenthrin to freshwater fish, Heteropneustes fossilis. The 24, 48, 72 and 96h LC50 values of bifenthrin to H. fossilis were 4.82, 4.47, 3.54 and 3.40 μg/l respectively. There was a significant variation (p<0.05) in the mortality of the treated fish exposed to bifenthrin with respect to the control at all the hours of exposure. A significant variation (p<0.05) between rate of mortality of H. fossilis and time slots (24-96h) was recorded for the final selected doses of bifenthrin except 4.0, 4.5 and 5.5 μg/l concentration of the toxicant. A vigorous mucous secretion and hyper-excitability was observed in the fish at the higher doses of bifenthrin specifically at 24h and 48h time slots. There was a profound loss in equilibrium of the treated fish particularly at the higher doses at 72h and 96h time slots.The gradual increase in dose of bifenthrin resulted in significant increase (p<0.05) in opercular movement of the fish with respect to the control. On the other hand, opercular movement showed a significant increase (p<0.05) with the advancement of time for all the treated doses. Therefore, bifenthrinis indicated to be very strongly toxic to fish. CONTACT Dip Mukherjee dip.m@rediffmail.com Department of Zoology, S.B.S. Government College, Hili, 733126, West


Introduction
Pesticides are being used indiscriminately to maintain a sustainable yield of various crops necessary to support the ever increasing animal population. They constitute a prime component of agricultural runoff which gets intermingled to the adjacent water bodies affecting thelife of differentnon-target organisms (Raina et al., 2009). The contamination of aquatic ecosystem by pesticides is a global problem (Hill, 1985;Sibley and Kaushik, 1991). Human beings are the worst victims of pesticide biomagnifications as they occupy the apex of the food pyramid (Sahai, 1992).
Bifenthrin is a type-I synthetic neopyrethroidhaving eight stereoisomers with the cis-isomer being the active ingredient (Khan et al., 2013). Pyrethroids act by exerting a time lag in the closing of sodium ion channelspresent on nerve cells even after an initial entry of sodium ions during the phase of depolarization of action potential (Saha and Kaviraj, 2008; Khan et al., 2013). This culminates in a sustained sodium ion flow. The absence of α-cyano-group assistsbifenthrin to bind to the sodium ion channels promoting the generation of after potentials followed bysustained firing of the axon, ineffective to the resting potential (Khan et al., 2013). Bifenthrinis also characterized by reduced environmental degradation and strong insecticidal effects (Mokry and Hoagland, 1989). It is also a popularstomach or contact insecticide and affects cellular ATPase production (Velisek et al., 2009;Roberts and Hutson, 1999). Though there are limited studies on the toxicity of Bifenthrin and its nanoencapsulated form to rainbow trout (Velisek et al., 2009), there is no report of its effects on air breathing fishes. Our study is the first of its kind and future chronic studies may be carried out to elucidate more detailed knowledge on the various aspects of toxicity to air breathing fishes.
The present research has the following objectives • To determine the acute toxicity of the pesticide bifenthrin to H. fossilis in order to ascertain its safe permissible levels for the water bodies of our country.

•
To determine the behavioural response and the alteration in respiratory rate of H. fossilis as a result of the toxic insult.

Materials And Methods
The Asian stinging catfish, Heteropneustes fossilis (Order: Siluriformes; Family: Heteropneustidae) was used as the test animal in the bioassay having a mean length of 11.7 ± 0.3 cm and a mean weight of 21.60 ± 0.7 g. It was procured from a nearby aquaculture farm followed by acclimatization under experimental ambience for three days prior to their use.  There was a significant variation (p<0.05) in the mortality of the treated fish exposed to bifenthrin with respect to the control at all the hours of exposure.

Fig. 2: Relationship between the concentrations of Bifenthrin and mortality of H. fossilis at 72h (left) and 96h (right)
The toxicity factors as calculated from median lethal toxicity (LC 50 ) values at different times of exposure are tabulated in Table 2. With the progress of time, the toxicity factor values for the tested fish species to bifenthrin increased gradually.
The estimated possible safe level of bifenthrin for the fish isstated in Table 3. In the current experiment, safe level estimated for bifenthrin ranged from 0.0341 -1.3628µg/l.
The ethological alterations noted in H. fossilis under exposure to various doses of bifenthrin are summarized in Table 4. The intensity of behavioral alterations increased with the increasing doses and progress of time of exposure (Table 4).

Discussion
Pesticides belonging to the group of pyrethroid present a risk for aquatic organisms, though they have low toxicity for aves and mammals (Bradbury and Coats, 1989). The current investigation shows the 96h median lethal concentration of bifenthrin as 3.407μg/l. The present value of 96h LC 50 of bifenthrin to the exposed fish (3.407μg/l) is much higher in comparison to rainbow trout (1.47 μg/l), common carp (2.08 μg/l) and tilapia (0.80 μg/l) (Velisek et al., 2009, Liu et al., 2005. This variation may be because of difference in physicochemical parameters of the experimental water, age, size, health and species of fish (having accessory respiratory organ) used in the present study (Farah et al., 2004;Diedrich et al., 2015;Patra et al., 2015). Temperature of the test water may be a key factor in determining the degree of toxicity since lower temperature increases the toxic potential of bifenthrin to fish (Mauck et al, 1976).
Living organisms in response to an adverse surrounding exhibit its defensive nature by means of a vital parameter called tolerance (Enuneku and Ezemonye, 2012). Toxicity factor (TF) is an important index for tolerance assay (Ayoola et al. 2011). In the current investigation, the toxicity factor for the neopyrethroid, bifenthrin increases in fish with the length of period of exposure (Table 2). This is corroborated by the study of Ayoola et al. (2011). In our present investigation, the estimated possible safe level of bifenthrin to fish showed large variation (0.0341 -1.3628 µg/l) due to different values of application factors (AFs) according to some International standard methods (Kennega, 1979) ( Table 3).
Various changes in the ethological responses are the primary indicators of signs of toxicity to a given xenobiotic. Likewise, our study considered changes in ethology as an important tool to assess bifenthrin toxicity in fish (Table 4). Initially, hyperactivity was noted in the treated fish with respect to the control. With the advancement of time and gradual rise in concentration H. fossilis exhibited the symptoms of stress build up which was manifested as erratic swimming, restlessness, gasping for air, surface adherence etc. Besides, somersaulting pattern in fish also observed at the upper dose limit. Probably, this behaviour was symbolic of anescape reaction from bifenthrin (Saha et al., 2018). The vigorous mucus secretion in H. fossilis may be attributed as an evading mechanism to bifenthrin from entering the body. It is an outcome of stress and irritating effect similar to that of many other neurotoxicants. Some organ ophosphates also elicit a similar response in fish (Rao et al., 2005;Pandey et al., 2008). At all the exposures the death of fish was characterized by wheezing, repeated turning of the opercula, loss of balance, disruption in buoyant behaviour, enhanced rigor and momentary cessation of ventilation. Restlessness and erratic swimming of treated fish were probably attributed to the adverse impact of bifenthrin on the cerebrospinal nervous system (Velisek et al, 2009).
In fish, the opercular movements are directly related to respiratory rate, which is often the first physiological response to be affected by the presence of toxicant in the aquatic environment (Dubeand Hosetti, 2010). In the present investigation, opercular movement in H. fossilis exposed to bifenthrin was found to increase significantly (p<0.05) in response to all doses of the pesticide (Figure 3). Gills are the principal respiratory organs of fish. The energy demand of different metabolic pathways is met up by the gills. Thus, any damage to this organ may culminate insevere respiratory ailments (Magare and Patil, 2000). The mechanism of toxicant uptake through gills probably occurs through simple diffusion (Opperhuizen et al., 1985). In the current investigation, gradual increase in the flapping of operculum of H. fossilis to bifenthrin may be a compensatory mechanism to overcome respiratory distress (Kumar et al., 2015).

Conclusion
This study reveals that bifenthrin is a potent toxicant and it may cause mortality in H. fossilis at very low concentration, even at short period of exposure.
The results of the current experimental work may provide some insights onlaying out an action plan for management of this pyrethroid including the marking of maximum permissible limit for this xenobiotic before discharging them tothe aquatic ecosystem having a myriad of aquatic organisms in it. In future, more studies on the chronic toxicity of bifenthrin to aquatic organisms may open up new vistas in the field of aquatic toxicology.