Evaluation of surface versus total permethrin content in permethrin-treated clothing: Implications for protection against mosquitoes

ABSTRACT

Permethrin is a synthetic pyrethroid insecticide used to treat uniforms (e.g. military, foresters) and other clothing to protect people against pests (e.g., mosquitoes, ticks). Pests contacting the surface of permethrin-treated clothing (PTC) are repelled and/or killed, depending on the dose and duration of exposure. Hence, it is important to assess the amount of permethrin on the surface of clothing. Fabric swatches prepared using two commercially available permethrin treatments (Insect Shield® & Sawyer Repellant) and one laboratory created treatment (4g permethrin/L) were tested. A Martindale Abrasion and Pilling Tester (MAPT) and gas chromatography were used to assess surface permethrin content (SPC) and total permethrin content (TPC). Sawyer PTC had the highest SPC (mean ± standard error) (32.68±14.55µg/g), followed by Insect Shield® (23.35±2.71µg/g) and lab-created 4g/L permethrin (8.7±0.78µg/g). SPC (after 1000 rubs on MAPT) for Insect Shield®, Sawyer, and 4g/L permethrin groups was significantly lower than TPC (P=0.011, P<0.001, and P=0.001, respectively). The SPC/TPC relationship varied widely between permethrin treatment methods and practical implications for this are discussed. Mosquito repellency tests indicate that Insect Shield®, Sawyer, and lab-created (4g/L) permethrin-treated fabrics showed a significantly higher repellency rate than control (untreated) fabrics (P=0.001, P<0.0001, P<0.0001, respectively). While Insect Shield® had the highest repellency rate against susceptible (53%) and exposed (48%) mosquitoes, differences between groups were not significant. Repellency rates indicate SPC plus other factors (e.g. treatment method) may contribute to mosquito repellency and should be considered in risk assessments for protection against pests.

Introduction

Permethrin, a commonly used synthetic pyrethroid and pesticide active ingredient (AI), has been registered with the Environmental Protection Agency (EPA) since 1979[1]. Pyrethroids impact the central nervous system of arthropods via ingestion and/or direct contact [2] and are used as insecticides/repellents[3]. Permethrin shows a low cancer risk in humans but is toxic to fish and aquatic invertebrates[1]. Permethrin can be applied on livestock housing/transportation vehicles and clothing worn by people and/or animals[1]. The first use of permethrin on clothing as a repellent was in 1990 by the United States (US) military[1]. In the 2000s, it was approved for consumers to spray on clothing and gear such as backpacks[1]. Factory-treated permethrin-treated clothing (PTC) became available to consumers in 2003[1]. Based on our investigation, permethrin is the only insecticide approved by the EPA for clothing application. Factory-treated PTC (e.g. shirts, jackets, pants, and socks) must be marketed and labeled as such (e.g. insecticide label)[1].

Treated clothing is useful as personal protective equipment (PPE) in many outdoor occupations such as forestry, where exposure to blood feeding pests (e.g. mosquitoes, ticks) occurs at a higher rate than the public[4]. Since PTC can be worn, it is ideal for protection in forested areas[5]. Foresters often work in rural areas which may not be accessible to insecticide application equipment. Wearing PTC in such areas can help reduce mosquito (and tick) biting; however, the concentration of permethrin in PTC decreases after being washed multiple times [6–8]. Long-lasting PTC is treated with a proprietary impregnation method that helps retain efficacy. Repellency of long-lasting PTC was assessed in outdoor workers over two years[9]. Findings indicate long-lasting PTC (up to 20 washes) protected wearers from tick and mosquito bites for up to one year without retreatment[9]. The same study demonstrated no difference in control and treatment groups in levels of antibodies to Aedes albopictus Skuse saliva in the second year of PTC wear, indicating a decrease in repellency (i.e. more mosquito bites) compared to the first year of wear.

Lyme disease and Rocky Mountain spotted fever are common tick-borne diseases in the US and ca. 75% of US vector-borne disease cases are caused by ticks. Mosquitoes vector pathogens causing diseases, such as malaria, chikungunya, dengue, West Nile encephalitis, and Zika via saliva inoculated while blood feeding [4,6]. Treated clothing can be used as a barrier and repellent since ticks and mosquitoes require direct contact to spread pathogens that cause diseases [5,10]. Vector-borne diseases (> 17% of all infectious diseases) cause >700,000 global deaths annually; hence, it is important to develop methods for public health protection[7].

Mosquitoes are expanding their geographic range due, in part, to climate change [11]. One way Integrated Mosquito Management Programs (IMMP) control mosquitoes is by applying insecticides [12]. Surveillance has shown a correlation between over-application of insecticides and development of insecticide resistance (IR) in mosquitoes [12]. Pre- and post-treatment efficacy surveillance conducted by IMMPs can help determine the extent to which IR exists in a mosquito population[12]. Aedes albopictus can transmit dengue virus, the cause of a globally prevalent vector-borne disease (dengue fever)[13]. Increased use of insecticides can also increase IR in Ae. albopictus and other mosquitoes [13] as resistance can be passed onto offspring [14]. With increasing IR, PPE used to deter pests such as PTC should be routinely assessed for effectiveness. Innovative development of PPE such as PTC and/or different types of breathable textiles with tight enough weave to prevent mosquito biting without the use of insecticides are needed [15].

The WHO Cone Bioassay is a common mosquito repellency/toxicity test [16,17]. Here, glass cones are placed over fabric (e.g. bed nets, fabric swatches) and female mosquitoes are transferred into cones [16,17]. Mosquitoes are exposed to treated fabrics for several minutes and then transferred to insecticide free containers and further observed for knockdown/mortality after 1 h and 24 h [16,17].

Arm-in-cage tests are another method for evaluating repellency. A study using 100% cotton PTC showed the greatest reduction in repellency against Ae. aegypti Linnaeus between 0 and 10 washes [6]. At 0 washes, a forearm covered with PTC gave 58.9% protection against landing/biting while PTC with 10 washes gave 18.5% protection rate compared to an arm with no PTC [6]. Hence, even with full arm protection, mosquitoes can still bite through clothing.

Fabric types can impact efficacy of PTC, likely due to retention qualities, weights, and other characteristics of different materials. Others tested two types of PTC fabrics (50% cotton/50% polyester and 100% cotton) against Ae. aegypti and Ae. albopictus [8]. Aedes albopictus showed no significant differences in knockdown or mortality rates (generally > 30% with efficacy decreasing with number of washes) among fabric types and the highest mortality/knockdown was achieved with unwashed swatches [8]. Aedes aegypti exposed to PTC (50% cotton/50% polyester) washed 0 and 5 times, had lower mortality/knockdown rates (2 ± 0.09% and 2 ± 0.07%, respectively). Exposure of Ae. aegypti to unwashed 100% cotton PTC resulted in no knockdown/mortality, indicating IR may have been a factor [8]. No significant differences in protection from Ae. aegypti were observed between factory-dipped (permethrin added to polymer coating prior to fabric treatment) and microencapsulated-treated (permethrin enclosed in pre-polymer or monomer shell) clothing after 3 min of mosquito exposure [7]. Another study assessed permethrin content and washing degradation of permethrin for five different types/brands of factory-treated PTC: 1) Battle Dress Uniforms (BDU), 2) ExOfficio, 3) Insect Shield®, 4) Labonal, and 5) Sol’s Monarch. Sol’s Monarch and Labonal had the highest initial permethrin concentration prior to washing, with 4,000 mg/m² and 4,300 mg/m², respectively [18]. After 100 washes, Insect Shield® and ExOfficio had the lowest concentrations of permethrin at 20 mg/m² and 40 mg/m², respectively [18]. The same study showed that Insect Shield®-treated clothing showed the greatest reduction in permethrin concentration after 100 machine washes, compared to the other brands, from 1300 mg/m² to 20 mg/m² (98.5% decrease). After 100 washes, Sol’s Monarch and Labonal demonstrated 570 mg/m² (86% decrease) and 1800 mg/m² (58% decrease) permethrin. As wash frequency increased, knockdown time (Ae. aegypti, Anopheles stephensi Liston, and Culex pipiens Linnaeus) also increased.

Mosquito repellency effects and permethrin content between home-dipped (permethrin kits used at home that are convenient but require frequent applications) kits and factory-treated fabric were similar prior to washing[19]. In this study, factory PTC was obtained from Insect Shield® and home-dipped PTC was treated using a Sawyer kit[19]. The arm-in-cage method was used to assess the repellency of both types of PTC over eight washes against Ae. aegypti[19]. Home-dipped PTC had 91.5% bite protection (i.e. 8.5 % of mosquitoes landed/probed on an arm wearing PTC compared to 91.5 % landing/probing on a bare arm), higher than factory PTC (79.9%); however, this difference was not significant[19]. Home-dipped (49.9%) also showed a higher degree of protection against landing of Ae. aegypti compared to factory (40.9%) PTC[19]. Others assessed the repellency of PTC against resistant and susceptible Ae. aegypti[3]. The same study (arm-in-cage method) showed a low mortality rate (0–11%) for both resistant and susceptible mosquitoes exposed to PTC. Landing rates for resistant and susceptible mosquitoes were 44% and 63%, respectively; however, the reduction in blood feeding (during a 1.5 min period) due to PTC for both resistant and susceptible colonies was 100%[3]. Studies indicate that, as PTC is washed and permethrin content decreases, the efficacy for repellency against susceptible and resistant mosquito populations decreases [6–8,18,19]. These studies also show that initial (relatively high) permethrin content of PTC (before washing) helped to decrease biting and landing rates; however, this effect decreased as the number of washes increased. Home-dipped PTC (i.e. Sawyer kit) demonstrated a higher degree of mosquito bite protection than factory-treated PTC (i.e. Insect Shield®)[3].

Pests contacting the surface of permethrin-treated clothing (PTC) are repelled and/or killed, depending on exposure dose, duration, and other factors. Hence, it is important to assess surface permethrin content (SPC) for repellency and/or control. The Collaborative International Pesticides Analytical Council (CIPAC) protocol for evaluating permethrin-treated mosquito nets describes a method for extracting total permethrin content (TPC) [20]. Another study evaluated back and forth rubbing [21] to determine SPC, but others [22] note that this method does not extract permethrin in a variety of directions like circular rubbing would. Others that used an improved method of multidirectional rubbing (as in the current study) have shown that, depending on fabric type/weave, TPC can be 200 times greater than SPC [22]. The ‘bioavailable’ surface of permethrin-treated fabrics must be included in risk assessments of arthropod exposure since the surface is what pests contact [23].

The objectives of the current study were to: 1) compare TPC and SPC for three types of PTC (Insect Shield®, Sawyer, laboratory-created [4 g permethrin/L]), 2) compare mosquito repellency rate for three types of PTC, and 3) compare the repellency rate of permethrin-susceptible Ae. albopictus with Ae. albopictus previously exposed to permethrin.

Materials and methods

Treatment methods for PTC

A diagram showing treatment methods for 4 PTC types is provided (Figure 1).

Figure 1.

Flowchart of treatment methods for PTC.

Insect shield®

Permethrin-treated clothing (two short-sleeved shirts [Hanes®], 100% cotton) was purchased from Insect Shield® (www.insectshield.com; Greensboro, NC). Circular swatches (N = 14 replicates for treated fabric, 8.5 cm diameter) were cut for use in experiments. Each swatch’s weight (in g) was recorded before processing.

Sawyer premium insect repellant

A single 710 mL spray bottle of Sawyer Premium Insect Repellant (SPIR) (Sawyer Products, Inc., Safety Harbor, FL) containing 0.5% permethrin was used to treat circular swatches (N = 14 replicates for treated fabrics, 8.5 cm diameter) that were cut from two untreated 100% cotton short-sleeve shirts [Hanes®]. Swatches were placed in individual petri dishes (8.5 cm diameter) and sprayed with SPIR three times on both sides of the fabric until completely damp, as per manufacturer instructions. These treated swatches were removed from their respective petri dishes, set on individual foil sheets and stored in a dark drawer (to avoid photodegradation) at room temperature until processed (ca. 24 h to allow fabric to air dry). Each swatch was weighed (in g) and data was recorded before/after processing.

Laboratory-created permethrin-treated clothing (4 g permethrin/L)

Circular swatches (N = 14, 8.5 cm diameter) were cut from two untreated 100% cotton short-sleeve shirts [Hanes®] for laboratory treatment via dipping process to create the 4 g/L permethrin swatches. Six bottles containing 250 mg of granulated permethrin (99.5% purity) (Chem Service, West Chester, PA) were used to create solutions for treatment. Each permethrin solution (4 g/L: 160 mg permethrin + 40 mL acetone) was created in 60 mL amber vials, hand shaken for 3 min and sonicated for 10 min using the 2.8 L ultrasonic bath (Fisher Scientific, Watham, MN) to ensure thorough mixing.

The dipping process used for laboratory treatment involved placing each swatch into an amber vial containing the 4 g/L permethrin solution. Amber vials containing the swatches were placed on a rack connected to a Belly Button Shaker (IBI Scientific, Dubuque, IA) and shaken for 1 h at 75 revolutions/min to maximize permethrin impregnation. Each swatch was removed from designated vials and weighed. Swatches were air dried in a dark drawer at room temperature and were reweighed after 24 h. Each swatch was weighed after being cut and after being dipped in permethrin solutions.

Control

An additional 14 swatches were cut from two 100% cotton, short-sleeve shirts [Hanes®] for use as untreated controls. These controls followed the same procedures as the other laboratory-created PTC except swatches were placed into 60 mL amber vials containing 40 mL of acetone containing no permethrin. Vials were shaken for 1 h at 75 revolutions/min on the Belly Button Shaker (IBI Scientific, Dubuque, IA) and then removed from their designated vials and allowed to air dry in a dark drawer at room temperature and reweighed after 24 h. These swatches were also weighed before and after being placed in acetone.

Gas Chromatographic analysis for permethrin content

Figure 2 shows a flow chart of exposure and testing methods for PTC. The TPC was calculated for each of the four clothing groups (control, Insect Shield®, Sawyer, and 4 g/L permethrin). Four 8.5 cm diameter swatches from each PTC type were inserted into four individual 60 mL amber vials containing 40 mL of acetone. Vials were placed into the 2.8 L ultrasonic bath (Fisher Scientific, Watham, MN) and sonicated for 60 min. The ultrasonic bath was used to further elute the permethrin from fabric into the acetone. After 60 min of sonication, 2 mL of solution was transferred into two replicate 2 mL glass amber vials for each sample. These vials were tested for TPC using the Agilent 6850 gas chromatograph (GC) (Agilent Technologies, Alpharetta, GA). This procedure was repeated for each type of PTC and control.

Figure 2.

Flowchart of testing methods.

The capillary column used was Agilent Technologies DB-5 MS (5% phenyl-methylpolysiloxane) with a 0.25 µm film thickness. The specific settings for the GC are adapted from another study [24, 25]. The injector temperature was set at 250°C and the detector temperature at 260°C. The oven temperature was programmed to run at 200–250°C at 10°C/min and held for 7 min. The total run time for each sample was 17 min. Nitrogen was used as both carrier and make-up gas at 32.6 mL/min and 10 mL/min, respectively. Hydrogen was used as the detector gas at 30 mL/min. The calibration standards were created using a permethrin stock with 0.01 g permethrin dissolved in 40 mL of acetone. A blank vial containing 2.0 mL of acetone was used between sample runs to avoid false peaks.

Martindale abrasion and pilling tester for surface permethrin content

To conduct the testing for SPC, the MAPT (Testex, Guangdong, China) was used (Figure 3). The head of this machine consists of five main parts: rubbing media, treated fabric, fabric holder, rod, and 9 kPa weights (Figure 3). The untreated rubbing media used here were 13.5 cm diameter circular swatches cut from untreated short-sleeve shirts [Hanes®; 100% cotton]. The rubbing media receives the permethrin from the treated fabric through the process of rubbing. The treated fabric is a smaller (3 cm diameter) circular swatch cut from the different types of PTC being tested. This size was needed so that the fabric could be placed within the fabric holder on the MAPT. The fabric holder is pressed/rotated against the rubbing media by the rod and weight (9 kPa set on top of rod). The MAPT rubbing rate setting used was 30 rubs/min. The designated number of rubs used on each test fabric included: 500, 1000, 2000, 3000 and 3500 rubs. After the designated number of rubs had been reached, the rubbing media was removed and placed into a 60 mL amber vial containing 40 mL of acetone to elute permethrin. The vial was then sonicated using the 2.8 L Fisher Scientific Ultrasonic Bath for 60 min. Sonication was done to extract the permethrin that had transferred to the rubbing media from the treated fabric. After sonicating, 2 mL of the solution was added into 2 mL amber vials (two replicate vials for each individual rubbing) and tested on the GC for SPC (permethrin rubbed from the surface of swatches). The MAPT test was conducted on three types of PTC (Insect Shield®, Sawyer, 4 g/L permethrin) and control fabrics. Two swatches of each PTC type were used at each designated number of rubs (10 swatches/PTC type). In total, there were 40 swatches tested using the MAPT.

Figure 3.

Schematic diagram of the martindale abrasion and pilling tester head.

Calculation of Permethrin Concentration

A five-point calibration curve was created using the GC and permethrin stocks at different concentrations: 12.5, 25, 50, 100, and 200 µg/L. The formula acquired from the five-point calibration curve is Y = 0.748(x) – 0.2218 with an R² of 0.999. The data area acquired from the GC testing was plugged into the calibration curve formula as (x). The results were then divided by the weight (g) of the swatch from which the acquired area came.

Development of permethrin-susceptible and -exposed mosquito colonies

The two Ae. albopictus colonies used in the cone bioassay were derived from the same colony originating from Louisiana (the LA colony) previously determined susceptible to permethrin (data not shown). Eggs of the LA colony were split into two separate colonies (susceptible and exposed) at generation F-38. The susceptible colony was created by placing F-38 egg strips into two separate plastic pans (33 x 28 cm) containing 2 L of tap water housed in an incubator at 28°C. As the eggs hatched, larvae were fed 2 mg of a 1:2 ratio of yeast:liver powder every 2 d. As the larvae turned into pupae, they were transferred into a 177 mL plastic cup containing 89 mL of tap water and placed into a holding cage (30.5 x 30.5 × 30.5 cm) until adulthood. Adult mosquito colonies were fed on cotton balls containing 20% sucrose solution ad libitum. One week old female adults were fed defibrinated bovine blood (Hemostat, Dixon, CA) warmed to 37°C using the Hemotek system (Hemostat, Dixon, CA). At 24 h before blood feeding, the sucrose-containing cotton ball was replaced with water (to promote blood feeding). At 48 h after the blood feeding session, three (2.5 x 7.6 cm) ovistrips (seed germination paper; Anchor Paper, St. Paul, Minnesota) were placed into 60 mL black plastic cups filled with tap water and positioned in the cages containing adult mosquitoes. Ovistrips were harvested 48 h later and another generation was propagated. The propagation process continued for nine generations until the F-47 LA colony was created.

The permethrin-exposed colony (i.e. we do not refer to this colony as resistant since it was classified as ‘developing resistance’ by CDC bottle bioassay) was created by placing egg strips containing F-38 LA colony eggs into two plastic pans (33 x 28 cm) filled with 2 L of tap water incubated at 28°C with 14:10 L:D cycle. As eggs hatched, larvae were fed in the same manner as the susceptible colony. When the larvae reached the 2^nd instar developmental stage, they were collected and separated between two pans containing 2 L of a 0.8 mg/L permethrin + tap water solution. These methods were adapted from another study which used a 50% lethal concentration (LC₅₀) of permethrin between 0.4 µg and 0.6 µg for Ae. albopictus [26]. Here, the permethrin solution was created from a mixture of granulated technical grade permethrin (ChemService, Westchester, PA) and tap water. Pupae were transferred to 177 mL clear plastic cups containing tap water and placed into a holding cage (30.5 x 30.5 × 30.5 cm). Sugar feeding, blood feeding, and egg strip harvesting processes for the exposed colony were the same as the susceptible colony to ensure comparability between groups. The same culturing process for the exposed F-38 LA colony was used on the exposed F-39-42 generations. However, at generation F-40 and F-41, the concentration of the solution used for 2^nd instar larvae was increased to 1.0 mg/L of permethrin. The permethrin concentration of the rearing water was further increased to 1.2 mg/L during propagation of the exposed F-42 LA colony. Mortality among generations F-38, and F-39 larvae ranged from ca. 10–15% after being exposed to 0.8 mg/L of permethrin. The increase in dose of permethrin from 1.0 mg/L and 1.2 mg/L increased the mortality rate of the larvae to approximately 25%.

Susceptible (Ae. albopictus F-47) and exposed (Ae. albopictus F-42) colonies were tested for resistance using a modified CDC bottle bioassay [26,27]. Briefly, three glass 250 mL Wheaton bottles were coated with a 2 mL solution of 4 µg/L of permethrin. The 4 µg/L permethrin solution was created with acetone and permethrin. After coating, bottles were stored uncapped in a dark/dry area for ca. 12 h. Another three 250 mL bottles designated as controls were coated with 2 mL of acetone and stored in a dark/dry area for ca. 12 h. One set of bottles for testing contained three control bottles and three 4 µg/L permethrin bottles. A total of two sets were created (one set for each mosquito group).

Susceptible and exposed mosquito bioassays were conducted separately (two sessions). Both sessions were conducted in a laboratory at room temperature (ca. 27°C). For the susceptible mosquito bioassay, ca. 15 female mosquitoes were aspirated into each of the three control (45–50 mosquitoes) and three permethrin-coated bottles (45–50 mosquitoes). Following standard protocols, mosquitoes in control and treatment groups were observed for mortality at the following time points: 0, 5, 10, 15, 30, 45, 60, 75, 90, 105, 120 min [26,27]. The same modified CDC bottle bioassay was used to assess resistance in the exposed group.

The concentration of permethrin used here was 4 µg/L for both susceptible and exposed colonies. The diagnostic time (DT) for 100% mortality at this diagnostic dose was 15 min (data not shown). The WHO recommends that mosquitoes experiencing a mortality rate > 96% at the DT are considered susceptible. A mortality rate < 90% at the DT is considered resistant, while a 90–96% mortality rate at the DT is considered as potential development of resistance [26,27].

Repellency bioassay with mosquitoes

The PTC (Insect Shield®, Sawyer, and 4 g/L permethrin) and control fabrics were also used for the cone bioassay. A modified WHO cone bioassay method was used to assess repellency [7,8]. Six swatches each from InsectShield®, Sawyer, 4 g/L permethrin, and control groups were tested against two groups (permethrin-susceptible and -exposed) of Ae. albopictus for repellency using the modified cone assay method. Briefly, six 8.5 cm diameter glass cones were placed on top of replicate 8.5 cm diameter swatches from each of the four fabrics for a total of 24 cones. Five to eight female mosquitoes were introduced into each cone and observed (total of approximately 120–192 mosquitoes). Within the first 3 min, the number of mosquitoes showing repellency to the fabric was recorded based on visual observation. Repellency for this test was defined as the mosquito flying away from the treated fabric or sporadic flying and landing onto the fabric. Proportion repellency was calculated by group: number of mosquitoes repelled/total number of mosquitoes. Knockdown (death or inability to fly after being exposed to the fabric) was also monitored/recorded and calculated by group: number of mosquitoes knocked down/total number of mosquitoes. Two cone bioassay sessions (one for each mosquito colony) were conducted for each fabric type. The same 8.5 cm diameter swatches of fabric used in cone bioassays were tested to assess TPC.

Table 1 summarizes the number of swatches used for each test conducted in this study, resulting in a total of 72 swatches tested.

Table 1.

Number of swatches used for each test

Number of Swatches (N) for Each Test
Test	Control	Insect Shield®	Sawyer	4 g/L Permethrin	Total # of Swatches
Total Permethrin Content	2	2	2	2	8
Martindale Abrasion: 500 rubs at 30rpm	2	2	2	2	8
Martindale Abrasion: 1,000 rubs at 30rpm	2	2	2	2	8
Martindale Abrasion: 2,000 rubs at 30rpm	2	2	2	2	8
Martindale Abrasion: 3,000 rubs at 30rpm	2	2	2	2	8
Martindale Abrasion: 3,500 rubs at 30rpm	2	2	2	2	8
Cone Bioassay (susceptible/exposed colonies)	6	6	6	6	24
Total # of Swatches	18	18	18	18	72

Statistical analysis

Analysis of variance (ANOVA) was used to evaluate the differences in TPC and SPC between the types of PTC, and to evaluate the proportion repellency between the susceptible/exposed mosquitoes. Bonferroni comparisons were used to compare means of permethrin content between fabric treatment types and between TPC and SPC. SPSS (Chicago, Illinois) was used to conduct the data analysis and P < 0.05 was considered significant.

Results

Modified CDC bottle bioassay

The DT used for this modified CDC bottle bioassay was 15 min. At 15 min, the ‘susceptible’ colony experienced 100% mortality (classified as susceptible), while the ‘exposed’ colony experienced 96% mortality (classified as developing resistance) [27,28]. No mortality was observed in control groups (hence no Abbott’s correction was needed)[29].

Total and surface permethrin content

Table 2 shows the mean ± standard error (SE) of TPC and SPC for the three treated PTC fabrics and controls. Sawyer PTC had the highest mean TPC per swatch (289.17 ± 0.51 µg/g), followed by Insect Shield® (53.90 ± 6.79 µg/g) and the lab-created 4 g/L permethrin swatches (12.67 µg/g ± 0.35). Sawyer PTC had the highest mean SPC with 32.68 ± 14.55 µg/g, followed by Insect Shield® (23.35 ± 2.71 µg/g) and the lab-created 4 g/L permethrin (8.7 ± 0.78 µg/g). We did not detect permethrin in the control swatches (0.00 µg/g).

Table 2.

Total permethrin content and surface permethrin content for different fabrics. *N = 2 replicate GC vials tested for each of 4 swatches per fabric treatment

	Total Permethrin Content			Surface Permethrin Content
Fabric Treatment	Mean (µg/g)	N*	SE	Mean (µg/g)	N	SE
Sawyer	289.17	2	5.07	32.68	10	14.55
InsectShield®	53.90	2	6.79	23.35	10	2.71
4 g/L Permethrin	12.67	2	0.35	8.69	10	0.78
Control	0.00	2	0	0.26	10	0.00

Table 3 shows differences of means of permethrin content between Insect Shield®, Sawyer, 4 g/L permethrin, and control. When comparing the differences in means of TPC for the three fabric treatments (Insect Shield®, Sawyer, 4 g/L permethrin) against control fabric treatment, Sawyer had the significantly highest difference in mean permethrin (288.90 µg/g; P < 0.01). The difference of means between Insect Shield® and control (53.64 µg/g) fabrics was also significant (P < 0.01).

Table 3.

Comparisons of total permethrin content (µg/g) by fabric treatment. Significant P-values indicated in bold text

Multiple Comparisons (Total Permethrin Content)
Dependent Variable: Permethrin Concentration
Bonferroni
(I) Fabric Treatment	(J) Fabric Treatment	Mean Difference (I-J)	P-value	95% Confidence Interval
				Lower Bound	Upper Bound
4 g/L Permethrin	Control	12.4035	0.646	−16.71	41.55
	Insect Shield®	−41.2355*	0.014	−70.34	−12.13
	Sawyer	−276.5000*	< 0.001	−305.61	−247.39
Control	4 g/L Permethrin	−12.4035	0.646	−41.51	16.71
	Insect Shield®	−53.6390*	0.005	−82.75	−24.53
	Sawyer	−288.9035*	< 0.001	−318.01	−259.79
Insect Shield®	4 g/L Permethrin	41.2355*	0.014	12.13	70.34
	Control	53.6390*	0.005	24.53	82.75
	Sawyer	−235.2645*	< 0.001	−264.37	−206.16
Sawyer	4 g/L Permethrin	276.5000*	< 0.001	247.39	305.61
	Control	288.9035*	< 0.001	259.79	318.01
	Insect Shield®	235.2645*	< 0.001	206.16	264.37

A one-way ANOVA was carried out for SPC of each fabric (Insect Shield®, Sawyer, 4 g/L permethrin) for each tested number of rubs. Significant (P < 0.01) differences were observed in SPC between groups. The control data set was omitted in this analysis to better assess the variance between fabric treatment and SPC (Control, TPC, and SPC were equivalent). When comparing SPC of Insect Shield®, Sawyer, and 4 g/L permethrin groups to SPC of control fabrics, Sawyer had the significantly (P < 0.01) highest difference in SPC (mean permethrin ± standard error) (75.17 ± 1.27 µg/g), followed by Insect Shield® (28.18 ± 1.27 µg/g). The significantly (P < 0.01) lowest mean difference in SPC was found between control and 4 g/L permethrin groups (9.09 ± 1.27 µg/g).

Total permethrin content vs surface permethrin content

A comparison was made between TPC and SPC for each type of fabric treatment. For Insect Shield®, the difference between TPC and SPC was significant for all tested rub frequencies except at 3,000 rubs, wherein the mean difference in permethrin (22.62 ± 4.90 µg/g) was not significant (P = 0.054). A significant (P < 0.01) difference in permethrin between TPC and SPC was observed in Insect Shield® at 3,500 rubs with a mean difference of 45.21 ± 4.90 µg/g.

For Sawyer, comparisons of TPC and SPC at all quantities of rubs were significant (P < 0.01). The highest mean difference in permethrin between TPC and SPC was at 2,000 rubs (283.11 ± 3.75 µg/g), while the lowest mean differences were at 500 and 3,500 rubs (169.76 ± 3.75 µg/g for both).

For the 4 g/L permethrin group, the highest mean difference in permethrin between TPC and SPC were at 1,000 rubs (6.86 ± 0.72 µg/g; P < 0.01), followed by 500 rubs (6.58 ± 0.72 µg/g; P < 0.01). The mean differences between TPC and SPC at 2000, 3000, and 3500 rubs were not significant, with the lowest mean difference (1.42 ± 0.72 µg/g) at 3,000 rubs.

Cone bioassay repellency testing

The cone bioassay results were compared between PTC fabric types (Figure 4). As expected, for the control fabric, neither mosquito group showed repellency. Insect Shield® performed the best against both susceptible and exposed mosquito groups, with 53% and 48% mean repellency, respectively. However, repellency rates for Insect Shield® for either mosquito group were not significantly different than Sawyer and 4 g/L permethrin. The Sawyer-treated swatches showed the second highest mean repellency against both mosquito groups (susceptible: 49% repellency; exposed: 46% repellency). Repellency rates for Sawyer-treated fabrics were not significantly different than those for Insect Shield® and 4 g/L permethrin-treated fabrics. The third highest repellency was observed in the 4 g/L permethrin group (susceptible: 42% repellency, exposed: 41% repellency). No significant differences in repellency rates were observed between the 4 g/L, Insect Shield®- and Sawyer-treated groups, except for rates between 4 g/L- and Insect Shield®-treated fabrics for the exposed group (P < 0.01). Repellency rates for Insect Shield®, Sawyer, and 4 g/L permethrin were significantly higher (P < 0.01) than the control group. Higher repellency rates were observed in the susceptible compared to the exposed mosquito group among all fabric types (e.g. InsectShield®: Susceptible 53%, Exposed 48%). However, the difference in repellency rates among PTC types against susceptible and exposed mosquitoes was not significant.

Figure 4.

Cone bioassay proportion repellency (± standard error) testing in Aedes albopictus by fabric treatment. Proportion repellency by treatment = number of mosquitoes repelled/total number of mosquitoes. The number of mosquitoes demonstrating repellency to the fabric was recorded during the first 3 min of exposure.

Discussion

Total permethrin content vs. surface permethrin content

Significant differences in TPC and SPC were observed for three PTC types (Insect Shield®, Sawyer, 4 g/L Permethrin). Sawyer-treated fabrics showed the highest mean TPC (289.17 µg/g) and mean SPC (32.68 µg/g), which were significantly (P < 0.01) higher than those for Insect Shield®, 4 g/L permethrin, and control groups. These findings indicate that PTC with higher TPC typically have a higher SPC. Sawyer-treated fabrics may have the highest TPC and SPC here due to the application method, i.e. permethrin was not impregnated into fabric, but sprayed on the surface. The dipping method for the 4 g/L permethrin group resulted in the lowest TPC and SPC. Variation in application method between different fabric types and human applicators (completely damp versus partially damp) for the Sawyer product could likely occur more frequently than the factory-treated Insect Shield® product which may be more uniformly applied. Permethrin from Sawyer-treated fabrics may have easily detached from the fabric (during elution prior to testing) compared to InsectShield® clothing where permethrin may be bound to the fabric due to their proprietary treatment method.

When comparing TPC and SPC, a significant difference indicated a relatively low SPC, while an insignificant difference indicated a relatively high SPC. The number of rubs in which these insignificant differences were found may indicate an optimal number of rubs that can be used in future assessments of SPC. For Insect Shield®, similar SPC and TPC was found at 3,000 rubs (P = 0.054). However, by 3,500 rubs, differences between SPC and TPC were significant, indicating the transfer of permethrin is lost/destroyed for this fabric after 3,000 rubs. Sawyer-treated fabrics showed similar SPC and TPC under all rubbing conditions and this may be due to the treatment method. Future studies should evaluate this further to determine an optimal number of rubs for assessing SPC. The 4 g/L permethrin group showed similar TPC and SPC for most rubbing conditions (2000: 2.69 µg/g, 3000: 1.42 µg/g, 3500: 2.32 µg/g). These results indicate that the optimal number of rubs for 4 g/L permethrin group was 3,000 rubs and the increase in mean difference after 3,000 rubs may indicate loss/destruction of permethrin (due to factors such as friction and fabric type). We used the same fabric type for all assays conducted in this study; however, it is important to note that different types of fabric will exhibit different amounts of SPC and TPC insecticide, depending, in part, on weaving pattern (e.g. tight, loose) (Dieval et al. 2017). The surface area and weave of a fabric also likely impacts the area that a mosquito tarsus (for example) contacts the fabric (i.e. tightly woven fabric may result in more mosquito contact than loosely woven fabric) [15,22]. These types of factors are important and should be considered when designing the PTC to optimize performance and public health protection.

Susceptible vs. Exposed mosquitoes

Two Ae. albopictus mosquito groups were used: 1) susceptible to permethrin and 2) previously exposed to permethrin (repeated exposure at low doses as larvae). CDC bottle bioassays were conducted to assess the level of susceptibility/resistance to permethrin for both mosquito groups. The exposed mosquito group showed 96% mortality at the DT (15 min) and was classified as developing resistance to permethrin. It is likely that continued (additional generations) repeated exposure to permethrin could have resulted in this population being classified as resistant and that will be investigated further, along with calculations of resistance ratios. It is important to create and/or have access to susceptible and resistant mosquito colonies to compare efficacy of PTC to different mosquito populations; hence the population developed here will be used in future studies.

Cone bioassay

A modified WHO cone bioassay was conducted to assess repellency for susceptible and permethrin-exposed groups of Ae. albopictus using PTC. No mortality was observed in control groups (hence no Abbott’s correction was needed)[29]. Insect Shield® had the highest repellency rate against susceptible (53%) and exposed (48%) mosquitoes. Insect Shield®-treated fabric repellency rates for susceptible and exposed groups were significantly (P < 0.01) higher than control fabrics. However, no significant differences were observed in repellency rates between Insect Shield®, Sawyer, and 4 g/L permethrin groups. Repellency rates found here indicate factors (e.g. treatment method) other than SPC may contribute to repellency rate. The (proprietary) impregnation process binds an AI (e.g. permethrin) to the fabric, hence minimizing degradation due to wash/wear. It should be considered that Insect Shield® clothing could have more permethrin than was detected here because it may not have eluted as easily as for the other (Sawyer and 4 g/L) fabrics (hence detected TPC and SPC could be lower) due to their proprietary impregnation process. Sawyer (0.5% permethrin, 99.5% other ingredients) PTC was treated in accordance with the label and it is unknown whether this product contains ingredients that assist with binding. The 4 g/L permethrin-treatment group used contained only acetone and permethrin (no other ingredients, such as binders). This may be the reason why the 4 g/L permethrin-treated fabrics had much lower TPC and SPC because permethrin did not bind with the fabric. Other have shown that fire-resistant uniforms do not uniformly absorb permethrin and different types of fabrics and treatments could also be evaluated in the future[30]. This type of information is important for those interested in improving the efficacy of PTC for public health protection.

Our findings agree with others [3] where a higher repellency rate was observed in mosquitoes previously exposed to permethrin in comparison to susceptible mosquitoes. While the findings of the cone assay comparing repellency rates between susceptible and exposed mosquitoes were not significant, results support that the exposed group of Ae. albopictus were developing resistance. It is possible that continued permethrin exposure through additional mosquito generations could lead to a permethrin-resistant colony and further studies are warranted to evaluate this. While beyond the scope of the current study, future studies could also conduct arm-in-cage tests using human volunteers to compare the extent to which mosquito repellency differs between fabrics and repellants such as DEET and others [30,31]. Furthermore, additional types of insecticide-treated fabrics and combinations of repellents and insecticides could be evaluated (e.g. tents, bednets) to improve PTC that could further protect health [30,32,33].

Future studies could focus on further refining the MAPT rubbing method for transferring SPC to other media (i.e. polyester, cotton, wool). Different populations and species of field-collected mosquitoes should also be assessed for resistance/repellency. Limited studies have used a cone bioassay to assess repellency rates for exposed/susceptible mosquitoes and the MAPT has rarely been used to assess SPC. More work is warranted to evaluate the relationship between SPC and repellency. Our findings showed that SPC is lower than TPC and this has important public health implications as the surface of fabrics are what pests contact when attempting to blood feed. Theoretically, only the surface layer of fabric needs to be treated and innovations in this area (including breathable fabrics impenetrable to mosquitoes) would reduce potential human exposure to permethrin. Additional studies should assess the MAPT’s ability to transfer insecticides and further refine this valuable assessment method. Studies could also further assess the relationship between washing of fabrics and SPC/TPC.

Funding Statement

The author(s) reported there is no funding associated with the work featured in this article.

Disclosure statement

No potential conflict of interest was reported by the author(s).