636.093:615.285.7:57.084

COMPARATIVE STUDY OF RESIDUAL EFFECT DURATION OF INSECTICIDES FROM DIFFERENT CHEMICAL GROUPS ON ADULT STAGE OF Siphonaptera

O.Yu. Eremina1, I.V. Ibragimkhalilova2, Yu.V. Lopatina3

The method of an estimation of action spot-on formulations insecticides by mouse-model against adult rat flea Xenopsylla cheopis (Roth) is developed. Residual action of 9 insecticides (1-100 mg/kg) from 5 groups of chemical compounds are defined. Residual toxic and repellent actions are estimated. The new method gives the chance to predict effective doses of studied preparations and to define residual activity on hematothermal animals, and to cut down expenses on preliminary tests.

Key words: insecticides, rat flea, spot-on, laboratory method, mice.

 

Chemical medications against blood-sucking insects and mites are widely used to protect farm animals, valuable fur-bearing animals in fur farms and small pets. There are various forms of these insecticides: spot-on drops, ear labels, sprays, etc. Active ingredients (AI) of these preparations - organophosphorus compounds (OPC) (diazinon, chlorpyrifos, phosmet, fenthion, etc.), pyrethroids (permethrin, d-phenothrin, zeta-cypermethrin, deltamethrin, cyhalothrin and others), neonicotinoids (imidacloprid), phenyl pirazoles (fipronil, piriprol), imines (amitraz); in recent years, insect growth regulators (IGR) are of an increasing interest - luphenuron (chitin synthesis inhibitor), S-methoprene and pyriproxyfen (analogues of juvenile hormone) (1-3).

Flea is a classical model object for studying the action of insecticides on ectoparasites since fleas can be easily kept in an insectarium. Effects of insecticides on fleas can be studied by several methods: the contact with impregnated surface - filter paper, cloth, carpeting (4) or with treated substrate (5), topical application on the insects of microdroplets the acetone solutions of insecticides (6); to assess a systemic action, insecticides are administered to white mice per os (7) or to white rats with feed (8), and then adult fleas are placed on these animals.

The technically simplest method is a forced contact of adult fleas with treated surface, though its difficult to assess the obtained results owing to uneven time of the contact, distinctions between used surfaces and flea species. Topical method is highly time-consuming. Besides, the described techniques have been developed for insecticides applied indoors and outdoors, whereas the preparations for treatment of warm-blooded animals require much more complicated and high-cost research.

There are many factors influencing the effectiveness of insecticides applied onto the skin of warm-blooded animals barrier function of the skin, solvent properties, enzymatic detoxification, hydrolysis, body temperature and humidity, dissolution in skin oil, deposition in the subcutaneous fat and etc. The degree of penetration can be estimated by comparing the toxicity at introduction per os and via the skin, as well as some physico-chemical characteristics - water solubility and the octanol-water partition coefficient. For example, pyrethroids and phenyl pirazoles are highly soluble in lipids and low-soluble in water, and most of them remain on animals skin (as it can be assumed upon the difference in pyrethroids toxicity depending on routs of their administration). OPC are more hydrophilic and highly soluble in organic solvents; they are almost equally toxic at dermal and oral administration, which indicates a pronounced skin-resorptive effect. Unlike other groups of flea medications, neonicotinoids have very low lipophilicity and relatively high hydrophilicity, which contributes to their penetration into the blood (Table 1).

1. Toxicity indices and physico-chemical properties of tested insecticides

Insecticide (AI)

LD50 for white laboratorial mice, mg/kg

STC

Solubility in water, mg/l

Lipophilicity, Log octanol/water

per os

on skin

Diazinon

96

500

95

40

3,81

Fenthion

145

500

57

55

4,09

Phenothrin

> 5000

> 10 000

> 3333

2

> 5,70

Permethrin

650

> 2500

> 3333

0,2

6,50

Alfa-cypermethrin

35

> 2000

2 7000

0,01

6,94

Cyphenothrin

350

> 5000

> 3100

< 0,1

6,20

Fipronil

100

400

2000

2

4,00

Imidacloprid

150

> 2000

910

510

0,57

Pyriproxyfen

> 5000

> 5000

a

< 1000

5,37

Note. STC (selective toxicity coefficient) = LD50 for hematothermal animals at on-skin application / LD50 for insects at topical treatment. STC was calculated upon the data of insecticides toxicity (N.N. Melnikov et al., 1995) and indices of their toxicity for insects (Methodological instructions MU 3.5.2.2358-08, S.A. Roslavtseva et al., 2009); the analogue of juvenile hormone, has no insecticidal action on insects).

Selective toxicity of preparations for animal protection against ectoparasites is quite important during the development of such medications. OPS were found to have a lowest STC indicating their potential danger for warm-blooded animals (selective toxicity coefficient (STC) = LD50 for warm-blooded animals at on-skin application / LD50 for insects at topical application) (Table 1).

The purpose of this study was to compare the duration of protective action of insecticides using the low-cost method of testing on warm-blooded animals developed by the authors.

Technique. Experiments were performed on adult fleas Xenopsylla cheopis (Roth.) 1-3-week-old, not fed, not separated by gender (the laboratorial flea culture from insectarium of the All-Russia Research and Development Institute of Disinfectology, Moscow).

Tested insecticides the spot-on preparations: the insecticide-acaricide drops SERKO (diazinon 15,0% by Grand Platinum, Russia), cyphenothrin (cyphenothrin 40%, pyriproxyfen 2% by Sentrypro XFC, USA), phenothrin (d-phenothrin 85,7% by Hartz, The Hartz Mountain Corp., USA), Frontline (fipronil 10,0% by Merial, France), Advantage (imidacloprid 10% by Bayer, Germany). Other tested insecticide solutions in propylene glycol ether contained AI - pyriproxyfen (5%), fenthion (15%), permethrin and pyriproxyfen (respectively, 45 and 5%), and alfa-cypermethrin and pyriproxyfen (respectively, 8 and 5%).

Insecticide (0,1 ml) of the desired concentration (obtained by serial dilution of preparations with propylene glycol ether) were evenly (dropwise) applied on both sides along the spine of white laboratory mice (average weight 25-30 g) which then were placed in mini cages (3x3x8 cm, a metal mesh with cells 1x1 cm). The used AI doses were 1-100 mg insecticide per 1 kg animal weight (respectively, 0,03-3 mg AI per mouse). The mini cages were set in Petri dishes on attached paper filters, and the dishes with cages were placed in high plastic containers. In 2 hours after the treatment, adult fleas were put on mice. During the following 120 minutes, the "repellent" effect was accounted at intervals of 15-30 min (fleas escape from a host animal), then insects were collected from the vessel and remaining fleas were combed off from the animal using a disposable plastic comb. Tubes with collected pests were kept in an incubator (28 C, relative air humidity 70%). The proportion of fed fleas, fleas gone from the animal and fleas remained on the body were determined. Residual effects of tested insecticides were investigated on previously treated mice kept in individual cages. The fleas were placed on animals at intervals of 1, 3, 7 days and then weekly until the termination of insecticidal activity (observation period 30-35 days). The tests were performed at least 3-fold, each replicate - using more than 30 insects (in each experiment - two controls: solvent-treated and untreated mice). The ability of insecticide to spread across the animal body was assessed by applying 0,1 ml preparation on tail and protecting it from further contact with fleas (other details of technique the same as above). To compare the results, duration of the repellent effect was expressed in days (time required for escape of 50% and 95% insects from treated animals - RT50 and RT95, resp.), as well as insecticidal action (time, during which 50 % and 95% fleas died - LT50 and LT95, resp.). It could be different factors causing fleas to leave treated animals: end of feeding (in control - up to 20% insects were gone from the host in first 120 minutes of a contact), repellent effect of preparation or toxicity of the insecticide (paralyzed fleas drop from an animal). Since its hard to distinguish these factors, the term "repellent" effect is mentioned in quotes.

The results were calculated using Abbotts formula (9). Statistical processing of data was performed in Microsoft Excel 7.0.Results. Spreading of insecticides on animal body. When fleas were placed on mice in 2 h after treatment with insecticide, indices of fleas escape from the animal ("repellent" effect) and insecticidal effect were less than in 1 day after treatment, which shows spreading of medication across the animal body during the 1st day (Table 2). To analyze this property, OPC and pyrethroids with varying duration of activity were studied. In particular, treatment with only fenthion 100 mg/kg resulted in an animal get rid of any fleas after 1 day. Repellent and insecticidal effects of fenthion applied only on tail were less pronounced and detected only at the dose of 100 mg/kg. Permethrin applied on the body performed a maximum efficiency in 1 day after treatment. Gradual spreading of permethrin on body surface was proved by applying it on mice tails: in this variant, the maximum effect of low doses of permethrin (10 mg/kg) was observed after 3 days, though at somewhat lower efficiency of the preparation. Probably, when the insecticide is applied on tail, it spreads in a superficial skin oil layer, and when applied on the body - in fur too owing to solvent spreading.

2. Dynamics of repellent and lethal effects of insecticides the various groups during the contact of insects with treated animals depending on method of treatment

Dose, mg/kg

Period of account, min

Body treatment

Tail treatment

2 h

24 h

72 h

2 h

24 h

72 h

Repellent effect (fleas escape, %)

Permethrin (pyrethroids)

10

15

75,8

100

100

6,7

56,3

90,0

30

80,0

100

100

6,7

65,6

95,0

120

95,7

100

100

16,7

68,8

100

100

15

93,8

100

100

64,7

100

100

30

100

100

100

82,4

100

100

120

100

100

100

100

100

100

Fenthion (OPC)

10

15

22,9

23,3

18,2

0

0

0

30

28,6

23,3

18,2

0

0

0

120

42,9

43,3

33,3

0

0

0

100

15

23,3

70,0

75,0

0

0

0

30

56,7

93,3

100

0

50,0

10,0

120

60,0

100

100

0

100

50,0

Diazinon (OPC)

10

15

25,7

7,1

0

0

0

0

30

25,7

7,1

0

0

0

0

120

42,9

10,7

0

9,5

10,7

0

100

15

25,7

14,3

3,5

0

7,1

0

30

64,3

21,4

7,0

0

10,7

0

120

84,3

89,3

10,0

13,0

36,0

0

Mortality of fleas, %

Permethrin (pyrethroids)

10

 

100

100

100

100

100

100

100

 

100

100

100

100

100

100

Fenthion (OPC)

10

 

100

50,0

24,2

100

39,3

15,6

100

 

100

100

100

100

100

90,0

Diazinon (OPC)

10

 

100

17,9

7,4

100

0

0

100

 

100

100

30,0

100

89,3

13,3

Note. OPC organophosphorus compounds.

The only exception diazinon, whose indices were higher after 2 h than in 1 day (Table 2). Possibly, this fact was connected with rapid penetration of diazinon into the organism of treated animals, following fast degradation and elimination as it can be concluded from significantly reduced mortality of fleas placed on mice in 1 day after treatment. Apparently, to assess distribution rates of such rapidly degrading insecticide, fleas should be placed on mice at smaller intervals (eg., in 2, 4 and 8 h).

The raise of insecticidal action after 1 day post application was found in medications based on fipronil and imidacloprid.

Generally, the maximum effect of almost all tested insecticides was reached in 1 day after treatment. This fact is consistent with literature data: insecticidal effect of imidacloprid on fleas was accounted in 3 and 8 h after treatment and found to increase from 26,9 to 82,8% (in cats), and from 22,2 to 95,7% (in dogs) (10). Application of 10 mg/kg imidacloprid on ferrets caused mortality of 95,3% fleas in 8 h and 100% - after 1 day (11).

Duration of action on adult fleas. Both repellent and insecticidal effects on adult fleas were found in almost all studied preparations (except the IGR pyriproxyfen), and its manifestation degree directly depended on chemical structure, dosage and duration of period from treatment to contact with fleas.

3. Duration of residual effects of insecticides the various groups depending on method of treatment

Active ingredient (AI)

Dose, mg/kg

Escape of fleas

Mortality of fleas

RT50, days

RN95, days

LT50, days

LT95, days

Body treatment

Fenthion

1

~1,0

N.r.

N.r.

N.r.

10

~1,0

N.r.

1,0

0,3

30

2,8

N.r.

4,0

1,9

50

5,0

2,2

6,2

4,3

100

8,2

4,8

8,6

5,0

Diazinon

1

N.r.

N.r.

N.r.

N.r.

10

N.r.

N.r.

0,6

0,3

50

~1,0

N.r.

2,3

1,5

100

2,3

0,8

2,7

1,6

Permethrin

1

1,6

< 1,0

1,5

< 1,0

10

27,0

13,0

13,0

3,5

100

> 30,0

20,5

> 30,0

24,0

Phenothrin

1

13,0

9,5

7,0

< 1,0

10

16,0

10,5

8,5

4,0

100

26,0

13,0

19,0

11,0

Cyphenothrin

1

9,0

2,8

< 1,0

< 1,0

10

> 30,0

> 30,0

> 30,0

28,0

100

> 30,0

> 30,0

> 30,0

30,0

Alfa-cypermethrin

1

16,0

10,0

3,5

< 1,0

5

25,0

18,0

9,5

2,8

10

> 35,0

25,0

17,0

11,0

100

> 35,0

> 35,0

> 32,0

21,0

Fipronil

1

N.r.

N.r.

5,0

1,4

10

1,0

N.r.

25,0

16,0

100

14,0

N.r.

> 30,0

> 30,0

Imidacloprid

1

N.r.

N.r.

1,0

N.r.

10

N.r.

N.r.

11,0

7,0

100

N.r.

N.r.

21,0

14,0

Pyriproxyfen

1

N.r.

N.r.

0

0

10

N.r.

N.r.

0

0

100

N.r.

N.r.

0

0

Tail treatment

Fenthion

1

< 1,0

N.r.

N.r.

N.r.

10

< 1,0

N.r.

< 1,0

N.r.

30

1,0

N.r.

4,4

2,0

50

1,7

N.r.

4,5

2,0

100

3,2

1,7

4,8

2,1

Diazinon

1

N.r.

N.r.

N.r.

N.r.

10

N.r.

N.r.

N.r.

N.r.

50

N.r.

N.r.

N.r.

N.r.

100

N.r.

N.r.

1,9

1,0

Permethrin

1

2,2

< 1,0

1,4

N.r.

10

7,0

2,5

10,0

3,0

100

18,0

11,0

14,0

3,4

Phenothrin

1

1,7

N.r.

8,0

4,0

10

2,0

N.r.

11,0

5,8

100

18,0

11,0

14,0

9,8

Cyphenothrin

1

1,0

N.r.

< 1,0

N.r.

10

20,0

2,0

4,0

1,9

100

> 30,0

26,0

21,0

4,0

Alfa-cypermethrin

1

11,5

5,8

1,0

N.r.

10

24,0

17,0

16,0

10,0

100

> 30,0

> 30,0

28,0

13,5

 

Fipronil

1

N.r.

N.r.

< 1,0

< 1,0

10

N.r.

N.r.

11,0

9,0

100

14,0

N.r.

26,0

20,0

 

Imidacloprid

1

N.r.

N.r.

N.r.

N.r.

10

< 1,0

< 1,0

3,2

1,7

100

< 1,0

< 1,0

4,5

2,0

 

1

N.r.

N.r.

0

0

10

N.r.

N.r.

0

0

100

N.r.

N.r.

0

0

Note. The account of fleas escape from mice was performed at body treatment in 120 minutes, at tail treatment in 2 hours, mortality in both variants in 24 hours after application. N.r. the level not reached.

"Repellent" effect of studied OPC was short-term and not as prolonged as insecticidal action (Table 3).

Toxic effects of OPC on insects are the result of irreversible inhibition of the active site of acetylcholine esterase by phosphorylation of serine leading to formation of the complex whose structure is stronger than acetylated serine. The obtained data show that the OPC fenthion and diazinon have a short-term residual effect on fleas. Animals get rid of the pest within 1-2 days after treatment with diazinon and about 8 days with fenthion. Apparently, this fact can be explained by OPC penetration via the skin leading to rapid oxidation and hydrolysis of AI, and then its elimination in the form of metabolites.

The results of these experiments are consistent with literature data. Diazinon (like other OPC insecticides) being ingested fairly quickly passes to elimination from the body of warm-blooded animals - by 90% after 7 days at its half-life of 7-12 h in rat organism (12). Reports about deaths of animals treated with fenthion contributed to development of new medications with low content of this insecticide (13). In opinion of foreign authors, fenthion doesnt correspond to modern standards for "spot-on preparations (14). In this study, fenthion and diazinon when applied on skin of mice at a dose of 100 mg/kg caused deaths of several experimental animals too.

Pyrethroids are highly lipophilic, so they can spread in skin oil layer and be deposited in the subcutaneous adipose tissue. When treated on skin, they are much less toxic than when administered orally. Main targets of their action are sodium channels of nerve cell membranes of presynapses in peripheral and central nervous system of insects. Pyrethroid insecticides have the highest STC (Table 1).

All tested pyrethroids performed a long-term repellent effect and a clear relationship between the dose of insecticide and intensity of fleas escape from treated animals (Table 3). "Repellent" effect of pyrethroids persisted much longer than their insecticidal action. Such dependence was observed at all methods of treatment - both on the body or on tail of a mouse. This fact confirms the feasibility of inclusion into flea preparations of IGR (particularly, pyriproxyfen) to prevent reproduction of insects left the host animal.

The residual effect of pyrethroids to flea was prolonged, although this period was somewhat shorter than the repellent action. When the maximum dose of pyrethroids (100 mg/kg) was applied on the body, mortality of the pest amounted to 50% - during 19-30 days or more and to 95% - in 11-30 days (Table 3).

These findings about a long-term residual effect of pyrethroids on fleas agree with literature data about their low penetration through the skin of warm-blooded animals. It has been shown that only 8-17% d-phenothrin was absorbed via the skin of an animal treated with condensed emulsion keeping 10 mg/kg AI (15). Unlike d-phenothrin, cyphenothrin molecule contains CN-group, and its much more toxic to warm-blooded animals when administered orally (LD50 = 350 mg/kg) than on the skin (LD50> 5000 mg/kg), which also indicates a weak absorption.

Alfa-cypermethrin is a mixture of two cys-isomers of cypermethrin. Being ingested, it is fairly fast excreted from the body of warm-blooded animals; when applied to intact skin, it shows low toxicity and can be deposited in fat layer. Alfa-cypermethrin causes local irritation of skin, which limits its use on animals (16).

The obtained data suggest that all medications based on pyrethroids remain on animal skin during a long time. The repellent action of pyrethroids significantly reduces the duration of insects contact with the insecticide, which was reflected by low mortality. Knockdown effect can be another reason for fleas escape from treated animals (paralyzed insects cant hold on the host); often this is a reversible process, which can explain the absence of insects mortality at low doses of these preparations. During the 1st week after treatment an animal with high doses of pyrethroids (100 mg/kg), poisoning of insects was lethal, but in 2-3weeks, the fleas gone by knockdown could return due to low dose of the insecticide after its biodegradation.

Phenyl pirazoles operate similar to diene insecticides and hexachlorocyclohexane (functional inhibition of nerve-muscle synapses mediated by g-amynobutyric acid). Fipronil is quickly absorbed in the organism of warm-blooded animals, gradually metabolized and excreted as non-destructed fipronil and its derivative sulfone. Fipronil is a highly lipophilic substance low soluble in water, whose maximum content was detected in animal tissues on the 7th day. In rats, about 40% and in mice about 60% of the administered dose remains in the body, mainly deposited in adipose tissue.

Fipronil revealed a weak repellent action: after the highest dose (100 mg/kg) was applied, 80% fleas left the host during the 1st day, and more than 50% - within 2 weeks, though not have reached the peak of RT50. Insecticidal effect of fipronil was high its residual effect lasted over 30 days (Table 3).

Neonicotinoids (imidacloprid etc.) are the antagonists of nicotine-acetylcholine receptors in postsynaptic membranes of neurons of insects. Penetration of low-lipophilic imidacloprid in flea organism occurs, apparently, via the non-sclerotized intersegmental membranes of the body.

Imidacloprid showed the absence of repellent effect at any doses, while the long-time insecticidal action was observed at the maximum dose 21 day (LT50) and 14 days (LT95).

It has been reported about mortality of adult fleas after the contact with fur of animals treated with imidacloprid, which indicates penetration of imidacloprid into the skin oil layer providing a lasting effect on fleas. Should this oil layer be removed from the skin with ethanol before treatment, the fleas placed on the treated host were feeding normally at usual rates of consumed blood and no symptoms of intoxication, like if it was a non-treated animal (18). It has been reported about imidacloprid presence in animal blood for less than 3 days, and about 4 weeks in fur (19).

Currently, high-efficient insecticides (pyrethroids, phenyl pirazoles etc.) are often combined with IGR whose targets are only immature larvae but not adults. IGR are used mainly for treatment in locations of larvae development. There are the data about the contact of the fleas C. felis with pyriproxyfen leading to suppressed egg-laying and non-viability of laid eggs (20-22). The authors study of pyriproxyfen revealed no repellent nor insecticidal action at all tested doses (1-100 mg/kg); its presence in medications didnt affect the duration of fleas contact with treated animal and didnt increase mortality of the pest.

Feeding of fleas on treated animals. Application of all preparations at all tested doses (1-100 mg/kg) on the body or on tail of host animals didnt hinder feeding of fleas. The obtain data just partially agree with results of other researchers. Earlier, it has been shown that treatment of animals with imidacloprid or fipronil didnt prevent fleas feeding during the 1st hour after treatment, and using the mixture fenithrothion / dichlorvos and permethrin protected against over 80% flea bites during, respectively, 3 and 7 days (23).

So, the duration of repellent action of studied AI ranks as follows: diazinon < fenthion < fipronil < phenothrin < cyphenothrin < alfa-cypermethrin. Imidacloprid and pyriproxyfen didnt cause fleas to leave the host. Pyrethroids were found to be the only group of insecticides whose repellent action was more prolonged than insecticidal effect.

The duration of insecticidal action of tested preparations grows in the line: diazinon < fenthion < phenothrin < imidacloprid < alfa-cypermethrin < fipronil < cyphenothrin. Cyphenothrin was found to perform a longest residual insecticidal effect 30 days (LT95). The authors technique for primary assessment of insecticidal spot-on drops includes testing wide range of doses (1; 10; 50; 100; 200 mg AI/kg animal body weight). The repellent effect (fleas escape) must be recorded in 15; 30; 45; 60; 90 and 120 min after treatment, mortality of insects in 2; 24; 48; 72 and 168 h after treatment. Insecticides with significant residual effect (pyrethroids, phenyl pirazoles and neonicotinoids) must be assessed by placing fleas on treated animals daily until the 50% reduce of tested indices.

The proposed method provides prognostication of effective doses of tested medications, determination of residual activity on warm-blooded animals and reducing costs of preliminary tests.

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1Research and Development Institute of Biocides and Nanotechnology, Moscow 119517, Russia,
-mail: eremina_insect@mail.ru;
2 Research and Development Institute for Disinfectology of the Federal Service on Customers' Rights Protection and Human Well-Being Surveillance of the RF, Moscow 117246, Russia ,
-mail: ilhamya@gmail.com;
3M.V. Lomonosov Moscow State University, Moscow 119992,
-mail: ylopatina@mail.ru

Received January 13, 2009