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Biochemical studies of [Na.sup.+], [K.sup.+]-ATPase and AChE sensitivity to insecticides and fertilization against Spodoptera littoralis larvae.

INTRODUCTION

The cotton leafworm, Spodoptera littoralis is considered as one of the most serious and destructive phytophagous Lepidopterous insect-pests in Egypt, not only for cotton plants but also for more than 70 cultivated field crops and vegetables [7,2]. The indiscriminating use of insecticides has caused a number of ecological, economical and social hazardous ecological agro-ecosystems around the world including Egypt. Besides, the resistance to pesticides appeared in several insect pests. Therefore, the results from this investigation could be attributed to the possibly right use of each fertilization as control mean in the Integrated Pest Management (IPM) of the Spodoptera littoralis to avoid the increasing use of conventional insecticides and reduce the occurring environmental pollution.

Fertilization might be good tool to produce a profitable cotton crop that competes with weeds and able to out-grow and overcome the possible occurrence of disease and insect damage, also corrected deficiencies of certain required nutrients in large amounts (macro-elements) and \ or required in trace amounts (microelements). [16,14].

The present investigation aimed to study the efficiency of insecticides, (Cyhalothrin and Chlorpyrifos) either alone or in their combination with fertilizer (Superphosphate, Potassium sulphate and Urea) on Spodoptera larvae. Also to study a two target in the insect to the knowleage about insecticide susceptibility, describe the development of a biochemical assay system for measuring the sensitivity of [Na.sup.+],[K.sup.+]-ATPase and AChE to Cyhalothrin and Chlorpyrifos respectively, and also provide enzyme kinetic data for in field strain and compared with data obtained of lab strain.

MATERIALS AND METHODS

1. Test insect:

The susceptible laboratory strain of cotton leafworm, Spodoptera littoralis was provided from centeral lab of pesticides. Agricultural Research Center (ARC) Cairo, Egypt which was reared for several years on artificial diet under standard laboratory conditions of 27 [+ or -] 2[degrees]C and 65-70% RH.

Field strain was obtained by the collection of the egg masses from cotton fields at Abeis area Alex. Proviance Egypt, the eggs were allowed to hatch larvae, chosen for bioassays, the 2nd , 3rd and 4th instar larvae and the 2nd larval instar chosen for biochemical assessments.

2. Chemicals and test insecticides:

Cyhalothrin provided as technical grade Pyrethroids insecticide from U.S.A. Environmental Protection Agency (EPA), U.S.A. Ouabain is a cardiac glycoside which specifically inhibits the [Na.sup.+], [K.sup.+]-ATPase (Mcllwain, 1963). A pure sample was obtained from Sigma Chem., Co. ST. loius. Dursban (Chlorpyrifos 48% EC) Organophosphate insecticide was obtained from Dow AngroSciences Co., (Dow England).

Fertilization, Urea fertilizer (46.5% N); Superphosphate fertilizer (15.5% [P.sub.2][O.sub.5]), and Potassium sulphate fertilizer (48% [K.sub.2]O), were supplied by Easterna Co. for Agriculture Development (Easterna, Egypt).

3. Bioassay tests:

3.1. Toxicity of the tested insecticides against S. littoralis:

Cyhalothrin and Chlorpyrifos were bioassayed against the larvae of S. littoralis. The castor leaves were dipped in different concentrations of the tested insecticide, Cyhalothrin concentrations were prepared in pure acetone, while Chlorpyrifos concentrations were prepared in distilled water. Treated and control plants were air-dried for 3 hrs, the treated leaves were placed in clean glass container at the laboratory conditions of 27 [+ or -] 2[degrees]C and 65-70 % RH, ten larvae (Lab and Field strains) were used for each test with three replicate at least. Number of alive and dead larvae per replicate was counted 24, 48, and 72hr, after treatment, concentrations -mortality percentages were calculated and corrected for natural mortality according to Abbott equation [1]. [LC.sub.50] values were calculated by using the of probit-analysis method of Finney [6].

3.2. Toxicity of tested insecticides in presence of fertilization against S. littoralis larvae:

Larvae of S. littoralis were starved for 6hrs before exposed test the selected larvae was bioassayed against fertilization, (Urea, Superphosphate, and Potassium sulphate) using three replicates for each concentration with ten larvae in each replicate.

Disc dipping technique was used since it has been proved to be the most common procedure for assessing mineral fertilization. Each castor leaves disc (2cm2) was dipped into the suspension of tested mineral fertilization for 10s. Tested concentration were prepared in glass distilled water (GDW) [21] disc were held vertically to allow excess solution to drip off and places on a rack to dry for at last 2 hr. Treated discs were offered to starved larvae (on disc per cup) and left under constant conditions (27 [+ or -] 2[degrees]C and 65-70 % RH). There after survivors were transferred with fresh castor oil plant leaves to clean cups and kept under the same conditions.

S. littoralis larvae (lab and field strains) were treated with solution of Cyhalothrin and Chlorpyrifos at different concentrations before 24, 48, and 72 hrs of feeding on discs of castor oil leaves discs treated with tested fertilization, joint action experiments have two controls. Larvae of the first control were allowed to fed castor oil leaf discs treated with tested fertilization alone, while larvae of the second control were fed with distilled water. Mortality counted and recorded daily for 3 days. Percentage of mortality were calculated according to Abbott [1] and subjected to probit analysis [6].

4. Biochemical studies:

4.1. AChE preparation and activity assay:

AChE was prepared from Spodoptera littoralis 2nd instar larvae was homogenized in Tris-HCl buffer (pH 7.4) at 30 larvae / 30 ml buffer, with polytron mixer (at 50 % power for 50 sec.), then subjected to low speed centrifuged at 5,000 rpm for 15 min at

4[degrees]C. The resulting supernatant was centrifuged at 15,000 rpm for 20 min at 4[degrees]C. The supernatant centrifuged at 25,000 rpm for 1 hr at 4[degrees]C. Pellets were resuspended in 1 ml of Tris-HCl buffer (pH 7.4) and stored at (-20[degrees]C) for used as enzyme source, the AChE activity measurements were done according to method reported by Ellman et al. [5].

4.2. [Na.sup.+], [K.sup.+]-ATPase preparation and activity assay:

[Na.sup.+], [K.sup.+]-ATPase was prepared from Spodoptera littoralis. 2nd instar larvae was homogenized in a solution of 0.32 M sucrose, 1 mM EDTA and 40 mM Tris-HCl buffer (pH 7.4). The homogenate was filtered through two layers of cheese cloth. Mitochondrial. ATPase was prepared according to the method reported by Koch [10], by differential centrifugation of the homogenate at 8000 Xg for 10 min. The supernatant was then centrifuged at 20000 Xg for 30 min. The formed pellets were then suspended in the same buffer and stored at (-20[degrees]C) for use.

ATPase activity was measured according to the method reported by koch [10] with slight modification by Morshedy [15]. Inorganic Phosphate (Pi) was determined according to the method, described by Taussky and Shorr, [20]. The activity of [Mg.sup.2+]-ATPase was measured after the addition of 1 mM Ouabain, whereas the activity of [Na.sup.+], [K.sup.+]-ATPase was calculated as the difference between the total ATPase and [Mg.sup.2+]-ATPase activities.

The protein content in prepared homogenates of S. littoralis was assayed spectrophotometrically by the method of Lowery et al. [12] at 1 750 nm using Bovine Serum Albumin (BSA) as a standard protein.

2.3. In vivo inhibition of AChE and [Na.sup.+], [K.sup.+]-ATPase activity:

In the inhibition studies, of AChE and [Na.sup.+], [K.sup.+]-ATPase activity, 10 [micro]l of the enzyme preparation was incubated with of the inhibitor for 30 min, the enzyme- inhibitor mixture was used to measure the remaining activity. The percent inhibition was calculated using the following formula:- %Inhibition = V-Vi x100 / V

Where:- (V) is the specific activity without inhibitor

([V.sub.i]) is the specific activity in presence of inhibitor.

2.4. In vitro inhibition and kinetics of AChE and [Na.sup.+],[K.sup.+]-ATPase activity:

The inhibition of [Na.sup.+], [K.sup.+]-ATPase activity was determined in 2nd instar larvae using the [LC.sub.50] values of the two tested compounds (Chlorpyrifos and Cyhalothrin). The inhibitor of AChE and [Na.sup.+], [K.sup.+]- ATPase were evaluated to determine enzyme kinetic parameters. The method of Dixon-plots by plotting 1/V versus concentrations of the inhibitor at two concentrations of the substrate, ATP (the substrate of ATPase) concentrations were 3.0 and 5.0 mM.

Estimation of [I.sub.50] value carried out by preincubating the enzyme with the inhibitor for 30 min, using the following concentrations 0.1, 1, 5, 10, 50 and 100 [micro]M. [K.sub.i] (the inhibition constant) values for each inhibitor were estimated from Dixon-plot.

Michaelis-Menten Kinetics ([K.sub.m] and [V.sub.max]) values were calculated by a linear regression of 6 points on each Lineweaver and Burk plot.

RESULTS AND DISCUSSION

Toxicity of tested insecticides pretreated with the tested fertilization against different Spodoptera larval instars:

The results of the toxicity of the Cyhalothrin and Chlorpyrifos in terms of [LC.sub.50] are given in Table (1) for 2nd, 3rd and 4th instar larvae of S. littoralis. [LC.sub.50] values were 0.039 and 0.055 ppm for Cyhalothrin and Chlorpyrifos respectively against 2nd instar larvae of Spodoptera lab strain, for field strains [LC.sub.50] values were 0.055 and 0.069 ppm for two tested insecticides respectively, while [LC.sub.50] values were 0.059 and 0.067 ppm for Cyhalothrin and Chlorpyrifos respectively against 3nd instar larvae of Spodoptera lab strain, for field strains [LC.sub.50] values were 0.073 and 0.079 ppm for two tested insecticides respectively, the [LC.sub.50] values were 0.075 and 0.078 ppm for Cyhalothrin and Chlorpyrifos respectively against 4th instar larvae of Spodoptera lab strain, for field strains [LC.sub.50] values were 0.086 and 0.088 ppm for two tested insecticides respectively. According to the [LC.sub.50] values, it is quite clear that the susceptibility of Spodoptera larvae to Cyhalothrin when pretreated with Superphosphate, and lab strain of Spodoptera larvae is more susceptible to Cyhalothrin when pretreated with Superphosphate in comparison to the field strain, general pattern was observed for the three instars, where the toxicity is decreased by the increasing in the insect instars. The second instar is more susceptible than the third and fourth instars respectively, While the lab strain of Spodoptera larvae is more susceptible to Chlorpyrifos when pretreated with Urea in comparison to the field strain. These results are in agreement with many investigators, [8,4,16,11,13,14].

Toxicity of tested insecticides alone or pretreated with tested fertilization against S. littoralis larvae:

Data in Table (2) show the [LC.sub.50] values of Cyhalothrin are 0.063, and 0.044 ppm after 24, and 48 hr for 2nd instar larvae of lab S. littoralis strain respectively, while the [LC.sub.50] values are 0.070, and 0.065 ppm against field Spodoptera strain respectively. The [LC.sub.50] values of Chlorpyrifos are 0.071, and 0.053 ppm after 24, and 48 hr against 2nd instar larvae of lab strain respectively, while the [LC.sub.50] values are 0.084, and 0.070 ppm against field strain respectively. The interaction of Cyhalothrin and Chlorpyrifos with tested fertilization against lab and field strains of Spodoptera larvae were studied larvae were allowed to feed on castor oil discs treated with [LC.sub.50] of the tested fertilization.

The [LC.sub.50] values of Cyhalothrin and Chlorpyrifos pretreated with the tested fertilization (Urea, Superphosphate, and Potassium sulphate) on lab and filed strains of Spodoptera larvae are presented in Table (2). The [LC.sub.50] values of Cyhalothrin and Chlorpyrifos when pretreated with tested fertilization were higher than [LC.sub.50] Cyhalothrin and Chlorpyrifos alone in lab or filed Spodoptera strains.

The enhancement of toxicity is calculated as a Potentiation factor (P.f.) Table (2). Potentiation factor (P.f.) values for Cyhalothrin are 2.1, 1.6, and 1.1 respectively, when pretreated with Superphosphate, Potassium sulphate, and Urea after 24 hr treatment for lab strain, while the P.f. values are 1.7, 1.5, and 1.1 respectively, when pretreated with tested fertilization 24hr treatment for field strain, the P.f. values are 3.1, 2.0, and 1.3 respectively, when pretreated with tested fertilization after 48hr treatment for lab strain, the P.f. values are 2.0, 1.6, and 1.2 respectively, when pretreated with tested fertilization 48hr treatment for field strain. Also when pretreated with Urea, Superphosphate, and Potassium sulphate the P.f. values are 1.8, 1.3, and 1.1 for Chlorpyrifos respectively, after 24hr for lab strain, while the P.f. values are 1.6, 1.2, and 1.1 respectively, after 24 hr treatment for field strain, the P.f. values are 2.2, 1.5, and 1.2 respectively, when pretreated with tested fertilization after 48 hr treatment for lab strain, the P.f. values are 1.6, 1.2, and 1.1 respectively, when pretreated with tested fertilization 48 hr treatment for field strain.

It is clear that the [LC.sub.50] values concentrations of tested fertilization enhancement the toxicity of the tested insecticides on S. littoralis larvae. Cyhalothrin when pretreated with Superphosphate were the most toxic treatments than pretreated with Potassium sulphate, and Urea respectively, while Chlorpyrifos when pretreated with Urea were the most toxic treatments than pretreated with Superphosphate, and Potassium sulphate respectively, these results are in agreement with which found by many [7,19,17].

In general the susceptibility of Spodoptera larvae to Cyhalothrin increase when treatment after Superphosphate, the observation that Cyhalothrin had the lowest effect when applied alone but it was the best when mixed with Superphosphate. The Superphosphate+Cyhalothrin, and Urea+Chlorpyrifos caused more toxic than effect single treatment. So may be these effect of tested fertilization in mixture and these are a good control of Lepidopterous larvae, it could be concluded that tested insecticide enhanced the toxicity effect of tested fertilization. Based on P.f. values, the lab strain of Spodoptera larvae is more susceptible comparison to the field strain. Generally, efficacy of tested fertilization have a very good additive toxicity for Pyrethroids (Cyhalothrin), and Organophosphate (Chlorpyrifos) either in lab or field Spodoptera strains.

In vivo inhibition of S. Littoralis [Na.sup.+], [K.sup.+]-ATPase andAChE activity:

The in vivo inhibitory effect of the [LC.sub.50] values of tested insecticides against to the Spodoptera 2nd instar lab and field strains larval [Na.sup.+], [K.sup.+]-ATPase and AChE are shown in the data given in Table (3 and 4). The data declared that Cyhalothrin and Chlorpyrifos exhibited the percentages of reduction of [Na.sup.+], [K.syp.+]-ATPase and AChE activity as values were 80.1 and 77.4 % respectively, for lab strain, while values was 77.3 and 70.7 % respectively, for field strain.

Data in Table (3 and 4) summarize the interaction of tested fertilization on the inhibitory effect of Cyhalothrin and Chlorpyrifos on the activity of [Na.sup.+], [K.sup.+]-ATPase and AChE. The resultes proved that pretreated of tested fertilization with Cyhalothrin induce increase the inhibition of enzyme activity. The inhibition of [Na.sup.+],[K.sup.+]-ATPase by Cyhalothrin alone were 80.1 and 77.3 % for lab and field strains respectively, while the inhibition increased to be 90.5 and 88.6 % for lab and field strains respectively when pretreated Superphosphate, the inhibition of AChE by Chlorpyrifos alone were 77.4 and 70.7 % for lab and field strains respectively, while the inhibition increased to be 86.6 and 83.5% for lab and field strains respectively when pretreated with Urea. This results agreement with Smagghe, and Degheele, [19] Casida and Quistad [3] and Saleem, [17].

The in vitro inhibition of S. Littoralis [Na.sup.+], [K.sup.+]-ATPase and AChE activity:

Table (5) The in vitro interaction of Cyhalothrin and Chlorpyrifos on [Na.sup.+], [K.sup.+]-ATPase and AChE activity of Spodoptera 2nd instar respectively. The [I.sub.50] values of Cyhalothrin for lab and field strains larval [Na.sup.+],[K.sup.+]-ATPase are 0.73 and 0.80 [micro]M respectively, these values of Chlorpyrifos for lab and field strains larval AChE are 0.81 and 0.88 [micro]M respectively. On the other hand the [K.sub.i] values of Cyhalothrin for lab and field strains larval [Na.sup.+],[K.sup.+]-ATPase are 51 and 60 [micro]M respectively, the values of These values of Chlorpyrifos for lab and field strains larval AChE are 63 and 75 [micro]M respectively.

Generally, it was noticed that pretreated of tested fertilizer with the tested insecticides clearly decreases values of [I.sub.50] and [K.sub.i] the lowest recorded [I.sub.50] and [K.sub.i] values of the mixture between fertilizer and insecticide reflect that the fertilizer may active the insecticide to inhibit the enzyme, in the other words, adding the fertilizer to the insecticide increased its inhibition potency. We study the in vitro biochemical interaction of them with the Cyhalothrin and Chlorpyrifos in vitro effects.

To characterize more details about the in vitro inhibition of [Na.sup.+],[K.sup.+]-ATPase and AChE by the inhibitor, the [I.sub.50] and [K.sub.i] values of each inhibitor were estimated from the graphical method of Dixon and Weeb, (Table 4). The obtained data proved that compounds competitive inhibition of [Na.sup.+],[K.sup.+]-ATPase activity and [K.sub.i] values were 30 and 44 [micro]M for lab and field strains in the case of Cyhalothrin pretreated with Superphosphate, while the obtained data proved that compounds competitive inhibition of AChE activity and K; values were 42 and 56[micro]M for lab and field strains respectively in the case of Chlorpyrifos pretreated with Urea.

It is concluded from the present results that the tested Pyrethroids and Organophosphate are potentially potent for control of S. littoralis however, with tested compounds, such as Fertilization currently in use, S. littoralis could be successfully included in the pest management programs, also, it is quite clear that when certain pairs of drugs or insecticides are administered together, the effects may be greater or less than might be expected from the sum of the activities of the components when administered separately. The phenomena involved, included under the term "synergism" "potentiation" and "antagonism", are becoming increasingly important in, for example, practical insect control and mammalian toxicology.

General, it could be concluded that the use of Pyrethroids (Cyhalothrin) and Organophosphate (Chlorpyrifos) and their mixtures with Fertilization (Superphosphate, Urea, and Potassium sulphate) instead of conventional hazardous insecticides; and these my reduce the environmental pollution and hazard effects on human health. tested compounds may play an important role in future insect pest management programs.

ARTICLE INFO

Article history:

Received 2 April 2014

Received in revised form

13 May 2014

Accepted 28 June 2014

Available online 23 July 2014

REFERENCES

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(1) Ismail Seham M, and (2) Nader Shaker

(1) Central Laboratory of Pesticides. Sabahia. Alexandria. A.R.C.

(2) Pesticide Chemistry Dept., Fac. Of Agric. Alex., Univ.

Corresponding Author: Nader Shaker, Pesticide Chemistry Dept., Fac. of Agric. Alex., Univ. E-mail: shaker_nader@yahoo.com
Table 1: [LC.sub.50] values of tested insecticides pretreated with
the tested fertilizer against different Spodoptera larval instars.

Compounds                           [LC.sub.50] (ppm)

                                    2nd

                                    Lab strain    Field strain

Cyhalothrin                            0.039          0.055
Superphosphate +Cyhalothrin            0.020          0.044
Potassium sulphate+ Cyhalothrin        0.025          0.047
Urea + Cyhalothrin                     0.034          0.050
Chlorpyrifos                           0.055          0.069
Urea + Chlorpyrifos                    0.042          0.054
Superphosphate+ Chlorpyrifos           0.048          0.057
Potassium sulphate+ Chlorpyrifos       0.053          0.064

Compounds                           [LC.sub.50] (ppm)

                                    3rd

                                    Lab strain    Field strain

Cyhalothrin                            0.059          0.073
Superphosphate +Cyhalothrin            0.044          0.060
Potassium sulphate+ Cyhalothrin        0.048          0.066
Urea + Cyhalothrin                     0.054          0.070
Chlorpyrifos                           0.067          0.079
Urea + Chlorpyrifos                    0.053          0.064
Superphosphate+ Chlorpyrifos           0.059          0.068
Potassium sulphate+ Chlorpyrifos       0.063          0.074

Compounds                           [LC.sub.50] (ppm)

                                    4th

                                    Lab strain    Field strain

Cyhalothrin                            0.075          0.086
Superphosphate +Cyhalothrin            0.062          0.074
Potassium sulphate+ Cyhalothrin        0.067          0.079
Urea + Cyhalothrin                     0.073          0.083
Chlorpyrifos                           0.078          0.088
Urea + Chlorpyrifos                    0.063          0.070
Superphosphate+ Chlorpyrifos           0.070          0.079
Potassium sulphate+ Chlorpyrifos       0.075          0.084

Table 2: Comparative toxicities of tested insecticides alone
or pretreated with tested fertilizer on Spodoptera larvae.

Compounds                          [LC.sub.50] (ppm)

                                          24hr

                                   Lab strain    P.f.*

Cyhalothrin                        0.063
Superphosphate +Cyhalothrin        0.030         2.1
Potassium sulphate+ Cyhalothrin    0.040         1.6
Urea + Cyhalothrin                 0.056         1.1
Chlorpyrifos                       0.071
Superphosphate+Chlorpyrifos        0.067         1.1
Potassium sulphate+ Chlorpyrifos   0.055         1.3
Urea + Chlorpyrifos                0.040         1.8

Compounds                          [LC.sub.50] (ppm)

                                           24hr

                                   Field strain    P.f.

Cyhalothrin                        0.070
Superphosphate +Cyhalothrin        0.042           1.7
Potassium sulphate+ Cyhalothrin    0.048           1.5
Urea + Cyhalothrin                 0.066           1.1
Chlorpyrifos                       0.084
Superphosphate+Chlorpyrifos        0.075           1.1
Potassium sulphate+ Chlorpyrifos   0.068           1.2
Urea + Chlorpyrifos                0.053           1.6

Compounds                          [LC.sub.50] (ppm)

                                           48hr

                                   Lab strain    P.f.

Cyhalothrin                        0.044
Superphosphate +Cyhalothrin        0.014         3.1
Potassium sulphate+ Cyhalothrin    0.022         2.0
Urea + Cyhalothrin                 0.035         1.3
Chlorpyrifos                       0.053
Superphosphate+Chlorpyrifos        0.046         1.2
Potassium sulphate+ Chlorpyrifos   0.035         1.5
Urea + Chlorpyrifos                0.024         2.2

Compounds                          [LC.sub.50] (ppm)

                                           48hr

                                   Field strain    P.f.

Cyhalothrin                        0.065
Superphosphate +Cyhalothrin        0.033           2.0
Potassium sulphate+ Cyhalothrin    0.042           1.6
Urea + Cyhalothrin                 0.055           1.2
Chlorpyrifos                       0.070
Superphosphate+Chlorpyrifos        0.065           1.1
Potassium sulphate+ Chlorpyrifos   0.057           1.2
Urea + Chlorpyrifos                0.044           1.6

* Potentiation factor (P.f.) = [LC.sub.50] tested insecticide
alone / [LC.sub.50] tested insecticide + tested fertilizer

Table 3: In vivo inhibition of Spodoptera larvae 2nd instar
[Na.sup.+], [K.sup.+] -ATPase activity by tested tested
fertilizer ([LC.sub.50]).

Compounds                       %Inhibition

                       Lab strain    Field strain

Cyhalothrin               80.1           77.3
Superphosphate            90.5           88.6
+Cyhalothrin
Potassium sulphate+       85.3           83.8
Cyhalothrin
Urea +                    83.2           79.1
Cyhalothrin

Table 4: In vivo inhibition of Spodoptera larvae 2nd instar
AChE activity by tested fertilizer ([LC.sub.50]).

Compounds                           %Inhibition

                       Lab strain   Field strain

Chlorpyrifos                77.4           70.7
Urea +                      86.6           83.5
Chlorpyrifos
Superphosphate              80.5           77.8
+ Chlorpyrifos
Potassium sulphate+         79.2           72.4
Chlorpyrifos

Table 5: In vitro inhibition of Spodoptera larvae [Na.sup.+],
[K.sup.+] -ATPase and AChE activity by tested insecticides.

Compounds                   [I.sub.50] ([micro]M)

                        Lab. strain    Field strain

Cyhalothrin                0.73            0.80
Chlorpyrifos               0.81            0.88
Superphosphate             0.50            0.64
+Cyhalothrin
Urea + Chlorpyrifos        0.63            0.71

Compounds                    [K.sub.i] ([micro]M)

                        Lab. strain    Field strain

Cyhalothrin                 51              60
Chlorpyrifos                63              75
Superphosphate              30              44
+Cyhalothrin
Urea + Chlorpyrifos         42              56
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Author:Seham M., Ismail; Shaker, Nader
Publication:Advances in Environmental Biology
Article Type:Report
Geographic Code:7EGYP
Date:Jul 1, 2014
Words:4440
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