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Enhanced Repellency of Binary Mixtures of Zanthoxylum armatum Seed Oil, Vanillin, and Their Aerosols to Mosquitoes Under Laboratory and Field Conditions HYUNG WOOK KWON,1 SOON-IL KIM,1 KYU-SIK CHANG,2 J. MARSHALL CLARK,3 1,4,5 AND YOUNG-JOON AHN

J. Med. Entomol. 48(1): 61Ð66 (2011); DOI: 10.1603/ME10042

ABSTRACT The repellency of Zanthoxylum armatum seed oil (ZA-SO), alone or in combination with vanillin (VA), its six major constituents, and another four major previously known Zanthoxylum piperitum fruit oil constituents, as well as aerosol products containing 5 or 10% ZA-SO and 5% VA, was evaluated against female Aedes aegypti in laboratory and Þeld studies. Results were then compared with those of N,N-diethyl-3-methylbenzamide (DEET) as a standard. Hand in cage laboratory tests showed that 0.2, 0.1, and 0.05 mg/cm2 ZA-SO resulted in ⬎92% protection through 30-min postexposure and was not signiÞcantly different than 0.05 mg/cm2 DEET. Skin treated with linalool and limonene (from Z. armatum) provided ⬎80% repellency to female Ae. aegypti at 10-min exposure, whereas cuminaldehyde, citronellal, geranyl acetate, and cuminyl alcohol (from Zanthoxylum piperitum) provided ⬎90% protection during this same time period. Only cuminaldehyde and citronellal provided complete protection comparable to DEET at 10-min postexposure. After that time, repellency of all plant constituents to mosquitoes was considerably decreased (⬍⬇65%). An increase in repellency and duration of effectiveness was produced by a binary 1:4 mixture of ZA-SO and VA (0.05:0.2 mg/cm2) that was signiÞcantly more effective than 0.05 mg/cm2 DEET through 90 min. In Þeld tests, an aerosol formulation containing 5 or 10% ZA-SO plus 5% VA gave 100% repellency at 60-min postexposure. Although these formulations were equal to the level of protection afforded by 10% DEET, repellency to the binary ZA-SO aerosol formulations at 90 min was signiÞcantly less effective than DEET. However, mixtures formulated from ZA-SO and VA merit further study as potential repellents for protection of humans and domestic animals from biting and nuisance caused by mosquitoes. KEY WORDS Zanthoxylum armatum, Zanthoxylum piperitum, natural mosquito repellent, vanillin, binary mixtures

Mosquito repellents can be effective tools for protecting humans from mosquito attack and mosquito-borne diseases (Peterson and Coats 2001, Moore et al. 2006). The most widely used mosquito repellent products are currently based on N,N-diethyl-3-methylbenzamide (DEET) (Fradin 1998), which continues to be an effective compound. It is estimated that approximately one-third of the people in the United States use DEET-containing products in various formulations (Fradin 1998). However, DEET can have an unpleas1 World Class University, Biomodulation Major, Department of Agricultural Biotechnology, Seoul National University, Seoul 151-921, Republic of Korea. 2 National Institute of Health, Korea Center for Disease Control and Prevention, Seoul 122-701, Republic of Korea. 3 Department of Veterinary and Animal Science, University of Massachusetts, Amherst, MA 01003. 4 Department of Plant Protection, Huazhong Agricultural University, Wuhan 430070, HuBei, P.R. China. 5 Corresponding author: Department of Agricultural Biotechnology, Seoul National University, Seoul 151-921, Republic of Korea (e-mail: [email protected]).

ant odor and may cause damage to certain plastics and synthetic rubber materials. Moreover, occasional toxic effects have been reported that include central nervous system depression, urticaria, contact dermatitis, or potential encephalopathic toxicity (Qiu et al. 1998, Katz et al. 2008). These problems highlight the need for the development of new products for repelling mosquitoes. Plant essential oils have been suggested as alternative sources for mosquito repellents largely because they constitute a potential source of bioactive chemicals that have been perceived by the general public as relatively safe and pose fewer risks to the environment with minimal impacts to animal and human health (Isman 2001, Panella et al. 2005). Additionally, essential oils are widely available, with some being relatively inexpensive compared with plant extracts (Isman 2006). Moreover, many essential oils are commonly used as fragrances and ßavoring agents for foods, beverages, and cosmetics (Lawless 2002). A number of products for repelling mosquitoes, based on

0022-2585/11/0061Ð0066$04.00/0 䉷 2011 Entomological Society of America

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essential oils, such as citronella, fennel, geranium, lavender, and rosemary, have been commercialized (Curtis et al. 1990, Brown and Hebert 1997). Recently, Zanthoxylum sp. in the family Rutaceae have drawn attention because they exhibit insecticidal activity against Aphis craccivora Koch and Plutella xylostella (L.) (Nissanka et al. 2001) or repellency against mosquitoes (Trongtokit et al. 2005, Choochote et al. 2007). Historically, Zanthoxylum armatum DC has long been considered to have medicinal properties and is used as a carminative, a stomachic, and an anthelmintic and for the treatments of disorders of the digestive organs (Tiwary et al. 2007). Additionally, vanillin (VA) has been shown to enhance mosquito repellency of certain essential oils (Khan et al. 1975). However, very little information exists on the repellency of Z. armatum seed oil (ZA-SO) alone or in combination with VA to mosquitoes, although essential oil of Zanthoxylum piperitum (L.) DC is known to have the repellency against mosquitoes (Trongtokit et al. 2005, Kamsuk et al. 2007). In this study, we assess the repellency of female Aedes aegypti (L.) to ZA-SO alone or in combination with VA, its six major constituents, and four previously identiÞed compounds from the essential oil of the fruit of Z. piperitum (Sakai et al. 1968) under laboratory condition for use as future commercial repellents. The repellency of these materials was compared with that of the currently available repellent DEET. Also, the repellency to mosquito Þeld populations of aerosol formulations containing ZA-SO (5 and 10%) alone or in combination with VA (5%) was compared with that of aerosol containing 10% DEET as an active ingredient because lotion, liquid, aerosol, or cream products containing ⱕ10% DEET are required for the registration of repellents for children at the Korea Food and Drug Administration for marketing in South Korea (Anonymous 2010). Materials and Methods Gas Chromatography-Mass Spectrometry. Major constituents of ZA-SO were identiÞed by gas chromatography-mass spectrometry using an Agilent 6890N gas chromatograph-Agilent 5973N MSD mass spectrometer (Santa Clara, CA). Analytes were separated with a 30 m ⫻ 0.25-mm inside diameter (df ⫽ 0.25 ␮m) DB-1 fused-silica capillary column (J&W Science, Folsom, CA). Helium carrier gas column head pressure was 15.7 psi (108.4 kPa). The oven temperature was programmed from 40⬚C, held for 1 min, and ramped at 6⬚C/min to 250⬚C, and held for 4 min. Chemical constituents were identiÞed by comparison of mass spectra of each peak with those of authentic samples from a mass spectra library (Anonymous 2000). Test Materials. ZA-SO was purchased from Seema International (Delhi, India). Six major constituents (1,8-cineole, limonene, linalool, linalool oxide, methyl cinnamate, and ␤-myrcene) identiÞed in ZA-SO used in this study were purchased from Sigma-Aldrich (St. Louis, MO). An additional four previously identiÞed

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compounds from the essential oil of the fruit of Z. piperitum (Sakai et al. 1968) were likewise evaluated (i.e., citronellal, cuminyl alcohol, cuminaldehyde, and geranyl acetate) and purchased from Sigma-Aldrich. DEET (97% purity) and vanillin (99% purity) were supplied by Sigma-Aldrich. All other chemicals used were of reagent-grade quality and commercially available. Aerosol Formulation Evaluation. Aerosols containing 5 and 10% ZA-SO (ZA-SO-5/VA-5% and ZA-SO10/VA-5% aerosol) were formulated using 5 or 10 g ZA-SO, 5 g VA, 20 g liqueÞed petroleum gas, and 70 or 65 g ethanol in aluminum cans, respectively. These aerosols were experimental formulations and packaged at the Central Research Center, Daeyuk Can Co. (Seoul, South Korea). ZA-SO, VA, and ethanol were put into the can and closed with a nozzle cap, and then weighed with liqueÞed petroleum gas to Þnal amount. A 10% DEET aerosol formulation (DEET-10% aerosol; Kukbo Science, Cheongju, Chungbuk Province, South Korea) was composed of 10 g DEET, 20 g liqueÞed petroleum gas, and 70 g ethanol. Subjects. Healthy human volunteers (male; 20 Ð24 yr old) were recruited from the students of the Department of Applied Biology and Chemistry, College of Agriculture and Life Sciences, Seoul National University. Before repellency tests of each material examined, all procedures and caution during test periods were properly informed and all human subjects were provided written informed consent. The ethical approval was permitted by the Internal Review Board, Seoul National University. Mosquitoes. Cultures of Ae. aegypti were maintained in the laboratory for 10 yr without exposure to insecticides (Perumalsamy et al. 2009). Adults were maintained on a 10% sucrose solution and blood fed on live mice. Larvae were reared in plastic trays (24 ⫻ 35 ⫻ 5 cm) containing 0.5 g of sterilized diet (40-mesh chick chow powder/yeast, 4:1 by weight). Larvae and adult mosquitoes were reared at 26 ⫾ 1⬚C, 65Ð75% relative humidity (RH), and a photoperiod of 16:8 (L:D) h. Laboratory Test. Test materials were evaluated on four human volunteers using the method of Kim et al. (2004). All bioassays were conducted between 16:00 and 20:30 h. Varying amounts (0.025, 0.05, 0.1, and 0.2 mg/cm2) of the test materials and DEET, each in 100 ␮l of ethanol, were directly applied to the exposed skin of the back of the left hand through a 5-cm-diameter hole made on back part of a rubber glove. Controls received 100 ␮l of ethanol only. After air drying for 1 min, the treated hand of each volunteer was exposed to mosquitoes for 5 min in the acrylic cage (35 ⫻ 35 ⫻ 35 cm) with wire screen mesh on four sides containing 200 Ð250 blood-starved, Ae. aegypti females (7Ð10 d old) (Rozendaal 1997). Tests were conducted at 26 ⫾ 2⬚C and 65Ð75% RH. VolunteersÕ hands were exposed at 5, 25, 55, 85, 115, and 145 min without retreatment of the hand. After one test cycle (⬍150 min) was terminated, the hands of volunteers were exposed alternately to new female mosquitoes and the test

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Table 1. Relative abundance of major constituents of ZA-SO identified by gas chromatography and gas chromatography-mass spectrometry Constituent

RT (min)

% area

␤-Myrcene 1,8-Cineole Limonene Linalool oxide Linalool Neral Geranial Methyl cinnamate Other compounds

9.20 10.11 10.16 11.41 11.86 14.84 15.49 17.87

1.63 6.05 11.05 0.91 56.82 4.63 5.36 7.91 5.64

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Data Analysis. Mean percentage of repelled mosquitoes was transformed to arcsine square root values, then analyzed with a general linear model using SAS procedures (SAS Institute 2004). The repellent index was calculated according to the following formula: % repellency ⫽ ([Ta ÐTb]/Ta) ⫻ 100, where Ta is number of mosquitoes in control and Tb is number of mosquitoes in the treated (Schreck et al. 1977). The Bonferroni multiple-comparison method was used to test for signiÞcant differences among treatments (SAS Institute 2004). Means ⫾ SE of untransformed data are reported.

Rt, retention time.

Results repeated. The numbers of biting females were recorded, and each assay was replicated three times. To determine an effective dose and mixture ratio for ZA-SO and VA, the repellency of ZA-SO (0.05 mg/ cm2) alone or in combination with VA (0.025, 0.05, 0.1, and 0.2 mg/cm2) in 100 ␮l of ethanol was examined, as described above. The treated hand of each volunteer was exposed for 1 min in the cage containing 400 Ð500 blood-starved, Ae. aegypti females to evaluate more clearly the enhanced repellency of the test materials and to check mosquito response much more quickly. Controls received 100 ␮l of ethanol only and each assay was replicated three times. Field Test. The repellency of three aerosol products was examined using six volunteers between mid-July and mid-September 2007, in a suburban area at Paju (Gyeonggi Province, South Korea), described previously by Kim et al. (2004). Each test was conducted between 19:00 and 22:30 h in 30-min test periods. The exposure time was enough to collect ⬎20 mosquitoes in every control volunteer. Throughout the test, there was little or no wind. Aerosols were applied for 4 s at ⬇20 cm distance in sufÞcient amounts to evenly cover the skin below the knees. Control volunteers received only liqueÞed petroleum gas and ethanol. All volunteers wore shorts, T-shirts, and shoes to protect against excessive attack by mosquitoes. Treatments and three collection sites were randomly selected among the six volunteers and were rotated during the experimental period. Mosquitoes that landed on exposed legs (below the knees) were captured using an aspirator. These mosquitoes were transferred to a plastic container, counted, and identiÞed. All treatments were replicated three times.

Table 2.

Repellency of ZA-SO and DEET to female Ae. aegypti using hand in cage exposure bioassays

Treatment

Concn (mg/cm2)

ZA-SO

0.2 0.1 0.05 0.025 0.05

DEET

Chemical Constituents of ZA-SO. ZA-SO was composed of eight major and 16 minor constituents by comparison of mass spectral data and coelution of authenticated samples after coinjection. The eight major constituents were linalool, limonene, methyl cinnamate, 1,8-cineole, geranial, neral, ␤-myrcene, and linalool oxide, and comprised 56.82, 11.05, 7.91, 6.05, 5.36, 4.63, 1.63, and 0.91% of the oil, respectively (Table 1). Together, they constituted ⬇94% of total constituents of the ZA-SO. Laboratory Repellency Test to Ae. aegypti. Effect of concentration (F ⫽ 125.64; df ⫽ 4, 54; P ⬍ 0.0001) and exposure time (F ⫽ 384.65; df ⫽ 5, 54; P ⬍ 0.0001) on repellence was signiÞcant (Table 2). The concentration by exposure interaction was also signiÞcant (F ⫽ 11.35; df ⫽ 17, 54; P ⬍ 0.0001). Concentrations at 0.05, 0.1, and 0.2 mg/cm2 resulted in complete (100%) repellency at 10 min and were just as effective as 0.05 mg/cm2 DEET against female Ae. aegypti. At 30 min, the highest ZA-SO concentration still provided significantly greater mosquito repellency compared with the other seed oil concentrations and DEET. At 60 min repellency of all treatments had considerably decreased to ⱕ80% with no signiÞcant difference between ZA-SO concentrations and DEET, with the exception of the seed oil at 0.025 mg/cm2, which proved to be signiÞcantly lower than the rest of the treatments. Repellencies were signiÞcantly different among treatments (F ⫽ 54.90; df ⫽ 10, 42; P ⬍ 0.0001; Table 3). Cuminaldehyde, citronellal, geranyl acetate, and cuminyl alcohol at 0.05 mg/cm2 resulted in ⱖ90% repellency at the 10-min posttreatment interval, but after that mosquito repellency of these compounds

% repellency (mean ⫾ SE) at minutes after exposure 10

30

60

90

120

150

100a 100a 100a 89 ⫾ 2.3b 100a

100a 93 ⫾ 1.5b 92 ⫾ 1.3b 40 ⫾ 1.8c 86 ⫾ 2.2b

80 ⫾ 2.0a 61 ⫾ 5.5a 46 ⫾ 6.4a 11 ⫾ 6.7b 73 ⫾ 1.2a

62 ⫾ 2.4a 47 ⫾ 4.9a 24 ⫾ 3.0b

49 ⫾ 3.2a 20 ⫾ 2.7b 8 ⫾ 2.9b

40 ⫾ 4.3a 5 ⫾ 3.3c 3 ⫾ 1.7c

51 ⫾ 5.5a

49 ⫾ 0.9a

28 ⫾ 7.8ab

Means within a column followed by the same letter are not signiÞcantly different (P ⫽ 0.05, Bonferroni method).

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Table 3. Repellency of six major constituents identified in ZA-SO, four compounds previously identified in Z. piperitum fruit essential oil, and DEET to female Ae. aegypti exposed at 0.05 mg/cm2 using hand in cage exposure bioassays

Compound Cuminaldehydea Citronellala Geranyl acetatea Cuminyl alcohola Linaloolb Limoneneb Methyl cinnamateb Linalool oxideb ␤-Myrceneb 1,8-Cineoleb DEET

% repellency (mean ⫾ SE) at minutes after exposure 10

30

60

100a 100a 96 ⫾ 2.0ab 90 ⫾ 7.2cd 84 ⫾ 3.8bc 83 ⫾ 5.8bc 48 ⫾ 4.2d 46 ⫾ 7.6d 43 ⫾ 2.1d 37 ⫾ 5.2d 98 ⫾ 2.3ab

66 ⫾ 9.6ab 49 ⫾ 2.9bc 27 ⫾ 9.8cÐg 37 ⫾ 6.7bÐe 21 ⫾ 4.4cÐf 39 ⫾ 2.6bÐd 6 ⫾ 1.5fg 3 ⫾ 2.7fg 10 ⫾ 8.1dÐg 0g 92 ⫾ 5.2a

26 ⫾ 8.8a 3 ⫾ 1.3ab 3 ⫾ 2.7b

78 ⫾ 5.7a

Means within a column followed by the same letter are not significantly different (P ⫽ 0.05, Bonferroni method). a Constituents of Z. piperitum essential oil (Sakai et al. 1968). b Major constituents of ZA-SO.

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Table 5. Repellencies of aerosols containing ZA-SO with VA and DEET to mosquito field populations

Aerosol

% repellency (mean ⫾ SE) at minutes after treatment

ZA-SO-5/VA-5% ZA-SO-10/VA-5% DEET-10%

100a 52 ⫾ 5.4b 47 ⫾ 2.1c 24 ⫾ 1.8b 100a 73 ⫾ 3.3ab 60 ⫾ 1.5b 42 ⫾ 6.9ab 4 ⫾ 1.9b 100a 93 ⫾ 3.3a 83 ⫾ 2.1a 64 ⫾ 6.8a 60 ⫾ 0.6a

60

90

120

150

180

Means within a column followed by the same letter are not significantly different (P ⫽ 0.05, Bonferroni method).

ment and was signiÞcantly less effective than the DEET-10% aerosol. The mosquito species and their relative abundance (percentage of total number collected) during the Þeld evaluations were as follows: Anopheles sinensis Wiedemann (75.6%), Anopheles pullus Yamada (12.7%), and Aedes vexans (Meigen) (11.7%). Discussion

decreased considerably over time (F ⫽ 244.26; df ⫽ 2, 42; P ⬍ 0.0001). Linalool and limonene at 0.05 mg/cm2 resulted in ⬇83% repellency at the 10-min posttreatment interval. However, all these compounds were less effective than DEET. The differences in repellency after the application of the seven treatments (ZA-SO, VA, four mixtures ZA-SO and VA, and DEET) were signiÞcant (F ⫽ 73.23; df ⫽ 5, 50; P ⬍ 0.0001; Table 4). Adding 0.2 mg/cm2 VA to 0.05 mg/cm2 ZA-SO resulted in significantly greater repellency than either ZA-SO, VA, or DEET alone through 90-min postexposure. Moreover, the repellency and duration of effectiveness of this 1:4 mixture against female Ae. aegypti were signiÞcantly more pronounced when compared with the rest of the other mixtures. However, the repellency of the mixtures was dramatically decreased according to time after exposure (F ⫽ 131.29; df ⫽ 7, 50; P ⬍ 0.0001). Field Repellency Test. Repellency was signiÞcantly affected by formulation (F ⫽ 127.62; df ⫽ 2, 36; P ⬍ 0.0001) and exposure time (F ⫽ 274.01; df ⫽ 5, 36; P ⬍ 0.0001; Table 5). The formulation by exposure time interaction was also signiÞcant (F ⫽ 17.08; df ⫽ 10, 50; P ⬍ 0.0001). ZA-SO-5/VA-5%, ZA-SO-10/VA-5%, and DEET-10% aerosols provided complete protection at 60-min posttreatment, but after that repellency substantially decreased through the 180-min posttreatTable 4.

Investigations on the physical and physiological characteristics of natural-occurring repellents are of practical importance for mosquito control because it may give useful information on the most appropriate formulations and delivery means to be adapted for their future commercialization. Because of their high volatility, essential oils are usually effective against arthropods only for a relatively short period, typically ⬍1 h (Rozendaal 1997, Isman 2006). Indeed, Choochote et al. (2007) reported that 3.3 ␮l of Z. piperitum essential oil gave a median complete protection time of 0.5Ð1 h against female Ae. aegypti, whereas 10% Zantoxylum limonella Alston seed and fruit oils gave complete protection time for 30 min (Trongtokit et al. 2005). Our current Þndings establish that the repellency of 0.05 mg/cm2 ZA-SO was comparable to that of DEET to female Ae. aegypti at 30-min posttreatment. Of the six major constituents identiÞed and evaluated from Z. armatum, we found that linalool and limonene were most active. Nevertheless, all the individual compounds identiÞed from this plant were less effective than either the whole extract of ZA-SO or DEET. It has been also reported that the repellency duration against mosquitoes was more pronounced in binary mixtures of a repellent essential oil and VA than the single oil (Tawatsin et al. 2001, Tuetun et al. 2005,

Repellency of ZA-SO alone or in combination with VA and DEET to female Ae. aegypti using hand in cage exposure bioassays % repellency (mean ⫾ SE) at minutes after exposure

Treatment (mg/cm2)

30

60

90

ZA-SO 0.05 ZA-SO:VA 0.05:0.2 ZA-SO:VA 0.05:0.1 ZA-SO:VA 0.05:0.05 ZA-SO:VA 0.05:0.025 VA 0.2 DEET 0.05

87 ⫾ 2.6b 100a 92 ⫾ 2.6b 94 ⫾ 1.3b 93 ⫾ 1.2b 91 ⫾ 3.2b 87 ⫾ 3.3b

82 ⫾ 3.9b 99 ⫾ 1.0a 81 ⫾ 1.2b 83 ⫾ 1.5b 56 ⫾ 4.4c 77 ⫾ 3.9b 69 ⫾ 2.1bc

37 ⫾ 3.7c 93 ⫾ 1.8a 45 ⫾ 4.9bc 48 ⫾ 0.9bc 36 ⫾ 3.5c 56 ⫾ 4.3b 46 ⫾ 2.1bc

120

150

180

210

240

90 ⫾ 3.8a 28 ⫾ 3.5b 31 ⫾ 2.7b

87 ⫾ 0.9

73 ⫾ 1.5

62 ⫾ 4.8

41 ⫾ 3.2

Means within a column followed by the same letter are not signiÞcantly different (P ⫽ 0.05, Bonferroni method).

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Kamsuk et al. 2007). For example, the median protection time for a binary mixture of Z. piperitum essential oil and 5% VA against Ae. aegypti increased from 1.5 to 2.5 h under laboratory conditions. We found that the repellency and duration of effectiveness produced by the binary mixture of ZA-SO and VA (1:4 by weight) against Ae. aegypti were comparable with either ZA-SO or DEET alone. The improved effectiveness of repellency might be attributed to the lower evaporation rate and better skin persistence of ZA-SO in the combined presence of VA, as described previously by Khan et al. (1975) and Tuetun et al. (2005). In our Þeld tests, aerosols containing 5 or 10% ZA-SO plus 5% VA or 10% DEET resulted in complete protection from mosquitoes at the 60-min posttreatment interval. The relatively lower repellency of the binary mixture aerosol beyond this time period may be attributed to the low VA content. The optimum VA content was determined to be ⬎10%, according to our laboratory results. However, our study indicates that binary mixtures of ZA-SO and VA may hold promise for the development of novel and effective mosquito repellent products. In conclusion, binary mixtures of ZA-SO and VA could be useful as repellents for protecting humans and possibly domestic animals from bites and nuisance caused by mosquitoes in light of global efforts to reduce the level of highly toxic synthetic repellents. For the practical use of the ZA-SO-derived products as novel mosquito repellents to proceed, further research is needed to establish their human safety. Additionally, detailed tests are needed to fully understand the modes of action of ZA-SO-derived materials, as well as how to improve repellency potency and stability for eventual commercial development. Acknowledgments We express our gratitude to the volunteers whose cooperation and patience were essential to the repellent trials. This work was supported by the Rural Development Administration (Biogreen Program [PJ007109]) and the Ministry of Education, Science, and Technology of Korean Government (World Class University Program [R31-10056]) (to Y.-J.A.).

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Tuetun, B., W. Choochote, D. Kanjanapothi, E. Rattanachanpichai, U. Chaithong, P. Chaiwong, A. Jitpakdi, P. Tippawangkosol, D. Riyong, and B. Pitasawat. 2005. Repellent properties of celery, Apium graveolens L., compared with commercial repellents, against mosquitoes under laboratory and Þeld conditions. Trop. Med. Int. Health 10: 1190 Ð1198. Received 18 February 2010; accepted 3 September 2010.