Simple Summary: Bed bugs cause health and economic impacts due to their hematophagous behavior. The tropical bed bug, Cimex hemipterus (F.) is predominant in tropical and subtropical regions and modern strains demonstrate high levels of insecticide resistance making them a major challenge to control. In this present study, we investigated the effect of exposure time and mortality assessment interval on bioassay results. Bed bugs were treated separately with six insecticide products at label rates using the surface contact method, with multiple exposure times (5 min, 10 min, 30 min, 1 h, 4 h, and continuous exposure for 96 h or 14 days for Phantom). Post-treatment mortalities of bed bugs were assessed daily for up to 4 days (Tandem, Temprid SC, Pesguard FG161, Sumithrin, and Sumithion) and 14 days for Phantom. Results showed that longer exposure times led to rapid knockdown and better killing effect in bed bugs. Exposure times and mortality assessment intervals should be carefully considered in resistance and efficacy studies with insecticide products. We evaluated the influences of insecticide exposure time and mortality assessment intervals on the bioassay results of three insecticide-resistant tropical bed bug Cimex hemipterus (F.) populations (Madam Mo, Tanjong Tokong, and Green Lane). This was achieved using the surface contact method and tested with six commercial insecticide products: Tandem, Temprid SC, Pesguard FG161, Sumithrin, Sumithion, and Phantom applied at label rate on glass petri dishes. Six exposure times (5 min, 10 min, 30 min, 1 h, 4 h, and continuous exposure for 4 or 14 days for Phantom) were tested. A susceptible common bed bug Cimex lectularius L. strain (Monheim) was used as the reference strain as no susceptible C. hemipterus strain exists. In treatment with Temprid SC, the Tanjong Tokong strain showed significantly higher KT50 values at 5, 10, and 30 min exposures than 1 h, 4 h, and continuous exposures. When all resistant populations were evaluated at continuous exposure to Sumithion, they demonstrated moderate resistance levels (RR50 ranged from 6.0 to 7.9), while KT50 and KT95 of other shorter exposure times failed to be generated due to low knockdown rate. Higher mortalities were observed in Tanjong Tokong and Green Lane strains when tested at longer exposure times with Temprid SC, Pesguard FG161, Sumithrin, Sumithion, and Phantom. Better killing effect was observed in the treatment with Temprid SC (Tanjong Tokong and Green Lane strains), Pesguard FG161 (Tanjong Tokong and Green Lane strains), Sumithrin (all C. hemipterus strains), Sumithion (all C. hemipterus strains), and Phantom (all strains tested) at longer mortality assessment intervals. We demonstrated that insecticide exposure time and mortality assessment interval could potentially affect outcomes of product performance evaluations, resulting in underestimation or overestimation of insecticide resistance levels in field populations.
Keywords: tropical bed bug; insecticide resistance; exposure time; mortality assessment interval
The global resurgence of bed bugs has been a growing concern over the last two decades. The two species of bed bugs, namely the common bed bug (Cimex lectularius) and the tropical bed bug (Cimex hemipterus), are frequently associated with humans [[
Insecticide resistance in bed bugs has been evaluated using technical grade insecticides [[
There have been limited studies on the effect of exposure time on treated surface and mortality assessment intervals on time response assays tested with a single insecticide application rate. Previous reports on insecticide formulations were evaluated at fixed exposure times on treated surfaces, ranging from the shortest being 5 min exposure [[
Various published sources or guidelines on insecticide resistance monitoring suggests different mortality assessment intervals for time response assays with a single insecticide application rate. WHO [[
The control of bed bugs relies heavily on residual insecticides. Nonetheless, the contact period of bed bugs on the treated surface is unknown, due to their nocturnal and cryptic behavior in nature. Insecticide resistance evaluation with different exposure times and mortality assessment intervals may need to be tested to better reflect insecticide efficacies in field condition. Similar studies have been conducted by Vander Pan et al. (2019) [[
This study evaluated the influence of exposure time (5 min, 10 min, 30 min, 1 h, 4 h, and continuous exposure) and mortality assessment interval on the bioassay results of different insecticide products when applied at the label rate. The evaluations were carried out using a surface contact method on adult field strains of C. hemipterus and a laboratory susceptible strain of C. lectularius. Knockdown responses and mortality were recorded for up to 4 days with the fast-acting insecticide products (Tandem, Temprid SC, Pesguard FG161, Sumithrin, and Sumithion) and up to 14 days for the slow-acting product (Phantom).
Three C. hemipterus field strains (Madam Mo, Tanjong Tokong, and Green Lane) collected in Penang, Malaysia were evaluated in this study (Table 1). A susceptible C. lectularius strain, Monheim, was chosen as the reference strain as no susceptible C. hemipterus strain could be sourced worldwide. All bed bug strains were reared in 0.5-L glass containers with folded craft papers as harborages, under conditions of 27 ± 2 °C, 70% ± 5% RH and a photoperiod of 12:12 (L: D). Bed bugs were fed defibrinated rabbit blood (No. of animal ethics approval: USM/Animal Ethics Approval/2016/(
Six insecticide products were tested in this study (Table 2). The products were diluted to label rate using deionized water. The diluted insecticide was applied onto a glass Petri dish (diameter: 90 mm) based on the application rate (Table 2) and spread evenly. Control Petri dishes were treated with deionized water only. The treated Petri dishes were placed in a fume hood and allowed to dry overnight. The wall of the treated Petri dishes was coated with a thin layer of Fluon (polytetrafluoroethylene suspension; BioQuip, Rancho Dominguez, CA) to prevent the insects from escaping.
Six experiment sets with different exposure times were used and included 5 min, 10 min, 30 min, 1 h, 4 h, and continuous exposure. For Tandem, Temprid SC, Pesguard FG161, Sumithrin and Sumithion, continuous exposure was up to 4 days, while for Phantom, it was 14 days. Ten adult bed bugs were introduced into each treated Petri dish (sex ratio 1:1). After the designated exposure time, the treated bed bugs were removed (except for continuous exposure) and placed into clean Petri dishes with folded filter paper as a harborage. Knockdown responses of the treated bed bugs were observed at regular time intervals (5 min interval for the first hour, 30 min interval for first 6 h and subsequently 6 h interval until experiment ended) for up to 4 days (for Tandem, Temprid SC, Pesguard FG161, Sumithrin, and Sumithion) and up to 14 days (for Phantom). A bed bug was considered knocked down when it could not right itself up after being gently probed with a pair of soft forceps. The knocked down bed bugs were kept in a clean container during the first 24 h in case any insects recovered. Mortalities of the knocked down insects (defined as death rate of tested insects) were scored after the 24 h period, and subsequently every day until the experiment ended. The knocked down bed bugs were considered dead when they showed no movement or were in a moribund state (on their back and with uncoordinated movement) after being gently probed with a pair of soft forceps after 24 h. Each experiment set was replicated three times.
Control knockdown and mortality were corrected using Abbott formula [[
Monheim and Madam Mo strains showed no significant differences between all KT
When exposed to pyrethroid only based products (Pesguard FG161 and Sumithrin), Monheim strain showed no significant differences between all exposure times tested (Table 4). Madam Mo strain demonstrated low resistance levels at all exposure times towards both Pesguard FG161 and Sumithrin, with 5 min exposure to Pesguard FG161 showed significantly larger KT
In treatment with Sumithion, the KT
Monheim strain demonstrated significantly lower KT
All strains exhibited no significant differences between mortalities tested at different exposure times with Tandem (Figure 1). Similarly, no significant differences between mortalities were observed in the Monheim and Madam Mo strains when treated with Temprid SC (Figure 2A,B). Nonetheless, significant differences were found between mortalities tested at different exposure times for Tanjong Tokong (Kruskal–Wallis test: 1 day: χ
For treatment with Pesguard FG161 and Sumithrin, the Monheim strain showed 100% mortalities towards all exposure times at 1 and 4 days post-treatment (Figure 3A and Figure 4A). When tested with Pesguard FG161, Madam Mo strain demonstrated no significant differences between all exposure times at 1 and 4 days post-treatment (Figure 3B). For Sumithrin, exposure times caused a significant effect on 1 day post-treatment mortalities of the Madam Mo strain (Kruskal–Wallis test: 1 day: χ
For Sumithion, no significant differences were observed between mortalities tested at different exposure times in the Monheim strain. Madam Mo (Figure 5B), Tanjong Tokong (Figure 5C), and Green Lane strains (Figure 5D) showed 100% mortalities in continuous exposure at 4 days post-treatment, while shorter exposure times resulted in <40% mortalities.
When treated with Phantom, exposure times significantly affected 1 day post-treatment mortalities of the Monheim strain (Kruskal–Wallis test: 1 day: χ
When treated with Tandem, all strains showed no significant differences between mortalities observed at different assessment intervals (Table S1). For Temprid SC, only the Tanjong Tokong strain exhibited significant differences between mortalities assessed at different intervals (Friedman test: 5 min: χ
This study investigated the influence of exposure time and mortality assessment interval on the bioassay results for three field strains of C. hemipterus collected in Penang, Malaysia. The Tanjong Tokong and Green Lane strains in this study showed higher knockdown responses at continuous exposure to Temprid SC, Pesguard FG161, Sumithrin, Sumithion, and Phantom. Higher mortalities were also observed in these strains when longer exposure times were tested, compared to those exposed for shorter times. In a study with Anopheles mosquitoes tested on Mosquitoes Contamination Device (MCD) bottle bioassay, longer exposure times also resulted in significantly higher knockdown rates and 24 h mortalities [[
It is also important to note that a longer exposure time could potentially kill all heterozygous resistant insects, hence potentially masking the detection of insecticide resistance. When a longer exposure time is used, this will lead to a higher amount of insecticide contact by the test insects. Moderate resistant strains, normally with a higher number of heterozygous resistant individuals, may have most of the test insects killed after the long exposure, hence masking the ability to detect resistance in these strains. On the other hand, if the exposure time is too short (which leads to lower amount of insecticide absorption), this may lead to overestimation of the resistance level [[
According to our study, the Green Lane strain showed 100% knockdown and mortality when tested with 4 h and continuous exposure to Phantom but KT
In the present study, KT
Besides, the slower knockdown response also could be due to the insecticide mode of action. For example, chlorfenapyr (Phantom) must first be converted into an active metabolite (AC 303268) through oxidative removal of the N-ethoxymethyl group before the metabolite could inhibit mitochondrial ability to produce ATP [[
Different exposure times may also give the appearance of discrepancies in the performance of a product [[
It is imperative to determine whether knockdown rate, mortality rate, or both are more suitable to reflect the bioassay results, as these could provide a different outcome [[
This study demonstrated that exposure time and mortality assessment intervals could influence time response bioassay results with bed bugs when tested at a single label rate. In our study, bed bugs were found to show differential responses with exposure time and mortality assessment intervals, resulting in different outcomes in RRs and mortalities. However, this may not apply to concentration-response bioassays that use a series of concentrations that result in mortalities between 0% and 100%. In a concentration-response bioassay, the time point for mortality assessment could affect lethal concentration (LC) and lethal dosage (LD) values. For example, a bioassay that records 24 h mortality is likely to show higher LC or LD values than that that registers mortality at 48 h. Nevertheless, the relative changes that occur in LC and LD values tested at different assessment intervals are likely similar for both susceptible and resistant strains. Hence, the assessment interval may unlikely affect the RR values of concentration-response bioassays significantly. More studies on this aspect are warranted.
Due to the lack of a known susceptible strain of C. hemipterus, we had to resort to the use of a susceptible strain of C. lectularius for comparison. It is assumed that both species do not have any inherent insecticide susceptibility differences, although we are unable to confirm this, and this may never be confirmed unless a susceptible C. hemipterus strain is established. In the past, other studies also had used a similar approach to evaluate insecticide resistance in C. hemipterus [[
We propose that all insecticide product evaluations on bed bugs should involve at least one susceptible and two insecticide-resistant strains for comparisons. Knockdown responses and mortalities data should be recorded up to 96 h for fast-acting insecticides and up to 14 days for slow-acting insecticides. Due to the cryptic nature of bed bugs, the insects may not spend much time on a treated surface in the field. Therefore, a shorter exposure time (probably <1 h) is recommended to better reflect the possible field exposure conditions on bed bugs.
Exposure times and mortality assessment intervals have significant influences on bioassays results of tests carried out using single label rates of insecticide products. Knockdown responses and mortalities of the resistant strains were substantially higher with longer exposure times on treated surfaces and longer time intervals for mortality assessment. Further investigations using technical grade active ingredients and more insecticide-resistant bed bug populations may be necessary to further substantiate the present findings.
Graph: Figure 1 Influence of exposure time on Tandem on percentage mortalities of a susceptible C. lectularius strain and three C. hemipterus strains at 1 and 4 days post-treatment. (A) Monheim, (B) Madam Mo, (C) Tanjong Tokong, (D) Green Lane strains. Bars with different letters are significantly different (Kruskal–Wallis test, p < 0.05).
Graph: Figure 2 Influence of exposure time on Temprid SC on percentage mortalities of a susceptible C. lectularius strain and three C. hemipterus strains at 1 and 4 days post-treatment. (A) Monheim, (B) Madam Mo, (C) Tanjong Tokong, (D) Green Lane strains. Bars with different letters are significantly different (Kruskal–Wallis test, p < 0.05).
Graph: Figure 3 Influence of exposure time on Pesguard FG161 on percentage mortalities of a susceptible C. lectularius strain and three C. hemipterus strains at 1 and 4 days post-treatment. (A) Monheim, (B) Madam Mo, (C) Tanjong Tokong, (D) Green Lane strains. Bars with different letters are significantly different (Kruskal–Wallis test, p < 0.05).
Graph: Figure 4 Influence of exposure time on Sumithrin on percentage mortalities of a susceptible C. lectularius strain and three C. hemipterus strains at 1 and 4 days post-treatment. (A) Monheim, (B) Madam Mo, (C) Tanjong Tokong, (D) Green Lane strains. Bars with different letters are significantly different (Kruskal–Wallis test, p < 0.05).
Graph: Figure 5 Influence of exposure time on Sumithion on percentage mortalities of a susceptible C. lectularius strain and three C. hemipterus strains at 1 and 4 days post-treatment. (A) Monheim, (B) Madam Mo, (C) Tanjong Tokong, (D) Green Lane strains. Bars with different letters are significantly different (Kruskal–Wallis test, p < 0.05).
Graph: Figure 6 Influence of exposure time on Phantom on percentage mortalities of a susceptible C. lectularius strain and three C. hemipterus strains at 1, 7, and 14 days post-treatment. (A) Monheim, (B) Madam Mo, (C) Tanjong Tokong, (D) Green Lane strains. Bars with different letters are significantly different (Kruskal–Wallis test, p < 0.05).
Table 1 Bed bug strains evaluated in this study.
Species Strain Location Collected Year Collected Monheim Lab colony, Monheim, Germany ≈Late 1960s Madam Mo Collected from apartment 2016 Tanjong Tokong Collected from foreign worker dormitory 2015 Green Lane Collected from nursing home 2015
Table 2 Insecticide products used in this study.
Insecticide Class Trade Name Active Ingredient (%) Application Rate (mg/m2) Pyrethroid-neonicotinoid mixture Tandem Thiamethoxam (11.6%), lambda-cyhalothrin (3.5%) 183.96 Temprid SC Imidacloprid (21%), betacyfluthrin (10.5%) 106.13 Pyrethroid Pesguard FG161 D-tetramethrin (4.4%), cyphenothrin (13.2%) 110 Sumithrin D-phenothrin (10%) 100 Organophosphate Sumithion Fenitrothion (20%) 250 Pyrrole Phantom Chlorfenapyr (24%) 300
Table 3 Classification of resistance level based on resistance ratio (RR
Resistance Ratio (RR50) Classification of Resistance Level ≤1 No resistance >1 to ≤5 Low resistance >5 to ≤10 Moderate resistance >10 to ≤50 High resistance >50 Very high resistance
Table 4 The KT
Product Strain Exposure Time KT50 (95% FL) (min) KT95 (95% FL) (min) Slope ± SE χ2 (df) RR50 Tandem Monheim 5 min 16.1 (15.3–17.0) 25.0 (22.9–28.4) 8.7 ± 1.0 2.5 (6) - 10 min 16.3 (15.3–17.4) 30.3 (27.2–35.0) 6.1 ± 0.5 3.0 (9) - 30 min 15.1 (14.2–16.2) 28.0 (24.5–34.0) 6.1 ± 0.6 4.0 (9) - 1 h 16.3 (15.1–17.6) 31.9 (27.8–39.2) 5.6 ± 0.6 2.4 (6) - 4 h 15.4 (14.3–16.6) 29.8 (26.1–35.9) 5.8 ± 0.6 1.8 (7) - Continuous 14.6 (13.7–15.8) 26.7 (23.3–32.4) 6.3 ± 0.7 1.3 (7) - Madam Mo 5 min 15.0 (14.3–15.8) 24.6 (22.5–27.9) 7.6 ± 0.7 4.7 (9) 0.9 10 min 16.2 (15.3–17.1) 29.5 (26.4–34.8) 6.3 ± 0.7 7.7 (9) 1.0 30 min 15.8 (15.0–16.7) 29.3 (26.4–33.8) 6.1 ± 0.5 4.1 (13) 1.0 1 h 15.6 (14.8–16.5) 28.9 (25.9–33.7) 6.2 ± 0.6 3.7 (10) 1.0 4 h 15.7 (14.8–16.6) 29.6 (26.3–34.9) 5.9 ± 0.6 2.9 (10) 1.0 Continuous 15.8 (14.9–16.8) 30.9 (27.4–36.7) 5.7 ± 0.6 5.6 (10) 1.1 Tanjong Tokong 5 min 33.6 (31.1–36.3) 104.3 (88.1–130.5) 3.3 ± 0.3 1.4 (19) 2.1 10 min 29.2 (26.8–32.0) 85.1 (70.4–110.8) 3.5 ± 0.3 1.9 (13) 1.8 30 min 28.2 (26.0–30.7) 82.0 (68.9–104.2) 3.6 ± 0.3 5.2 (14) 1.9 1 h 27.9 (25.7–30.4) 80.6 (67.6–102.4) 3.6 ± 0.3 2.3 (14) 1.7 4 h 31.6 (28.8–34.6) 91.0 (76.2–115.8) 3.6 ± 0.3 2.5 (12) 2.1 Continuous 29.2 (26.6–32.0) 97.8 (81.2–125.5) 3.1 ± 0.3 3.3 (15) 2 Green Lane 5 min 57.6 (53.3–61.5) 124.0 (109.2–148.7) 4.9 ± 0.5 4.3 (11) 3.6 10 min 49.7 (45.9–53.4) 115.6 (100.7–140.6) 4.5 ± 0.4 3.6 (11) 3.0 30 min 43.3 (40.1–46.6) 101.1 (87.8–122.9) 4.4 ± 0.4 3.0 (11) 2.9 1 h 39.5 (37.4–41.6) 80.6 (73.1–91.5) 5.3 ± 0.4 2.5 (17) 2.4 4 h 39.2 (37.0–41.4) 83.7 (74.8–97.5) 5.0 ± 0.4 4.6 (16) 2.5 Continuous 39.2 (37.3–41.2) 86.7 (77.7–100.0) 4.8 ± 0.4 4.5 (21) 2.7 Temprid SC Monheim 5 min 15.9 (15.1–16.6) 26.7 (24.5–29.9) 7.3 ± 0.6 2.9 (11) - 10 min 17.0 (16.1–17.9) 28.3 (25.5–33.3) 7.4 ± 1.0 3.6 (7) - 30 min 16.2 (15.2–17.3) 29.4 (26.2–34.5) 6.4 ± 0.6 2.4 (7) - 1 h 16.4 (15.3–17.4) 32.3 (28.5–38.7) 5.6 ± 0.6 3.7 (9) - 4 h 17.3 (16.3–18.3) 31.2 (28.3–35.6) 6.4 ± 0.6 2.1 (10) - Continuous 16.7 (15.6–17.9) 31.7 (28.2–37.2) 5.9 ± 0.6 4.4 (8) - Madam Mo 5 min 16.3 (15.5–17.0) 26.3 (24.3–29.2) 7.8 ± 0.7 1.5 (11) 1.0 10 min 17.0 (16.1–17.9) 28.6 (25.7–34.0) 7.3 ± 0.9 6.1 (7) 1.0 30 min 16.5 (15.7–17.4) 27.8 (25.2–32.1) 7.3 ± 0.8 3.0 (8) 1.0 1 h 15.8 (14.9–16.6) 27.8 (25.2–31.7) 6.7 ± 0.6 1.5 (10) 1.0 4 h 16.1 (15.2–16.9) 28.0 (25.5–31.9) 6.8 ± 0.6 1.7 (10) 0.9 Continuous 16.1 (15.3–16.9) 26.5 (24.1–30.6) 7.5 ± 0.8 1.8 (9) 1.0 Tanjong Tokong 5 min 168.2 (133.3–218.3) 2890.9 (1572.8–7402.3) 1.3 ± 0.2 1.4 (11) 10.6 10 min 201.3 (160.8–259.0) 4770.9 (2633.3–11159.0) 1.2 ± 0.1 3.5 (16) 11.8 30 min 183.5 (139.9–255.6) 4295.4 (2009.2–14846.2) 1.2 ± 0.1 0.8 (10) 11.3 1 h 52.3 (47.2–57.8) 174.0 (144.2–222.6) 3.1 ± 0.2 6.6 (13) 3.2 4 h 60.1 (51.3–68.6) 225.2 (183.8–299.7) 2.9 ± 0.3 7.5 (9) 3.5 Continuous 68.4 (61.0–75.7) 217.1 (118.6–278.2) 3.3 ± 0.3 4.8 (11) 4.1 Green Lane 5 min 691.2 (520.5–1056.8) 7437.7 (3539.9–28224.0) 1.6 ± 0.2 1.0 (7) 43.7 10 min 638.3 (503.0–915.0) 5080.6 (2644.2–17058.0) 1.8 ± 0.3 2.5 (7) 37.5 30 min 415.2 (339.1–519.7) 3353.0 (2113.0–6820.6) 1.8 ± 0.2 0.9 (10) 25.2 1 h 279.2 (232.0–342.5) 2273.3 (1482.2–4265.5) 1.8 ± 0.2 0.9 (10) 17.7 4 h 60.4 (56.5–64.4) 124.0 (110.3–145.3) 5.2 ± 0.5 2.2 (11) 3.8 Continuous 56.4 (52.4–60.4) 130.9 (115.4–155.1) 4.5 ± 0.4 2.5 (13) 3.5 Pesguard FG161 Monheim 5 min 8.0 (7.5–8.5) 14.5 (13.2–16.6) 6.3 ± 0.6 3.4 (9) - 10 min 7.4 (6.8–7.9) 14.2 (12.6–17.2) 5.8 ± 0.7 3.1 (7) - 30 min 7.6 (7.0–8.1) 14.2 (12.6–16.9) 6.0 ± 0.7 2.4 (7) - 1 h 7.7 (7.1–8.2) 16.4 (14.4–20.0) 5.0 ± 0.5 3.5 (9) - 4 h 8.0 (7.5–8.5) 14.2 (12.9–16.3) 6.7 ± 0.7 0.7 (8) - Continuous 8.1 (7.5–8.6) 15.1 (13.5–17.7) 6.1 ± 0.7 0.8 (8) - Madam Mo 5 min 13.0 (11.7–14.1) 23.6 (20.8–28.9) 6.3 ± 0.8 1.8 (4) 1.6 10 min 10.0 (8.9–11.1) 21.1 (17.5–29.3) 5.0 ± 0.8 1.2 (3) 1.4 30 min 9.1 (8.4–9.8) 18.6 (16.0–23.4) 5.3 ± 0.6 3.5 (8) 1.2 1 h 10.1 (9.5–10.8) 18.3 (16.2–22.1) 6.4 ± 0.7 2.5 (7) 1.3 4 h 9.1 (8.5–9.8) 17.3 (15.2–21.0) 5.9 ± 0.6 3.1 (8) 1.1 Continuous 9.4 (8.8–9.9) 17.3 (15.5–20.2) 6.2 ± 0.6 2.0 (9) 1.2 Tanjong Tokong 5 min >5760.0 >5760.0 ND ND >720.0 10 min >5760.0 >5760.0 ND ND >778.4 30 min >5760.0 >5760.0 ND ND >757.9 1 h >5760.0 >5760.0 ND ND >748.1 4 h >5760.0 >5760.0 ND ND >720.0 Continuous 778.1 (336.6–2594.6) >5760.00 0.5 ± 0.1 2.8 (5) 96.1 Green Lane 5 min >5760.0 >5760.0 ND ND >720.0 10 min >5760.0 >5760.0 ND ND >778.4 30 min >5760.0 >5760.0 ND ND >757.9 1 h >5760.0 >5760.0 ND ND >748.1 4 h >5760.0 >5760.0 ND ND >720.0 Continuous >5760.0 >5760.0 ND ND >711.1 Sumithrin Monheim 5 min 11.0 (10.3–11.6) 19.2 (17.2–22.3) 6.8 ± 0.7 2.1 (8) - 10 min 11.1 (10.5–11.7) 19.8 (18.1–22.4) 6.5 ± 0.6 1.5 (11) - 30 min 10.7 (10.1–11.3) 19.3 (17.5–22.2) 6.4 ± 0.6 2.0 (10) - 1 h 10.3 (9.7–10.9) 18.6 (16.7–21.8) 6.4 ± 0.7 3.0 (9) - 4 h 10.7 (10.1–11.3) 19.1 (17.1–22.5) 6.5 ± 0.7 2.1 (8) - Continuous 10.8 (10.2–11.4) 17.9 (16.4–20.3) 7.5 ± 0.8 1.6 (8) - Madam Mo 5 min 30.6 (28.0–33.3) 103.9 (86.3–134.1) 3.1 ± 0.3 0.4 (17) 2.8 10 min 28.0 (25.5–30.5) 94.8 (76.8–129.4) 3.1 ± 0.3 1.5 (15) 2.5 30 min 28.4 (26.3–30.5) 77.4 (66.3–94.8) 3.8 ± 0.3 1.4 (16) 2.7 1 h 27.3 (25.6–29.0) 62.1 (55.3–72.4) 4.6 ± 0.4 3.8 (16) 2.7 4 h 28.6 (26.9–30.4) 66.8 (59.3–78.0) 4.5 ± 0.3 3.3 (17) 2.7 Continuous 25.8 (23.3–28.3) 64.5 (55.7–79.3) 4.1 ± 0.4 1.5 (9) 2.4 Tanjong Tokong 5 min >5760.0 >5760.0 ND ND >523.6 10 min >5760.0 >5760.0 ND ND >518.9 30 min >5760.0 >5760.0 ND ND >538.3 1 h >5760.0 >5760.0 ND ND >559.2 4 h >5760.0 >5760.0 ND ND >538.3 Continuous 5367.7 (3642.1–10435.0) >5760 1.1 ± 0.2 3.9 (7) 497.0 Green Lane 5 min >5760.0 >5760.0 ND ND >523.6 10 min >5760.0 >5760.0 ND ND >518.9 30 min >5760.0 >5760.0 ND ND >538.3 1 h >5760.0 >5760.0 ND ND >559.2 4 h >5760.0 >5760.0 ND ND >538.3 Continuous >5760.0 >5760.0 ND ND >533.3 Sumithion Monheim 5 min 157.2 (145.7–167.3) 294.4 (264.9–344.4) 6.0 ± 0.7 3.7 (9) - 10 min 133.3 (112.3–147.9) 306.8 (264.3–402.1) 4.5 ± 0.7 2.4 (8) - 30 min 137.1 (129.2–143.8) 243.8 (224.3–274.7) 6.6 ± 0.7 2.3 (14) - 1 h 128.5 (119.9–136.7) 207.1 (189.5–234.2) 6.2 ± 0.6 1.3 (8) - 4 h 113.5 (106.0–121.1) 221.5 (197.6–259.7) 5.7 ± 0.5 2.3 (9) - Continuous 116.4 (109.5–123.5) 211.3 (189.0–248.9) 6.4 ± 0.7 1.7 (8) - Madam Mo 5 min >5760.0 >5760.0 ND ND >36.6 10 min >5760.0 >5760.0 ND ND >43.2 30 min >5760.0 >5760.0 ND ND >42.0 1 h >5760.0 >5760.0 ND ND >44.8 4 h >5760.0 >5760.0 ND ND >50.7 Continuous 698.8 (653.6–743.8) 1207.3 (1087.2–1405.9) 5.8 ± 0.5 1.9 (6) 6.0 Tanjong Tokong 5 min >5760.0 >5760.0 ND ND >36.6 10 min >5760.0 >5760.0 ND ND >43.2 30 min >5760.0 >5760.0 ND ND >42.0 1 h >5760.0 >5760.0 ND ND >44.8 4 h >5760.0 >5760.0 ND ND >50.7 Continuous 921.7 (812.8–1035.3) 3713.7 (3045.2–4823.5) 2.7 ± 0.2 1.4 (13) 7.9 Green Lane 5 min >5760.0 >5760.0 ND ND >36.6 10 min >5760.0 >5760.0 ND ND >43.2 30 min >5760.0 >5760.0 ND ND >42.0 1 h >5760.0 >5760.0 ND ND >44.8 4 h >5760.0 >5760.0 ND ND >50.7 Continuous 702.7 (638.1–767.3) 1450.2 (1250.0–1819.3) 5.2 ± 0.6 3.4 (5) 6.0
Table 5 The KT
Strain Exposure Time KT50 (95% FL) (h) KT95 (95% FL) (h) Slope ± SE χ2 (df) RR50 Monheim 5 min 24.0 (23.9–24.2) 24.9 (24.8–25.2) 104.7 ± 12.9 1.0 (5) - 10 min 23.7 (23.6–23.9) 24.8 (24.6–25.2) 85.88 ± 12.1 3.8 (4) - 30 min 23.2 (23.0–23.4) 25.3 (24.9–25.9) 44.0 ± 4.6 4.2 (7) - 1 h 22.6 (22.3–22.9) 25.7 (25.0–26.7) 30.2 ± 3.7 2.4 (8) - 4 h 22.8 (22.4–23.1) 24.9 (24.3–25.9) 41.9 ± 4.3 9.4 (7) - Continuous 21.4 (20.2–22.2) 25.6 (24.3–29.1) 21.1 ± 3.1 5.0 (4) - Madam Mo 5 min 36.1 (32.5–39.6) 84.9 (72.8–106.8) 4.4 ± 0.5 3.3 (7) 1.5 10 min 30.4 (26.7–34.4) 91.8 (73.1–130.5) 3.4 ± 0.4 2.7 (6) 1.3 30 min 32.0 (29.0–35.1) 77.6 (66.2–96.9) 4.3 ± 0.4 6.3 (8) 1.4 1 h 31.6 (27.5–35.7) 79.2 (64.5–108.7) 4.1 ± 0.5 7.2 (7) 1.4 4 h 25.8 (21.2–29.7) 95.8 (75.9–143.2) 2.9 ± 0.4 2.7 (8) 1.1 Continuous 23.9 (19.7–27.7) 102.9 (75.9–175.6) 2.6 ± 0.4 1.9 (7) 1.1 Tanjong Tokong 5 min 32.6 (28.7–36.6) 108.7 (95.3–154.5) 3.1 ± 0.4 2.0 (8) 1.4 10 min 38.4 (34.8–42.5) 102.6 (85.1–133.9) 3.8 ± 0.4 0.9 (8) 1.6 30 min 22.4 (19.1–25.1) 60.4 (47.8–95.8) 3.8 ± 0.6 1.0 (5) 1.0 1 h 20.8 (16.8–23.9) 67.6 (53.0–106.8) 3.2 ± 0.5 3.6 (6) 0.9 4 h 21.6 (16.5–25.5) 95.8 (67.7–199.5) 2.5 ± 0.5 1.6 (6) 0.9 Continuous 18.9 (16.1–21.3) 57.0 (45.4–84.2) 3.4 ± 0.5 2.5 (6) 0.9 Green Lane 5 min >336.0 >336.0 ND ND >14.0 10 min >336.0 >336.0 ND ND >14.2 30 min 332.0 (235.0–736.3) >336.0 1.5 ± 0.4 2.0 (6) 14.3 1 h 284.9 (202.3–769.1) >336.0 1.2 ± 0.4 0.8 (6) 12.6 4 h 48.4 (39.2–57.8) 378.9 (272.2–612.8) 1.8 ± 0.2 1.1 (11) 2.1 Continuous 28.3 (22.4–33.9) 81.2 (58.8–173.4) 3.6 ± 0.5 6.4 (5) 1.3
Conceptualization, X.-Y.L. and C.-Y.L.; methodology, X.-Y.L.; formal analysis, X.-Y.L.; resources, C.-Y.L., G.V.S., S.L.D., A.C.S.-C.; writing—original draft preparation, X.-Y.L. and C.-Y.L.; writing—review and editing, X.-Y.L., G.V.S., A.C.S.-C., S.L.D. and C.-Y.L.; supervision, G.V.S., A.C.S.-C. and C.-Y.L.; project administration, A.C.S.-C., G.V.S. and C.-Y.L.; funding acquisition, C.-Y.L. All authors have read and agreed to the published version of the manuscript.
X.-Y.L. was financially supported by a Universiti Sains Malaysia Fellowship. This study was supported by Ministry of Education (MOE) Malaysia through Fundamental Research Grant Scheme FRGS/1/2018/STG03/USM/02/2, and Semco Co. (Japan).
The authors declare no conflict of interests.
We thank Kai Dang and Dae-Yun Kim for their advice and assistance during the planning and development of this research.
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By Xin-Yeng Leong; G. Veera Singham; Alexander Chong Shu-Chien; Stephen L. Doggett and Chow-Yang Lee
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