In total, 6 herb essential oils were purchased from Jin-Ah Aromatics (Anyang, Kyunggi Province, Korea); pennyroyal (Mentha pulegium), ylang ylang (Cananga odorata), citronella (Cymbopogon nardus), lemon grass (Cymbopogon citratus), tea tree (Melaleuca alternifolia), and rosemary (Rosmarinus officinalis). Negative control was applied ethanol only and positive control used was a commercial acaricidal product. The major active components of the positive control are permethrin 0.98 g/100 g, pthalthrin 0.294 g/100 g, and fragrance.

The mites D. farinae and D. pteronyssinus, which have been maintained in our veterinary parasitology laboratory for 5 years, without exposure to any known acaricide, were used. Mites were reared in plastic containers (12.5 × 10.5 × 5.0 cm) at 25.0 ± 1.0℃ and 75 % relative humidity in dark chamber and fed a laboratory diet (powdered rat feed plus Ebioze® powder 1:1 by weight).

A filter paper contact bioassay was used to evaluate toxicities of the herb essential oils and acaricides to D. farinae and D. pteronyssinus. In preliminary experiments with pennyroyal, ylang ylang, citronella, lemon grass, tea tree, and rosemary essential oils and the positive control, 0.1 µl/cm² appeared an appropriate starting dose for primary screening. If an herb essential oil had similar or better activity than the positive control further bioassays were conducted. First, 40 µl of ethanol were applied to all testing filter papers. After drying under a fume hood for 1 min, each filter paper was placed at the bottom of a Petri dish (5 cm in diameter, 1.2 cm in height). Batches of about 300 D. farinae and D. pteronyssinus were placed in each Petri dish. The negative control filter papers received 40 µl of ethanol and about 300 mites only. Amounts (0.1, 0.05, 0.025, 0.0125, and 0.00625 µl/cm²) of each herb essential oil and the positive control were applied to filter papers (Whatman No.2, 4.25 cm in diameter) and lids were then fitted.

In a separate experiment, vapor phase toxicity against D. farinae and D. pteronyssinus was investigated according to the method described by Kwon and Ahn (2002). Briefly, batches of about 300 D. farinae and D. pteronyssinus were placed on the bottom of Petri dishes (5 cm diameter × 1.2 cm) and covered with a lid containing a fine wire sieve (200 mesh, 4 cm diameter) attached to the middle part of the Petri dish. Filter papers (4 cm in diameter), treated with pennyroyal oil concentrations (0.1, 0.05, 0.025, 0.0125, and 0.00625 µl/cm²) were placed over the wire sieve, which prevented direct contact between mites and test oils. Each Petri dish was then covered with a lid (method B) to investigate the vapor phase toxicities of the test compounds or left uncovered (method A). Control Petri dishes were exposed to filter paper containing 40 µl of ethanol only. Mortalities were determined at 5, 10, 20, 30, and 60 min after treatment under a binocular microscope. Mites were considered dead if appendages did not move when the mite was prodded with a pin.

Chromatographic analyses were performed using a Hewlett-Packard 5890II (Palo Alto, California, USA) series gas chromatograph, equipped with an injector and a flame ionization detection (FID) system. Separation and quantitation were performed using a DB-WAX column, 30 m × 0.252 mm ID, with a film thickness 0.25 µm (J & W Scientific Inc., Folsom, California, USA).

The GC temperature program used was as follows: initial temperature 40℃, held for 5 min then ramped at 6℃/min to 240℃, and held for 5 min. Helium was used as the carrier (l ml/min). The FID gases were hydrogen and air at flow-rates of 500 and 45 ml/min, respectively. The detector temperature was set at 270℃ and the injector block temperature at 240℃.

GC-MS was performed using a GC (HP 5890II) - Mass spectrometer (JMS-600W, JEOL, Tokyo, Japan), using the GC column and temperature conditions described above. Helium was used as the carrier gas at a flow of 1 ml/min. The GC/MS interface was held at 230℃ and mass spectra were obtained at 70 eV. The effluent of the capillary column was introduced directly into the MS ion source. The mass analyzer was set to scan from 50 to 550 amu every 0.7 sec. Constituents of pennyroyal oil were identified by comparing of MS data with data in a MS library (Wiley Registry of Mass Spectral Data, 6th ed.) and these findings were confirmed by comparing GC retention times with those of authentic samples.

Direct contact testing showed that pennyroyal at a dose of 0.025 µl/cm² for 5 min had the greatest acaricidal effect (98.6% mortality) followed by ylang ylang (50.4%), lemon grass (9.8%), tea tree (0%), and rosemary (0%). Mortality depended on exposure time and dose (Table 1). The negative control had a mortality of 0% at this test dose and exposure time and the positive control had a mortality of 0% at a dose of 0.1 µl/cm² for 5 min.

The method A was less effective than the method B (Table 2). For the method A at a dose of 0.1 µl/cm², mortalities for pennyroyal oil ranged from 0% to 42.6% (time from 5 to 60 min did not much affect these results), whereas for the method B, mortalities of pennyroyal ranged from 2.1% to 100% and these were time- (from 5 to 60 min) and dose-dependent (from 0.1 to 0.025 µl/cm²). For the method B, after exposure for 30 and 60 min, mortalities ranged from 90.4% to 100% at 0.1 µl/cm². The minimum dose required to kill house dust mites was 0.025 µl/cm² for 60 min (Table 2). The other doses and time had no acaricidal effects.

GC-MS showed that pennyroyal essential oil composed almost entirely of pulegone component (99%) (Fig. 1). Five other constituents were identified, i.e., cyclohexanone, 8-hydroxy-δ-4(5)-p-methen-3-one, 3-octanol, dl-limonene, and β-pinene. Pulegone had a retention time on our system of 14.55 min, and β-pinene was the first eluted at 4.15 sec.