Efficacy of Germinated Cereals as Bait Carrier for Zinc Phosphide and Bromadiolone against Field and Commensal Rodent Pests : A Laboratory Evaluation

Both sexes of rodent pests such as Bandicota bengalensis, Millardia meltada, Mus booduga, and Rattus rattus were subjected to toxicity tests (acute rodenticide: 1.5% and 2% zinc phosphide and chronic rodenticide: bromadiolone (0.005%), under no-choice and choice tests) by using their preferred germinated cereals, namely, paddy, pearl millet, and fingermillet, as bait base, individually. The results indicated that the poison baits in the germinated cereals induced all the chosen four species of rodent pests to consume greater quantities of bait perhaps due to the bait carrier’s palatability and texture. Besides these, the chosen three germinated cereals proved themselves that they are also capable of acting as suitable bait base for both selected rodenticides in bringing maximum mortality among the tested rodent pests under both no-choice and choice tests. Therefore, these germinated cereals may be recommended as a bait carrier for both zinc phosphide (2%) and bromadiolone (0.005%) poisons for the control of all these four species of rodent pests under field conditions. However, this requires field based trials with rodenticides for making a final recommendation.


Introduction
Rodents are economically important organisms and some of them are reported to be serious pests destroying crops, fruit gardens, orchards, and stored food grains.Moreover, they cause damage to the properties of various kinds belonging to men which results in huge economic losses.They have a high breeding rate and many show periodic increase in the population which coincides with the availability of food [1].In India, they are responsible for 10-15% of loss to total national produce [2].
In Tamil Nadu, four species of field rodent pests are found in Cauvery delta, "the Granary of South India." According to Sivaprakasam and Durairaj [3], Neelanarayanan et al. [4], and Neelanarayanan [5] the rodent pests such as Bandicota bengalensis, Millardia meltada, and Mus booduga are known to inhabit the crop fields and Tatera indica is found in barren lands around the crops fields.Neelanarayanan [5][6][7] and Neelanarayanan et al. [4,[8][9][10][11] reported that these rodent pests inflict damage to different stages of various crops of this area.Brown rats (Rattus norvegicus) and house mice (Mus musculus) along with the roof rats (Rattus rattus) are known as commensal rodents; that is, they are usually found in association with humans.
Rodents also pose a serious health risk as transmitters of several diseases to human and domestic animals such as leptospirosis, salmonellosis, trichinosis, hantavirus pulmonary syndrome, hantavirus renal syndrome, Argentine hemorrhagic fever, and lymphocytic choriomeningitis [12][13][14].Commensal rodents can carry many diseases transmissible to humans [15].Some of them, such as plague, are carried mainly by noncommensal rodents and may have to be passed into a commensal species before they can become important in public health field [16].Thus, it becomes indispensable to 2 Advances in Zoology bring down the population of both crop field and commensal rodents in animal husbandry and human environments to minimize these health risks.Several methods are being employed by farmers to control the rodent pests, such as cultural methods (habitat management, clean cultivation), physical methods (burrow digging, rat hunting by men), mechanical methods (trapping), biological methods (using biological agents such as predators/parasites), nonlethal agents (repellents), and chemicals (rodenticides).Poisoning or chemical control is the most common, expedient, and humane method to control rodent populations.Rodents often prefer soft or finely divided foods to harder and coarser ones.Earlier, Carlson, and Hoelzel [17] reported that the laboratory rats tended to eat the softer parts of grains and to leave the harder parts, and on the other hand they stated that if the grains were soaked in water, they were eaten whole.Soaking, however, may influence taste, through the formation of sugars.According to Sridhara [18] food selection among rodents is influenced by energy value, water content, physiological effects, and flavour of the food.Further, she opined that the rodent control measures could be successful only with the poison baits that are readily accepted by the target species.The preferred bait carrier (nongerminated grains in different forms) for 14 species of rodent pests under laboratory conditions has been compiled and reported by Prakash and Mathur [19].
From the literature review it is understood that there is no published information on the preferred germinated grains' use as bait base under laboratory situations and hence the present study was designed with the following objectives: to study and record the mortality rate of four species of rodents (individually) by using the most preferred germinated bait bases (paddy, pearl millet, and finger millet) at 1.5% and 2% concentrations of zinc phosphide and 0.005% concentration of bromadiolone (independently) under laboratory conditions under both no-choice and bi-choice tests.

Procurement of Test Animals.
Three species of field rodent pests, namely, B. bengalensis, M. meltada, and M. booduga, which are found in the crops fields of Cauvery delta, South India and one species of commensal rodent, R. rattus, which is a serious pest in grain godowns, houses, and shops, were chosen as test animals for the present study.The first three field rodent pests were live trapped with the help of traditional rodent trappers from the nearby crop fields in and around Puthanampatti villages, Tiruchirappalli district, Tamil Nadu, India, by burrow digging method as suggested by Sivaprakasam and Durairaj [3].They were brought to the laboratory, weighed, sexed, and lodged in individual cages (60 cm × 30 cm × 30 cm).They were placed in the live animal keeping rooms of the Rodent Laboratory of our college.The commensal rats (R. rattus) were trapped alive from residential, office, godowns, and hostel premises in and around Kottathur and Puthanampatti villages, Tiruchirappalli district, Tamil Nadu, India, by using wooden live traps and wonder traps.These traps were set with bait in the evening and placed on the runways of rats [20].In order to avoid trap shyness for wooden traps, the traps were set in "off " position for first two days with chosen baits placed inside the traps and on the third day onwards they were set in "on" position [21].In the next day morning the traps were checked and the trapped rodents were brought to the laboratory to be weighed, sexed, and lodged in the rodent cages.

Acclimation of Experimental Animals.
The individually lodged animals in rodent cages were acclimatized to laboratory conditions for ten days.During this period ad lib quantities of nongerminated paddy and water were provided to them.For each set of experiments ten healthy animals (5 males + 5 females), individually, were used.

Toxicity Tests.
Zinc phosphide (1.5 and 2%), an acute rodenticide, and bromadiolone (0.005%), chronic rodenticide powder formulations, were chosen for the present study to evaluate their efficacy by using germinated cereals as poison bait carrier against rodents under laboratory conditions.The acute toxicity studies were carried out with two different concentrations of zinc phosphide, that is, 2% for B. bengalensis [22] and 1.5% for M. meltada [23] and M. booduga.However, Prakash [24] recommended zinc phosphide at 2% for all rodents in general, including R. rattus.The bromadiolone was used at 0.005% concentration.
The grains preferred by the test animals under two-choice and multiple choice tests, namely, germinated paddy, pearl millet, and finger millet and because of their low cost and easy availability when compared to pulses, were used as bait carrier for the chosen rodenticides as suggested by Sakthivel [25], Sakthivel and Neelanarayanan [26], and Sakthivel et al. [27].These tests were conducted under both no-choice and choice conditions, individually.All these tests were conducted by following the method suggested by Baskaran et al. [28].

2.3.1.
No-Choice Tests.In these tests, no optional food was given to the experimental animals and they were fed only with poison bait prepared individually.Both poison baits were prepared in the following proportions: 96 g of germinated cereals (paddy/pearl millet/finger millet) mixed with 2 g of zinc phosphide poison/2 g of bromadiolone and 2 gram of coconut oil.The 1.5% concentration of zinc phosphide was prepared by mixing 97 g of germinated cereals (paddy, pearl millet and finger millet), 1.5 g of zinc phosphide and 1.5 g of coconut oil.All the caged animals were famished for 24 hrs before being offered with poison bait.Then, each caged rodent was offered a cup of 20 g poison bait and a cup of water.At the end of every 24 hours, poison bait consumption was recorded, individually.In order to calculate the hours to death as far as zinc phosphide is concerned the experimental animals were inspected every one-hour interval and mortality rate was calculated.As far as bromadiolone poison bait is concerned the bait was offered for two consecutive days and same was replenished at 24 hrs interval.The animals fed with bromadiolone were inspected once in 24 hrs and the days to death were calculated and recorded.a Mean and standard deviation values were obtained from observation of ten animals in each test (numbers in brackets are the range values).

Choice Tests.
All the caged animals were starved for 24 hrs before being fed with poison bait.Then, 20 g of poison prepared in the germinated cereals and 20 g of plain bait (its counterpart in nongerminated form-as the alternative food does not exist in germinated form in field situations) along with a cup of water were offered to the caged rodents, individually.
The baits were offered 24 hrs for zinc phosphide and 48 hrs (two consecutive days) for bromadiolone, for both no-choice and two-choice tests.The survived rodents were provided with plain bait for 72 hrs for zinc phosphide fed rodents and 14 days for bromadiolone rodents.All the baits were replenished with fresh bait once in 24 hrs.At the end of every 24 hours, poison/plain bait consumption was recorded.All these experiments were carried out from January 2007 to December 2007.

Statistical Analysis.
All the data were analysed by using SPSS version 16.0 as described by Rajathi and Chandran [29].Data were subjected to -tests (two-tailed) to establish significant differences in consumption between germinated poison bait and nongerminated plain bait for zinc phosphide poisoning under choice tests.Paired "" test was used to find the difference between the poison bait consumption on first and second days under no-choice tests and poison and plain bait ingestion by the experimental animals (bi-choice tests) under laboratory conditions for bromadiolone poisoning.

Acute Toxicity Tests:
No-Choice Tests.The results of nochoice tests on acute toxicity studies with four species of rodents fed with different geminated cereals (paddy, pearl millet, and finger millet) mixed with zinc phosphide are provided in Table 1.

The Soft-Furred Field Rat (M. meltada).
The poison consumption by the test animals was found to be maximum for bait prepared by using germinated paddy a Mean and standard deviation values were obtained from observation of ten animals in each test (numbers in brackets are the range of values).b The difference between poison and plain bait intake is statistically significant based on paired "" test.*  < 0.05; * *  < 0.01; * * *  < 0.001; NS  > 0.05.

The Indian Field Mouse (M. booduga).
The highest poison consumption by the test animals could be observed for the bait prepared by using germinated paddy (14.31 ± 3.77 g/100 g body wt.) and lowest for pearl millet (1.9 ± 0.7 g/100 g body wt.).Hundred percent mortality of M. booduga was observed on feeding with 1.5% concentration of zinc phosphide prepared by using two germinated cereals, namely, pearl millet and finger millet.Mean hours to death of experimental animals were 7.6 ± 3.7 (paddy), 5.2 ± 1.2 (pearl millet), and 4.6 ± 1.0 (finger millet) (Table 1).

House Rat (R. rattus).
The high intake of poison by the test animals was recorded in germinated paddy (6.16 ± 2.20 g/100 g body wt.) and less in finger millet (1.9±0.8 g/100 g body wt.).Cent percent mortality of R. rattus was observed on feeding with 2% concentration of zinc phosphide prepared by using germinated cereals such as paddy and pearl millet.
Hours to death of experimental animals ranged between 4 and 12 hours irrespective of the germinated cereals used in the bait (Table 1).
The results under no-choice tests revealed that hundred percent mortality was observed in all four species of rodents on feeding with zinc phosphide baits mixed with germinated cereals.However, 90% mortality was observed in M. booduga (feeding with zinc phosphide in germinated paddy) and R. rattus (feeding with zinc phosphide in germinated finger millet).

Acute Toxicity Tests: Choice Tests.
The results of choice tests on acute toxicity studies with four species of rodents fed with different geminated cereals (paddy, pearl millet, and finger millet) mixed with zinc phosphide are given in Table 2.

Lesser Bandicoot Rat (B. bengalensis).
The experimental animals were found to feed more quantity of plain bait than the poison bait and the difference between them was found to be statistically significant ( < 0.05; 0.01; 0.001).However, cent percent mortality of test animals could be observed only with the animals fed on the bait prepared by using pearl millet and finger millet.Hours to death of experimental animals ranged between 4 and 18 (Table 2).

Soft Furred Field Rat (M. meltada).
All the M. meltada were found to feed more quantity of poison bait than the plain bait; however, the difference between them (paddy and finger millet) was found to be statistically insignificant ( > 0.05).
Cent percent mortality of test animals could be observed only with the animals fed on the bait prepared by using paddy.Hours to death of experimental animals ranged between 3 and 12 (Table 2).

Indian Field Mouse (M. booduga).
Although the experimental animals were found to feed more quantity of poison bait (paddy) than the plain bait, insignificant difference was observed between them ( > 0.05).Hundred percent mortality of test animals could be observed only with the animals fed on the bait prepared by using pearl millet.Hours to death of experimental animals ranged between 4 and 18 (Table 2).

House Rat (R. rattus).
The experimental animals were found to feed more quantity of poison bait than the plain bait and the difference between them was found to be statistically significant ( < 0.05: finger millet;  < 0.01: pearl millet).
Cent percent mortality of test animals could be observed with the animals fed on the bait prepared by using all the three cereals.Hours to death of experimental animals ranged between 4 and 14 (Table 2).Under choice tests, cent percent mortality of rodents was observed in B. bengalensis (fed with zinc phosphide bait prepared by using germinated pearl millet and finger millet), M. meltada (fed with zinc phosphide bait by using germinated paddy), M. booduga (fed with zinc phosphide bait by using germinated pearl millet), and R. rattus (fed with zinc phosphide bait by using germinated paddy, pearl millet, and finger millet).

Chronic Toxicity Tests:
No-Choice Tests.The results of nochoice tests on chronic toxicity studies with four species of rodents fed with different geminated cereals (paddy, pearl millet, and finger millet) mixed with 0.005% bromadiolone on two days exposure are provided in Table 3.

Lesser Bandicoot Rat (B. bengalensis).
The difference between the poison bait intake on first and second days was found to be statistically significant for pearl millet and finger millet ( < 0.01).The highest intake of active ingredient (mg/kg −1 ) in the test animals was 6.6 ± 4.7 on the second day of feeding (paddy bait).Cent percent mortality of B. bengalensis was observed on feeding with 0.005% concentration of bromadiolone prepared by using all germinated cereals.Mean days to death of experimental animals were 5.2 ± 2.5 (paddy), 2.1 ± 0.5 (pearl millet), and 3.4 ± 0.8 (finger millet) (Table 3).

Soft-Furred Field Rat (M. meltada).
It is evident from Table 3 that significant difference could be observed between the poison bait intake on the first and second days for paddy and finger millet ( < 0.01).The highest intake of active ingredient (mg/kg −1 ) in the test animals was 10.4 ± 3.8 on the second day of feeding (paddy bait).Cent percent mortality of M. meltada was observed.Mean days to death of experimental animals were 4.6 ± 2.5 (paddy), 8.5 ± 4.1 (pearl millet), and 8.2 ± 2.2 (finger millet) with an overall range of 1-14.

Indian Field Mouse (M. booduga).
The highest poison consumption by the test animals could be observed for the bait prepared by using germinated finger millet (2.5 ± 0.3 g) and lowest for paddy (1.6 ± 0.2 g).The difference between the poison bait intake on first and second days was found to be statistically significant for paddy ( < 0.01) and pearl millet ( < 0.05).The maximum consumption of active ingredient (mg/kg −1 ) in the test animals was 14.8±3.7 (paddy bait).Hundred percent mortality of M. booduga was observed on feeding with 0.005% concentration of bromadiolone bait prepared by using two germinated cereals, namely, paddy and finger millet.Mean days to death of experimental animals were 3.6 ± 1.5 (paddy) and 4.4 ± 3.9 (finger millet) (Table 3).

House Rat (R. rattus).
It is apparent from the results that poison consumption by the test animals was found to be high for the bait prepared by using germinated paddy (11.4 ± 3.0 g) and less for finger millet (7.1 ± 1.0 g).The observed difference between the poison bait intake on first and second days was found to be statistically significant for all the three cereals.The ingestion of active ingredient (mg/kg −1 ) in the test animals was found to be high in paddy bait (8.1 ± 3.2).However, cent percent mortality of R. rattus was not observed on feeding with 0.005% concentration of bromadiolone bait prepared by using germinated paddy.The baits prepared by using germinated cereals such as pearl millet and finger millet yielded hundred percent mortality.Days to death of experimental animals ranged between 3 and 14 (paddy, pearl millet, and finger millet) (Table 3).
In the no-choice tests, cent percent mortality of rodents could be observed in B. bengalensis (fed with bromadiolone in all germinated cereals bait), M. meltada (fed with bromadiolone in all germinated finger millet bait), M. booduga (fed with bromadiolone in germinated paddy and finger millet bait), and R. rattus (fed with bromadiolone in germinated pearl millet and finger millet bait) (Table 3).

Chronic Toxicity
Tests: Choice Tests.The results of chronic toxicity studies on two-day exposure of 0.005% bromadiolone mixed with different geminated cereals (paddy, pearl millet, and finger millet) against the four species rodents under choice tests are presented in Table 4.

Lesser Bandicoot Rat (B. bengalensis).
The experimental animals were found to feed more quantity of poison bait than the plain bait and the difference between them was found to be statistically significant ( < 0.05; 0.01; 0.001).The intake of active ingredient (mg/kg −1 ) in the dead rodents ranged from 1.0 to 5.8 and in the survived animals from 0.7 to 1.8 in all the three cereals used in the bait.A maximum of ninety percent mortality of test animals could be observed only with the animals fed on the bait prepared by using pearl millet and finger millet.Days to death of experimental animals ranged between 1 and 11 (Table 4).

Soft Furred Field Rat (M. meltada).
The experimental animals were found to feed significantly more quantity of poison bait prepared in germinated finger millet than the plain bait ( < 0.05) on both days of feeding.The intake of active ingredient (mg/kg −1 ) ranged from 0.8 to 8.5 in the dead rodents in all the three cereals used in the bait.Cent percent mortality of test animals could be observed only with the animals fed on the bait prepared by using pearl millet.Days to death of experimental animals ranged between 2 and 14 (Table 4).

Indian Field Mouse (M. booduga).
Insignificant difference was observed between the consumption of poison prepared in all the cereals and plain bait ( > 0.05) excepting germinated paddy bait consumption ( < 0.01).The intake of active ingredient (mg/kg −1 ) ranged from 0.4 to 17.8 in the dead animals in all the three cereals used in the bait.The observed mortality was 80% for paddy and 70% each for pearl millet and finger millet.Days to death of experimental animals ranged between 3 and 10 (paddy), 3 and 7 (pearl millet), and 1 and 11 (finger millet) (Table 4).

House Rat (R. rattus).
Definite trend could not be seen in the poison and plain bait consumption by the test animals and the difference between them was found to be statistically not significant ( > 0.05).The intake of active ingredient (mg/kg −1 ) in the dead animals ranged from 1.9 to 8.6 in all the three cereals used in the bait.The pearl millet and finger millet bait produced a maximum of 90% mortality among the house rats.The paddy bait resulted in sixty percent mortality of test animals.Days to death of experimental animals ranged between 2 and 14.
The results of the toxicity studies of zinc phosphide and bromadiolone mixed with germinated cereals against four species were highly susceptible.The oral LD 50 of Zn 3 P 2 for rodents is usually 40 mg kg −1 or less.Zn 3 P 2 is a broad-spectrum toxin; there are some differences in the susceptibility of animal species [30].The onset of poisoning is rapid following ingestion of Zn 3 P 2 and usually occurs within 15 min to 4 h after ingestion of a toxic amount.Death from large doses usually occurs between 3 and 5 h [31].The results of the present study also corroborate these earlier observations and the mortality of the test animals started from 3 hrs onwards and it might be due to high quantity of consumption of poison in the bait.Parshad et al. [32] reported that, in the assessment of brodifacoum poison bait palatability in choice feeding test, rodent species (namely, M. meltada, T. indica, and B. bengalensis) consumed significantly less poison bait than the plain bait alternative.Similar results have been reported for Mus musculus [33], T. indica, and M. hurrianae [34].The results of the present study corroborate these earlier results.Though more consumption was observed in paddy bait, the toxic consumption was comparatively less because some amount of chemicals was removed along with outer husk while eating.Similar trend has been observed by Chaudhary and Tripathi [35] with various species of rodents with difethialone (0.0025%) treated with pearl millet bait.
A good-quality cereal is normally, on its own, sufficiently attractive to rodents to produce excellent results when used with an effective rodenticide.However, various attractants are sometimes added to bait [36].Certain edible oils are known, however, to enhance bait uptake by rodents and for this reason one of them, corn (maize) oil, is component in a challenge diet advocated by the US Environmental Protection Agency (EPA) and frequently used in palatability testing.Cereal grains, either whole, broken rolled, or ground, produce satisfactory rodenticide baits and are widely used by both large manufacturers and small-scale formulators [37].Besides these, the chosen three germinated cereals in the present study proved themselves that they are also capable of acting as suitable bait base for both selected rodenticides in bringing maximum mortality among the tested rodent pests under both no-choice and bi-choice tests in laboratory situations.The palatability of rodenticide bait to rodent species was found to be influenced by age/sex [38], quality of bait [3,[39][40][41], and concentration of rodenticides [42].According to Krishnakumari [43], less nourishing foods are accepted if they are "tasty" with flavours or soft texture.Food selection in rats is determined by factors such as texture, odour, and taste cues [44,45].In the present study the germinated grains would have provided soft texture and palatability and hence the rodents might have preferred them.

Conclusions
The results suggests that the poison baits in the germinated cereals induced all the chosen four species of rodent pests to consume greater quantities of bait perhaps due to the bait carrier's palatability and texture.Therefore, these three germinated cereals may be recommended as a bait carrier for both zinc phosphide (2%) and bromadiolone (0.005%) poisons for the control of all these four species of rodent pests under field conditions.However, this requires field based trials with these rodenticides for making a final recommendation.

Table 1 :
Mortality of four rodent species after feeding on zinc phosphide (1.5%-2%) baits of germinated cereals in no-choice tests.

Table 2 :
Mortality of four species of rodents after being fed on zinc phosphide (1.5%-2%) baits of germinated cereals in choice tests.

Table 3 :
Mortality of four rodent species after feeding on 0.005% bromadiolone baits of germinated cereals for two days in no-choice tests.
a Mean and standard deviation values were obtained from observation of ten animals in each test (numbers in brackets are the range of values).bThedifference between the poison bait consumption on first and second days is statistically significant (paired "" test).*  < 0.05; * *  < 0.01; NS  > 0.05.

Table 4 :
Mortality of four species of rodents after being fed on 0.005% bromadiolone baits of germinated cereals for two days in choice tests.
a Mean and standard deviation values were obtained from observation of ten animals in each test (numbers in brackets are the range of values).bThedifference between poison and plain bait intake is statistically significant based on paired "" test.*