Parasitol Res (2008) 103:405–412 DOI 10.1007/s00436-008-0988-2
ORIGINAL PAPER
A novel molluscicidal formulation of niclosamide Jian-rong Dai & Wei Wang & You-sheng Liang & Hong-jun Li & Xiao-hong Guan & Yin-chang Zhu
Received: 11 December 2007 / Accepted: 2 April 2008 / Published online: 3 May 2008 # Springer-Verlag 2008
Abstract Snail control by molluscicides is an important strategy for schistosomiasis control in China. Currently, only one chemical molluscicide, niclosamide, which is used as 50% wettable powder of niclosamide ethanolamine salt (WPN), is commercially available for field snail control in China. However, WPN is costly, toxic, and has a lower dispersibility and precipitates rapidly. In this paper, we describe the development of a novel formulation of niclosamide, a suspension concentrate of niclosamide (SCN). The efficacy of SCN was evaluated both in the laboratory and field. SCN showed better molluscicidal effects than conventional formulation of WPN, as determined by LC50 for adult snails, young snails, and snail eggs. The acute toxicity of SCN to Brachdanio rerio hamiton was less than WPN. In conclusion, the novel formulation of SCN suspension is physically more stable, more effective, and less toxic. Therefore, it can be more useful for controlling snails in endemic areas of schistosomiasis in China.
Introduction Schistosomiasis is a major tropical disease infecting over 207 million people in 76 countries with a further 779
J.-r. Dai : W. Wang : Y.-s. Liang (*) : H.-j. Li : Y.-c. Zhu Jiangsu Institute of Parasitic Diseases, Wuxi 214064, People’s Republic of China e-mail:
[email protected] J.-r. Dai : X.-h. Guan Department of Parasitology, Nanjing Medical University, Nanjing 210019, People’s Republic of China
million at risk of infection (Steinmann et al. 2006). As the infection with Schistosoma japonicum in China is a zoonosis, control involves both the treatment of humans and animals with praziquantel and the killing of the snail intermediate host Oncomelania hupensis (Chen et al. 2005). The only chemical available for snail control, niclosamide (WHO 1992), suffers from the problem of being difficult to dissolve in both water and organic solvents. Since 1992, the World Bank Loan Project for Schistosomiasis Control has developed a 50% wettable powder of niclosamide ethanolamine salt (WPN) that is the only commercial molluscicide available for use in China (Chen et al. 2005). Experience with the product over a 10-year period has shown that WPN has physical problems that include rapid precipitation and being difficult to disperse as well as being toxic to aquatic animals and expensive (Tadros 1980; Dai et al. 1998). To ensure rapid dispersion in water as well as to reduce the surface tension involved in transition from the solid to the liquid phase, easily absorbed surfactants are added to the WPN preparation that result in very small suspended particles (Zhang et al. 1996; Guido and Tadros 2000). However, since the surfactants readily absorb water, the WPN preparation can be damaged during transport and storage with small clumps of niclosamide forming that do not disperse evenly in the water. When the particle size is greater than 44 μm, there is reduced contact with the snail resulting in lowered molluscicidal activity (Liu et al. 1998; Huang et al. 2003). To try to overcome these problems, larger amounts of WPN have been used in the field; however, this has resulted in increased cost and environmental toxicity (Xiu et al. 1996; Eduardo et al. 2000; Zhang and Jiang 2002). To overcome these problems, a novel formulation of niclosamide has been produced and evaluated in the laboratory and field.
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Materials and methods SCN preparation A mixture, containing 50.4 g niclosamide (99.1% purity, made in Jiangsu Institute of Materia Medica), 8.0 g alkyl polyglycosides (APG-0810, Sinopec Jinling Petrochemical Corporation), 3.0 g glycerol (AR, Shanghai Reagent Co., LTD.), 0.2 g sodium carboxymethyl cellulose (AR, Shanghai Chemical Reagent), 0.6 g sodium benzoate (AR, Shanghai Chemical Reagent), and 137.8 ml distilled water were ground in a ball miller (QM-ISP04, Nanjing University) containing ten 20 mm, twenty 10 mm and forty 6 mm diameter steel balls at 180 r/min for 2 h. The mixture was poured through a 44 μm mesh sieve and resulted in a 25% suspension concentrate of niclosamide (SCN). Determination of the physical and chemical characteristics of the SCN preparation The pH of the new preparation was determined by a HI 8424 microcomputer pH meter (Hanna) and viscosity was measured by a rotation viscometer NDJ-1 (Shanghai Jingke Scale Factory). For particle size determination, 800 ml sterile water was added to the measuring cup of a Mc 2000 laser diffraction micrometer–diameter determinator (Mastersizer 2000, Malvern Instruments, UK) followed by addition of SCN drop by drop to the required concentration. SCN was weighed, diluted at a ratio of 1:104, surface potential of SCN was measured with a Zeta potential determinator (Zetasizer 3000HSA, Malvern Instruments Ltd, UK). A sample of niclosamide or a 50 mg sample of the SCN preparation were dissolved in 80 ml methanol in a 100 ml flask and treated at 100 Hz for 10 min in an ultrasound bath (KQ250DE ultrasound instrument, Kunshan). Methanol was added to 100 ml and the preparation thoroughly mixed then centrifuged at 3,000 r/min for 5 min. The supernatant fluid was analyzed on a Shimadzu LC-10A high performance liquid chromatography (HPLC) using a Kromasil® C18 column (250 mm×4.6 mm). Mixtures of methanol and water at ratio of 90:10 (v/v; phosphoric acid buffered pH 3.64), was filtered and degassed with 0.22 μm microspore filter membrane and used as the mobile phase. Flow velocity was 1.0 ml/min, column temperature 40°C, detection wavelength 236 nm and a sample size of 20 µl was used. SCN and WPN (0.2 g, respectively) were weighed, a 100 ml suspension was prepared (active content 0.5 g/l for SCN, and 1.0 g/l for WPN) with dechlorinated water and held in lab at 25°C. At 0, 0.5, 1, 2, 4, 8 and 24 h intervals, a 1 ml sample was collected from the intermediate section of the SCN and WPN suspensions, respectively, added to 10 ml of methanol, and analyzed by HPLC as described
Parasitol Res (2008) 103:405–412
above. The suspension rate of the niclosamide content at 0 h was considered to be 100%. The concentration was compared with the sample at at 0 h. Molluscicidal activity Adult Oncomelania hupensis snails were collected by individual picking with forceps from the rural marshland of Longtan Town, Nanjing City, Jiangsu Province, China. After feeding in the laboratory for 24 h, active adult snails with 7∼8 spirals were randomly divided into groups for the molluscicidal test. Small snails were collected with plastic nets in July and August, 2004, and those with a length WPN> niclosamide ethanolamine salt (Table 6).
Discussion At present, schistosomiasis remains endemic in seven provinces of China, with a population of 66.41 million at risk and an estimated 0.80 million infected people (Hao et al. 2006). Controlling the O. hupensis snails, the only intermediate host of Schistosoma japonicum, is one of the most important measures for schistosomiasis control in China. The young snails live in water, but the adults are amphibious being able to live on wet soil and marshland or
Concentration (mg/l)
1.0 0.5 0.0 1.0 0.5 0.0
Mortality rate of snails (%) 1days
2days
3days
56 72 2 80 89 3
82 92 0 92 95 2
98 100 2 98 99 5
(28/50) (36/50) (1/50) (104/130) (85/96) (3/108)
(41/50) (46/50) (0/50) (181/197) (98/103) (2/112)
(49/50) (50/50) (1/50) (308/315) (145/146) (3/66)
0.01 0.01 188.16
0.01 7.62
0.01
0.01
0.01
14.79
30.21
765.03
(157/203) (155/220) (88/109) (89/153) (281/337) (264/403) (4,450/4,772) (1,249/1,866) (10/322)
80
∗
60
∗
∗
∗
∗
∗
∗ SCN WPN
40 20 0
3d
7d 15d 3d
Nanjing
7d 15d 3d
Zhengjiang
7d 15d 3d
Yueyang
7d 15d
Wuhu
Fig. 5 The mortality rates of Oncomelania hupensis snails after spraying in Nanjing and Zhenjiang cities of Jiangsu Province, Wuhu City of Anhui Province and Yueyang City of Hunan Province
in water unlike the intermediate hosts of S. mansoni and S. haematobium. It is a common practice to kill snails in water by immersion and to kill snails in soil surface of wet marshland by spraying with chemical molluscicide (Wang and He 2005). Snail control has been shown to be an essential part of the overall control of schistosomiasis. In 2006, the total area of O. hupensis snail habitats was 380,151.74 hm2, and the area of snail control with chemical molluscicides was 95,635.31 hm2 per year (Hao et al. 2007). Since the 1950s, more than 2000 chemicals have been screened for molluscicidal activity in China, but only a few have shown to be effective (Xu et al. 2005). Sodium pentachlorophenate, which was used as a molluscicide in China before 1990s, was banned because of its toxicity to human and livestock and carcinogenesis. Since 1990s, WPN has been used for snail control in China on the recommendation of WHO (Chen et al. 2005). However, because of its insolubility, it has been less effective than hoped. The SCN preparation described here for the first time, with good dispersion and suspension, is suitable for spraying at a high concentration directly with a sprayer in various environments (such as marshland and mountainous areas). As SCN can be mixed with water in any ratio, it is more suitable for use than the preparation of WPN. The suspension concentrate is also small in size and dispersed evenly, so SCN has an extensive coverage and can reach the target organisms thoroughly, thus improving the
0.5 1.0 0.5 1.0 0.5 1.0 0.5 1.0 0.0
77 70 81 58 86 66 93 67 3
Mortality rate (%)
77.64
>0.05 0.02
0.01 37.01
0.01
(215/290) (95/140) (198/218) (177/250) (189/249) (224/293) (1,362/1,377) (1,015/1107) (2/171) 74 68 91 71 76 76 99 92 1 2.15 >0.05 2.59
83 78 86 64 88 77 99 91 1
(276/331) (143/183) (227/265) (246/384) (251/286) (322/416) (2,972/3,007) (1,749/1,929) (4/375)
χ2
Table 6 Comparison of LC100, LC50 and LC0 of Brachydanio rerio Hamiton among SCN, niclosamide, WPN and niclosamide ethanolamine salt (mg/l) LC100 LC50
Control
Wuhu
Yueyang
Nanjing
SCN WPN SCN WPN SCN WPN SCN WPN –
Formulation
Zhenjiang
Mortality rate (%) P value χ2 Mortality rate (%)
∗
100
>0.05
29.33
>0.05
120
Mortality rate (%)
1.85
411
P value
15days 7days 3days Dose (g/ l m2) Formulation Test site
Table 5 Comparison of the mortality rates of Oncomelania hupensis snails sprayed with different concentrations of SCN and WPN in field at 3, 7, and 15 days
χ2
P value
Parasitol Res (2008) 103:405–412
SCN Niclosamide WPN Niclosamide ethanolamine salt
0.845 0.385 0.296 0.134
LC0
24h
48h
96h
0.427 0.246 0.185 0.095
0.288 0.246 0.150 0.095
0.281 0.210 0.138 0.095
0.228 0.134 0.104 0.061
412
efficiency, saving the consumption, and reducing the cost. SCN costs 4,000 US$ per ton compared with WPN at 5,000 US$ per ton (Third Pharmaceutical Company of Huainan), which is also an advantage. The new SCN formulation has been accepted for use by the Chinese government, and four training courses for staff from 45 counties in seven provinces have already taken place. In the last 3 years, 190 tonnes have been applied to 9,500 hm2. The present formulation may be able to further improved by use of synergists such as chlorphoxim (Dai et al. 1997, 2001) and additional reduction in particle size may also be an advantage by increasing stability. Due to its biodegradation niclosamide should not cause long term environmental effects (Graebing et al. 2004a, b). It should be investigated whether the new preparation may be more effective for the control of the intermediate hosts of S. mansoni and S. haematobium than the existing preparations. Acknowledgements This study was funded by grants from the HiTech Research and Development Program of China (863 Program; No.2004AA2Z3540) and the Department of Health of Jiangsu Provincial Government (No. 200219). We are grateful to Dr G. C. Coles, University of Bristol, UK for the help during the preparation of this manuscript. The experiments in this study are in compliance with the current laws and regulations in China.
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