lable at ScienceDirect

Crop Protection 67 (2015) 235e242Contents lists avaiCrop Protection

journal homepage: www.elsevier .com/locate/croproEffectiveness and persistence of cinnamic aldehyde as an antifeedanton rats under storage conditions

B.K. Babbar a, *, Jagwinder Kaur a, Neena Singla a, Amrit Kaur Mahal b

a Department of Zoology, Punjab Agricultural University, Ludhiana 141004, Punjab, Indiab Department of Mathematics, Statistics & Physics, Punjab Agricultural University, Ludhiana 141004, Punjab, Indiaa r t i c l e i n f o

Article history:Received 1 June 2014Received in revised form11 October 2014Accepted 16 October 2014Available online

Keywords:RodentsCinnamic aldehydeAntifeedantCommensal situations* Corresponding author. Tel.: 91 9915636711; fax:E-mail address: bhupinder@pau.edu (B.K. Babbar)

http://dx.doi.org/10.1016/j.cropro.2014.10.0090261-2194/ 2014 Elsevier Ltd. All rights reserved.a b s t r a c t

The house rat, Rattus rattus (Linnaeus), is responsible for causing severe damage in outdoor commercialgrain stores where rodent proofing is not possible. Rodenticides are the preferred option for preventingrodent attacks. However, secondary poisoning and development of bait shyness and resistance amongrodents after exposure to toxic chemicals has increased the search for new, safe, ecofriendly and effectivecontrol methods, which can prevent damage for a long duration. In the present study, the effectivenessand persistence of cinnamic aldehyde as an antifeedant against R. rattus were evaluated. Cinnamicaldehyde treated bait at 5% concentration was effective as an antifeedant and secondary repellent againstR. rattus in bi-choice feeding tests. The antifeedant effect was retained for at least 14 days after treatment.There was no significant difference between consumption of treated and untreated bait during the firstfour hours of exposure of rats under bi-choice feeding tests, indicating absence of a primary repellenteffect of cinnamic aldehyde. Feeding bait mixed with 5% cinnamic aldehyde continuously for 27 days inbi-choice feeding tests revealed the persistence of the antifeedant effect. This study demonstrated that5% cinnamic aldehyde can be used under field conditions to prevent the damage caused by house rats forlong durations. A formulation of 5% cinnamic aldehyde containing sodium bicarbonate as an emulsifierprevented rodent damage in terms of consumption of wheat grains, number of cuts on bags, quantity ofgrains spilled from bags cut by rats and percent damage due to rat urine under simulated storageconditions in laboratory pens for durations up to the experimental period of 15 days. Study of feedingbehaviour in feed scale consumption monitoring also confirmed the secondary repellent effect of cin-namic aldehyde against the house rat. Our results indicate that the sodium bicarbonate emulsifiedformulation of 5% cinnamic aldehyde has the potential to protect stored products from rodent damage ina manner that is effective, persistent and environmentally acceptable.

2014 Elsevier Ltd. All rights reserved.1. Introduction

Rodents are significant economic pests that devastate crops,gardens, orchards, landscape plantings, and damage commercialforest plantations or impede reforestation efforts (Tobin and Fall,2004; Singla and Babbar, 2010, 2012; Singla and Parshad, 2010;Singla, 2011). A national study made by the Indian Grain StorageManagement and Research Institute (IGMRI) revealed a total post-harvest loss of 4.75% to wheat grains with rodents accounting for0.59% (Rao, 2003). Overall losses of grains due to rodents in Indiawere approximately 25% at pre-harvest and 25e30% at post-harvest situations, bringing the loss to at least US$5 billion annu-ally in stored food and seed grain in India (Hart, 2001). The house91 161 2400945..

rat, Rattus rattus (also called the black rat, ship rat, or roof rat), is anative of the Indian sub-continent and is now foundworldwide andnominated as among 100 of the world's worst invaders (Gillespieand Myers, 2004). It causes severe damage in outdoor commer-cial grain stores by consuming the stored food items and alsocontaminates the food material by urination and defecation, thusmaking it unfit for human consumption (Prakash and Ghosh, 1992;Drummond, 2001; Brown et al., 2007).

Chemical control is the preferred option worldwide to preventrodent attacks. However, environmental pollution, secondarypoisoning and health problems caused by the use of rodenticidesand development of bait shyness and resistance among rodentsafter exposure to toxic chemicals have increased the search for new,safe, ecofriendly and sustainable control methods (Rao, 2005).Natural compounds provide protection against pests with differentmodes of action i.e. by either killing the pest or by acting as primaryor secondary repellents for pests (Sbeghen-Loss et al., 2011). Over

Delta:1_given nameDelta:1_surnameDelta:1_given nameDelta:1_surnamemailto:bhupinder@pau.eduhttp://crossmark.crossref.org/dialog/?doi=10.1016/j.cropro.2014.10.009&domain=pdfwww.sciencedirect.com/science/journal/02612194http://www.elsevier.com/locate/croprohttp://dx.doi.org/10.1016/j.cropro.2014.10.009http://dx.doi.org/10.1016/j.cropro.2014.10.009http://dx.doi.org/10.1016/j.cropro.2014.10.009

B.K. Babbar et al. / Crop Protection 67 (2015) 235e242236the last 50 years, thousands of plants have been screened as po-tential sources of repellents (Sukumar et al., 1991). Repellents usedfor the management of birds and mammals often rely on thedevelopment of a conditioned taste aversion (CTA) in response topost-ingestional malaise (Johnson et al., 1982) or alternatively,repellency is mediated through sensory irritation by contact orolfactory avoidance (Mason et al., 1991). CTA is acquired through anassociation between the taste of the food and feeling of illnessexperienced after ingestion and thus avoidance of that food infuture (Garcia et al., 2001; Provenza, 1995). Repellents whichinduce CTA are more effective because they promote long termavoidance responses that are proportional to the severity of themalaise caused by the compound. Antifeedants or secondary re-pellents causing CTA have been proposed as alternatives to syn-thetic pesticides (Ballesta-Acosta et al., 2008). Pyrethrum,rotenone, neem and plant essential oils have been used for cen-turies to protect stored commodities or to repel pests from humanhabitations (Isman and Machial, 2006).

Many plant derived extracts have been found to have repellentproperties against rodents. Methanol extracts of Piper nigrum L.(Piperaceae), Aucklandia lappa Dene. (Compositae), Cinnamomumcassia Pres l. (Laurac eae), Illicium verum Hook. (Magnoliaeceae),Rheum coreanumNakai. (Polygonaceae), and Pinus densiflora Sieb. etZucc. (Pinaceae) showed the potent antignawing activity againstmice (Yun et al., 1998). Many cinnamomum species are rich inessential oils and tannins. The main components of the essential oilobtained from the bark of Cinnamomum zeylanicum are cinnamicaldehyde, eugenol and linalool. While C. cassia bark contains cin-namic aldehyde, cinnamic acid, cinnamyl alcohol and coumarin (Heet al., 2005). Cinnamic aldehyde is reported to repel dogs and othercanids (Mason,1998). It also acted as an irritant for brown tree snake(Clark and Shivik, 2002). Antignawing activity of cinnamic aldehydeis also reported against mice (Lee et al., 1999). Systematic work oncinnamic aldehyde as an antifeedant against rodents, particularlyagainst R. rattus, is lacking. Moreover no report is available on thepersistence of antifeedant effect of cinnamic aldehyde and devel-opment of any formulation using antifeedant for field applicationagainst rodents. The objectives of the present study were (i) Todetermine the effective concentration of cinnamic aldehyde as anantifeedant against thehouse rat. (ii) Todetermine thepersistenceofan effective concentration of cinnamic aldehyde in laboratory cagesand (iii) To determine the persistence of different formulationscontaining an effective concentration of cinnamic aldehyde undersimulated storage conditions in laboratory pens.2. Materials and methods

The present study was carried out at the Animal House andRodent Research Laboratory, Department of Zoology, Punjab Agri-cultural University (PAU), Ludhiana, Punjab, India (30550 N; 75540

E). Animals were used and maintained as per the guidelines ofAnimal Ethics Committee. Approval of Institutional Animal EthicsCommittee, Guru Angad Dev Veterinary and Animal Sciences Uni-versity, Ludhiana, Punjab, India was obtained for the usage of ani-mals. Adult rats were collected from poultry farms at Ludhianausing multi catch rat traps and acclimatized for 10e15 days on foodand water provided ad libitum.AFI % Consumption of untreated bait Consumption of treateConsumption of untreated bait Consumption of treate2.1. Determination of effective concentration of cinnamic aldehyde

Cinnamic aldehyde was evaluated as an antifeedant againstR. rattus in the present studies. It was purchased from S.D Fine-chem limited, Mumbai, India. Three different concentrations ofcinnamic aldehyde (1, 2.5, 5%) were tested against adult house rats.Treated baits containing different concentrations of chemical wereprepared by dissolving the required chemical in a known volume ofmethanol and then mixing in a known quantity of plain WSO(Wheat:Sugar:Oil e 96:2:2) bait. Rats of treated (3 groups) groups(n 10, 5 males and 5 females in each group) were fed on differentconcentrations of cinnamic aldehyde (1, 2.5, 5%) in bi-choicefeeding tests for three consecutive days during treatment period1 (D0) after recording pre-census bait consumption. Treatmentswere repeated again after 7 days (treatment period 2 e D7) oftreatment period 1 and after 14 days (treatment period 3 e D14) oftreatment period 2 to test whether rats could retain the memory ofthe compounds after 7 and 14 days of treatment. Simultaneously,rats of untreated group (n 10, 5 males and 5 females) were fed onplain WSO bait mixed with known volume of methanol. Bait con-sumption (g) of both untreated and treated bait was recorded dailyto calculate mean daily bait consumption in g/100 g body weight(bw) as per the formula given below:

Daily=hourly bait consumption g=100 g bw

Daily=hourly bait consumption by rat gWeight of rat g 1002.2. Determination of primary repellent effect of cinnamic aldehyde

Experiments were also conducted in laboratory cages to recordthe hourly consumption of groups of adult rats (n 10, 5 males and5 females) treated with effective dose of cinnamic aldehyde(determined from Section 2.1) up to 4 h under bi-choice conditionsin order to determine the existence of primary repellent effect. Forthis treated group was fed on known weight of WSO bait mixedwith 5% cinnamic aldehyde dissolved in methanol in bi-choicefeeding test for 4 h consecutively to observe the hourly consump-tion of treated bait.2.3. Determination of development of habituation in rats toeffective dose of cinnamic aldehyde

In order to confirm whether the rats treated with effective doseof cinnamic aldehyde became habituated to treated bait, con-sumption of bait treated with the effective dose of compound anduntreated bait was recorded daily for up to 27 days under bi-choiceconditions using adult rats of both sexes (n 10, 5 males and 5females).2.4. Calculation of antifeedant index (AFI)

Percent antifeedant index (AFI) was calculated as per themethod described in Singla and Parshad (2007) which is givenbelow:d baitd bait

100

B.K. Babbar et al. / Crop Protection 67 (2015) 235e242 2372.5. Development and testing of formulations using effectiveconcentration of cinnamic aldehyde

Three different bait formulations containing the effective doseof cinnamic aldehyde (determined from Section 2.1) were prepared.Formulation I was prepared by mixing 5% cinnamic aldehyde, 2%starch and 1% sodium bicarbonate (NaHCO3) in water. As cinnamicaldehyde is immiscible with water, 1 g of NaHCO3 was added toform an oil emulsion inwater. Starch was added in this formulationto increase the stickiness of this spray solution on bags. Formula-tion II was a chalk formulation, prepared by first mixing 5 ml cin-namic aldehyde in 25mlmethanol. This solutionwas thenmixed in100 g of Plaster of Paris (POP). Formulation III was prepared bymixing 5% cinnamic aldehyde and 1% NaHCO3 in water.

A total of six laboratory pens (each of size 252 100 72 cm)containing one rat each at a time, were used to test the effective-ness and persistence of each formulation. Each pen consisted ofthree chambers of equal size. A rat released in each pen had freeaccess to all the three chambers and nest boxes attached to thesechambers on opposite sides. Two chambers at the extreme ends ofthe laboratory pens were provided with 12 bags of wheat kept in astack on an aluminium tray. Each bag was filled with 500 g ofwheat. Bags were placed on trays in three layers (each layer havingfour bags) in both the extreme end chambers of laboratory pens toform stacks. Bags of one chamber were treated whereas the bags ofsecond chamber remained untreated. Formulations I and III ofcinnamic aldehyde were used as a spray on bags and formulation IIof cinnamic aldehyde was in the form of a chalk, which was used todraw lines around and on the bags. Each spray solutionwas sprayedon the twelve bags of treated chamber of laboratory pen placed inform of stack. Rodent damage was recorded in terms of consump-tion of wheat grains (g/100 g body weight (bw)/day), number ofcuts on bags kept in treated and untreated chamber was recordedboth after 7 and 15 days of treatment, quantity of grains (g) spilledfrom bags cut by rats was recorded after 7 and 15 days of treatment,% contamination due to rat urine was recorded after 15 days oftreatment. Rat urine fluoresces under ultraviolet (UV) light. Thenumber of wheat grains fluorescing under UV light was recorded ina UV chamber to determine the contamination caused by rats tograins with their urine. For this 50 spilled wheat grains as well as 50wheat grains each from upper, middle and lower layers of bagswere collected from both treated and untreated chambers. Thesegrains were kept in UV chamber and number of grains shining withfluorescent light was counted. Total weight of 50 grains and thoseshining under fluorescent light was recorded. From this percentcontamination of grains with urine was calculated.

2.6. Maze experiments under camera connected to ethovision videotracking system

This experiment was conducted to confirm the presence/absence of a primary repellent effect of the effective formulation ofcinnamic aldehyde (determined from Section 2.5) against thehouse rat. No visit of rats in the treated zone was considered as aprimary repellent effect of the compound. An I-Maze consisting oftwo arms (each of length 47 cm and height 18.5 cm) on oppositesides and one central hub was used for this purpose. This maze waskept under the camera connected to computer installed with thesoftware used (Ethovision Video Tracking System) to record themovement of rats. During treatment, in one arm of the I-Maze(called treated zone), a spray was applied of formulation III on thebag (containing wheat grains) placed at the end of arm, whereas nospray was applied to the bag placed in other arm as well as thecentral hub (called untreated zones). For the first three days of theweek, the behaviour of the rat was recorded in I-Maze for2 hwithout spray is having been applied in any of the arm. This wasdone in order to reduce the fear developed in rats on exposure tonew environment followed by the spray on the bag kept in one ofthe arms on the fourth day. Again, behaviour of the rat was recor-ded for 2 h in a day from the fourth to the sixth day. Each day, themovement of the rat in the I-Maze was recorded in the form oftracks and the observations recorded for quantitative data were ofzone frequency, total distance moved, frequency of rearing andmobility in both treated and untreated zones. A total six rats (3males and 3 females) were used for this experiment.

2.7. Experiments in feed scale consumption monitor

Feeding behaviour of rats was recorded using a Feed ScaleConsumption Monitor (FSCM). This system consists of three ratchambers, each kept on an electronic balance provided with a foodbowl and connected to a computer having software for recordingfeeding behaviour of rats. This experiment was conducted toconfirm the existence of secondary repellent effects to the house ratin response to effective formulation of cinnamic aldehyde (deter-mined from Section 2.5). For this purpose, rats kept hungry over-night were released in chambers of FSCM and their feedingbehaviour was recorded using plain WSO bait for 2 h a day for sixconsecutive days. This was also done in order to reduce the feardeveloped in rats on exposure to new environments. A total of sixrats was used. Three rats were released in three chambers of FSCMat a time, and their feeding behaviour was recorded for 2 h per day.It was then followed by exposure of all the rats to WSO bait mixedwith formulation III of cinnamic aldehyde for 2 h a day for sixconsecutive days. Each day, quantitative data on feeding boutevents, feeding bout time (s) and feed consumed (g) was recorded.

2.8. Statistical analyses

Values were calculated as mean SE. Significance of differenceswas determined at the P 0.05 level. Significance differencesamong various parameters were determined by using factorialcompletely randomized design (CRD). Various factors taken forcomparison in different experiments were sex (male, female),treatments (control, 1%, 2.5% and 5%), treatment periods (D0, D7and D15), consumption of plain and treated bait, antifeedant index,hours of treatment (1st to 4th hour), days of treatment (1e27 days),formulations and treated and untreated zones. Analysis was doneusing general linear model (GLM) in SAS version 9.3. All pair wisetreatment comparisons were made using Tukeys' HSD test.

3. Results and discussion

3.1. Determination of effective concentration of cinnamic aldehyde

A significant difference (P < 0.0001) in overall mean daily foodconsumption (g/100 g bw) between the two sexes was recordedwith a higher effect of treatment in male rats. In male rats, averageconsumption (g/100 g bw) during treatment period 1 was signifi-cantly less from treated bait (P at 1% 0.0004; 2.5% and5%

Table 1Antifeedant index (AFI) and average mean daily consumption of bait treated with different concentrations of cinnamic aldehyde during three treatment periods.

Sex Concentrationstested

First treatment period Second treatment period Third treatment period

Mean daily baitconsumption (g/100 g bw)

AFI (%) Mean daily baitconsumption (g/100 g bw)

AFI (%) Mean daily baitconsumption (g/100 g bw)

AFI (%)

Plain Treated Plain Treated Plain Treated

Male* (n 5) 1% 6.4 0.451 1.22 0.342 68.42 7.95 5.04 0.611 1.14 0.352 65.38 9.80 6.52 0.651 1.94 0.522 58.17 7.632.50% 7.64 2.111 0.97 0.462 79.94 5.29 5.14 0.401 1.66 0.4412 53.45 10.29 6.38 0.851 1.51 0.622 66.51 7.825% 7.34 0.761 0.64 0.282 85.94 5.48 7.09 1.001 1.94 0.382 55.13 6.96 5.37 0.531 1.57 0.592 57.16 14.99Control 6.39 0.761 5.38 0.241 9.33 1.241

Female** (n 5) 1% 5.21 0.44a 2.15 0.69ab 56.69 6.99 6.93 0.99a 3.77 0.98ab 34.46 10.15 7.52 0.99a 3.77 0.59ab 33.50 7.472.50% 6.79 0.40a 2.95 0.23ab 41.60 8.44 9.13 0.32a 4.13 0.51b 43.07 13.19 10.49 1.65a 5.82 2.13b 40.62 13.75% 8.28 1.10a 1.8 0.80b 72.82 10.63 7.71 0.44a 2.69 0.60b 50.6 8.01 8.05 1.23a 2.29 0.49b 54.79 8.89Control 6.89 0.67a 7.93 1.25a 6.36 0.87a

Values are Mean SE, Means followed by same superscript or no superscript in a column or in a row do not differ significantly at P 0.05.*, **Significant difference (P 0.05) in overall average food consumption between the two sexes.1,2Significant difference (P 0.05) in consumption between plain and treated bait in male rats at each concentration tested.a,bSignificant difference (P 0.05) in consumption between plain and treated bait in female rats at each concentration tested.

Fig. 1. Comparison in overall antifeedant index among different treatments of cin-namic aldehyde.

B.K. Babbar et al. / Crop Protection 67 (2015) 235e242238cinnamic aldehyde but the antifeedant index was maximum(85.94 5.48% in male rats and 72.82 10.63% in female rats) at 5%concentration.

During treatment period 2, in male rats, average food con-sumption (g/100 g bw) was found to be significantly lower from thetreatment side as compared to untreated side in groups of ratstreated with 1 (P 0.0232) and 5% cinnamic aldehyde (P 0.0004)but non-significantly different in group of rats treated with 2.5%cinnamic aldehyde. However in female rats, average food con-sumption (g/100 g bw) was found to be significantly lower from thetreatment side as compared to the untreated side in groups of ratstreated with 2.5 (P 0.0010) and 5% (P 0.0009) cinnamic alde-hyde but was not significantly lower in group of rats treated with1% cinnamic aldehyde (Table 1).

During treatment period 3, in male rats, average food con-sumption (g/100 g bw) was found to be significantly lower from thetreatment side (P at 1% 0.0063; 2.5% 0.0018; 5% 0.044) ascompared to the untreated side in all groups of rats treated withdifferent concentrations of cinnamic aldehyde. However in femalerats, average food consumption (g/100 g bw) was found to besignificantly (P 0.05) lower from the treatment side as comparedto the untreated side in groups of rats treated with 2.5 (P 0.0049)and 5% (P < 0.0001) cinnamic aldehyde but not significantlydifferent in group of rats treated with 1% cinnamic aldehyde. Therewas no significant difference in overall antifeedant index amongdifferent groups of rats treated with different concentration ofcinnamic aldehyde (Fig. 1). Consumption of treated bait by groupsof rats treated with 5% cinnamic aldehyde remained minimum andwas significantly lower from untreated bait in both sexes fromtreatment periods 1 to 3 (Table 1). All the experiments demon-strated that cinnamic aldehyde treated bait at 5% concentration iseffective as an antifeedant or secondary repellent against the houserat. Lee et al. (1999) also reported an antignawing effect of cinnamicaldehyde at 5% concentration for approximately 15 days againstmice. The present study also demonstrated that antifeedant effectof different concentrations of cinnamic aldehyde retained both atan interval of 7 (treatment period 2) and 14 (treatment period 3)days of treatment, although its effect reduced as compared totreatment period 1 in both sexes. There was a significant reductionin overall antifeedant index between treatment periods 1 (D1) and2 (D7) as well as between treatment periods 1 and 3 (D14) but therewas a non-significant reduction in overall antifeedant index be-tween treatment periods 2 (after 7 days of treatment period 1) and3 (after 14 days of treatment period 2) indicating an increase in theantifeedant effect of cinnamic aldehyde up to 7 days after treatmentwhich stabilized afterwards from 7 to 14 days (Fig. 2).3.2. Determination of primary repellent effect of cinnamic aldehyde

The experiment conducted to determine the existence of aprimary repellent effect of cinnamic aldehyde against R. rattusrevealed a significant difference (P 0.0219) in overall averagehourly consumption (g/100 g bw) between both sexes in bi-choicetests. There was no significant difference between overall averageconsumption of untreated and treated bait in both the sexes. Alsothere was no significant difference in the mean consumption oftreated as well as untreated bait from the 1st to the 4th hour in bothmale and female rats (Table 2). This experiment demonstrated thatrats showed no repellency towards treated bait during the first fewhours of their exposure to treated bait, indicating absence of aprimary repellent effect in house rats towards effective dose ofcinnamic aldehyde but because the consumption of 5% treated baitwas significantly less than that of untreated bait after 24 h of theirexposure to cinnamic aldehyde (Table 1) during treatment period 1,

Fig. 2. Comparison of overall antifeedant index among different treatment periods.Fig. 3. Comparison of overall consumption of plain and treated bait among differentday durations.

B.K. Babbar et al. / Crop Protection 67 (2015) 235e242 239rats might have started avoiding cinnamic aldehyde after experi-encing some problem in gastrointestinal tract post-ingestion, thussuggesting a secondary repellent effect of cinnamic aldehyde. Thesecondary repellent effect of 5% cinnamic aldehyde treated baitmight have developed in house rats sometime between 4 and24 h of exposure of rats to treated bait. A secondary repellent effectof some products inducing gastrointestinal malaise has also beenreported earlier (Sayre and Clark, 2001). Dolbeer et al. (1994) re-ported that secondary repellents are more effective than primaryrepellents because they promote long term avoidance responsesthat are proportional to the severity of the malaise caused by thecompound.

3.3. Determination of development of habituation to effective doseof cinnamic aldehyde

There was a significant difference (P < 0.0001) between overallaverage consumption of plain and treated bait in both the sexes butthere was no significant difference in mean consumption of treatedTable 2Hourly consumption of plain and treated bait (5% cinnamic aldehyde) in bi-choicefeeding test.

Hours oftreatment

Consumption of bait (g/100 g bw)

Male* Female**

Plain1 Treated1 Plain1 Treated1

1st hr 0.04 0.02a 0.04 0.00a 0.06 0.11b 0.04 0.01b

2nd hr 0.02 0.01a 0.03 0.00a 0.02 0.01b 0.04 0.02b

3rd hr 0.14 0.07a 0.18 0.12a 0.17 0.04b 0.12 0.06b

4th hr 0.08 0.05a 0.03 0.00a 0.07 0.05b 0.03 0.03b

Values are Mean SE.*, **Significant difference (P 0.05) in overall average hourly consumption betweenboth sexes.1Non-significant difference (P 0.05) in overall average consumption of plain andtreated bait.aNon-significant difference (P 0.05) in consumption of bait along the columns inmale rats.bNon-significant difference (P 0.05) in consumption of bait along the columns infemale rats.as well as untreated bait from days 1e3 to day 25e27 (Fig. 3). Alsothere was non-significant difference in the antifeedant index fromdays 1e3 to days 25e27 (Table 3; Fig. 4). This experimentdemonstrated that rats were not habituated to bait treated with aneffective dose of cinnamic aldehyde and avoided the treated baitthroughout the experimental period of 27 days. One of theimportant problems related to practical application of antifeedantsis habituation to them. Previous studies revealed that long-termassays are essential to screen chemicals for practical use (Usheret al., 1988). Long term assays indicate the possibility of habitua-tion to antifeedants. If animals show habituation to an antifeedant,it may then be excluded from practical use under field conditions. Itwas reported that attenuation of CTA is slower under choice feedingtests (Batsell and Best, 1993). This may be due to the presence ofalternative food, which leads the animal to sample treated food lessfrequently. It is reported that a choice feeding test is more sensitivefor detecting an aversion than a no-choice feeding test and it alsoremoves the complications associated with hunger (Riley andTable 3Antifeedant index (AFI) and consumption of plain and treated bait (5% cinnamicaldehyde) for 27 days in bi-choice feeding test.

Days Consumption (g/100 g bw) of bait AFI (%)

Plain* Treated**

1e3 5.96 0.54a 0.76 0.19b 77.12 5.264e6 5.94 0.44a 0.37 0.09b 86.90 1.507e9 7.70 0.75a 1.90 0.50b 65.40 4.8710e12 6.79 0.54a 1.85 0.57b 64.20 2.5213e15 6.82 0.48a 1.50 .40b 67.34 2.1816e18 8.05 0.44a 1.16 0.38b 77.16 5.2019e21 8.02 1.36a 1.74 0.40b 66.27 10.5222e24 8.22 0.66a 0.90 0.50b 84.80 10.7425e27 6.80 0.94a 0.66 0.20b 84.96 5.57

Values are Mean SE, BW e 122 6.86 g.Means followed by same or no superscript in a column do not differ significantly atP 0.05.a, bSignificant difference (P 0.05) in consumption of plain and treated baits in a rowfor different day durations.

Fig. 4. Comparison of antifeedant index (AFI) among different day durations.

B.K. Babbar et al. / Crop Protection 67 (2015) 235e242240Mastropaolo, 1989). This experiment demonstrated that animalswere not habituated to 5% concentration of cinnamic aldehyde, so,it can be used under field conditions to prevent the damage causedby house rats under commensal situations.Table 4Antifeedant index (AFI) and consumption (g/100 g bw/day) of wheat from untreated bagsaldehyde.

Formulations After 7

Consugrains

Untre

Formulation I (5 ml cinnamaldehyde, 2 g starch, 1 g NaHCO3 in 100 ml water) 3.62 Formulation II (5 ml cinnamaldehyde, 25 ml methanol, 100 g POP) 3.26 Formulation III (5 ml cinnamaldehyde, and 1 g NaHCO3 in 100 ml water) 6.62

Values are Mean SE, Number of rats 6 (3 males and 3 females).a, bSignificant difference (P 0.05) in food consumption in a row as well as in a column1, 2Significant difference (P 0.05) in food consumption in a row as well as in a column

Table 5Number of cuts and spilled grains from untreated bags and bags treated with different fodue to rat urine recorded in UV chamber after 15 days of treatment.

Formulations Number of cuts Spilled

After 7 days After 15 days After 7

Untreated Treated Untreated Treated Untrea

Formulation I (5 mlcinnamaldehyde, 2 gstarch, 1 g NaHCO3 in100 ml water)

3.33 0.63 1.66 0.47 4.83 0.981 2.66 0.5012 0

Formulation II (5 mlcinnamaldehyde, 25 mlmethanol, 100 g POP)

1.50 0.51 1.50 0.08 4.00 1.101 2.66 0.5012 5.00

Formulation III (5 mlcinnamaldehyde, and1 g NaHCO3 in 100 mlwater)

1.67 0.19 0.67 0.19 4.00 0.331 1.16 0.272 33.33

Values are Mean SE, Number of rats 6 (3 males and 3 females).Means followed by same or no superscripts in a row or in a column do not differ signifi1,2Significant difference (P 0.05) in number of cuts in a row as well as in a column afta,bSignificant difference (P 0.05) in percent contamination in a row as well as in a colu3.4. Testing of formulations developed using effective concentrationof cinnamic aldehyde

For application of cinnamic aldehyde in grain stores and tomaintain its effectiveness under storage conditions, three formu-lations of cinnamic aldehyde using the effective concentrationweredeveloped and applied under simulated grain storage conditions inlaboratory pens. Application of Formulations I and II in treatedchambers had no significant effects on food consumption, numberof cuts on bags, spillage of grains and contamination due to urine ascompared to that in untreated chamber. From the above results, it isclear that spraying of formulations I and II could not prevent thedamage caused by house rats.

Using formulation III, there was significantly more consumptionfrom untreated chamber after 15 (P 0.0038) days of treatment.However there was no significant difference in consumption fromtreated bags between the 7th and the 15th day of treatment(Table 4). Number of cuts was significantly less (P 0.0121) on bagstreated with formulation III after 15 days of treatment (Table 5).Daily observations revealed no cuts on treated bags until the 6th dayafter spraying. The quantity of spillage does not differ significantlybetween treated and untreated side for up to 15 days of treatment(Table 5). Contamination due to rat urine was significantly less ontreated side as compared to untreated side.

This experiment clearly revealed that damage due to rats interms of consumption of wheat, cuts on bags and contamination ofwheat grains due to rat urine was significantly less in chambertreated with formulation III as compared to the chambers treatedwith formulations I and II. Sodium bicarbonate mixed inand bags treated with different formulations of effective concentration of cinnamic

days After 15 days

mption of wheat(g/100 g bw/day)

AFI (%) Consumption of wheatgrains (g/100 g bw/day)

AFI (%)

ated Treated Untreated Treated

1.12a 7.17 1.99a e 3.06 1.011 7.38 1.191 e1.19a 3.64 0.73ab e 5.64 0.591 3.78 0.7712 27.69 11.121.54a 1.12 0.54b 68.05 14.36 8.52 0.691 1.12 0.322 77.25 6.07

after 7 days of treatment.after 15 days of treatment.

rmulations after 7 and 15 days of treatment and percent (%) contamination of grains

grains (g) Percent contamination dueto urine

days After 15 days Untreated Treated

ted Treated Untreated Treated

84.83 73.27 5.00 4.20 108.33 87.93 10.75 0.51a 10.25 0.97a

3.11 1.33 0.82 20.00 7.49 7.16 3.56 8.66 1.07a 8.33 0.84a

16.10 1.66 1.52 41.33 27.72 7.33 3.41 9.83 1.07a 2.67 0.42b

cantly at P 0.05.er 15 days of treatment.mn after 15 days of treatment.

Table 6Repellent behaviour of Rattus rattus in response to formulation III of cinnamicaldehyde in I-Maze under bi-choice conditions.

S.No.

Parameter Treatment/days

Untreated zone(n 6)

Treated zone(n 6)

1. In zone frequency Pretreatment

171.22 102.07 79.84 31.39

Day 1 oftreatment

40 20.02 43.83 16.52

Day 2 oftreatment

67.33 40.99 21.83 8.75

Day 3 oftreatment

41.50 15.97 32 15.82

Posttreatment

151 96.72 44.94 29.16

2. Total distancemoved (cm)

Pretreatment

6117.39 2300.92 6162.90 2016.62

Day 1oftreatment

18,013.12 2115.97 6087.65 932.33

Day 2 oftreatment

15,107.82 5865.88 4302.62 1609.67

Day 3 oftreatment

12,473.99 5541.12 3915.65 157.98

Posttreatment

93,769.15 2523.34 4331.93 1796.90

3. Frequency ofrearing

Pretreatment

369.50 157.72 754.11 79.29

Day 1 oftreatment

1009.5 123.86 549 203.17

Day 2 oftreatment

1411.5 513.94 367 158.86

Day 3 oftreatment

1326 664.91 268 46.90

Posttreatment

532.33 212.84 334.88 125.78

4. Frequency ofmobility

Pretreatment

599.37 254.46 944.08 256.20

Day 1 oftreatment

857 250.32 726.5 265.52

Day 2 oftreatment

1760 640.16 502.5 223.79

Day 3 oftreatment

1848.16 633.53 286.16 134.06

Posttreatment

707.16 263.32 461.38 223.85

Values are Mean SE.Means followed by no superscript in a column or in a row do not differ significantlyat P 0.05.

Table 7Antifeedant behaviour of Rattus rattus in response to formulation III of cinnamic aldehyd

Gender Days Pre-treatment period*

Number offeeding bouts

Feeding bout length (s) Feed consumed (g)

Male# Day 1 6.33 2.12 327 134.77 0.72 0.3Day 2 17.67 11.57 543 354.61 1.07 0.69Day 3 10.33 7.22 580 371.34 1.33 0.62Day 4 8.33 3.21 405.33 108.96 1.67 0.54Day 5 8.67 2.33 455.33 160.75 1.7 0.42Day 6 21.33 4.45 537.67 169.51 2.07 0.38

Female# Day 1 4 0.47 456.66 50.40 0.64 0.11Day 2 8 2.49 483 65.64 3.33 0.82Day 3 10.67 1.78 557.66 189.44 2.45 0.60Day 4 11.67 2.68 665.66 95.15 2.28 0.85Day 5 11.67 1.65 522 186.23 2.86 1.3Day 6 8.67 3.34 430.67 229.35 3.91 2.27

Values are Mean SE BW of male rats e 147 4.54 g, BW of female rats e 139.33 4.2*, **In overall, significant difference between pre-treatment and treatment periods.#In overall, non-significant difference between male and female rats.Means followed by no superscript in a column and in a row do not differ significantly at

B.K. Babbar et al. / Crop Protection 67 (2015) 235e242 241formulation III not only acted as an emulsifier but might also havereduced degradation of cinnamic aldehyde, increased stickiness ofcinnamic aldehyde on bags and synergized the antifeedant effect ofcinnamic aldehyde. Previous studies have revealed that thoughessential oils are effective when freshly applied, their protectiveeffects dissipate quickly (Trongtokit et al., 2005). In order to in-crease the efficiency of essential oils, some fixative materials suchas liquid paraffin, vanillin, salicyluric acid, mustard and coconut oilshave been used (Das et al., 1999; Tawatsin et al., 2001; Oyedeleet al., 2002; Blackwell et al., 2003)

3.5. Repellent behaviour in response to formulation III of cinnamicaldehyde in I-maze

Results revealed no significant difference in zone frequency,total distance moved (cm), frequency of rearing and frequency ofmobility between untreated and treated zones. Rats visited bothzones indicating absence of primary repellent or olfactory repellenteffect in response to spray with formulation III of cinnamic alde-hyde. Rats showed movement in both zones but the activities of ratand distancemovedwas less in the treated zone as compared to theuntreated zone (Table 6). Kalandakanond-Thongsong et al. (2010)reported a decrease in the number of visits of rats in response torepellents to the treated side as compared to untreated side.

3.6. Antifeedant behaviour in response to formulation III ofcinnamic aldehyde in the feed scale consumption monitor

Results revealed that there was no significant difference in thenumber of feeding bouts and feeding bout length but significantdifference (P 0.0397) in feed consumed (g) between both thesexes but there was a significant reduction (P 0.0001) in thevalues of all the three parameters during the treatment period ascompared to the pre-treatment period (Table 7). This experimentdemonstrated that rats did not find the compound immediatelyrepellent, consumption of treated bait declined almost completelyonly after day 3 and remained almost depressed for the rest of thetreatment period. The response of house rats to cinnamic aldehydeis indicative of learned aversion due to gastrointestinalmalaise. Thetaste/odour of the food that made the animal ill acts as a cue, so thatfood is avoided by the animal in later encounters (Rozin and Kalat,1971). This experiment also demonstrated that rats not onlyreduced their consumption of treated bait but there was also ae in feed scale consumption monitor.

Treatment period** Antifeedantindex (%)

Number of feedingbouts

Feeding boutlength (s)

Feed consumed (g)

0.67 0.18 15 6.34 0.15 0.11 87.92 8.322.67 1.18 123.33 50.67 0.29 0.12 73.46 16.894 1.88 227.33 110.69 0.74 0.41 76.63 12.111 0.82 15 12.24 0.04 0.03 98.4 1.300 0 0 100 00.33 0.27 9 7.35 0.01 0.08 99.64 0.28

2.67 1.78 106 81.29 0.49 0.23 33.33 27.223.33 2.72 109 88.99 0.62 0.51 81.77 14.882.67 1.78 90.67 44.92 0.56 0.38 66.13 25.372.33 1.51 98.67 59.88 0.79 0.40 65.96 22.872 1.25 48 32.89 0.31 0.24 69.90 24.430.33 0.27 4.67 3.81 0.02 0.02 97.75 1.83

5 g.

P 0.05.

B.K. Babbar et al. / Crop Protection 67 (2015) 235e242242decline in the mean number of feeding bouts followed by a declinein the mean length of feeding time during the trial period. So ratsalso handled the treated bait less frequently. This might be due toan irritant effect of cinnamic aldehyde.

In conclusion, the present study revealed that bait treated with5% cinnamic aldehyde is effective as an antifeedant against thehouse rat, R. rattus. Results suggested a secondary repellent effect ofcinnamic aldehyde which persisted for the entire experimentalperiod. The rats were not habituated to bait treated with an effec-tive dose of cinnamic aldehyde and avoided the treated baitthroughout the experimental period of 27 days. This study alsodemonstrated the effectiveness of formulation containing 5% cin-namic aldehyde and 1% sodium bicarbonate in preventing rodentdamage to wheat grain for a long duration. Sodium bicarbonate,being edible, has no adverse effects if sprayed on bags containinggrains. For outdoor usage of this compound as an antifeedant,further research is required to increase the persistence of theeffective formulation by adding some photostabilizers or throughmicroencapsulation and then application of effective formulationin grain stores in integration with other methods of rodent control.

Acknowledgements

Authors are thankful to the Professor & Head, Department ofZoology, Punjab Agricultural University, Ludhiana for the facilitiesprovided and Indian Council of Agricultural Research (ICAR-52),New Delhi for providing financial assistance.

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Effectiveness and persistence of cinnamic aldehyde as an antifeedant on rats under storage conditions1. Introduction2. Materials and methods2.1. Determination of effective concentration of cinnamic aldehyde2.2. Determination of primary repellent effect of cinnamic aldehyde2.3. Determination of development of habituation in rats to effective dose of cinnamic aldehyde2.4. Calculation of antifeedant index (AFI)2.5. Development and testing of formulations using effective concentration of cinnamic aldehyde2.6. Maze experiments under camera connected to ethovision video tracking system2.7. Experiments in feed scale consumption monitor2.8. Statistical analyses

3. Results and discussion3.1. Determination of effective concentration of cinnamic aldehyde3.2. Determination of primary repellent effect of cinnamic aldehyde3.3. Determination of development of habituation to effective dose of cinnamic aldehyde3.4. Testing of formulations developed using effective concentration of cinnamic aldehyde3.5. Repellent behaviour in response to formulation III of cinnamic aldehyde in I-maze3.6. Antifeedant behaviour in response to formulation III of cinnamic aldehyde in the feed scale consumption monitor

AcknowledgementsReferences