Fenoxycarb, an Insect Growth Regulator (IGR)

Fenoxycarb, an insect growth regulator (IGR),

induces anemia in mammals A.A. Bazzaz

Department of Environmental and Evolutionary Biology, University of Liverpool, Liverpool L69 3BX, UK

Abstract

Fenoxycarb, an Insect growth regulator (IGR), has been investigated to determine its toxic effect on mammalian life by analyzing blood samples as an indicator. One hundred and eight male mice were used in

this study (24 control and 84 experimental animals). The mice were sacrificed by head dislocation, and blood samples were collected into heparinised tubes for haematological tests. Haematological changes in blood parameters have been measured in mice given fenoxycarb in

water at three different dose (1, 5 and 10 ppm) for 4 weeks. Fenoxycarb primarily induced a mild to moderate anemia as early as the first week, at all three doses by causing a significant (p<0.05-0.0001) decline in the levels of haemoglobin (Hb%), packed cell volume (PCV%)

leucocytes (WBC) and erythrocytes (RBC) population. It also caused a steady decline in both macrophages and neutrophils. However, changes in other leucocyte counts were inconsistent. It has been concluded that fenoxycarb, at the doses used in these experiments, is not directly toxic, but long term exposure could lead to hypoxia

stress and an increased risk of infection.

Introduction

It has been found that insect growth regulators (IGRs) are less dangerous than pesticides to non-target animals and to humansi. The

hazards of the pesticides and IGRs to aquatic organisms have well been documented2,3. Recently, a new compound with juvenile-hormone [JH] activity, ethyl (2-(4-phenoxy-phenoxy) ethyl} carbamate [fenoxycarb],

was reported to exhibit a strong juvenile-hormone activity against a variety of insects^-io. it is a non-terpenoid, non-neurotoxic carbamate and has been shown to interact with juvenile hormone esterase activityn. The activity of IGRs is reported to be via different modes of

374 Air Pollution Monitoring, Simulation and Control

action, and various methods have been employed to control the growth of insects and arthropods. Moreover, the IGRs have greater field stability and, against some insects, higher potency and control potential than the earlier terpenoidis.ia. However, when these IGRs run off to adjoining aquatic ecosystem, they cause some hazards to aquatic animals as well as to non-target animals by accidental contact with surface water of industrial and agricultural areas^. One of the principal advantages of using IGRs instead of pesticides in insect control is their very low mammalian toxicityio. They are not necessarily immediately toxic but eventually cause abnormalities that impair survival or reproduction of insects and arthropods! 5-17. To the best of my knowledge, nothing is known about the effect of fenoxycarb on the non-target animals which might drink contaminated shallow ground water. The present study has been designed to investigate the haematological effects of fenoxycarb to laboratory animals (mice) as an example of non-target mammals.

Materials and Methods

Preparation of Fenoxycarb Doses: One hundred and eight male albino

Swiss mice (Mus musculus) weighing (25±2 gm) were used in this investigation. Their age ranged from 3 weeks old at the beginning of the experiment up to 2 months old at the end of week four. They were maintained under controlled environmental conditions of 25±2°C. Woodshavings were used as bedding material. Normal laboratory chow and tap water (except for treated animals) were available ad lib and were replenished three times a week. Fenoxycarb was obtained as a gift from Maag Agrochemicals as a pure solution, and was stored at 4 C. Three concentrations: 1, 5 and 10 ppm were emulsified with tap water and were prepared freshly three times a week and used as replacement for drinking water for the experimental animals. Haematological Tests: At the end of each week, twenty one experimental animals (7 per group) in addition to six (control mice) were scarified by cervical dislocation (without anesthesia). Blood samples were collected from the cervical artery into heparinised tubes for haematological tests. Levels of haemoglobin in (g/dl), packed cell volume (PCV%, haematocrit) and differential counting of leucocytes (absolute numbers was performed according to method described by

BatraiG. Counting of leucocytes and erythrocytes was done using haemocytometer. Blood smears were prepared on slides and stained with Lieshman's stain. The blood cell counting performed using Olympus light microscope (100x objective oil lens). Biostatistical analysis were carried out using student T-test.

Air Pollution Monitoring, Simulation and Control 375

I Control/W1 Experl./W1 Cont./W2 Experi./W2 Control/W3 Experl/W3

CO Control/W4 0) Experl./W4 O)

0) 2 0) Q.

Hb% (1ppm) Hb% (Sppm) Hb% (10ppm)

Figure 1: Dose and time-dependent decrease in Hb%.

Control/W1 40 - Experl./W1 Cont./W2 - 30 H Experi./W2 o a. Control/W3 •5 20- Experl/W3 0) o> «#COw Control/W4 10 - Q) Exper!./W4 2

Figure 2: Dose & time-dependent decrease in PCV%.

376 Air Pollution Monitoring, Simulation and Control

0) o>

• Control/W1 C3 Exper!./W1 • Cont./W2 0 Experi./W2 o 10- o H Control/W3 Q Experl/W3 H Control/W4 H Experl./W4 0

CD 0 * WBC% (1p|WBC% (5pWBC% (10ppm)

Figure 3: Decrease in WBC mean numbers of treated vs control animals

• ControlAVI Q Experl.W1 H Cont./W2

El Experi./W2 • Control/W3 Ei Experl/W3 • Control/W4 H Experl./W4

RBC (1ppm)RBC (SppmRBC (10ppm)

Figure 4: Dose and time dependent decrease in number of RBC.

Air Pollution Monitoring, Simulation and Control 377

Tablel: Blood parameters of Mice given 1 ppm fenoxycarb in the drinking water. (Hb%) percentage of Haemoglobin; (PCV%) percentage of Packed Cell Volume; (WBC) number of leucocytes; (RBC) number of erythrocyte; (SD) standard deviation; (p) Student T-test. Animals Mean Hb% Mean PCV% Mean WBC & Mean RBC & Weeks(W1-W4) ISO ISO & ±SD & ±SD (x1 03/u/) (x1 QG/u/)

Control (n=6) 13.55±0.696 44.50±2.22 9.0510.333 9.5010.327 Treated (n=7) 13.38+0.042 42.00±2.097 7.8010.335 9.6410.320 P< NS NS 0.005 NS Control (n=6) 13.7010.541 43.80±3.16 9.1010.333 9.7010.34 Treated (n=7) 11.18±1.772 37.35±0.599 7.8110.362 9.6410.373 0.05 0.005 0.0005 0.005 P< Control (n=6) 11.20±0.42 39.2011.24 10.012.72 9.8910.343 Treated (n=7) 9.85±0.407 34.6310.953 8.1210.137 8.2010.907 FK 0.05 0.005 0.005 0.013 Control (n=6) 13.82±1.12 44.6710.55 8.3013.82 9.3110.17 Treated (n=7) 9.6±0.314 33.9111.297 7.5211.34 7.2210.323 0.0005 0.005 0.005 P< 0.005

Table 2: Blood parameters of Mice given 5 ppm fenoxycarb in the drinking water.

(Hb%) percentage of Haemoglobin; (PCV%) percentage of Packed Cell Volume; (WBC) number of leucocytes; (RBC) number of erythrocyte; (SD) standard deviation; (D) Student T-test. Animals Mean Hb% Mean PCV% Mean WBC & Mean RBC & Weeks(W1-W4) ISO 1SD &1SD & ISO (x1 03/t//) (X106/CI/)

Control (n=6) 13.5216.96 44.5012.22 9.0510.333 9.5010.327 Treated (n=7) 12.7010.357 40.8011.326 7.5010.368 8.6610.338 P< NS 0.01 0.0005 0.004 Control (n=6) 13.7310.541 43.8013.16 9.1010.333 9.7010.34 Treated (n=7) 11.1510.55 36.3511.32 7.7510.83 8.2810.31 0.05 0.05 0.0005 0.005 P< Control (n=6) 11.2010.42 39.2011.24 10.012.72 9.8910.343 Treated (n=7) 9.6010.365 36.9311.289 7.4710.37 7.5210.157 0.005 P< 0.05 0.005 0.005 Control (n=6) 13.8211.12 44.6710.55 8.3013.82 9.3310.17 Treated (n=7) 9.4010.19 32.0210.821 7.8710.188 6.4210.219 0.005 0.005 0.005 0.0005 P<

378 Air Pollution Monitoring, Simulation and Control

Table 3: Blood parameters of Mice given 10 ppm fenoxycarb in the drinking water. (Hb%) percentage of Haemoglobin; (PCV%) percentage of Packed Cell Volume; (WBC) number of leucocytes; (RBC) number of erythrocyte; (SD) standard deviation; (p) Student T-test.

Animals Mean Hb% Mean PCV% Mean WBC & Mean RBC & Weeks(W1-W4) ISO ISO &±SD & ±SD (X1Q3/W/) (X106/U/) Control (n=6) 13.52±6.96 44.50±2.22 9.0510.333 9.5010.327 Treated (n=7) 11.30±0.681 36.67±3.15 7.1710.17 8.1810.14 0.0005 0.001 0.0005 0.0005 P< Control (n=6) 13.73±0.541 43.80±3.16 9.1010.333 9.7010.34 Treated (n=7) 9.38±0.552 32.78+1.297 7.3010.227 7.3010.33 P< 0.0005 0.0005 0.005 0.005 Control (n=6) 11.20±0.42 39.2011.24 10.012.72 9.8910.343 Treated (n=7) 9.50±0.224 32.65±1.732 7.3010.153 7.0810.397 0.05 0.05 0.005 0.05 P< Control (n=6) 13.82±1.12 44.67±0.55 8.3313.82 9.3310.17 Treated (n=7) 9.40±0.224 31.4011.36 7.7210.414 6.2710.345 P< 0.0005 0.005 0.005 0.0005

Table 4: Differential leucocyte counting (100u/) of Mice given 1 ppm fenoxycarb in the drinking water. (L) lymphocyte; (M) monocyte; (N) neutrophil; (E) eosinophil; (B) basophtl; (NS) not significant; (SD) standard deviation; (p) Student-T test.

Animals Lymphocyte Monocyte Neutrophil Eosinophil Basophi Weeks & ISO & ISO & ISO &1SD & SD (W1-W4) Control (n=6) 680 1 6 23 1 0.12 192 1 2.04 9 1 0.12 ' Treated (n=7) 505 1 7.6 19 1 0.2 184 12.5 11 1 0.07 P< 0.005 0.01 NS 0.05

Control (n=6) 670 1 21 27 1 1.3 156 1 2.48 7 1 2.2 ' Treated (n=7) 660 1 22.7 22 1 0.1 130 1 2.4 8 1 0.07 P< NS 0.05 0.05 NS Control (n=6) 647 1 19 21 1 0.9 197 1 2.9 11.4 1 0.2 ' Treated (n=7) 528 1 13 17 1 0.08 170 1 3.2 12 1 0.06 P< 0.01 0.01 0.05 NS

Control (n=6) 730 1 11 9 1 0.12 174 11.77 8 1 3.4 Treated (n=7) 585 1 11 16 1 0.2 140 1 3.2 9 1 0.03 P< 0.0001 0.005 NS NS

Air Pollution Monitoring, Simulation and Control 379

Table 5: Differential leucocyte counting (100u/) of Mice given 5 ppm fenoxycarb in the drinking water. (L) lymphocyte; (M) monocyte; (N) neutrophil; (E) eosinophil; (B) basophil; (NS) not significant; (SD) standard deviation; (p) Student T-test. Animals Lymphocyte Monocyte Neutr oph il Eosinoph i I Basophil & ±SD & ±SD & ±SD &±SD & SD Weeks (W1-W4) Control (n=6) 680 ± 6 23 ± 0.12 192 ± 2.04 9 ± 0.12 Treated (n=7) 591 ± 19.5 15 ± 0.9 132 ±3.4 12 ± 0.09 P< 0.01 0.005 0.021 0.01 156 ± 2.48 7 ± 2.2 Control (n=6) 670 ± 21 27 ± 1.3 Treated (n=7) 628 ± 24.6 18 ± 0.1 121 ± 4.7 8 ± 0.07 0.024 NS P< NS 0.05 Control (n=6) 647 ± 19 21 ± 0.9 197 ± 2.9 11.4 ± 0.2 Treated (n=7) 576 ± 12 13 ± 0.09 148 ± 3.1 10 ± 0.05 P< 0.05 0.005 0.01 NS 8 ± 3.4 Control (n=6) 730 ± 11 9 ± 0.12 174 ±1.77 Treated (n=7) 606 ± 14 17 ± 0.06 125 ± 3.08 6 ± 0.02 0.05 P< 0.001 0.005 0.005

Table 6: Differential leucocyte counting (100u/) of Mice given 10 ppm fenoxycarb in the drinking water. (L) lymphocyte; (M) monocyte; (N) neutrophil; (E) eosinophil; (B) basophil; (NS) not significant; (SD) standard deviation; (p) Student T-test. Animals Lymphocyte Monocyte Neut roph il Eosi noph il Basophil Weeks & +SD & ±SD & ±SD &±SD & SD (W1-W4)

Control (n=6) 680 ± 6 23 ± 0.12 192 ± 2.04 9 ± 0.12 Treated (n=7) 583 ± 11.3 12 ± 0.05 109±2.1 10 ± 0.04 P< 0.0005 0.005 0.0005 NS Control (n=6) 670 ± 21 27 ± 1.3 156 ± 2.48 7 ± 2.2 Treated (n=7j 586 ± 7.3 17 ± 0.13 112 ± 3.14 12 ± 0.45 0.05 0.05 0.01 NS P< Control (n=6) 647 ± 19 21 ± 0.9 197 ± 2.9 11.4 ± 0.2 Treated (n=7) 597 ± 11.1 18 ± 0.09 105 ± 1.6 10 ± 0.1 P< 0.05 0.01 0.005 NS Control (n=6) 730 ± 11 9 ± 0.12 174 ±1.77 8 ± 3.4 Treated (n=7) 632 ± 10 13 ± 0.2 118 ± 1.89 13 ± 0.05 0.01 0.05 0.005 0.01 p<

380 Air Pollution Monitoring, Simulation and Control

Results

The haematological parameters of experimental animals showed significant changes (p<0.05-0.0005) as early as the first week at all three doses compared with control levels (Figures 1-4). In general, the effects appeared to be time and dose dependant. There was a general decline in almost all the parameters a week after the use of the lowest dose but was insignificant (NS) except for mean number of leucocytes (WBC, p<0.005) [table 1]. However, at this dose there was a significant decline in leucocytes (WBC) counts (p<0.005), particularly, in the absolute number of lymphocytes (p<0.005), monocytes (p<0.01), and eosinophils (p<0.05). However, the change in neutrophil number was insignificant [table 4]. By the end of the experiments the levels of haemoglobin (g/dl), packed cell volume (PCV%), leucocytes (WBC), and erythrocytes (RBC) had shown a regular decline as had the differential count of leucocytes which ranged from non- to highly significant with the higher doses (up to p<0.0001). Similar changes, described for 1ppm, continued with the higher doses (tables 2 & 3), but the effect was greater (p<0.005-0.0005). Lymphocytes (L), at a dose 5 ppm, showed fluctuating changes when compared with the control group, but showed a steady increase with time at dose 10 ppm. In higher doses, similarly, monocytes (M) and neutrophils (N), showed significantly (p<0.05-0.0005) fluctuating changes particularly at doselO ppm.

Eosinophils (E) significantly (p<0.05) increased in week two and three at dose 1 ppm however, levels also continued to fluctuate (ranged from insignificant upto p<0.0.5) at the other two higher doses. No basophils (B) were ever detectable.

Discussion

A moderate to heavy anemia developed in experimental animals using heavy metals"! 9.20 indicated by a decline in blood parameter measurements. Similar results were also detected using fenoxycarb. Therefore, it seems more important to interpret the blood picture after exposure to Fenoxycarb as the blood represents the direct response of the body fluid to the toxic effects of any chemical that enters the body.

A careful and systematic examination of the blood has been carried out to detect an accurate measurement of many different parameters following the emphasis of O'Connor and Bunch2i. Haematological examinations taken from the blood of the experimental animals chronically treated with three doses of fenoxycarb have shown a significant decline (p<0.05-0.0005) in haemoglobin and other blood components, as early as the first week and these changes increased with both, time and dose. Similar results have been detected using zinc phosphide22, lead monoxide^ and potassium dichromate2o. The latter

Air Pollution Monitoring, Simulation and Control 381 parallels the dose dependant effects of fenoxycarb on the blood. In all cases, anemia occurs. In the case of fenoxycarb, there was a marked decline in white blood cell numbers. It is known that a moderate fall in the haemoglobin level (or red cell count) below the normal range is an indictor of anemiazs. in fact most chronic illnesses, such as infection, malignant disease, renal disease are accompanied by a moderate fall in the haemoglobin level which may occur within a few weeks of the onset of the illness24. The mechanism of anemia is complex and multifactorial. Even certain drugs produce important adverse effects on blood cell formation and survival, as well as on the function of leucocytes and plateletzs. An example of the side effects of some chemicals is the presence of renal failure in patients receiving methotrexate as this drug is eliminated poorly under these circumstancesss. However, it seems possible, in case of fenoxycarb, that its toxic effects might occur through damage to the renal tubules within the kidney. To confirm this some histological work would be necessary. One possible explanation of the effects is that the action of fenoxycarb on blood parameters is similar to the action of lead monoxide and potassium dichromat and is due to the failure of bone marrow to maintain its proliferation of blood cells26,27, and failure to produce enough red cells to keep pace with destruction of red cells20.

Such anemia would lead to tissue hypoxia due to the reduction in the oxygen carrying capacity of the blood28. The leucocyte data showed a significant and consistent dose dependant decline in all cases, with the exception of eosinophils which increased slightly. By the end of the four week period, particularly at higher dose of fenoxycarb, lymphocytes had declined to 92% of control values, however, macrophages had declined to 57% and neutrophils to 61%. Without evidence of disease levels it is difficult to make a categoric statement as to the impact of fenoxycarb on the animals health. However, as the data suggest an increase in effect with both time and dose, it is very likely that long term chronic exposure to fenoxycarb would lead to a decline in the immune response of wild populations to infection. In general terms, neutrophils in particular have been shown to recover following withdrawal of drugs25. This suggests that short acute treatment with fenoxycarb, would have a lesser environmental impact on wild mammalian populations than larger chronic exposures, assuming the effects of this preparation are reversible. In summary, fenoxycarb at the doses used in this study causes anemia and a steady decline in certain leucocyte populations particularly macrophages and neutrophils. Although at these doses the compound is not directly toxic, long term exposure could lead to hypoxia stress and an increased risk of infection.

382 Air Pollution Monitoring, Simulation and Control

References

1. Thind, B. & Edwards, J. Stimulation of egg production in the grain mite Ascaris siro by the juvenile-hormone analogue fenoxycarb Experim. Appl. Acaralo\ 1990, 9, 1-10.

2. Murty, A. S. Toxicity of Pesticides to Fish. CRC Press Boca Raton, Florida, USA 1986.

3. Ali, A. Perspectives on management of pestiferous Chiromidae (Diptera), an emerging global problem. J. Am. Mosq. Cont. Assoc; 1991; 7, 260-281.

4. Dorn, S.; Friscknecht, M; Martinez, V.; Zurfluh, R. & Fischer, U. A noval non- neurotoxic insecticide with a broad activity spectrum. Z Pflanzenker.Pflanzenschutz; 1981; 88, 269-275.

5. Edwards, J & Short, J. Evaluations of three compounds with juvenile-hormone activity as grain protectants against insecticides-susceptible and resistant strains of Sitophilus species (coleoptera, Curculionidae). J. Stored Prod. Res. 1984; 20, 11-15.

6. Kramer, K.; Henrick, L; Wojciak, J & Fyler, J. Evaluation of fenoxycarb, Bacillus thringgiensis, and Malathion as grain protectants in small bins. J. Econ. Entomol. 1985, 78, 632-635.

7. Thind, B.B. & Edwards, J. Laboratory evaluation of the juvenile-hormone analogue fenoxycarb against some insecticides-susceptible and resistant stored product beetles. J. Stored Prod. Res. 1986, 22, 235-241.

8. Edwards, J, Short, J & Abraham, L. Symposium on stored products pest control, Br. Crop. Prot Counc. Reading GBR, 37, 197, 1987.

9. Marchiondo, A., Riner, J., Sonenshine, D., Rowe, K. & Slusser, J. Ovicidal and larvicidal modes of action of fenoxycarb against the cat flea (Siphonaptera: Pulicidae). J. Med. Entomol. 1990, 27, 913-921.

10. Reid, B; Bennett, G, & Yonker, J. Influence of fenoxycarb on German cockroach (dictyoptera: Blattellidae) populations in public housing. J. Ecol. EntomoL; 1993, 83, 444-450.

11. Masner, P., Dorn, S., Vogel, W., Kalin, M., Graf, O & Gunthart, E. Types of responses of insects to a new IGR and to proven standards. Scientific papers of the Institute of Organic and Physical Chemistry of Wrolclaw Technical University

Conferences; 1981, 7, 809-818.

12. Philips, S.A. & Thornvilson, H.G. Use fenoxycarb for area-wide management of red imported fire ants (Hymenoptera: Formicidae). J. Econ. Entomol. 1989, 82, 1646-1649.

Air Pollution Monitoring, Simulation and Control 383

13. Solomon, M. G. & Fitzgerald, J.D. Fenoxycarb, a selective insecticide for inclusion in integrated pest management systems for pear in the U.K. J. Hort. Sci.; 1990; 65: 535-539.

14. Bazzaz, A.A., Madhloom, 1.1., AI-Aluci, R.S., Muhsin, S.S. & Chelebi, N.A. The toxic effects of the new insect growth regulator (IGR), fenoxycarb on mice. Proc. 3rd World Conf. Environ. Health Hazards of Pesticides, Cairo, Egypt (Abstract);

1989, 11-5-0.

15. Staal, G.B. Insect growth regulator with juvenile hormone activity. Annu. Rev. Entomol. 1975, 20, 417-460.

16. Edwards, J. and J. Menn; The Use of Juvenoids in Insect Pest Management, Von R. Wegler (Ed), Springer, New York, USA;1981; 185-224.

17. Sehnal, F. Juvenile hormone analogue, pp: 657-672. In R.G. Downer & H. Laufer [Eds], Endocrinology of Insects] R.G. Downer and H. Laufer (Eds), Alan R. Liss, New

York, 1983, 657-672.

18. Batra, N. Clinical Pathology for Medical Students, Vikas Publisher, Bombay, India; 1982, 12-42.

19. Bazzaz, A.A., Muhsin, S.S. & Sulaiman, N.M. Haematological changes in blood of mice induced by subchronic ingestion of lead monoxide. J. Biol. Sci. Res.; 1988, 20,

31-40.

20. Bazzaz, A.A., Sulaiman, N.M. & Muhsin, S.S. Histopathological and haematological effects of the ingested potassium dichromates on the mouse. Proc. 5th Sci. Conf./SRC-lraq, 1989, 5: 297-310.

21. O'Connor, N., Bunch, C. Laboratory diagnosis in haematology. Med. Internal',

1992; 96, 3984-3989.

22. Mukhta-Bai, K., Krishnakumari, M.K., Ramesh, M., Shivandappa, T. & Majunder, S. Short term toxicity study of zinc phosphide in albino rats Rattus norvegicus. Ind. J. Exp. Biol.; 1980, 18, 854-857.

23. McGee, J.O'D., Isaacson, P.G. & Wright, N.A. Haematology, (Oxford Text-book of Pathology) vol. 2b, Pathology of Systems; Oxford University Press1992.

24. Bunch, C. The blood in systematic disease. Medicine International, 1991, 96, 3990-3995.

25. Firkin, F.C. Haematological side-effects of drugs. Medicine International, 1991,

99, 4026-4029.

26. Hardistry, R.M. & Weatherall, D.J. Blood and Its Disorders. In Blackwell Scientific, Oxford (A Comperhansive and Authoritative Multiauthor Text) 1974.

384 Air Pollution Monitoring, Simulation and Control

27. Dagg, J., Lee, F.G.The blood and bone marrow. In Muir's Textbook of Pathology; J. R. Anderson, 10th ed., Edward Arnold Press; 1976.

28. Anderson, J.R. Urinary System. In Muir's Textbook of Pathology; J. R.

Anderson, 10th ed., Arnold Press; 1976