Efficacy of several insecticide formulations against Periplaneta americana (L.) (Blattaria: Blattidae) in sewers

Objective: Periplaneta americana (L.) is the most important invasive urban pests in sewers with high significance of public health. The aim of the study was to evaluate the efficacy of several insecticide formulations against American cockroaches in sewers. Methods: The population density of cockroaches was estimated before and after 1 and 5 months of the use of insecticides in sewer manholes. About 10 manholes were allocated for treatment of each insecticide formulation. Twenty manholes were allocated to controls (without the use of insecticides). Insecticides were considered effective achieving more than of 90% mean density reduction of cockroach population per a manhole. Results: The effectiveness of insecticide formulations in sewer manholes were considered effective after 1 month of insecticide treatments while the effectiveness of diazinon EC 5% and 0.05% formulations considered insufficient including 47.% and 19.5%mean density reduction of cockroaches, respectively. The effectiveness of chlorpyrifos EC 5% and Fog, and diazinon EC 5% were observed about 80.5%, 81.5%and 53.5% mean density reduction of cockroaches, respectively after 5 months of treatment while the other formulations did not achieve effectiveness. Conclusions: The insecticide formulations were considered effective and ineffective after 1 and 5 months of the use of insecticides, respectively. It would be more satisfied if insecticide formulations repeated every three months. A reasonable manner is using a combination of integrated pest management strategies recommending for successful P. americana control.


Introduction
Cockroaches demonstrate to have miraculous adaptation to a wide range of habitats as important pest of various public places, especially in areas of the tropics and subtropics. In addition to the great medical importance included potential vectors of the opportunistic or pathogenic bacteria and fungi found in drug resistant nosocomial infections and foodborne poisoning their involving as intestinal parasites of human and intermediate hosts, and playing a role as allergens may cause asthma and other long-term health issues in human [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16] . Cockroaches are one of the substantial insect pests in urban sewers 17 . Among the most common of worldwide cockroaches  10,18,19 .The appropriate conditions of the sewers such as presence of plenty of food and water, and suitable darkness, humidity and temperature have also caused P. americana cockroach as the most important pests of sewer environments. Currently, the American cockroaches are considered the most frequent species in sewers. Periplaneta americana colonize in sewers as suitable environments for its growth and reproduction with high significance of human public health. Although, researchers are achieving new methods, and effective and appropriate insecticides for control of cockroaches 22 . In sewers the use of spraying insecticides is the most common method. Fogging and fire have been also used to control the American cockroaches in sewers. Because of the high population density of the American cockroaches in Esfahan sewer as a suitable and warm environment, the determination of an appropriate and effective insecticide formulation is necessary for their control. Insecticide formulations of baits, dusts, and sprays have been applied for control of the American cockroaches. In this regard few studies are available about the formulation efficacy of insecticides for control of the American cockroaches in sewers 20,21,23,24 . Therefore, a study designs to evaluate the efficacy of insecticide formulations against the American cockroaches in sewer of Esfahan. The aim of the study was to evaluate the efficacy of insecticide formulations against P. americana cockroaches in Esfahan sewer.

Materials and methods; Study area
Esfahan is an area of 250 km 2 with 1,695,789 residents. Its altitude is 1,571 m, latitude 32° 38' 30'' N and longitude 51° 39' 40'' E. After Tehran, Esfahan is the second most populous metropolitan city located in a dry region in the central of Iran. It has a semidesert climate with an annual rainfall of 102 mm, average summer maximum and winter minimum temperatures of 37.1°C and -5.8°C, respectively 25 . Description and site selection Esfahan sewer have over 55,375 manholes with an average depth of 9 meter on the main lines and 1 meter on lateral lines. Most of the manhole covers were cast irons with two holes, and the average diameter of the holes was three cm. The shafts of the manholes were made of brick and cement coverings, and their stables moderate warmth and high humidity which provide excellent conditions for cockroaches. According to the field surveys, the most abundant cockroaches were reported from the central regions of Isfahan sewer. The sewer system in the central regions also was old and the most popular complaints were from the central regions. Therefore, three areas were selected from the central regions of Isfahan sewer with permission from Esfahan's Water & Wastewater Company. In total, 164 manholes were selected for this study.

1.1.
Insecticide application 1.1.1. Cockroach density estimation The population density of cockroaches per manhole was estimated periodically before, and after 1 and 5 months of treatment with insecticide formulations. The cast-iron covers were removed from each manhole, then the adults and nymphs of cockroaches were counted with a strong flashlight and recorded. The shafts with more than 300 cockroaches were estimated by counting of the 0.25 m 2 shaft section and multiplying by the number of infested sections. 1.1.2. Insecticide application procedure About 10 manholes were allocated for treatment of each insecticide formulation. Each sewer shaft was randomly assigned to one insecticide formulation treatment in an ascending order after estimation of cockroach population density and coding the treatment method with paint on the lower part of the wall near the manhole cover. The manhole shafts were then inspected for cockroach infestation after 1 and 5 months of treatment. All of the insecticide formulations were prepared according to Bueno-Marí et al. (2013) description 21 . Aqueous sprays were applied with HUDSON X-Pert®110 power compression sprayer equipped with a nozzle, flexible hose, stainless steel tubular spray tip extension on the handle grip, delivering 757 cm 3 /minute. The extension tube was lowered to the bottom of the shaft and the tip was raised upwards at a uniform rate of 0.5 m/s while the spray was applied. A 5% spray, therefore delivered about two grams of the active ingredient/m 2 . Boric acid baits in petri dishes were placed in a dry ledge at the bottom of the shaft. Thermal fog with solutions were applied from a "Light-weight Model" TF 30. The TF 30 was operated at a combustion chamber temperature of 600 °C, a dial flow setting of four liters/hour. Each manhole was treated for five seconds per m 3 . Fire with oil (10 g/m 2 ) was used to burn manhole shafts. Fire was applied with HODSON X-Pert®110 filled with oil, the extension tube was lowered almost to the bottom of the shaft and spray was applied raising the tip upwards at a uniform rate of 10 g/m 2 . Oxygen also was blown into the manhole with an electrical fan equipped with a flexible hose. Twenty manholes were allocated to controls (untreated) which were not used any insecticide formulations there.

1.2.
Statistical analysis IBM SPSS Statistics Data Editor Version 24 was used for statistical analyses. Analysis of variance (ANOVA) was used to compare the insecticides and formulations. For any significant of ANOVA, Post Hoc Tests (Tukey) analysis was used to compare the insecticides with control (untreated) and between formulation types. Population density of P. americana cockroaches before and after treating of insecticide formulations in manholes of sewer and approximate efficacy of insecticide formulations against P. americana were calculated using Microsoft office excel 2013. P < 0.05 was considered significant. To estimate the approximate efficacy of the insecticide formulations, an equation was designed and calculated as follows: Approximate efficacy = 10 × (t -c)/(10c) Where is percentage treatment density reduction and c is percentage control (untreated) density. Insecticide formulations were considered effective when more than 90% of cockroach population density reduction was achieved after 1 or 5 months treatment in the manholes of sewer.

Density of cockroaches
In the present study, the efficacy of several insecticide formulations was investigated against American cockroaches in Isfahan sewer. Tables 1-2show the data from the application of insecticide formulations against P.americana in the manholes of sewer. The population density of P. americana cockroaches which was estimated about between 66 and 282 cockroaches before treatment in the manholes of sewer while after 1 month of treatment with the insecticide formulations (except boric acid bait) was decreased to about ≤ 10 cockroaches. The cockroach density in the manholes which was treated with boric acid bait decreased from 106-115 before treatment to 15-20 after 1 month of treatment. But the cockroach adult density was decreased from 20-26 to < 2 cockroaches. The density of cockroaches after 5 months of treatment with the insecticide formulations were different results. The density of cockroaches decreased for a few insecticide formulations, did not change for some insecticide formulations and increased for some insecticide formulations (Tables 1-2).     Efficacy of several insecticide formulations against Periplaneta americana (L.) (Blattaria: Blattidae) in sewers Tables3-4 show the population density of P. americana cockroaches before and after application of insecticide formulations in the manholes of sewer. The mean population density of P. americana cockroaches in the manholes of sewer which was estimated from 7.7 to 20.0% before treatment while was decreased to about ≤ 3.0% after 1 month of treatment with insecticide formulations (except boric acid bait). The mean cockroach density decreased from 20.0% before treatment to 16.1% after 1 month of treatment in the manholes which was treated with boric acid bait. But the mean cockroach adult density which were treated with boric acid bait was decreased from 20% to 3.5%. The mean density of cockroaches after 5 months of treatment with chlorpyrifos Fog, diazinon EC 5%, diazinonEC 0.05% and chlorpyrifosEC 5% formulations decreased from 10.0, 7.7, 20.0 and 8.3% to 1.6, 2.1, 6.8 and 0%, while the mean population density of P. americana cockroaches which was treated with the other insecticide formulations did not change (Tables3-4).          Table 5 shows the approximate efficacy of insecticide formulations against P. americana in manholes of sewer. Insecticide formulations were considered effective when more than 90% of cockroach population density reduction was achieved. Although the diazinon EC5%, diazinon EC0.05% and boric acid bait formulations which their effectiveness considered insufficient including 47.0, 19.5 and 39.0%, respectively after 1 month of treatment in the manholes of sewer while the other insecticide formulations were considered effective. The effectiveness of chlorpyrifos EC5%, diazinon EC5% and chlorpyrifos Fog formulations were observed about 80.5, 53.5 and 81.5%, respectively after 5 months of treatment in the manholes of sewer while the other formulations did not achieve effectiveness (Table 5).

Analysis of insecticide applications
Tables 4-5 show the ANOVA and followed Post Hoc tests (Tukey) analysis between applications of insecticide formulations against P. americana in the manholes of sewer. There were significance differences between insecticidal effectiveness and formulation types including WP, EC, SC, Fog and Fire (P = 0.0001). After ANOVA analysis, Post Hoc tests (Tukey) showed significance differences between treated manholes with insecticides and untreated (control) manholes (P = 0.0001) ( Table  6). Post Hoc tests (Tukey) also showed significance differences between manholes which treated with diazinonEC5% and boric acid bait, and the manholes treated with the other insecticide formulations (P = 0.0001) ( Table 7).

Discussion
Like present study which was observed the P.americana, the most prevalent cockroaches in the Esfahan sewer, P. americana is the most prevalent insect pests in urban sewers 26 of areas of tropics and subtropics. Large-scale of P. americana cockroaches can invade to human apartments through breathing sewer manholes. Unlike P. americana being the most important pests of sewer, it is noteworthy that the German cockroach (Blattella germanica) is the most invading human dwellingspecies [27][28][29][30][31][32][33][34] .
Irrespective of considering insufficiency effectiveness of diazinon EC5%, diazinon EC0.05% and boric acid bait formulations observed 47.0, 19.5 and 39.0 percent efficacy, respectively after 1 month of sewer treatment which confirmed by observing a significance differences between formulation typesincluding WP, EC, SC, Fog and Fire (P = 0.0001) and then between manholes which treated with diazinonEC5% andboric acid bait, and the manholes treated with the other insecticide formulations (P = 0.0001) followed by Post Hoc tests (Tukey) ( Table 7). It may be due to insecticide resistance 35 and inadequate insecticide doses. While the other insecticide formulations were considered effective after 1 month of treatment in the manholes of sewer. These results confirmed with observing a significance differences between insecticidal effects (P = 0.0001). Post Hoc tests (Tukey) also showed a significance differences between treated manholes with insecticides and untreated (control) manholes (P = 0.0001) ( Table 6). These results concordance with results of the previous studies 21,23,36 . Obviously, the effectiveness of the insecticide formulations for 1 month control is very little time that this time should be increased.
The effectiveness of chlorpyrifos EC5%, diazinon EC 5% and chlorpyrifos Fog formulations were also observed about 80.5, 53.5 and 81.5 percent, respectively after 5 months of treatment in the manholes of sewer which they are also unsatisfied results. While the other formulations did not achieve effectiveness after 5 months of treatment in the manholes of sewer (Table 5). Even though there was a significance differences between treated manholes with insecticides and untreated (control) manholes (P = 0.0001) by Post Hoc tests (Tukey) ( Table 6). There are several solutions to solve the problem. A solution is to reduce the duration time to about 3 months. It would be expected that the results lead up to satisfied results. Paints with insecticide also provided interestingly an optimal reduction of P. americana populations for three months 21 . Another appropriate choice is to use baits particularly gel baits. Although the use of bait formulations were considered to be effective against the cockroaches 37-41 while due to their application problems in the sewer failed to provide consistent control 23 and the use of spraying insecticides is the most popular method in the sewers. An important factor that was reported to affect baits performance against American cockroaches is sanitation 42 . Although sanitation has not been having a direct relationship with the domiciliary cockroach infestation levels, its role on bait performance against American cockroaches is unknown. A reasonable strategy is using a combination of integrated pest management strategies with strong vector control management such as sanitation, spraying and bait formulations which are recommended for successful cockroach pest control including P. americana 10,40 .
The effectiveness of chlorpyrifos Fog formulation was also observed about 81.5 percent, respectively after 5 months of treatment in the manholes of sewer which they are also unsatisfied results. It would be more satisfied if it repeated every three months. Thermal fogging was observed to be the best and simplest method for cockroaches control in sewer system. As thermal fogs of this pesticide penetrated deeply into the hiding places and they were particularly useful in basements of buildings, sewers and drainage systems, it would be more successful requiring specialized equipment. The insecticide formulations are considered effective when more than 90% of cockroach population density reduction is achieved. There are many problems in the control of cockroaches with the use of pesticides. The commonly used insecticides may lead to resistant in cockroaches. To complete the American cockroach life cycle, it takes between 4-12 months. The generations of the American cockroach per year are not as many as of other cockroach species, therefore the expression of resistant genes may take more time. Many pesticides are repellents to cockroaches. Control by chemical provides only temporary control, it is recommended that it accompany by other control methods 43 .
It is also well known that efficacy and persistence of insecticides in sewers depends on several factors such as organic matter amounts, conditions of climates, the cockroach population, the presence of cockroach resistant population, and the kind of active matter and formulation. It seems emerging resistant to insecticides is the most factors for the efficacy and persistence of insecticides. Although the insecticide resistance in the German cockroaches has been well studied and emerged to a wide range of old and new insecticides including organophosphates, carbamates, pyrethroids, phenyl pyrazoles, spinosad, neonicotinoids and oxadiazines [29-32, 34, 41, 44-54] , while in the American cockroaches less studied. Recently has found P. americana cockroach resistant to malathion 35 and previously to trichlorfon and diazinon. According to the study results, aqueous wettable powder of carbaryl and diazinon sprays provides suitable results in the control of P. americana population at least for one month. However, the effects of these pesticides on the cockroaches lasted for less than five months. This may be due to the inability of the pesticides to penetrate the egg sacs (oothecae) and the long life cycle and breeding of cockroaches in winter. Some insecticides lose their effectiveness after one month of application, as we observed the population of cockroaches after 5 months not only decreased but also increased. Results show that chlorpyrifos EC 5% and diazinon EC 5% were suitable pesticides for cockroach control which provided a suboptimal reduction of P. americana population density for five months respectively. These pesticides provide 80.5 and 53.5 percent efficacy, respectively for five months. Spraying emulsion with chlorpyrifos in water (EC) provides a quick and temporary knockdown of cockroaches, and gives semi-long-term control relatively. Use of fire with oil as a physical control method which was applied caused a good efficacy against P. americana after one month. It would be more satisfied if it also repeated every three months. Because of destroying the oothecae of cockroaches, a suitable strategy in the control of cockroaches in manholes of sewers is using the fire by proper equipment. Briefly the application of insecticide formulations was considered effective after 1 month of treatment, while did not achieve effectiveness after 5 months of treatment in sewer. It would be more satisfied if it repeated every three months. As insecticide resistance occurs due to the application of insecticides in very long period without fieldmonitoring 55 . A reasonable manner is using a combination of integrated pest management strategies with strong vector control management recommending for successful cockroach pest control including P. americana.