TOXICOLOGICAL STUDIES ON BORIC ACID, IMIDACLOPRID AND FIPRONIL AND THEIR BINARY MIXTURES AS INSECTICIDES ON GERMAN COCKROACH Blattellagermanica (L.) (DICTYOPTERA: BLATTELLIDAE)

By

FATMA SHERIF AHMED B.Sc. Agric. Sci. (Pesticides), Fac. Agric., Cairo Univ., 2007

THESIS Submitted in Partial Fulfillment of the Requirements for the Degree of

MASTER OF SCIENCE

In

Agricultural Sciences (Pesticides)

Department of Economic Entomology and Pesticides Faculty of Agriculture Cairo University EGYPT

2015

١ INTRODUCTION The German cockroaches, Blattellagermanica (L.), (Dictyoptera, Blattellidae) are the most common indoor pests, especially in multiple-family housing and the most significant pest in many parts of the world (Goddard, 2003). German cockroaches prefer warm, wet locations with high humidity such as kitchens, bathrooms and laundry areas. These conditions are available in several places as homes, apartments, restaurants, supermarkets, hospitals and other buildings where food are stored. Cockroaches are not only corrupt food but also transfer pathogens such as Salmonella, Shigella, Escherichia coli, Staphylococcus aureus and Bacillus cereus (Baumholtz et al., 1997 and Tachbeleet al., 2006). Medically important parasites such as bacteria, fungi and molds, protozoans, viruses were isolated from external and internal surface of cockroach (Brenner, 1995). Cockroaches can also transfer both gram-positive and negative bacteria (El-Sherbini and El- Sherbini, 2011). A large number of neurotoxic and non-neurotoxic insecticides were used for German cockroach control, as this pest has a considerable ability to develop resistance to a variety of chemical insecticides(Cochran, 1989 and 1995a; Scott et al., 1990; Rust and Reierson, 1991; Rust et al., 1993; Holbrook et al., 1999; Espinosa-Islas et al., 2002 and Rahayuet al., 2012). The toxicity of some new neurotoxic insecticides is either very limited (i.e., neonicotinoids) or not well established (i.e.,phenylpyrazole) against German cockroach in Egypt.

Boric acid (H3BO3) has been used as an insecticide since 1948 (See et al., 2010). It was the standard chemical used for the control of some pests until the chlorinated hydrocarbon pesticides become available (Lal and Srivastava, 1950). Moreover, it is not classified as carcinogen or skin irritant (Langford et al., 1954). One advantage of using boric acid is that, it destroys the cellular lining of the foregut. This effect induces death to the insects by starvation and

٢ the cockroaches have difficult to show any resistance to boric acid (Cochran, 1995b). A phenylpyrazole insecticide fipronil discovered by Bayer Crop Science in 1987, and introduced in 1993 as a form of solid (e.g., insect bait), liquid spray, or as a granular product (Anonymous, 2000), and registered in the U.S. in 1996 (Ware, 2000 and Jeong Lee et al., 2010). It controls cockroaches, mosquitoes, ticks, and fleas at both larval and adult stages (Chantonet al., 2002). Cole et al. (1993) found that fipronil interferes with the δ-aminobutyric acid (GABA) channel, where it disrupts normal nerve influx transmission (e.g., passage of chloride ions) this causes excessive neural excitation, paralysis, and finally insect death (Gant et al., 1998). Fipronil gel baits have been widely used to control German cockroach in America since several years due to german cockroach resistance to several insecticides and fipronil gel bait consider more safe in application than other treatment methods (Ahmed and Suliyat, 2011). A neonicotinoid insecticide, imidacloprid belonging to the chloronicotinyl compounds. It was first proposed in 1991. It exerts toxic effects after oral ingestion; it is also suitable for use in bait formulations (Nasirian, 2007). The main factor of chloronicotinyl being favorable is their low affinity to vertebrate compared to insect nicotinic receptors. They have high selectivity; have a quick knock down effect and long residual activity. Worldwide, it is considered to be one of the insecticides used in the largest volume (Cox, 2001). Nasirian (2008) reported that fipronil and imidacloprid gel baits completely killed the German cockroaches under laboratory conditions in ingested bait method. Poisoned baits, in the last two decades, have largely displaced other formulations for controlling of German cockroaches and other pests (Anaclerio and Molinari, 2012). The poisoned baits are much less translocatable, so it is less hazardous than the spray formulations (Gore and Schal, 2004). So that the tested insecticides used as a bait to be safer and environmentally friendly than

٣ insecticide sprays. The toxicity of fipronil baits has been estimated from mortality rates (Scott, 1991 and Ross, 1993) and consumption rate (Wang et al., 2006). The three tested compounds (boric acid, imidacloprid and fipronil) were selected on the basis of their high efficacy against German cockroach. Although fipronil hasn't been used in Egypt against German cockroach yet, it widely used in all developed countries as a highly effective tool for controlling the German cockroach especially in bait form. The present study evaluated the efficacy of fipronil, imidacloprid and boric acid on the nymphal instars and adult males of German cockroach. On the other hand, German cockroach didn't appear to be resistant to boric acid compound although this compound is used against this insect for ages. Also, the application of neonicotinoid insecticide imidacloprid in bait form is easy to apply in out-of- way places and is stable under a wide range of conditions. The objectives of current study are: 1. Determination of the oral toxicity of boric acid, imidacloprid and fipronil as poisoned baits against different stages of B. germanica (L.). 2. Evaluation of the palatability and the toxicity of fipronil incorporated into different food baits on the 6th instar nymphs of German cockroach B. germanica (L.). 3. Comparing the toxicity of imidacloprid as topical application and stomach poisoned bait against the different stages of the tested insect.

4. Estimation of the lethal times (LT50) of the two tested compounds on the different stages. 5. Evaluation of the effect of binary mixtures of the three tested insecticides on 6th instar nymphs.

٤ MATERIALS AND METHODS

Tested insect (Rearing and Manipulation the Insect): German cockroach, BlattellaGermanica (L.) Family: Blattellidae Order: Dictyoptera Cockroaches are insects with three stages: eggs, nymphs, and adults, males and females. About one week after mating, the female cockroach deposits eggs into a protective covering called the oetheca or egg case which protects the eggs from drying. The egg case looks like a tiny, brown, purse-shaped capsule, it is about 8 mm long, 3 mm high, and 2 mm wide (Tanaka, 1976). The eggs hatch into a small but fully-developed nymphs that grow through a series of molts, shedding its old cuticle (exoskeleton) at each molt. The range of head capsule widths in the lst to 6th instars nymphs are, respectively: 0.74-0.85 mm; 0.88-1.05 mm; 1.02-1.35 mm; 1.20-1.62 mm; 1.40-1.86 mm; and 1.84-2.25 mm (Murray, 1967). Nymphal survival percentages from egg hatch to the adult stage was estimated at 85- 87% (Willis et al., 1958 and Archboldet al., 1987). The nymphs of cockroaches emerge as fully-winged adults after the final sixth molt. Newly emerged adults appear white, but become fully pigmented within 24 hours (Ross and Mullins, 1995). As the nymphs develop faster, they gain more body mass and the adults are heavier, the females produce more fertile oethecae (Gemenoet al., 2001). German cockroaches are semi-social insects; all their stages and ages are found at the same place. Colony of German cockroach BlattellaGermanica (L.) used for the present study was obtained from Aburwash, Giza Governorate, reared for two years and the all bioassay tests were carried out in the Department of Economic Entomology and Pesticides, Faculty of Agriculture, Cairo University, Egypt. The average number of (eggs per capsule, capsules per female), period between egg case formation to hatching, number of molts,

٥ nymphal stage (in days), life cycle and average longevity of adults (in days) are given in the following.

Cockroaches insects were reared in plastic containers (1 liter capacity) and maintained at 27 ± 1°C, 60±5% relative humidity in an incubator at a photoperiod of 12:12 dark-light. A wet piece of cotton was placed in each jar as a water source for the cockroaches with some of modifications as described by (Nalyanya and Schal, 2001; Nasirianet al., 2006a and 2011; Sulaimanet al., 2007 and Kilani-Morakchiet al., 2009). The cockroaches for rearing were fed on a source of protein and a sugar solution coated on a piece of cotton, and a cardboard or a plastic tubes as a shelter (Nasirianet al., 2006a,c). The present investigations were carried out on the 2nd, 6th instar nymphs (two days old) and adult males (2-3weeks old). Only adult males, not adult females, were used in the experiments because their weight and physiology are more uniform than adult female (Appelet al., 1983). The females were also needed for further reproduction. Insecticides tested: Three commercial formulations were used in the toxicological study. These compounds are representing boric acid (inorganic compound), imidacloprid(neonicotinoid insecticide group) and fipronil (phenylpyrazole group). The common name, trade name and properties of the tested compounds (boric acid, imidacloprid and fipronil) are given in Table 2. a. Boric acid insecticide:

Boric acid (H3BO3) was used as an insecticide for control of cockroaches, termites, fire ants, fleas, silverfish, and many other insects(See et al., 2010). It is considered to be safe to use in household kitchens to control cockroaches and ants.The dry powder is abrasive to the insect and may also have some toxic effects on the nervous system of insects (Habeset al., 2005). It acts as a stomach poison (affecting the insect’s metabolism) and as a contact poison.

٦ b. Imidacloprid insecticide: Imidacloprid is an insecticide belonging to the neonicotinoid class of compounds. It was first proposed in 1991 and registered for use by the United States Environmental Protection Agency (U.S. EPA) in 1994. It is used to control a wide variety of agricultural, urban, public health, and veterinary pests and is estimated to account for 11-15% of the total global insecticide market (Tomizawa and Casida, 2005). c. Fipronil insecticide: Fipronil is a phenylpyrazole insecticide discovered by Bayer Crop Science Company), and introduced as form of solid (e.g., insect bait), liquid spray, or as a granular product (Anonymous, 2000). It was registered in the U.S. in 1996 (Ware, 2000; Connelly, 2001 and Jeong Lee et al., 2010). It was made available in 1999 in bait formulations for use against the German cockroach, BlattellaGermanica(L.). Tested application treatments: a. Toxicity studies of fipronil, imidacloprid and boric acid against the 6thnymphal stage: The three tested compounds fipronil, imidacloprid and boric acid were tested to know their toxicity on the last 6thnymphal stage of German cockroach, B. Germanica. Each compound was incorporated into the diet at different gradual concentrations. Experimental baits with 0.1% sodium benzoate to prevent fungal growth were placed in each glass jar (in control diet and treated diet jars). All tested insecticides used as bait were prepared in water solution that it is important to note that cockroaches drink regulatory and survive longer in higher humidity than in lower humidity percentage (Cochran, 1983 and Dambach&Goehlen, 1999).

Cockroaches were not fed before any experiments for 3 days in order to increase their hungry level and optimize their feeding on the toxic baits

٧ (Durierand Rivault, 2000). According to Young et al. (1999) nymphs consumed a large ration of food after being starved, then it return to the normal feeding pattern, and quickly resume normal development. Boric acid, imidacloprid and fipronil were separately incorporated with diet and orally administrated to the tested cockroaches. Eight different gradual concentrations of boric acid (1.25, 3.5, 7.0, 12.5, 19.0, 25.0, 37.5 and 50%) were tested. Also imidacloprid was incorporated into diet and orally administered at different diluted seven concentrations (1250, 2500, 5000, 10000, 15000, 20000 and 28000 ppm a.i.). Fipronil was also incorporated into the diet at 9 serial diluted concentrations 1.5, 3.0, 6.0, 12.5, 25.0, 50.0, 75.0, 100 and 125 ppm a.i. Each concentration of boric acid, imidacloprid and fipronil was applied separately to 30 individuals of the last nymphal (6th instar nymph) stage of B. Germanica (L.) divided into three replications (10 individuals/ replicate). The tested nymphs were exposed to the poisoned baits for 7 days in case of boric acid and imidacloprid, but for fipronil the exposure period to the poisoned bait was only 3 days. The check cockroaches nymphs (control group) provided with untreated diets (beef+ powdered milk+ sugar solution), coated on a piece of cotton and cardboard on a plastic tubes as a shelter (Nasirianet al., 2006c). Thirty untreated 6th instar nymphs (control groups) were divided into 3 replicates (10 individuals/ replicate) and provided with untreated food and lifted for 3 or 7 days, after these periods the dead nymphs were recorded. The mortality percentages in the treated nymphs groups (i.e.fipronil, imidacloprid and boric acid) were also recorded and corrected for the natural mortalities by using the Abbott’s formula (Abbott, 1925) and calculated to obtain the Net % kill as following formula:

٨ X - Y Net % kill = ------× 100 100- Y

Where: X= Mortality percentage in treatment. Y= Mortality percentage in control group

The LC50 and LC90 values were calculated. b. Palatability of tested baits treated with fipronil on 6thnymphal stage: In this study, four laboratory prepared baits were used. These baits contain different components and administrated to the cockroaches to choice the most palatable and toxic baits depending on their consumption rate and mortality percentages. Such baits were: a. Beef bait: (beef, powdered milk and sugar 2:1:1, weight/ weight). b. Onion bait: (onion, sugar, flour and industrial margarine 2:1:1:2, w/w). c. Strawberry gel bait: (strawberry gel, sugar and flour 2:1:1, w/w). d. Sweet honey bait: (sweet honey, rusk and flour 2:1:1, w/w).

The palatability of the four tested different baits (i.e. beef, onion, strawberry gel or sweet honey bait) treated with fipronil was estimated by calculating consumption rate and mortality percentage. The consumption rate was conducted by weighing the tested bait before and after treatment. Fipronil was selected on the base of being highly effective insecticide against German cockroach depending on the bioassay tests in the present study. The mortality was conducted from adding the LC50 and LC90 value of fipronil, 22.5 and 107.5 ppm, respectively, as calculated by the Probit toxicity line. Thirty insects of last nymphal stage or adult males of B. Germanica were transferred to each treatment and divided into three replicates (10 individuals for each) for the different concentrations of fipronil were conducted. The control insects were divided into two groups, the first one had each type of the tested diets with cockroaches, and the second control group had the diet without cockroaches for estimation of water loss to be excluded from the

٩ consumption rate (Wang et al., 2006). Consumption rate of bait was calculated by the following:

(W1- W2) - WC2 Consumption rate % = ------X 100 Wc1

Where, W1 and W2 are the weight of bait treatment groups before and after exposure to cockroaches, respectively. Wc1 is the weight of bait in control groups with untreated bait, whereas Wc2 is the weight of tested diets without cockroaches for estimation of water loss. c. Toxicity of the three tested insecticides on different nymphal stage and adult males:

As mentioned before the selected individuals, nymphs and adult males were left to habituate for 3 days before the treatments. Fipronil was evaluated on 3 different instars of nymphal stage (2nd, 4th and 6th instar) or adult males of the German cockroach. The methods of treatments were the incorporation into diet technique. Fipronil was prepared at 9 serial concentrations (1.5- 125 ppm) against 2nd, 4th and 6th instar nymphal stage and adult males. Thirty individuals were divided into three replicates, 10 cockroaches for each. Control groups received only untreated diet. The mortality percentages were recorded after 24, and 72 hours. The LC50 and LC90 were conducted after 24 and 72 h. Imidacloprid and boric acid compounds were tested against different stages, 2nd and 6th instar nymphs and adult males of B. Germanica. Thirty individuals of cockroaches were transferred to each treatment in 3 replicates. Each replicate of 10 cockroaches was hold in a glass jar covered with muslin and rubber band. Boric acid was incorporated only as a stomach poison into the diet and orally administrated at eight different concentrations from 1.25 to 50.0%.

١٠ Imidacloprid was evaluated on three different stages of the German cockroach (2nd and 6th instar nymphs and adult males), using two techniques 1) orally treatment by incorporating into the treated food baits, and 2) obligatory contact with treated glass-jars. Thirty of 2nd and 6th instar nymphs or adult males of B. Germanica were selected to each treatment, divided into three replicates (10 individuals for each) of the different concentrations. For application of imidacloprid insecticide as a stomach poison, it was incorporated into the diet and orally administrated at 7 different diluted concentrations (2500- 28000 ppm) against 2nd and last nymphal stage and adult males of the German cockroach. The insects were exposed to the insecticide for 7 days, after that the insects was allowed to eat untreated food for other 7 days.

The LC50 and LC90 were estimated after 7 and 14 days, respectively. For application of imidacloprid insecticide as a contact poison, the inside surface of jars (1 liter capacity) was covered with imidacloprid by residual film technique. Different concentrations of imidacloprid were prepared by dilution with acetone (10 ml for each replicate), the treated jars were rolled horizontally until all of the acetone had evaporated, so that the insecticide was deposited evenly over the inner surface of the jar (Nasirianet al., 2006c). The insects were exposed to the treated jars with 7 different diluted concentrations (1250- 28000 ppm). The insects were exposed to the treated jars for 7 days, then the survival individuals were moved to clean jars and exposed to untreated diet for other 7 days. The LC50, LC90 were estimated after 7 and 14 days, respectively.

Determination of the lethal time concentrations (LT50 and LT90 values):

The LT50 and LT95 values were estimated every two days for the different concentrations after treatments along two weeks (i.e. 3, 5, 7, 9, 11 and 14 days) and recoded to measure the effect of the period of exposure (lethal time concentrations) in the tests of boric acid and imidacloprid insecticides against the 2nd and 6th instars nymphs and adult males of B. Germanica (L.). The insects

١١ were exposed to the treated baits for 7 days, after that the survival individuals were transferred to clean jars and exposed to untreated diet. The mortality responses were recorded after 7 and 14 days and the LC50, LC90 were estimated. Control groups were fed only on untreated diet.

Effect of binary mixture of the three different tested insecticides against the 6thnymphal stage of German cockroach:

In order to assess the joint action of the tested compounds (fipronil, imidacloprid and boric acid), different binary combinations of these compounds th at their LC25 values were tested on the 6 instar nymphs. The binary mixtures were prepared before treatment at ratios of 1: 2, 1: 1 and 2: 1. For each treatment, there were 30 insects in three replicates (10 individuals for each) were used. The mortality percent of each mixture were recorded after 6 days as observed mortality. The combined action of the different mixtures was expressed as the co- toxicity factor which was estimated according to the equation given by Mansour et al., (1966). Observed mortality – Expected mortality Co- Toxicity factor = ------X100 Expected mortality

Generally, the co- toxicity factor of value 20 or above indicates potentiation effect, while the negative co-toxicity values from -20 or less indicates antagonism (inhibition) effect. The co- toxicity factor values between > -20 and < 20 indicates an additive effect. Statistical analysis of the obtained data The mortalities of tested insects were recorded for fipronil after 24 and 72 h, but for boric acid and imidacloprid after 7 and 14 days. All mortalities were

١٢ corrected using Abbott’s formula (Abbott, 1925). The LC50 and LC90 were estimated after treatments of tested compounds. Also, consumption rate of the untreated and poisoned baits was calculated by the formula of (Wang et al., 2006).

The lethal time (LT50 and LT90) were also estimated for the different concentrations of the tested insecticides, boric acid and imidacloprid and recoded after 14 days. The LC50 was estimated every two days after the treatment of boric acid and imidacloprid through two weeks after treatments and plotted to measure the significance of the increased period of exposure. The combined action of the different binary mixtures was expressed as the co-toxicity factor which was estimated according to the equation given by Mansour et al. (1966). Results are expressed as means ± standard error (SE). The significance between control and treated series was estimated using t-test at 5% level. The toxicity lines were plotted according to Finney analysis (Finny, 1971). Results from all treatments were tested with χ2 (calculated Chi – Square test) for Heterogeneity. The software program Micro origin was used for plotting the histograms.

RESULTS

1. Evaluation of different poisoned baits on the last nymphal stage (6th) of german cockroach B. germanica

a. Determination of the toxicity (Bioassay tests) of fipronil, imidacloprid and boric acid against the last nymphal stage of B. germanica(L.)

According to the LC50 and LC90 values, it could be generally concluded that fipronil proved to be highly toxicagainst German cockroaches, which gave the lowest LC50 and LC90 (22.5 and 107.5 ppm, respectively) followed

١٣ ascending by imidacloprid (9228 and 24980 ppm, respectively) and then boric acid (9.87% and 46.3 %, respectively).

b. The food consumption rates and mortality percentages of different components baits: Four different baits contains different food constituents were tested for evaluate their attractancy and mortality percentages resulted by adding either the LC50 or LC90 of fipronil. At the LC90 level, the beef bait with fipronil caused the highest rate of consumption (97.3%) and the highest mortality percentage (93.3%). According to the calculated mortality percentage, the baits effectiveness at the LC90 level of fipronil could be arranged in a descending order as follow: beef bait (93.3%) onion bait (80.0%)> strawberry gel bait

(60.0%)> sweet honey bait (46.7%).˃Finally, it could be concluded that the beef bait had the highest consumption rate and mortality%. So beef poisoned bait could be recommended to control german cockroaches especially in high infested area.

2. Toxicity of fipronil as a poisoned bait against different nymphal instars (2nd,4th and 6th) and adult males of german cockroach B. germanica after 24 and 72 hours of exposure.

nd For 2 instar nymph, the LC50 and LC90 values were 9.5 and 44.9 ppm after 24 hours, these values were 2.3 and 24.7 ppm after 72 hours, respectively. th For 4 instar nymphs, the LC50 and LC90 values after 24 hours were 15.9 and 69.8 ppm and they were 6.5 and 56.7 ppm after 72 hours, respectively. For the th last 6 instar nymph, the LC50 and LC90 were 22.5 and 107.5 ppm after 24 hours and these were 12.5 and 53.9 ppm after 72 hours, respectively.

The LC50 and LC90 of fipronil against German cockroach adult males were 38.9 and 221.5 ppm after 24 hours, and they were 14.2 and 107.6 ppm after72 hours, respectively.

١٤ nd The median lethal time LT50 of fipronil against 2 instar nymph was 37.4, 12.9 and 6.6 hours for the concentrations of 6, 12.5 and 25 ppm, respectively. th For the 4 instar nymphs, The LT50 value of concentrations 12.5, 25 and 50 ppm were 42.9, 16.7 and 8.4, 33.9 h, respectively. For the 6th instar nymphs, The

LT50 value of concentrations 25, 50, 75 and 100 was 22.7, 15.05, 13.02, and 6.63 h, respectively.

For adult males, The LT50 values of concentrations 25, 50, 75 and 100 ppm were 42.16, 28.4, 20.23 and 15.15 hours, respectively.

3. Toxicity of imidacloprid against different stages of german cockroach B. germanica a. Assessing mortalities after 7 days from treatment. (1) as a stomach poison nd The LC50 and the LC90 of imidacloprid against 2 instar nymphs were 4604 and 16261 ppm, respectively. These values against 6th instar nymphs were 9228 and 24980 ppm, respectively.

The LC50 and LC90 values of imidacloprid as a stomach poison against adult males were 5441 and 17777 ppm, respectively. (2) as a contact poison nd The LC50 and the LC90 of imidacloprid against 2 instar nymphs were 3740 ppm and 13108 ppm, respectively. These values against last nymphal stage were 5646 and 32350 ppm, respectively.

The LC50 and LC90 of imidacloprid as a contact poison against adult males were 4350 and 14009 ppm, respectively. b. The mortalities of treated cockroaches after 14 days. (1) as a stomach poison nd For the 2 instar nymphs, the LC50 and LC90 after 14 days were 1539 and 5413 ppm, respectively. These values against the 6th instar nymphs were 3656 and 10659 ppm, respectively.

١٥ The LC50 and LC90 of imidacloprid as a stomach poison against adult males were 2881 and 10565 ppm, respectively. (2) as a contact poison nd The LC50 and LC90 of imidacloprid against 2 instar nymphs after 14 days were 920 and 3714 ppm, respectively. These values against the last nymphal stage after 14 days were 2566 and 11618 ppm, respectively.

The LC50 and LC90 of imidacloprid as a contact poison against adult males were 1844 and 5090 ppm, respectively.

4. Toxicity of boric acid against different stages of german cockroach B. germanica.

After 7 days of exposure, the LC50 and LC90 values of boric acid against the 2nd instar nymphs were 8.82 % and 35.38 %, respectively. These values against 6th instar nymphs were 9.87% and 46.34 %, respectively. The LC50 and LC90 (%) of boric acid against the adult males were 12.3% and 77.3%, respectively, after seven days of inspection.

nd The LC50 and LC90 of boric acid against the 2 instar nymph After 14 days of exposure were 3.14% and 11.96%, respectively. These values against the 6th instar nymph were 4.7% and 17.6 %, respectively. The LC50 and LC90 of boric acid against adult males were 6.97% and 23.71%, respectively.

The toxicity of boric acid after 14 days could be arranged at a descending order as follow, 2nd instar nymphs 6th instar nymphs adult males. 5. The lethal effect (LT ) of boric acid and imidacloprid during 14 days 50 ˃ ˃ after treatment.

a. The lethal effect (LT50) of boric acid The concentrations of 19.0, 25.0 and 37.5 % of boric acid lasted 2.99, 2.63 and 2.48 days to kill 50% of the treated 2nd instar nymphs.

١٦ The concentrations of 19.0, 25.0 and 37.5 % caused 50 % mortality to 6th instar nymphs after 3.76, 2.95 and 2.62 days, respectively. Boric acid lasted 5.42, 3.22 and 2.77 days to kill the half treated adult males for the concentrations of 19.0, 25.0 and 37.5 %, respectively.

b. The median lethal time (LT50) of imidacloprid nd The LT50 of imidacloprid as a contact poison against 2 instar nymph after 14 days were 4.72, 2.99, and 1.94 days for the concentrations of 5000, 10000, and 15000 ppm. These values were 5.99, 3.98 and 3.21 days for the same concentrations of imidacloprid as a contact poison against 6th instar nymphs, respectively. The LT50 values of imidacloprid as a contact poison against adult males were 5.75, 3.25 and 2.65 days, respectively. nd The LT50 of imidacloprid as a stomach poison against 2 instar nymph after 14 days were 5.30, 3.85 and 3.16 days for the concentrations of 5000, 10000, and 15000 ppm, respectively. These values were 10.65, 5.81 and 3.79 days, respectively, when imidacloprid treated as a stomach poison against 6th instar nymphs. These values were 96, 4.28 and 3.38 days, respectively, when imidacloprid treated as a stomach poison against adult males. The toxicity of Imidacloprid as a stomach poison could be arranged by ascending order as follow: 2nd instar nymphs adult males 6th instar nymphs.

The same order of toxicity could be followed˃ when imidacloprid˃ treated as a contact poison.

6. Joint action of binary mixtures of the three tested insecticides against the last nymphal stage of German cockroach

The joint action of the three compounds was tested by adding the LC25 of each according to the following ratio 1:1, 1:2 and 2:1. The co-toxicity factor was calculated for all mixtures to know their joint action.

١٧ a. Boric acid and fipronil mixtures: The mixture of boric acid and fipronil at ratio of 1:1 show a potentiation effect. On the other hand this mixture showed an additive effect at ratio of 1:2, but it produced antagonism effect at ratio of 2:1. b. Boric acid and imidacloprid mixtures: The ratio of 1: 2 of boric acid+ imidacloprid mixture showed an additive effect. The other two tested ratios of boric acid and imidacloprid mixtures (1:1 and 2:1) showed an antagonism effect, the co-toxicity factors were -42.86 and - 52.4, respectively. c. Fipronil and imidacloprid mixtures: The joint action of the obtained results of that all fipronil and imidacloprid mixtures at ratios of 1:1, 2:1 and 1:2 led to an antagonism effect. The co-toxicity factor was calculated for all mixtures to know their joint action. Out of the nine tested mixtures the fipronil+ boric acid mixture at ratio 1:1 had a potentiation effect. The rest mixtures; two of them had an additive effect however six mixtures had an antagonism effect. In conclusion, our findings showed the efficacy of fipronil for control of cockroaches in laboratory after 24 hrs of exposure. The application of fipronil bait was not only found more convenient but was also easy to apply (and was very stable under a wide range of conditions) and there was minimal disruption of the home or workplaces. Treatment with binary mixture of fipronil with boric acid 1:1 as a cockroach bait was found to be highly efficacious and safe for the user and the local environment.

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