Permethrin-treated clothing included in the study were either purchased as already impregnated retail products from Insect Shield (T-shirts and socks) or purchased untreated and then sent to Insect Shield for impregnation (pants and shoes) (Fig. 1). Hereafter, T-shirts are referred to simply as shirts. The Insect Shield shirts were 100% cotton, whereas the Insect Shield socks were a blend of cotton (49%), polyester (49%), rubber (3%), and spandex (1%). The pants (Carhartt, Inc., Dearborn, MI) were 100% cotton, and the ‘tongue’ portion of the high top shoes (Nike, Inc., Beaverton, OR) used in the bioassays was 100% cotton canvas. For shirts, pants, and socks, tick challenge bioassays were conducted with pieces of textile that were: 1) treated but neither worn nor washed/dried (designated Treatment 1, T1, representing pristine treated clothing); 2) treated and washed/dried but not worn (Treatment 2, T2); and 3) treated, worn, and washed/dried (Treatment 3, T3, representing normal use). The shoe tongue textiles challenged in the bioassays were either treated but neither worn nor washed/dried (T1) or treated and worn but not washed (designated T3 as normal use because shoes are worn but rarely washed). The nontreated control (designated C) was always a 100% cotton shirt textile.

Laboratory-reared I. scapularis ticks used in bioassays were second-generation nymphs (CT14) originating from females collected in Fairfield County, CT, and maintained at the Centers for Disease Control and Prevention, Division of Vector-Borne Diseases, Fort Collins, CO. Prior to being used, the nymphs were held within desiccators (90–95% RH) in a growth chamber maintained at 21–22°C with a 16:8 (L:D) h cycle. Laboratory-reared nymphs used in the bioassays were, with a few exceptions, 3.2- to 5.2-mo postmolt. Field-collected nymphal ticks (CT-field) of unknown age used in the bioassays originated from three collection locations within Fairfield County, CT, in May–June 2016. Field-collected nymphs were collected by dragging a 1 m by 0.5 m flannel tick flag through the leaf litter for 30-s intervals and then inspecting the flag after each flag sample. Field-collected nymphs were held in the laboratory at room temperature in desiccators (85–95% RH) for ≥2 wk before being used.

As noted above, the T3 shirts, pants, and socks were both worn and washed/dried, whereas the shoes were worn but not washed/dried. The T3 category included clothing worn by four different individual female human subjects (age range, 22–30) wearing similar clothing for outdoor fieldwork activities on 2 d/wk over an 8-wk period (total of 16 d) in late spring and early summer coinciding with the peak activity period for I. scapularis nymphs in Connecticut (Stafford 2007). Subjects conducted similar field activities while together in the same locations on the days wearing the treated clothing. Field sites included multiple locations in western Connecticut (Fairfield County), in public lands consisting primarily of deciduous or mixed deciduous and coniferous tree species with minimal to moderate understory vegetation. The shirts, pants, and socks were washed/dried twice weekly (16 total occasions) in a household washing machine using the medium heat setting (range 24.2–25.1°C, pH 6.4–6.5, chlorine 0–0.5 ppm, alkalinity 0–70 ppm) and All Free and Clear (Sun Products, New Providence, NJ). After being washed, the clothing was dried in a household dryer using the medium heat setting (approximately 57°C, per the manufacturer). The T2 shirt, pants, and socks were not worn but washed/dried 16 times using the same methods described above. The number of ticks detected on the clothing or body of human subjects was recorded daily. The component of this study involving humans wearing permethrin-treated clothing in the field was approved by the Institutional Review Board at Western Connecticut State University and by the Human Investigations Committee at the Connecticut Department of Public Health.

The contact irritancy and toxicity bioassays used to evaluate the impact of test textiles on I. scapularis nymphs were conducted using methods described in detail previously (Eisen et al. 2017b). Briefly, the contact irritancy assay employed a playing card (64 mm wide by 89 cm tall) covered by a solid piece of test textile (Fig. 1) and positioned at a 45° angle. Groups of 4–10 ticks were introduced in the center of the textile-covered card and the number of ticks still remaining on the textile was recorded at 1-min intervals over a 5-min period. Strong contact irritancy manifests as nymphal ticks becoming visibly agitated (‘hot-foot’ effect) and actively dislodging from treated textile by flipping over and tumbling downward along the textile-covered card until they fall off the bottom of the card. For trials with laboratory-reared nymphs, each test textile was challenged with 5 groups of 10 ticks, for a total of 50 ticks (grand total of 1,390 ticks). Following the completion of the assays, ticks were held at room temperature in desiccators (90–95% RH) until scored for vigor (see section called ‘Classification Scheme for Tick Vigor After Exposure to Test Textiles’) at 1 and 24 h after exposure to the test textile. Assays with <80% of matched control ticks, originating from the same colony storage vials and tested on the same date, displaying normal activity at 24 h after exposure were excluded from data analysis relating specifically to tick vigor at 24 h after exposure to treated textile. For trials with field-collected nymphs, each test textile was challenged with groups of 4–5 nymphs, testing up to 40 ticks per textile (grand total of 338 ticks). Due to a shortage of field-collected nymphs, fewer ticks were tested upon sock and shoe textiles than for the shirt and pants textiles.

The toxicity assay exposed ticks to continuous contact with a test textile, cut into a 100-mm-diameter circle (Fig. 1) and placed within a horizontal petri dish, for a predetermined period of time. In this study, ticks were held in continuous contact with test textiles (prevented from moving off of the test textile) for 30, 60, or 120 s. These contact durations were found previously to severely impact the vigor of I. scapularis nymphs (Eisen et al. 2017b). For trials with laboratory-reared nymphs, each combination of test textile and exposure time was challenged with three groups of 10 ticks, for a total of 30 ticks (grand total of 2,320 ticks). Due to shortage of laboratory-reared ticks of uniform source and age, only T1 and T3 test textiles were challenged in the toxicity assay. Following the completion of the assays, ticks were held at room temperature in desiccators (90–95% RH) until they were scored for vigor at 1 and 24 h after exposure to the test textile. Assays with <80% of matched control ticks, originating from the same colony storage vials and tested on the same date, displaying normal activity at 24 h after exposure were excluded from data analysis relating specifically to tick vigor at 24 h after exposure to treated textile. Preliminary data analysis (not shown) revealed no consistent trends for a given type of clothing toward decreased proportions of ticks displaying normal behavior or willingness to ascend onto a finger with increasing exposure times within the 30–120 s exposure time range. The presented bioassay data therefore are combined across the 30, 60, and 120 s exposure times. For trials with field-collected nymphs, each test textile was challenged over a 60-s period with groups of 3–5 ticks, testing up to 21 ticks per textile (grand total of 120 ticks). Due to a shortage of available field-collected nymphs, toxicity assays were conducted only for shirt and pants textiles.

Our classification scheme for tick vigor at different time points after exposure to permethrin-treated textiles was described in detail previously (Eisen et al. 2017b) and is summarized here. Tick vigor following introduction onto a nontreated surface and stimulation of activity via gentle physical prodding and human breath was scored across four categories of capacity for movement: 1) tick completely motionless; 2) tick capable of some movement of the legs but unable to right itself or walk; 3) tick capable of righting itself but not able to move in a coordinated way or readily orient toward a stimulus; and 4) tick displaying normal movement and response to a stimulus. Ticks that were scored as displaying normal movement (4) were further assessed to determine if they would ascend onto a finger when presented the opportunity. Our previous study (Eisen et al. 2017b) showed that I. scapularis nymphs exposed to permethrin-treated textiles may display normal movement but nevertheless be unwilling to ascend a human finger.

Previous studies have expressed the concentration of permethrin present in a textile as mg permethrin per cm² or m² of textile (Schreck et al. 1978, 1982; Evans et al. 1990; Faulde et al. 2003, 2006, 2016; Faulde and Uedelhoven 2006; DeRaedt Banks et al. 2015; Richards et al. 2017). However, this is problematic when comparing textiles of different thickness or weave, or clothing made from different types of materials. We therefore prefer to express the concentration permethrin present in a textile as mg permethrin per g of textile. Pristine Insect Shield clothing are marketed as containing 0.52% permethrin (w/w), which equates to 5.2 mg of permethrin per g of textile.

Quantification of permethrin in treated test textiles used in the tick bioassays was done at the Colorado State University Proteomics and Metabolomics Facility (Fort Collins, CO). Three 1-cm² samples were cut from each treated test textile and their mass (g) was recorded. Three samples from a nontreated 100% cotton textile were included as a negative control. Aiming to recover ≥95% of the permethrin in the textile samples, a series of four consecutive extractions using 1 ml of solvent containing 100% methanol labeled with an internal standard, transpermethrin-(phenoxy-d5) (Sigma-Aldrich, St. Louis, MO) at 0.05 mg/ml, was performed. The samples were sonicated at room temperature for 15 min after each round of extraction. The average rate of recovery of permethrin from treated samples was estimated at 98.6%. Samples (4 ml total volume) were then stored at −80°C until further analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) performed using an Acquity UPLC system (Waters, Milford, MA) coupled with a Xevo TQ-S Tandem Quadrupole Mass Spectrometer (Waters). Chromatographic separation was achieved using a UPLC T3 C18 stationary phase (1 × 100 mm, 1.7 μM) column (Waters).

Mobile phases were 100% methanol (A) and water with 0.1% formic acid (B). The analytical gradient was as follows: time = 0 min, 80% B; time = 0.5 min, 80% B; time = 1 min, 99.9% B; time = 5 min, 99.9% B. The column was flushed to avoid carryover and re-equilibrated with a subsequent injection of 100% hexane with the following gradient: time = 0 min, 99.9% B; time = 2 min, 99.9% B; time = 2.5 min, 80% B; time = 5 min, 80% B. The flow rate was 220 μl/min and the injection volume was 1 μl for each sample and 5 μl for the hexane blank. Samples were held at 4°C in the autosampler and the column was maintained at 60°C. The mass spectrometry was operated in selected reaction monitoring and positive ionization modes, with the capillary voltage set to 3.2 kV. Interchannel delay was set to 3 ms. Transition ions for permethrin and trans-permethrin-d5 are 408 > 183, 355 and 413 > 188, 360, respectively. Collision energy was set at 10 volts and cone voltage was set to 30 volts for both target analyte and deuterated internal standard. The source temperature was 150°C and the desolvation temperature was 350°C. Desolvation gas flow was 1,000 liter/h, cone gas flow was 150 liter/h, and collision gas flow was 0.2 ml/min. The nebulizer pressure (nitrogen) was set to 7 Bar and argon was used as the collision gas. A calibration curve was generated for permethrin, ranging from 0 to 312 μg/ml, using the authentic permethrin standard in 100% methanol solution. The internal standard concentration in the methanol curve was held constant at 0.05 mg/ml.

Resulting raw data files were imported into the Skyline open source software package (MacLean et al. 2010) and analytes were visually inspected for retention times and peak area integration. Peak areas were integrated between 1.5 and 2 min for all samples. Permethrin detected in treated test samples was normalized to the peak area of the deuterated internal standard present in each sample. Normalized peak areas were then quantified using the linear regression equation generated for permethrin from the calibration curve. Limits of detection (LOD) and limits of quantification (LOQ) represent the lower limits of detection and quantification for each compound in the matrix of this study. Two regression curves were plotted for this data set in order to accurately represent lower concentrations (i.e., less than 5 μg/ml). The permethrin LOD (0.05 μg/ml) and LOQ (0.16 μg/ml) were calculated as 3 times or 10 times the standard deviation (0.00022) of the blank divided by the slope (0.0135) of the low-range calibration curve (Broccardo et al. 2013). Finally, based on the mass (g) of each test sample, the uniform extraction volume (4 ml), and the permethrin concentrations in the extraction for the test samples (μg/ml), we calculated the concentration of permethrin in the test samples expressed as mg permethrin per g of textile. Outliers in the data for a given type of clothing (shirt, pants, sock, or shoe) and treatment were defined, using Tukey fences, as: values below Quartile₁ − 1.5(Quartile₃ − Quartile₁) or above Quartile₃ + 1.5(Quartile₃ − Quartile₁).

Pairwise statistical comparisons among treatments for a given type of clothing (shirt, pants, sock, or shoe) for the probability of ticks to remain on a test textile, display normal movement, or display normal movement and willingness to ascend onto a finger were based on likelihood ratio test, conducted in JMP Pro 13 (Cary, NC). Results were considered significant when P < 0.05, with Bonferroni correction to account for multiple pairwise comparisons for a given treatment and outcome measure.

The average concentrations of permethrin per g of textile cut from the pieces of pristine (T1), washed/dried (T2), or worn and washed/ dried (T3) treated clothing that were used in the bioassays are shown in Table 1. Nontreated control samples were below the limit of quantification for permethrin. Although the sample sizes were judged too small for statistical analysis, it is evident from the data that permethrin was lost from shirts, pants, and socks due to washing/drying of treated clothing. After 16 rounds of washing/drying (T2), approximately 90% of the permethrin appeared to have been lost, compared with pristine treated textile (T1), from shirts, with smaller percentage losses due to washing/drying for pants (~60%) and socks (~50%). Additional loss of permethrin that could be attributed to 16 d of wear, by comparing worn and washed/dried clothing (T3) with washed/dried but unworn clothing (T2), was not observed for either socks or shoes, whereas it was minimal (<2%) for shirts and limited (<15%) for pants.

Samples from pristine treated shirts, pants, and socks (T1) were found to contain, on average, 8–13 mg permethrin per g of textile (Table 1). After 16 washes and 16 d of wear, the average concentration of permethrin per g of textile remained above 5.2 mg for the socks but had fallen below 2.2 mg for the pants and below 1.2 mg for the shirts. Moreover, the concentration of permethrin per g of textile tended to be more variable (higher coefficient of variation) among worn and washed/dried samples (T3) compared with pristine samples (T1) from the same type of treated clothing (Table 1). High- or low-end outliers in the data for concentration of permethrin per g of worn and washed/dried textile were recorded for pants and shoe samples, which were purchased untreated and then sent in for treatment, but not for shirts and socks, which were purchased already impregnated.

Results for assessments of tick vigor at 1 and 24 h after forced exposures (30–120 s) to textiles in the horizontal toxicity assay are shown in Tables 6 and 7. Following exposure to nontreated textile (C), all 580 examined ticks displayed normal movement 1 h later and 93–99% still displayed normal movement after 24 h. At 24 h, 77–84% of the ticks exposed to nontreated textile (C) displayed normal movement and ascended onto a human finger. In striking contrast, none of 360 ticks held in contact with pristine permethrin-treated shirt, pants, sock, or shoe textile (T1) displayed normal movement at 1 h (Table 6). By 24 h, only 1 of these 360 ticks (0.3%) displayed normal movement, and this tick was not willing to ascend a finger (Table 7). Across shirt, pants, and sock textile, worn and washed/dried treated clothing (T3) were significantly less toxic than pristine treated clothing (T1) but still significantly more toxic than nontreated textile (C), regardless of whether the outcome measure was ticks displaying normal movement (at 1 or 24 h) or displaying normal movement and ascending onto a human finger (24 h) (Tables 6 and 7). The increase in percentage points of ticks displaying normal activity after exposure to worn and washed/dried treated clothing (T3), compared with pristine treated clothing (T1), ranged from 59% for the shirt at 1 h to 29–42% for the shirt at 24 h and the pants and sock at 1 or 24 h. On the other hand, the decrease in percentage points of ticks displaying normal activity after exposure to worn and washed/dried treated clothing (T3), compared with nontreated textile (C), ranged from 58–69% for the shirt at 24 h and the pants and sock at 1 or 24 h, and to 41% for the shirt at 1 h. The toxicity of the T3 shoe textile, which was worn but not washed, was similar to the pristine (T1) shoe textile.

Two notable observations from the vertical assay also held true in the horizontal assay. At the 1 h time point most ticks displaying normal movement were unwilling to ascend onto a finger (Table 6). Moreover, when comparing tick vigor at 1 and 24 h after exposure, the percentage of ticks displaying normal movement decreased distinctly from 1 to 24 h, due to slowly developing toxic effects, after exposure to the washed and worn shirt (from 59 to 29%).

Results for assessments of tick vigor at 1 and 24 h after forced exposures (60 s) to shirt and pants textiles in the horizontal toxicity assay are shown in Tables 8 and 9. Exposure to pristine treated clothing (T1) or worn and washed/dried treated clothing (T3) resulted in significant reductions in the proportion of ticks displaying normal movement compared with nontreated textile (C), both for shirt and pants at 1 and 24 h.

The relationships, across all individual examined treated test textiles, between the concentration of permethrin per g of test textile and contact irritancy or tick vigor in the vertical and horizontal assays are illustrated in Figs. 3–5. These relationships should be interpreted with caution as the underlying data include multiple types of clothing (shirts, pants, socks, and shoes) and treatments (pristine, washed/dried, and worn and washed/dried). Nevertheless, some clear patterns emerge. First, data points for socks consistently are outliers as the socks appear to underperform in terms of irritancy and impact on tick vigor in relation to the concentration of permethrin they contain (Figs. 3–5). Disregarding the socks, the observed distributions across shirt, pants, and shoe textiles tend to be L-shaped with consistently high irritancy and low tick vigor after exposure to test samples with >5 mg permethrin per g of textile but variable irritancy and tick vigor when the permethrin concentrations fell below 5 mg per g of textile (Figs. 3–5).