benzoylation. Substitution of 2-phenyl moiety on the indole ring in stead of side aliphatic carboxylic group increased the larval mortality in case of compound **6** more than in indole-3-acetic acid (**1**). Substitution with 1-acetyl on 2-phenylindole multiplied the lethality against the two tested larval instars, while substitution with 1-benzoyl in compound **8** enhanced the toxicity only against the 4th larval instar. The most effective compound was indole-3-butyric acid (**2**) with 70.9 and 39.7 g/gm LC50 values on the 4th instar after 9 and 13 days, while 1 acetylindole-3-butyric acid **(3)** and 1-acetyl-2-phenylindole (**7**) were more effective with 151.4 and 80.6 g/gm LC50 values against the 6th instar. So**,** compounds **2, 3** and **7** were

The larval weight of the 4th instar (after 7 days) was differently affected with the applied derivatives. Benzoylation of indole-3-acetic acid in compound **4** affected the larval weight in non systematic arrangement with concentrations. Acetylation of indole-3-butyric acid in compound **3** reduced the larval weight at 50 and 100 µg/gm, followed by an increase at higher concentrations. On the contrary, its benzoyl derivative (compound **5)** increased the larval weight at lower concentration, followed by reduction at the two higher concentrations. Light reduction occurred at low concentrations, followed by gradual activation with increasing the concentration, which was exhibited by compound **6**. Substitution with 1-acetyl moiety in compound **7** increased the larval weight at low concentration followed by inhibition percents ranging from 3.2 to 18.5% of control at 100- 1000 µg/gm. Benzoylation of 2-phenylindole in compound **8** decreased the reduction effect more than compound **7**. Comparing with the untreated 6th larval weight (0.77 gm) after two days, all the tested compounds reduced the treated larval weight at all concentrations with different degrees and arrested their development to 7 days after treatment. Compounds **1** and **2** showed narrow differences among their concentrations with less reducing effect, followed by compounds **8, 6**, **5** and **7**. Compounds **3** and **4** were the most active derivatives in weight reduction. From these results, the hormonal effect was obviously clear through the activation of larval weight in most cases when applied earlier at the 4th instar more than at

Untrated 4th instar larvae developed to pupal and adult stages after 6-7 and 9-10 days, respectively. Indole-3-acetic acid (**1**) at 10 µg/gm delayed this development to 29 and 45 days, respectively. However, the other compounds were less effective causing developing of 50, 10, 75, 92, 13, 63, and 83% of the treated larvae to pupae in case of compounds **2**, **3**, **4**, **5**, **6**, **7** and **8**, respectively after 21 days. While, compounds **5** and **7** caused complete transformation of the treated population to adults, compounds **2**, **4**, **6** and **8** caused developing of 75, 75, 55 and 67 % of pupae to adults. Compound **3** (1-acetylindole-3-butyric acid) was the most effective structure blocking adult emergence to 25% of the treated population after 45 days. Regarding 6th larval instar, its control completely developed to the pupal and adult stages after 2-3 and 7-8 days, respectively. All compounds arrested the Comparing with the untreated larvae, compounds **1** and **2** exhibited 14.6% and 16.7% malformation in the intermediates of the treated 4th larval instar at 200 µg/ml, with no effect on 6th larvae. Acetylation of indole-3-butyric acid in compound **3** affected the intermediates at lower concentrations in the 6th larval instar, while its benzoylation increased this effect against the 4th instar only. Acetylation of 2- phenylindole caused 32.6 and 61.1% intermediate malformation at 100 and 200 µg/ml in treated 4th instar larvae. 1-Benzoyl-2 phenylindole affected 4th larvae at 10 µg/ml with 7.6% malformation. However, its effect was as high as 10.1% at the higher concentrations against 6th instar intermediates. These malformation symptoms appeared as larval-pupal intermediates in which the posterior portion of the body only exhibited the pupal shape, while the anterior portion had larval head capsule and thoracic legs (Figure **3**). Malformation of the produced pupa (forming abnormal pupa without wings or that failed to shed the larval cuticle) resulted from the 4th

larval instar, which was more sensitive than that from 6th larval instar to treatment with compounds **1-3**, 2-phenylindole (**6**) and 1-benzoyl-2-phenylindole (**8**). The effects of compounds **4** and **5** depended on the applied concentration. Benzoylation of 2-phenylindole increased the pupae malformation. Adult malformation (adult failed to shed the pupal cuticle or adult with dwarf wings) was affected with the tested compounds, concentration and larval instar. Adult emergence from both treated instars was affected. Compounds **1**, **2**, **3** and **5** blocked the adult emergence to 10.3 - 47.4%, 16.7 - 50%, 20.2 - 50.6% and 10.6 - 55.7% in systematic arrangement, respectively from 4th larval populations comparing with 100% of control. The blocking effect was reduced with increasing the concentration. They blocked adult emergence to 25.9-43.7%, 36.8-57.0%, 31.9-40.9% and 32.5-66.9%, respectively in non systematic arrangement in case of the 6th larval population. Compound **4** caused 9.5-20.8% and 22.9-69.5% adult emergence in case of the treated 4th and 6th larval instars, respectively. Although 2-phenylindole and its 1-acetyl derivative affected the adult emergence from both treated instars in non systematic arrangement, its 1-benzoyl derivative blocked the adult emergence with increasing the concentration. Adult emergence was more inhibited from 4th larval instar treatment indicating that treatment of the lower larval instars gave good results of control (Figure **4**).

#### *Effect on eggs*

Egg hatchability was inhibited increasingly in systematic arrangement with concentrations. Both 1-acetylindole-3-butyric acid (**3**) and 1-acetyl-2-phenyl-indole (**7**) completely stopped hatching when mixed at 100 g/gm with the medium. As the untreated egg mass hatched completely within 24 hours, treated eggs took 48-96 hours and 6-7 days at high concentrations of compound **2** and compounds **3** & **7,** respectively. After 48 hours, only dipping the egg masses in solutions of compound **2** inhibited hatching with IC50 value equaled 29.1 g/ml and killed the produced larvae with LC50 value equaled 26.2 g/ml. Transferring treated eggs to the poisoned medium enhanced the toxicity to IC50 equaled 13.2 g/gm and LC50 equaled 15.2 g/gm. Although acetylation of compound **3** decreased larval mortality in dipping technique with or without transferring the eggs to the poisoned medium, it enhanced egg-hatching inhibition when dipped only in the toxic solutions. Although compound **7** was less effective when egg masses were dipped in it, its mixing with the used medium greatly enhanced the effect with IC50 value equaled 15.3 g/gm on egg-hatching and LC50 value equaled 7.5 g/gm on larval mortality. In conclusion, mortality of 4th instar larvae was increased with increasing the aliphatic side chain. Substitution of N-H of 2-phenylindole raised the toxicity, vice versa in case of indole-3 butyric acid against the same instar. The tested compounds affected larval weight, pupation and adult emergence indicating that treatment induced an effect typical to juvenile hormone excess. These effects varied according to the tested compound. These delayed effects are expressed as developmental abnormalities in the adult stage. These effects may be due to oxidative decarboxylation forming 3-methylene-2-oxindole, which may conjugate with DNA bases and protein thiols [34]. It may be also due to inhibition of cholinesterase [40]. Its effect is associated with cell phenoloxidase (PO) and peroxidase activities [6, 41]. Phenoloxidase (PO) is believed to be a key mediator of immune function in insects.

76 Pesticides – Advances in Chemical and Botanical Pesticides

of control (Figure **4**).

*Effect on eggs*

larval instar, which was more sensitive than that from 6th larval instar to treatment with compounds **1-3**, 2-phenylindole (**6**) and 1-benzoyl-2-phenylindole (**8**). The effects of compounds **4** and **5** depended on the applied concentration. Benzoylation of 2-phenylindole increased the pupae malformation. Adult malformation (adult failed to shed the pupal cuticle or adult with dwarf wings) was affected with the tested compounds, concentration and larval instar. Adult emergence from both treated instars was affected. Compounds **1**, **2**, **3** and **5** blocked the adult emergence to 10.3 - 47.4%, 16.7 - 50%, 20.2 - 50.6% and 10.6 - 55.7% in systematic arrangement, respectively from 4th larval populations comparing with 100% of control. The blocking effect was reduced with increasing the concentration. They blocked adult emergence to 25.9-43.7%, 36.8-57.0%, 31.9-40.9% and 32.5-66.9%, respectively in non systematic arrangement in case of the 6th larval population. Compound **4** caused 9.5-20.8% and 22.9-69.5% adult emergence in case of the treated 4th and 6th larval instars, respectively. Although 2-phenylindole and its 1-acetyl derivative affected the adult emergence from both treated instars in non systematic arrangement, its 1-benzoyl derivative blocked the adult emergence with increasing the concentration. Adult emergence was more inhibited from 4th larval instar treatment indicating that treatment of the lower larval instars gave good results

Egg hatchability was inhibited increasingly in systematic arrangement with concentrations. Both 1-acetylindole-3-butyric acid (**3**) and 1-acetyl-2-phenyl-indole (**7**) completely stopped hatching when mixed at 100 g/gm with the medium. As the untreated egg mass hatched completely within 24 hours, treated eggs took 48-96 hours and 6-7 days at high concentrations of compound **2** and compounds **3** & **7,** respectively. After 48 hours, only dipping the egg masses in solutions of compound **2** inhibited hatching with IC50 value equaled 29.1 g/ml and killed the produced larvae with LC50 value equaled 26.2 g/ml. Transferring treated eggs to the poisoned medium enhanced the toxicity to IC50 equaled 13.2 g/gm and LC50 equaled 15.2 g/gm. Although acetylation of compound **3** decreased larval mortality in dipping technique with or without transferring the eggs to the poisoned medium, it enhanced egg-hatching inhibition when dipped only in the toxic solutions. Although compound **7** was less effective when egg masses were dipped in it, its mixing with the used medium greatly enhanced the effect with IC50 value equaled 15.3 g/gm on egg-hatching and LC50 value equaled 7.5 g/gm on larval mortality. In conclusion, mortality of 4th instar larvae was increased with increasing the aliphatic side chain. Substitution of N-H of 2-phenylindole raised the toxicity, vice versa in case of indole-3 butyric acid against the same instar. The tested compounds affected larval weight, pupation and adult emergence indicating that treatment induced an effect typical to juvenile hormone excess. These effects varied according to the tested compound. These delayed effects are expressed as developmental abnormalities in the adult stage. These effects may be due to oxidative decarboxylation forming 3-methylene-2-oxindole, which may conjugate with DNA bases and protein thiols [34]. It may be also due to inhibition of

**Figure 2.** Effect on *S. littoralis* 4th larval development at 10 g/gm; **1:** Indole-3-acetic acid; **2:** Indol-3 butyric acid; **3:** 1-Acetylindole-3-butyric acid; **4:** 1-Benzoylindole-3-acetic acid; **5:** 1-Benzoylindole-3 butyric acid; **6:** 2-Phenylindole; **7:** 1-Acetyl-2-phenyl indole; **8:** 1-Benzoyl-2-phenyl indole

Malformations in the prod uced pupae comparing with control

**I** Normal pupa (control), **II,** juvenilized larval-pupal intermediates, **III,** abnormal pupae failed to shed the larval cuticle

Different abnormal forms of the produced adults comparing with control **I,** Normal adult (control), **II,** abnormal adults failed to shed the cuticle, **III, a**dult with dwarf wings

**Figure 3.** Maleformations effects of the tested indole derivatives

This notice may clarify the effect of the tested compounds on adult emergence and pupation. N-H and N-substituted indole-2- and 3-carboxamide showed a strong inhibitory (95-100%) effect on superoxide anion (SOD). Substitution on 1-position of the indole ring caused significant differences between the activity results regarding lipid peroxidation inhibition [42] emphasizing the differences in effects due to the derivative structure.

**Figure 4.** Emergence percents of *S. littoralis* adults produced from treated larvae. **Upper,** from treated 4th instar**; Lower,** from treated 6th larval instar**;** Concentrations (g/gm). **1:** Indole-3-acetic acid; **2:** Indol-3-butyric acid; **3:** 1-Acetylindole-3-butyric acid; **4:** 1-Benzoylindole-3-acetic acid; **5:** 1-Benzoylindole-3 butyric acid; **6:** 2-Phenylindole; **7:** 1-Acetyl-2-phenyl indole; **8:** 1-Benzoyl-2-phenyl indole

#### **3.3. Pesticidal activities of some pyrazole derivatives [5]**

78 Pesticides – Advances in Chemical and Botanical Pesticides

Malformations in the prod uced pupae comparing with control **I** Normal pupa (control), **II,** juvenilized larval-pupal intermediates, **III,** abnormal pupae failed to shed the larval cuticle

Different abnormal forms of the produced adults comparing with control **I,** Normal adult (control), **II,** abnormal adults failed to shed the cuticle, **III, a**dult with dwarf wings

This notice may clarify the effect of the tested compounds on adult emergence and pupation. N-H and N-substituted indole-2- and 3-carboxamide showed a strong inhibitory (95-100%) effect on superoxide anion (SOD). Substitution on 1-position of the indole ring caused significant differences between the activity results regarding lipid peroxidation

inhibition [42] emphasizing the differences in effects due to the derivative structure.

**Figure 3.** Maleformations effects of the tested indole derivatives

**I**

Due to antimicrobial activity of some 3,5-dimethylpyrazole derivatives [43], 3,5 dimethylpyrazole (**1**), 1-Benzoyl-3,5-dimethylpyrazole (**2**), 3-methyl-1-phenylpyrazol-5-one (**3**) and 3-methyl-1-(2,4-dinitrophenyl)-pyrazol-5-one (**4**) were prepared, structurally confirmed and studied for their effects against *Fusarium oxysporum*; *Pythium debarianum Rhizoctonia solani* and *Macrofomina phaseoli*. Metalaxyl (Radomil), methyl- N-(2,6 dimethylphenyl-N-methoxyacetyl)-DL-alaninate (**6**) was used as a standard fungicide. Their phytocidal effects were determined on both wheat (*Triticum aestivum*) and squash (*Cucurbita pepo*) seedlings comparing with metribuzin (sencor), 4-amino-6-tert.butyl-4,5-dihydro-3 methylthio-1,2,4-triazin-5-one (**5**). Insecticidal effects were evaluated on the 4th instar of cotton leaf worm, *S. littoralis* Boisid. Their fungitoxic effects as IC50 values illustrated that comparing with metalaxyl, *R. solani* was less affected than the other fungi. 3,5- Dimethylpyrazole (**1**) proved to be moderately toxic with470, 380 and 330 µg/ml IC50 values against *P. debarianum*, *F. calmorum* and *M. phaseoli*, respectively after 6 days exposure, whereas 1-benzoyl-3,5-dimethylpyrazole (**2**) reduced the activity against all the tested fungi but 3-methyl-1-phenylpyrazol-5-one (**3**) enhanced the activity against *R. solani* with IC50

value of 155 µg/ml after 4 days exposure, *P. debarianum* and *M. phaseoli* with IC50 values of 68 and 170 µg/ml after 6 days exposure, respectively whereas it was inactive on *F. calmorum* as its IC50 was >500 µg/ml. On the other hand, 3-methyl-1-(2,4-dinitrophenyl)pyrazol-5-one (**4**) caused the toxic effect against *R. solani*, *F. calmorum* and *M. phaseoli* with 100, 440 and 140 µg/ml IC50 values, respectively after the same exposure time. From these results, some of the prepared compounds exceeded the standard fungicide in their effects against the tested fungi under the used experimental conditions.

*Chemical structures of pyrazole derivatives and used standard pesticides*

Pyrazole derivatives inhibited the growth of root and shoot systems of wheat and squash seedlings differently. Benzoylation of 3,5-dimethylpyrazole (**1**) decreased its inhibition on wheat shoot system growth, vice versa against its root system. Introducing the 2,4-dinitro- moiety enhanced the toxicity of 3-methyl-1-phenyl pyrazol-5-one (**3**) on wheat shoot and root systems. Compounds 1, 2, 3 and 4 inhibited cucumber seedlings root system with 95, 109, 95 and 58 µg/ml and its shoot system with 38, 90, 60 and 78 µg/ml, comparing with 115 and 86 µg/ml of metribuzin, respectively. It gave 81 and 52 µg/ml on wheat shoot and root systems. The standard herbicide was less effective than the tested compounds on quash shoot system. Compound **1** was inactive against *S. littoralis* (Boisid.), its activity slightly increased to 10% mortality by substitution with 1-benzoyl- moiety. The effect became 23% mortality with reduction of palatability to 8.5-50 % of control in case of phenylpyrazol-5-one in. Substitution with 2,4-dinitrophenyl- moiety decreased the activity to 14 % mortality and 50-67 % palatability.

#### **3.4. Pesticidal effects of some imidazolidine and oxazolone derivatives [6]**

Actually we were interested to evaluate pesticidal actions of some imidazolidine and oxazolone derivatives as some of them are insecticides, herbicides and fungicides [44]. So three other derivatives of imidazolidine: 5,5-dimethylimidazolidin-2,4-dione, 5,5 diphenylimidazol-idin-2,4-dione and 5,5-diphenylimidazolidin-2-thione-4-one and two oxazolone derivatives: 4-Benzylidine-2-methyloxazol-5-one and 4-Benzylidine-2-phenyloxazol-5-one were prepared and checked for their structure. Their fungicidal, phytocidal and insecticidal effects were carried out as in case of pyrazole derivatives.

#### *Fungicidal activity*

80 Pesticides – Advances in Chemical and Botanical Pesticides

fungi under the used experimental conditions.

N H N CH3

H3C N

**1 2**

H3C

NN

SCH3 (H3C)3C

decreased the activity to 14 % mortality and 50-67 % palatability.

N O

NH2 **5**

*Chemical structures of pyrazole derivatives and used standard pesticides*

N CH3

C O

value of 155 µg/ml after 4 days exposure, *P. debarianum* and *M. phaseoli* with IC50 values of 68 and 170 µg/ml after 6 days exposure, respectively whereas it was inactive on *F. calmorum* as its IC50 was >500 µg/ml. On the other hand, 3-methyl-1-(2,4-dinitrophenyl)pyrazol-5-one (**4**) caused the toxic effect against *R. solani*, *F. calmorum* and *M. phaseoli* with 100, 440 and 140 µg/ml IC50 values, respectively after the same exposure time. From these results, some of the prepared compounds exceeded the standard fungicide in their effects against the tested

> N NH CH3

> > H

N NH CH3

NO2

NO2

O

**3 4**

H CH3 3C H2 H3COC C

O

N C CH CH3 COOCH3

**6**

H

O

Pyrazole derivatives inhibited the growth of root and shoot systems of wheat and squash seedlings differently. Benzoylation of 3,5-dimethylpyrazole (**1**) decreased its inhibition on wheat shoot system growth, vice versa against its root system. Introducing the 2,4-dinitro- moiety enhanced the toxicity of 3-methyl-1-phenyl pyrazol-5-one (**3**) on wheat shoot and root systems. Compounds 1, 2, 3 and 4 inhibited cucumber seedlings root system with 95, 109, 95 and 58 µg/ml and its shoot system with 38, 90, 60 and 78 µg/ml, comparing with 115 and 86 µg/ml of metribuzin, respectively. It gave 81 and 52 µg/ml on wheat shoot and root systems. The standard herbicide was less effective than the tested compounds on quash shoot system. Compound **1** was inactive against *S. littoralis* (Boisid.), its activity slightly increased to 10% mortality by substitution with 1-benzoyl- moiety. The effect became 23% mortality with reduction of palatability to 8.5-50 % of control in case of phenylpyrazol-5-one in. Substitution with 2,4-dinitrophenyl- moiety

**3.4. Pesticidal effects of some imidazolidine and oxazolone derivatives [6]**

and insecticidal effects were carried out as in case of pyrazole derivatives.

Actually we were interested to evaluate pesticidal actions of some imidazolidine and oxazolone derivatives as some of them are insecticides, herbicides and fungicides [44]. So three other derivatives of imidazolidine: 5,5-dimethylimidazolidin-2,4-dione, 5,5 diphenylimidazol-idin-2,4-dione and 5,5-diphenylimidazolidin-2-thione-4-one and two oxazolone derivatives: 4-Benzylidine-2-methyloxazol-5-one and 4-Benzylidine-2-phenyloxazol-5-one were prepared and checked for their structure. Their fungicidal, phytocidal Imidazolidine derivatives appeared more effective than the oxazol-5-one derivatives on *R. solani* depending on the substituent on position 5. 5,5-Dimethyl moiety increased the toxicity of compound **1**, 5,5-dimethylimidazol-idin-2,4-dione than 5,5-diphenyl moiety in compound **2**, 5,5-diphenylimidazol-idin-2,4-dione with IC50 values of 191.8 and 447.6 g/ml, respectively. Replacing sulfur in compound **3,** 5,5-diphenylimidazolidin-2-thione-4-one instead of oxygen at position 2 increased its toxicity with 148.4 IC50 value exceeding the standard fungicide (233.8 g/ml). 2-Phenyl moiety enhanced the toxicity of compound **5**, 4 benzylidine-2-phenyl oxazol-5-one more than 2-methyl moiety in compound **4**, 4 benzylidine-2-methyl oxazol-5-one with 542.0 and 785.3 g/ml, respectively. Vice versa against *P. debarianum*, compound **5** was the most effective among the other tested compounds with IC50 of 76.9 g/ml. Imidazolidine derivatives were nearly similar or more active than standard in its effect. Compound **3** was more toxic than compounds **1**, **2**, **4** and the standard fungicide against *P. debarianum* with IC50 values 156.4, 357.1, 318.7, 516.5 and 334.3 g/ml, respectively. Compounds **1** and **3** were more effective against *F. calmorum* than others with 306.7 and 314.1 g/ml IC50 values. The standard fungicide exceeded all compounds against *F. calmorum*. Compound **3** was the most effective against *M. phaseoli* with 139.0 IC50 value surpassing all compounds including the standard fungicide.

*Preparation scheme of compounds 1-5*

From the obtained results, fungitoxic activities proved to be a function of both the tested compound and the used fungus. In general, through analysis of variance (ANOVA) of hyphal growth inhibition percents, compound **3,** 5,5-diphenylimidazolidin-2-thione-4-one was the most active against the tested fungi with Mean SE of growth inhibition equaled 34.69e. The other tested compounds were arranged as Mean SE was 32.742.53d, 28.672.79c , 25.082.44b and 24.652.29b and 19.932.00a, respectively in case of standard fungicide, compound **5,** compounds **1** and **2**, compound **4**. *P. debarianum* was more sensitive than *R. solani, F. calmorum* and *M. phaseoli* with Mean inhibition% SE of 29.552.23d, 27.882.07c , 27.301.98b and 25.772.01a, respectively.

#### *Phytocidal activity*

The tested compounds inhibited germination and shoot growth of treated *T. aestivum* seeds. Compound **5** inhibited shoot growth exceeding the other prepared compounds with EC50 value equaled 98.6 g/ml. Compound **2** surpassed compounds **1**, **3** and **4** with EC50 values equaled 154.1, 177.9, 282.6 and 703.4 g/ml, respectively. The tested compounds inhibited germination of treated seeds with EC50 values ranged from 517.3 to 726.8 g/ml. Metribuzin as a standard herbicide was the most effective inhibiting germination and shoot growth with EC50 values of 92.4 and 54.8 g/ml. As a result of being these compounds more effective on seedling shoot growth than on germination process, they were tested against both the root and shoot systems of pregerminated seeds of wheat (*T. aestivum*) as a narrow leaf plant and squash (*C. pepo*) as abroad leaf plant. Compound **1** showed the lowest effect, followed by compound **4** against both the root and shoot systems of *T. aestivum*. Compound **3** exhibited the strongest effect with EC50 values equaled 25.2 and 35.6 g/ml on root and shoot comparing with 53.6 and 60.6 g/ml of the used standard herbicide. Differences in the tested compounds controlled their effect on the broad leaf plant. The standard herbicide proved to be the most active with EC50 values equaled 104.1 and 113.7 g/ml and compound **5** was the next with 274.1 and 203.6 g/ml EC50 values on its root and shoot systems growth. While compound **4** was less effective with 886.9 and 613.7 EC50 values against the root and shoot systems, the other tested compounds affected this plant with EC50 values ranged from 320.2 to 437.7 g/ml.

#### *Insecticidal activity*

The tested compounds exhibited low mortality on 24 hours treated *S. littoralis* larvae. Among the studied imidazolidine derivatives, compound **2** affected it with LC50 value equaled 867.3 g/ml inhibiting the feeding activity with effective concentration on 50% (EC50) equaled 31.78 g/ml. The other two derivatives caused very weak mortal effects and inhibited feeding with EC50 values equaled 3200 and 3489.9 g/ml in case of compounds **1** and **3**, respectively. Regarding the oxazolone derivatives, although compound **4** exhibited mortality percent as high as 24%, it reduced the feeding with EC50 value equaled 376.8 g/ml. The other oxazolone derivative (compound **5)** caused LC50 value equaled 659.7 g/ml and reduced the feeding activity with EC50 equaled 982.5 g/ml. So these compounds affected as antifeedants more than as killers against the studied insect and compound **2** was the most effective structure among them (Figure **5**).

The tested compounds exhibited phytocidal and fungicidal activities higher than their insecticidal effects. Differences in these compounds could be referred to chemical structure as in imidazolidine derivatives, presence of the 2-thione in compound **3** increased its fungitoxic effect nearly against all of the tested fungi. Substitution of 5,5-dimethyl moiety in compound **1** increased the toxicity more than 5,5- diphenyl moiety in compound **2** against *R. solani* and *F. calmorum* fungi. On contrary against *P. debarianum* and *M. phaseoli*, they showed almost the same effect. Their insecticidal effects were changed against the treated larvae as compound **2** exceeded the effects of the two other imidazolidine derivatives. Regarding the

**Figure 5.** Feeding inhibition on *S. littoralis* Boisd. **1**, 5,5-Dimethylimidazolidin-2,4-dione**; 2,** 5,5 diphenylimidazolidin-2,4-dione; **3,** 5,5-di-phenylimidazolidin-2-thione-4-one; **4**, 4-Benzylidine-2 methyloxazol-5-one**; 5,** 4-benzyl-idine-2-phenyloxazol-5-one.

prepared oxazolones, compound **5** appeared more effective than compound **4** against the tested fungi and larvae. This difference between oxazolone derivatives could be due to the substituted moiety on C-2 position [45] as they revealed that substitution of functional group (s) at C-4 and C-2 positions plays a vital role in oxazolone series activity. They also revealed that oxazolone derivatives demonstrated excellent *in vitro* tyrosinase inhibitory. Fungitoxic effects of oxazolone derivatives maybe due to their mutagenic potential during *in vitro* DNA synthesis inducing mainly dAMP insertion [46]. The effect may referred to inhibition of fungal RNA synthetase [47]. In conclusion, compound **3**, 5,5 diphenylimidazolidin-2-thione-4-one was the most useful fungitoxic structure among the prepared compounds and so, it might be useful in controlling plant pathogenic fungi after suitable formulation and helping in integrated management programmes. It also proved to be the most suitable structure for phytotoxicity, especially for the narrow leaf weeds.

#### **3.5. Fungicidal effects of certain benzotriazole and coumarin derivatives [48]**

To extend the spectrum of newly discovered antifungal compounds facing continuous fungal infections, six benzotriazole derivatives as well as two coumarin derivatives were synthesized, confirmed for their structure and evaluated on *F. oxysporum*; *R. solani*; *M. phasoli*; *Helminthosporium sp* and *Alternaria alternata.* Triazole ring was chosen due to discovering some effective fungicidal triazole derivatives [49-51].

#### *In vitro fungitoxicity effects*

82 Pesticides – Advances in Chemical and Botanical Pesticides

The tested compounds inhibited germination and shoot growth of treated *T. aestivum* seeds. Compound **5** inhibited shoot growth exceeding the other prepared compounds with EC50 value equaled 98.6 g/ml. Compound **2** surpassed compounds **1**, **3** and **4** with EC50 values equaled 154.1, 177.9, 282.6 and 703.4 g/ml, respectively. The tested compounds inhibited germination of treated seeds with EC50 values ranged from 517.3 to 726.8 g/ml. Metribuzin as a standard herbicide was the most effective inhibiting germination and shoot growth with EC50 values of 92.4 and 54.8 g/ml. As a result of being these compounds more effective on seedling shoot growth than on germination process, they were tested against both the root and shoot systems of pregerminated seeds of wheat (*T. aestivum*) as a narrow leaf plant and squash (*C. pepo*) as abroad leaf plant. Compound **1** showed the lowest effect, followed by compound **4** against both the root and shoot systems of *T. aestivum*. Compound **3** exhibited the strongest effect with EC50 values equaled 25.2 and 35.6 g/ml on root and shoot comparing with 53.6 and 60.6 g/ml of the used standard herbicide. Differences in the tested compounds controlled their effect on the broad leaf plant. The standard herbicide proved to be the most active with EC50 values equaled 104.1 and 113.7 g/ml and compound **5** was the next with 274.1 and 203.6 g/ml EC50 values on its root and shoot systems growth. While compound **4** was less effective with 886.9 and 613.7 EC50 values against the root and shoot systems, the other tested compounds affected this plant with EC50 values ranged from 320.2

The tested compounds exhibited low mortality on 24 hours treated *S. littoralis* larvae. Among the studied imidazolidine derivatives, compound **2** affected it with LC50 value equaled 867.3 g/ml inhibiting the feeding activity with effective concentration on 50% (EC50) equaled 31.78 g/ml. The other two derivatives caused very weak mortal effects and inhibited feeding with EC50 values equaled 3200 and 3489.9 g/ml in case of compounds **1** and **3**, respectively. Regarding the oxazolone derivatives, although compound **4** exhibited mortality percent as high as 24%, it reduced the feeding with EC50 value equaled 376.8 g/ml. The other oxazolone derivative (compound **5)** caused LC50 value equaled 659.7 g/ml and reduced the feeding activity with EC50 equaled 982.5 g/ml. So these compounds affected as antifeedants more than as killers against the studied insect and compound **2** was the most

The tested compounds exhibited phytocidal and fungicidal activities higher than their insecticidal effects. Differences in these compounds could be referred to chemical structure as in imidazolidine derivatives, presence of the 2-thione in compound **3** increased its fungitoxic effect nearly against all of the tested fungi. Substitution of 5,5-dimethyl moiety in compound **1** increased the toxicity more than 5,5- diphenyl moiety in compound **2** against *R. solani* and *F. calmorum* fungi. On contrary against *P. debarianum* and *M. phaseoli*, they showed almost the same effect. Their insecticidal effects were changed against the treated larvae as compound **2** exceeded the effects of the two other imidazolidine derivatives. Regarding the

*Phytocidal activity*

to 437.7 g/ml.

*Insecticidal activity*

effective structure among them (Figure **5**).

Effect of the tested 1,2,3-triazole and coumarin derivatives on three soil fungi and two foliar fungi based on their structure differences. 5,6- Dichlorobenzotriazole proved to be highly toxic against *R. solani*, *A. alternata* and *Helminthosporium sp* with IC50 values of 12, 20 and 27 µg/ml and moderately fungitoxic against both *M. phasoli* and *F. oxysporum* with IC50 values equaled 53 and 56 µg/ml. Toxicity categories are devised by [52]. However benzotriazole

3, 3 -

–Methelene bis –4–hydroxy coumarin, 2,3–Dihydro–2-(2-hydroxybenzoyl) (**Dicoumarol**) – 4H Furo[3,2 –C] [1] benzopyran-4- one, (**Furopyrone**)

**Scheme 2.** Chemical structure of tested triazole and coumarin derivatives

and 1-acetyl-5,6-dimethylbenzotriazole caused moderate effects against *M. phasoli* and *F. oxysporum* as soil fungi, respectively with equal IC50 values (155 µg/ml). 5,6-Dimethyl-, 1 benzoyl-5,6-dimethyl- and 1-benzoyl- benzotriazoles as well as dicoumarol and furopyrone (coumarin derivatives) needed increasing their highest concentration (200 µg/ml) to get 50% inhibition against hyphal growth of all fungi, and in case of 1,2,3-benzotriazole against *F. oxysporum* and *R. solani*; and 1-acetyl-5,6-dimethylbenzotriazole against *R. solani* and *M. phasoli*. *Helminthosporium sp* (foliar fungus) was moderately affected by 1-acetyl-5,6 dimethyl- and 1-benzoyl-5,6-dimethyl- benzotriazoles with IC50 values of 150 and 165 µg/ml, respectively. The other compounds could not reach 50% fungitoxicity against it at the concentration range. *A. alternata* was also moderately affected by benzotriazole; 1-benzoyl-5,6 dimethyl-; 1-acetyl-5,6-dimethyl- and 5,6-di-methylbenzotriazoles with IC50 values equal 150; 155; 165 and 170 µg/ml, respectively. This fungus was less sensitive to 1 benzoylbezotrtiazole and both coumarin derivatives. However, 5,6-dichloro substituent highly improved the fungitoxic effect of benzotriazole against all tested fungi, comparing with other benzotriazoles in addition to dicoumarol and furopyrone (coumarin derivatives). 1-Benzoylbenzotriazole and coumarin derivatives were less effective against all tested fungi. The weak effects of coumarin derivatives may be due to their classification as mammalian poisons. So, it could be concluded that 5,6-dichlorobenzotriazole was very good fungicide against all tested fungi. On the other hand, *A. alternata* (as a foliar fungus) was moderately affected by the other tested benzotriazoles except 1-benzoylbenzotriazole and coumarin derivatives.

#### *In Vivo biochemical effects*

84 Pesticides – Advances in Chemical and Botanical Pesticides

O O O O

3, 3 -

derivatives.

 Chemical structure of tested triazole and coumarin derivatives

–Methelene bis –4–hydroxy coumarin, 2,3–Dihydro–2-(2-hydroxybenzoyl) (**Dicoumarol**) – 4H Furo[3,2 –C] [1] benzopyran-4- one,

and 1-acetyl-5,6-dimethylbenzotriazole caused moderate effects against *M. phasoli* and *F. oxysporum* as soil fungi, respectively with equal IC50 values (155 µg/ml). 5,6-Dimethyl-, 1 benzoyl-5,6-dimethyl- and 1-benzoyl- benzotriazoles as well as dicoumarol and furopyrone (coumarin derivatives) needed increasing their highest concentration (200 µg/ml) to get 50% inhibition against hyphal growth of all fungi, and in case of 1,2,3-benzotriazole against *F. oxysporum* and *R. solani*; and 1-acetyl-5,6-dimethylbenzotriazole against *R. solani* and *M. phasoli*. *Helminthosporium sp* (foliar fungus) was moderately affected by 1-acetyl-5,6 dimethyl- and 1-benzoyl-5,6-dimethyl- benzotriazoles with IC50 values of 150 and 165 µg/ml, respectively. The other compounds could not reach 50% fungitoxicity against it at the concentration range. *A. alternata* was also moderately affected by benzotriazole; 1-benzoyl-5,6 dimethyl-; 1-acetyl-5,6-dimethyl- and 5,6-di-methylbenzotriazoles with IC50 values equal 150; 155; 165 and 170 µg/ml, respectively. This fungus was less sensitive to 1 benzoylbezotrtiazole and both coumarin derivatives. However, 5,6-dichloro substituent highly improved the fungitoxic effect of benzotriazole against all tested fungi, comparing with other benzotriazoles in addition to dicoumarol and furopyrone (coumarin derivatives). 1-Benzoylbenzotriazole and coumarin derivatives were less effective against all tested fungi. The weak effects of coumarin derivatives may be due to their classification as mammalian poisons. So, it could be concluded that 5,6-dichlorobenzotriazole was very good fungicide against all tested fungi.  Effect on polyphenoloxidase and peroxidase activities

5,6-Dichlorobenzotriazole at 0.1, 0.25, 0.5, 1 and 2 IC50 rates in µg/ml affected both polyphenoloxidase (PPO) and peroxidase (PO) enzymes for each tested fungi. Their activities were in non-systematic response depending on the type of fungus and concentration. The activity of polyphenoloxidase was highly increased in *F. oxysporum*; slightly increased in *R. solani*; weakly increased in *M. phasoli* with increasing the tested concentration; whereas, in *Helmintho-sporium sp* its activity weakly increased until 0.5 IC50 then weakly inhibited by IC50 and 2 IC50 rates. In case of *A. alternata* this enzyme weakly inhibited by 33.2% inhibition at 0.5 IC50 value, then the enzyme was slightly activated with increasing the tested rates to IC50 and 2 IC50. Concerning peroxidase, its activity weakly increased in *F. oxysporum* at all the tested rates, weakly inhibited in *R. solani* at the lower two rates then weakly increased. In *M. phasoli* and *A. alternata*, peroxidase enzyme was highly inhibited with I50 values equal 39.64 and 5.78 µg/ml, respectively. 5,6-Dichlorobenzotriazole was very effective to inhibit peroxidase enzyme in *A. alternata* through all the tested rates.

#### b. Effect on DNA and RNA contents

5,6-Dichlorobenzotriazole at several rates of its IC50 values affected DNA and RNA contents in each tested fungus. In *F. oxysporum*, DNA and RNA contents as compared with control were reduced at the two lower rates (0.1 and 0.25 of IC50) of 5,6-di-chlorobenzotriazole then increased with increasing the tested rates. On the other hand, their contents in *R. solani* highly increased more than control reaching the maximum peak at 0.5 IC50 then decreased but still more than control. DNA and RNA contents in both *M. phasolina* and *Helminthosporium sp* were decreased with increasing the tested rates of IC50 values. The contents in *A. alternata* were highly increased with increasing the tested rates of IC50 values. In conclusion, 5,6 dichlorobenzotriazole may be useful as a good fungi-cide against all the tested fungi. The 5,6-dichloro- substituent was required to improve benzotriazoles effects against all treated fungi. So, it was highly effective against the activities of polyphenoloxidase, peroxidase and DNA and RNA contents.

#### **3.6. Rodenticidal activity of certain benzotriazole and coumarin derivatives [53]**

The previously explained benzotriazole and coumarin derivatives were studied also for their rodenticidal effects against the white Noway rat. In fact the two coumarin derivatives might be expected in their effects, while the benzotriazole derivatives were tested to stand on their toxicity related to studied coumarin comparing with Coumachlor**,** 3-(-acetonyl-4 chlorobenzyl)-4-hydroxy-coumarin as standard anticoagulant rodenticide.

  Mortality percents caused by synthesized

dicoumarol, furopyrone and 1-acetyl-5,6-dimethylbenzotriazole increased with increasing the dosages; their LD50 values were 64, 400 and 580 mg/kg body weight, respectively. So, both dicoumarol and furopyrone were moderately toxic, whereas 1-acetyl-5,6 dimethylbenzotriazole was slightly toxic [52]. The average times to death were ranged between 6.3 and 5.5 days. However, EP50 and EP98 (Effective periods of 50% and 98% mortalities) of 100 mg/kg were 4.5 and 11.5 days, respectively. These compounds exhibited good rodenticidal properties on three consecutive dosages in a week when compared with coumachlor with LD50 equal to 50-100 mg/kg if applied on five consecutive days [54].

#### *Biochemical effects*

Benzotriazole derivatives weakly affected the haemoglobin and haematocrit of both males and females within the tested doses (10-300 mg/kg) with ED50 of >300 mg/kg. While, dicoumarol and furopyrone were highly and moderately toxic against haemoglobin of male and female rats with ED50 values of 24 & 27 mg/kg and 90 & 130 mg/kg body weight, respectively. Furopyrone and dicoumarol were moderately active on haematocrit of males with ED50 values of 53 and 65 mg/kg but on females with 135 and 195 mg/kg respectively. Red blood cells (RBC's) of females were found to be more sensitive to coumachlor, dicoumarol, furopyrone, 5,6-dimethylbenzotriazole, benzotria-zole followed by 1-benzoyl-5,6-dimethylbenzotriazole as highly toxic compounds reducing RBC's of males with ED50 values of 7, 12, 19, 28, 40 and 44 mg/kg, respectively (Loomis, 1976). Coumachlor, dicoumarol, furopyrone and 5,6-dimethylbenzotriazole were also highly toxic against RBC's of females with ED50 value of 10.5, 25, 32 and 40 mg/kg, respectively. However the other compounds moderately reduced the RBC's counts of males and females. White blood cells (WBC's) of males were highly sensitive to 5,6- dichlorobenzotriazole, coumachlor and dicoumarol with ED50 values of 12, 13, and 37 mg/kg, whereas dicoumarol and 5,6 dichlorobenzotriazole were highly toxic in reducing it in females with ED50 values of 32 and 42 mg/kg, respectively. The other compounds proved to be moderately toxic in both males and females except benzotriazole, 1-acetylbenzotriazole and furopyrone. 5,6- Dichlorobenzotriazole was nearly equal to coumachlor in reducing males WBC's, whereas dicoumarol and 5,6-dichlorobenzotriazole were more effective than coumachlor against female WBC's.

Benzotriazole derivatives as well as furopyrone weakly affected sALT enzyme in both males and females. 5,6-Dimethylbenzotriazole was more potent reducing sAST enzyme activity in both males and females with ED50 values of 8.8 and 13.5 mg/kg, respectively. Dicoumarol was also highly toxic compound against sAST in males and females with ED50 of 24 and 32 mg/kg, respectively whereas coumachlor was highly toxic against females and moderately against males with 24 and 54 mg/kg ED50 values, respectively. 5,6-Dimethylbenzotriazole was more potent reducing sAST activity in both males and females with ED50 values of 8.8 and 13.5 mg/kg, respectively. Dicoumarol was also categorized as highly toxic against sAST in males and females with ED50 of 24 and 32 mg/kg, respectively whereas coumachlor was highly toxic against females and moderately against males with 24 and 54 mg/kg ED50 values, respectively. 1-Acetylbenzotriazole was moderate reducing sAST activity in males with ED50 equalled 160 mg/kg. The other derivatives and furopyrone weakly affected it in both sexes.

#### **3.7. Pesticidal activity of some benzylidine derivatives [4]**

Actually ,- unsaturated ketones were prepared according to Claisen Schmidt reaction (CSR) mechanism for searching their potency controlling some pests based on their biological history [55-56]. These biological activities of chalcone derivatives (as benzylidine derivatives) directed the attention to prepare some chaclone derivatives; dibenzylidineacetone, dibenzylidineacetylacetone and benzylidineacetophenone (chalcone).

#### *Phytocidal Effects*

86 Pesticides – Advances in Chemical and Botanical Pesticides

*Biochemical effects*

female WBC's.

dicoumarol, furopyrone and 1-acetyl-5,6-dimethylbenzotriazole increased with increasing the dosages; their LD50 values were 64, 400 and 580 mg/kg body weight, respectively. So, both dicoumarol and furopyrone were moderately toxic, whereas 1-acetyl-5,6 dimethylbenzotriazole was slightly toxic [52]. The average times to death were ranged between 6.3 and 5.5 days. However, EP50 and EP98 (Effective periods of 50% and 98% mortalities) of 100 mg/kg were 4.5 and 11.5 days, respectively. These compounds exhibited good rodenticidal properties on three consecutive dosages in a week when compared with

coumachlor with LD50 equal to 50-100 mg/kg if applied on five consecutive days [54].

Benzotriazole derivatives weakly affected the haemoglobin and haematocrit of both males and females within the tested doses (10-300 mg/kg) with ED50 of >300 mg/kg. While, dicoumarol and furopyrone were highly and moderately toxic against haemoglobin of male and female rats with ED50 values of 24 & 27 mg/kg and 90 & 130 mg/kg body weight, respectively. Furopyrone and dicoumarol were moderately active on haematocrit of males with ED50 values of 53 and 65 mg/kg but on females with 135 and 195 mg/kg respectively. Red blood cells (RBC's) of females were found to be more sensitive to coumachlor, dicoumarol, furopyrone, 5,6-dimethylbenzotriazole, benzotria-zole followed by 1-benzoyl-5,6-dimethylbenzotriazole as highly toxic compounds reducing RBC's of males with ED50 values of 7, 12, 19, 28, 40 and 44 mg/kg, respectively (Loomis, 1976). Coumachlor, dicoumarol, furopyrone and 5,6-dimethylbenzotriazole were also highly toxic against RBC's of females with ED50 value of 10.5, 25, 32 and 40 mg/kg, respectively. However the other compounds moderately reduced the RBC's counts of males and females. White blood cells (WBC's) of males were highly sensitive to 5,6- dichlorobenzotriazole, coumachlor and dicoumarol with ED50 values of 12, 13, and 37 mg/kg, whereas dicoumarol and 5,6 dichlorobenzotriazole were highly toxic in reducing it in females with ED50 values of 32 and 42 mg/kg, respectively. The other compounds proved to be moderately toxic in both males and females except benzotriazole, 1-acetylbenzotriazole and furopyrone. 5,6- Dichlorobenzotriazole was nearly equal to coumachlor in reducing males WBC's, whereas dicoumarol and 5,6-dichlorobenzotriazole were more effective than coumachlor against

Benzotriazole derivatives as well as furopyrone weakly affected sALT enzyme in both males and females. 5,6-Dimethylbenzotriazole was more potent reducing sAST enzyme activity in both males and females with ED50 values of 8.8 and 13.5 mg/kg, respectively. Dicoumarol was also highly toxic compound against sAST in males and females with ED50 of 24 and 32 mg/kg, respectively whereas coumachlor was highly toxic against females and moderately against males with 24 and 54 mg/kg ED50 values, respectively. 5,6-Dimethylbenzotriazole was more potent reducing sAST activity in both males and females with ED50 values of 8.8 and 13.5 mg/kg, respectively. Dicoumarol was also categorized as highly toxic against sAST in males and females with ED50 of 24 and 32 mg/kg, respectively whereas coumachlor was highly toxic against females and moderately against males with 24 and 54 mg/kg ED50 values, respectively. 1-Acetylbenzotriazole was moderate reducing sAST activity in males Benzylidineacetophenone (chalcone), dibenzylidineacetone and dibenzyli-dineacetylacetone were active against the root system of wheat seedlings (*Triticum aestivum*) with EC50 values equal 54, 55 and 68 µg/ml, respectively. On the other hand, their shoot systems were less sensitive, since benzylidineacetophenone (chalcone) was less effective, followed by dibenzylidineacetylacetone and dibenzylidine-acetone. It could be concluded that, the three prepared compounds proved to be highly toxic on root system whereas dibenzylidine acetone and dibenzylidineacetylacetone proved to be moderately toxic against the shoot system of wheat seedlings. On the other hand, root system of squash seedlings (*Cucarbita pepo*) was highly affected by dibenzylidineacetylacetone, benzylidineacetophenone and dibenz-ylidineacetone. These prepared compounds also affected the shoot system of squash seedlings. However the standard herbicide, Granstar was more effective against both wheat and squash seedlings than the prepared compounds. Although the obtained results revealed that the prepared compounds could be considered as a moderate phytotoxicants against wheat and squash seedlings but they were more specific on root system of wheat seedlings.

Chemical structures of the tested compounds and standard pesticides

**1**, dibenzylidineacetone (1,5-diphenylpenta-1,4-dien-3–one); **2**, benzylidine acetophenone (1,3-diphenyl propen-3–one) (Chalcone); **3**, dibenzylidine acetylacetone (1,7-diphenyl hepta-1,6-dien-3,5–dione); **4**, tribenuron methyl, (2-[4-methoxy-6-methyl-1,2,3-triazin-2-yl] methyl carbamoyl sulfamoyl benzoic acid) (Granstar); **5**, methomyl, S-methyl-N-(methyl carbamoyl -oxy) thioacetimidate

#### *Insecticidal and molluscicidal effects*

Comparing with methomyl (Lannate), Dibenzylidineacetone and lannate proved to be highly toxic against the cotton leaf worm (*S. littoralis*) with LC50 values < 10 µg/ml. Dibenzylidineacetylacetone and benzylidineacetophenone slightly affected it with LC50 values equalled 510 and > 2000 µg/ml, respectively. Dibenzylidineacetone weakly affected the tested snails, whereas benzylidineacetophenone was very weak against *E. Vermiculata* but it was not mortal on *T. pisana*. Dibenzylidineacetylacetone showed no lethal effects against the two terrestrial snails.

Generally, the prepared compounds caused moderately phytotoxic effects on both wheat and squash seedlings but they were specific on root system of wheat seedlings. Dibenzylidineacetone caused nearly the same effects as methomyl against cotton leaf worm. So, it could be concluded that dibenzylidineacetone after different biological tests may be safe as an insecticide against cotton leaf worm as it was previously prepared as a sun protection cream [57].

#### **3.8. Evaluation of certain benzylidine and pyrazole derivatives against wood decay fungi [58]**

Wood decay fungi are destructive agents of wood industry. They degraded the used fungicides [59,60]. Due to their importance and the activities of benzylidine and pyrazole derivatives, their toxic effects were evaluated on the white rot fungus *Coriolus versicolor* and the brown rot fungus *Gloeophyllum trabeum*.

*In Vitro* fungitoxic effects were dependent on their concentrations, chemical structures and the treated fungus. 1,5-Diphenylpenta-1,4-dien-3–one (compound **1)** exhibited its fungitoxicity with IC50 of 295.4 and 976.9 g/ml against *C. versicolor* and *G. trabeum*, respectively. While, the toxicity was diminished because of the CH2CO- moiety in 1,7 diphenylhepta-1,6-dien-3,5-dione (compound **2**) with IC50 of 317.1 and 1995.4 g/ml in case of the two studied fungi. Fungitoxicity was more than three nine times against *C. versicolor* and *G. trabeum*, respectively without -CH=CH- moiety in 1,3-diphenylpropen-3-one (Chalcone) **(**compound **3)**. 3,5-Dimethylpyrazole **(c**ompound **4)** was less effective against the tested fungi with IC50 values of 867.7 and 944.8 g/ml against *C. versicolor* and *G. trabeum*, respectively. Substitution with 1-phenyl moiety changing to pyrazol-5-one ring in 3-methyl-1-phenylpyrazol-5-one **(**compound **5)** increased the effects with IC50 values of 744.2 and 632.4 g/ml against the treated fungi.. Higher enhancement was achieved by replacing the substituted 1-phenyl ring with 2,4-dinitrophenyl moiety in 3-Methyl-1-(2,4-dinitro-phenyl) pyrazol-5-one **(**compound **6)**, the most active with IC50 of 19.6 and 112.7 g/ml against the white and brown rot fungi. In general, significantly *C. versicolor* appeared more sensitive than *G. trabeum* with general mean ± SE of mycelium growth inhibition percents of 39.18 ± 3.12 and 32.7 ± 2.58, respectively. Additionally, compound **6** was the most effective followed by compound **3**, exceeding boric acid as a standard compound with mean mycelium growth inhibition percents of 61.0, 46.9 and 40.9%, respectively. While, the other tested compounds were less effective than the standard (Table **2**).

Results in the same column with the same superscript are not significantly different (p < 0.05), DF = 4

**Table 2.** Fungicidal effects of certain benzylidine and pyrazole compounds on *Coriolus versicolor* and *Gloeophyllum trabeum* fungi

#### *In vivo antifungal activity*

88 Pesticides – Advances in Chemical and Botanical Pesticides

Comparing with methomyl (Lannate), Dibenzylidineacetone and lannate proved to be highly toxic against the cotton leaf worm (*S. littoralis*) with LC50 values < 10 µg/ml. Dibenzylidineacetylacetone and benzylidineacetophenone slightly affected it with LC50 values equalled 510 and > 2000 µg/ml, respectively. Dibenzylidineacetone weakly affected the tested snails, whereas benzylidineacetophenone was very weak against *E. Vermiculata* but it was not mortal on *T. pisana*. Dibenzylidineacetylacetone showed no lethal effects

Generally, the prepared compounds caused moderately phytotoxic effects on both wheat and squash seedlings but they were specific on root system of wheat seedlings. Dibenzylidineacetone caused nearly the same effects as methomyl against cotton leaf worm. So, it could be concluded that dibenzylidineacetone after different biological tests may be safe as an insecticide against cotton leaf worm as it was previously prepared as a sun

**3.8. Evaluation of certain benzylidine and pyrazole derivatives against wood**

Wood decay fungi are destructive agents of wood industry. They degraded the used fungicides [59,60]. Due to their importance and the activities of benzylidine and pyrazole derivatives, their toxic effects were evaluated on the white rot fungus *Coriolus versicolor* and

*In Vitro* fungitoxic effects were dependent on their concentrations, chemical structures and the treated fungus. 1,5-Diphenylpenta-1,4-dien-3–one (compound **1)** exhibited its fungitoxicity with IC50 of 295.4 and 976.9 g/ml against *C. versicolor* and *G. trabeum*, respectively. While, the toxicity was diminished because of the CH2CO- moiety in 1,7 diphenylhepta-1,6-dien-3,5-dione (compound **2**) with IC50 of 317.1 and 1995.4 g/ml in case of the two studied fungi. Fungitoxicity was more than three nine times against *C. versicolor* and *G. trabeum*, respectively without -CH=CH- moiety in 1,3-diphenylpropen-3-one (Chalcone) **(**compound **3)**. 3,5-Dimethylpyrazole **(c**ompound **4)** was less effective against the tested fungi with IC50 values of 867.7 and 944.8 g/ml against *C. versicolor* and *G. trabeum*, respectively. Substitution with 1-phenyl moiety changing to pyrazol-5-one ring in 3-methyl-1-phenylpyrazol-5-one **(**compound **5)** increased the effects with IC50 values of 744.2 and 632.4 g/ml against the treated fungi.. Higher enhancement was achieved by replacing the substituted 1-phenyl ring with 2,4-dinitrophenyl moiety in 3-Methyl-1-(2,4-dinitro-phenyl) pyrazol-5-one **(**compound **6)**, the most active with IC50 of 19.6 and 112.7 g/ml against the white and brown rot fungi. In general, significantly *C. versicolor* appeared more sensitive than *G. trabeum* with general mean ± SE of mycelium growth inhibition percents of 39.18 ± 3.12 and 32.7 ± 2.58, respectively. Additionally, compound **6** was the most effective followed by compound **3**, exceeding boric acid as a standard compound with mean mycelium growth inhibition percents of 61.0, 46.9 and 40.9%, respectively. While, the other tested compounds

*Insecticidal and molluscicidal effects*

against the two terrestrial snails.

the brown rot fungus *Gloeophyllum trabeum*.

were less effective than the standard (Table **2**).

protection cream [57].

**decay fungi [58]**

After six weeks exposure to fungal attack, the average mass loss in control was 41.27 and 41.53% for poplar (*Populus nigra*) and Scots pine sapwood (*Pinus sylvestris*), respectively. Regarding poplar, compounds **3** and **6** reduced the mass loss to 30.43% and 29.23% (75% and 71% of control) at the lowest concentration. This effect was significantly increased reaching 23.87% (57.7% of control) and 13.67 % (33.1% of control) mass losses in systematic arrangement in un-leached samples in case of compound **3** and **6**, respectively at the highest concentration (10 IC50 value). Leaching reduced antifungal effects of the two compounds to 28.10 % and 28.63% mass loss at the highest concentration with a narrow range of difference with their lowest concentration (0.5 IC50) as the mass loss was 33.03% and 31.13% in compound **3** and **6**, respectively. The tested compounds protected the Scots pine sapwood samples in the same manner as compound **3** reduced its mass loss to (30.83% - 24.80%) while compound **6** reduced its mass loss to (30.0% - 14.47%) at concentration used in comparison to 41.53% of control samples. Leaching of the used blocks decreased the effect to (34.87% – 29.0%) and (33.30% – 27.5%), respectively (Table **3**).

Results in the same column with the same superscript are not significantly different (p < 0.05).

**Table 3.** Average of retention (kg/m3) and mass losses (%) of Poplar (*P. nigra)* and Scots pine sapwood (*P. sylvestris*) mini-blocks treated with compounds 3 and 6 and exposed to *C. versicolor* and *G. trabeum*, respectively.

The effect of compound **6 was reduced by leaching** samples more than compound **3**  ensuring that the former is easily leached due to its hygroscopic nature. Descriptive analysis proved compound **6** more significantly effective with general mean of mass loss ± SE of 25.12% ± 2.58 in comparison to 29.98% ± 1.63 of compound **3** in case of un-leached poplar samples, while no significant differences between them in leached samples were observed. In Scots pine sapwood, significance appeared in both cases as compound **6** achieved general mean of mass loss ± SE of 26.49% ± 2.44 and 32.59% ± 1.31 comparing with 30.33 % ± 1.61 and 33.76 % ± 1.16 of compound **3** in un-leached and leached samples. Differences between the benzylidine derivatives in their fungicidal activity could be referred to the conjugation among carbonyl groups, phenyl rings and double bonds, so compound **2** was less effective due to lack of this conjugation because of CH2 moiety. Compound **3** was more effective than compound **1** may be due to the lipophilicity [61]. The effect of benzylidine derivatives (benzaldhyde derived compounds) was greatly inhibited against *G. trabeum* than *C. versicolor*, which may be due to degradation as benzaldehyde and its metabolic intermediates were effectively degraded by *G. trabeum* to 3,4-dihydroxybenzoic acid. This was further metabolized via the decarboxylation reaction to yield 1,2,4-trihydroxybenzene, which is susceptible to the ring-fission reaction [62]. Compound **3** was retained approximately in the same amount in both wood specimens. The retained amount of compound **6** in *P. sylvestris* was four times more than in *P. nigra*. On the other hand, compound **6** was retained in about one fifth of compound **3** in *P. nigra*, although it was more effective. So it could be concluded that compound **6** was found to be more effective than compound **3** in all cases and it was more toxic against *C. versicolor* than on *G. trabeum*. Moreover, these compounds need to be applied at higher concentrations to enter wood preservatives clique.

#### **3.9.  Average of retention (kg/m3) and mass losses (%) of Poplar (*P. nigra)* and Scots pine sapwood (*P. sylvestris*) mini-blocks treated with compounds 3 and 6 and exposed to *C. versicolor* and *G. trabeum*,

The effect of compound **6 was reduced by leaching** samples more than compound **3**  ensuring that the former is easily leached due to its hygroscopic nature. Descriptive analysis proved compound **6** more significantly effective with general mean of mass loss ± SE of 25.12% ± 2.58 in comparison to 29.98% ± 1.63 of compound **3** in case of un-leached poplar samples, while no significant differences between them in leached samples were observed. In Scots pine sapwood, significance appeared in both cases as compound **6** achieved general mean of mass loss ± SE of 26.49% ± 2.44 and 32.59% ± 1.31 comparing with 30.33 % ± 1.61 and 33.76 % ± 1.16 of compound **3** in un-leached and leached samples. Differences between the benzylidine derivatives in their fungicidal activity could be referred to the conjugation among carbonyl groups, phenyl rings and double bonds, so compound **2** was less effective due to lack of this conjugation because of CH2 moiety. Compound **3** was more effective than compound **1** may be due to the lipophilicity [61]. The effect of benzylidine derivatives (benzaldhyde derived compounds) was greatly inhibited against *G. trabeum* than *C. versicolor*, which may be due to degradation as benzaldehyde and its metabolic intermediates were effectively degraded by *G. trabeum* to 3,4-dihydroxybenzoic acid. This was further metabolized via the decarboxylation reaction to yield 1,2,4-trihydroxybenzene, which is susceptible to the ring-fission reaction [62]. Compound **3** was retained approximately in the same amount in both wood specimens. The retained amount of compound **6** in *P. sylvestris* was four times more than in *P. nigra*. On the other hand, compound **6** was retained in about one fifth of compound **3** in *P. nigra*, although it was more effective. So it could be concluded that compound **6** was found to be more effective than compound **3** in all cases and it was more toxic against *C. versicolor* than on *G. trabeum*. Moreover, these compounds need to be applied at higher concentrations to enter wood

Results in the same column with the same superscript are not significantly different (p < 0.05).

Conc

(IC50 values)

Treatment

1,3-Diphenylpropen-3-one Chalcone) (3)

3-Methyl-1-(2,4 dinitro-phenyl) -pyr-azol-5-one (6)

respectively.

preservatives clique.

Phytocidal effects of five-memberred heterocyclic derivatives were studied on monocotyledonous (*Triticum aestivum* L.) and dicotyledonous (*Cucurbita pepo*) plants. Some activities of nitrogen heterocycles as herbicides [64-65] helped growing this idea.

In seed treatment, wheat seedlings growth was more sensitive to the tested compounds than seed germination. Pyrazole derivatives were less effective than both indole and benzotriazole derivatives against seed germination, while their effects against vegetation depended on the structure. 5,6-Dichlorolbenzotriazole was more potent than the standard herbicide, metribuzin against both seed germination and growth of seedlings. 1- Acetylindole-3-butyric acid caused nearly the same effect of metribuzin on seed germination, whereas its effect on seedling growth was less than it. However, the other benzotriazole, indole and pyrazole derivatives were less effective than it against both seed germination and seedling growth. Screening effects of the tested compounds on root and shoot systems of squash (*C. peppo*) and wheat (*T. aestivum*) proved 5,6-dichlorolbenzoriazole the most effective inhibiting squash root and shoot systems with EC50 values equaled 8.6 and 16.8 g/ml exceeding the standard herbicides with 86.2 and 97.2 g/ml, respectively. 1- Acetyl-5,6-dimethylbenzotriazole and 3-methyl-1-(2,4-dinitrophenyl)pyrazol-5-one were also more potent than metribuzin, with EC50 values equaled (26.2 and 47.2) and (72.2 and 77.1) g/ml against root and shoot systems of squash seedlings. 5,6-Dimethylbenzotriazole, 1-benzoylbenzotriazole and 1-acetylindole-3-butyric acid were more effective than it against squash root system with 63.1, 71.2, and 80.1 g/ml EC50 values. Indole-3-butyric acid inhibited squash shoot system with EC50 value equaled 63.7 g/ml. The other tested benzotriazoles, indole and pyrazole derivatives were less effective than the standard herbicide. Comparing with 55.8 and 68.2 g/ml EC50 values of the standard herbicide against root and shoot systems of wheat, 5,6-dichlorolbenzotriazole, indole-3-butyric acid, indole-3 acetic acid, 1-acetylindole-3-butyric acid, benzotriazole, 1-benzoylindole-3-acetic acid and 3 methyl-1(2,4-dinitrophenyl)pyrazol-5-one were more potent with 16.9, 6.0, 3.3, 2.86, 1.82, 1.8 and 1.45 times against root system of wheat. While 5,6-dichlorolbenzotriazole was 5.3 times more effective than metribuzin on wheat seedlings shoot growth, the other benzotriazoles, indole and pyrazole derivatives were less effective than it. All the tested derivatives were more effective inhibiting growth of root system than shoot growth in squash (*C. peppo*) seedlings except indole-3-acetic acid with EC50 values equaled 311.4 and 201.4 g/ml and 3 methyl-1-(2,4-dinitrophenyl) pyrazol-5-one with EC50 values equaled 47.2 and 77.1 g/ml. The same trend was obtained in case of wheat seedlings by benzotriazole and indole derivatives except 1-benzoylindole-3-butyric acid with EC50 values equaled 611 and 544 g/ml and 1-benzoyl-2-phenylindole with EC50 values equaled 453 and 503 g/ml, respectively. However pyrazole derivatives proved to be more effective against root than shoot depending inhibition degree on the chemical structure differences among the applied derivatives except 3,5-dimethylpyrazole with EC50 values equaled 208 and 172 g/ml, respectively. Due to high effects of both 5,6-dichlorobenzotriazole and 3-methyl-1-(2,4 dinitrophenyl)pyrazol-5-one, they were applied in post emergence treatment to study their effects against some plant active sites of action. Pre emergence treatment with 5,6-

dichlorobenzotriazole inhibited both fresh and dry weights of wheat seedlings. Fresh weight of the emerged wheat seedlings was reduced with 45.1 - 94.7% at a concentration of 2 - 30 g/ml with EC50 values equaled 2.9 g/ml, while their dry weight was reduced increasingly with increasing the concentration with 30.6 - 94.2% reduction with EC50 value equaled 3.6 g/ml. At 50 g/ml, it completely prevented seeds emergence. Post-emergence treatment of wheat seedlings with 5,6-dichlorobenzotriazole and 3-methyl-1-(2,4-dinitrophenyl)pyrazol-5-one affected their dry weight increase depending on concentration and time after treatment. Both two compounds reduced this increasing rate in comparison to control at all times. The highest effect was obtained during the first three days after treatment at all concentrations. 3-Methyl-1-(2,4-dinitrophenyl)pyrazol-5-one highly affected it during seven days after treatment. It was more potent than 5,6-dichlorobenzotriazole nearly at all the tested concentrations (Figure **6**).

**Figure 6.** Effect of post emergence treatment on wheat seedlings dry weight. **Left**, 5,6-dichlorobenzotriazole; **Right**, 3-methyl-1-(2,4-dinitrophenyl)pyrazol-5-one

#### *Concentrations in µg/ml*

Single post-emergence treatment with both 5,6-dichlorobenzotriazole and 3-methyl-1-(2,4 dinitro-phenyl)pyrazol-5-one affected the total soluble sugars contents in a function of concentration and time after treatment (Figure **7**). Both reduced and non-reduced sugars alternatively changed regarding the time after treatment. 100 *µ*g/ml was the most effective concentration reducing TSS increasingly with time after treatment. Low activity at 200 *µ*g/ml might be referred to its difficult penetration. However 3-methyl-1-(2,4-dinitrophenyl)pyrazol-5-one showed its maximum activity after 7 days at 100 *µ*g/ml. The highest effect of 5,6-dichlorobenzotriazole on chlorophyll was after 3 days. At 15 days after treatment, chlorophyll contents were enhanced at all concentrations. Chlorophyll **a** was more sensitive than **b** Vice versa, 3-methyl-1-(2,4-dinitrophenyl)pyr-azol-5-one affected chlorophyll **a** less than **b.** Enhancement was noticed at low concentrations at all the tested periods. The most reducing concentration was 100 µg/ml. Treatment with 5,6-dichlorobenzotriazole reduced the soluble phenolics content mostly until 3 days after treatment at all concentrations systematically with increasing the tested concentration. This effect was fluctuated according to the applied concentration at 7 days after treatment. The most effective concentration was 100 µg/ml. At 15 days after treatment, it was too long to keep its effectiveness in reducing their content. While 3-methyl-1-(2,4-dinitrophenyl) pyrazol-5-one caused reduction of their content up to 15 days after treatment. Effects on chlorophyll content disturb several physiological processes in plants. The effect on soluble phenolics interferes in the protective compounds [66]. Fluctuated results of chlorophyll and soluble phenolics may be due to the interactive effects of temperature and the accumulated soluble phenolics [67]. 5,6 dichlorobenzotriazole may inhibit cell division and protoporphyrinogen oxidase leading to membrane disruption and inhibiting photosynthesis [64]. Benzotriazoles are effective in blocking photosynthetic electron transfer [68], slowing down the growth and decreasing plant size emphasizing our results on fresh and dry weight [69]. They may affect through inhibition of protein kinases [70]. Indole derivatives effects varied based on structure and concentrations inducing growth abnormalities leading to desiccation, tissue necrosis, and decay. They also increased H2O2 levels, which contributes to the induction of cell death, deoxyribonuclease (DNase) activity and chlorophyll loss as sensitive indicators for tissue damage [65]. Pyrazole derivatives are considered as branched chain amino acid synthesis (ALS or AHAS) inhibitor stopping cell division and plant growth.

**Figure 7.** Effect of post emergence treatment on wheat seedlings sugars. **Left**, 5,6-dichlorobenzotriazole; **Right**, 3-methyl-1-(2,4-dinitro-phenyl) pyrazol-5-one; Concentrations in *µ*g/ml

#### **4. Conclusion**

92 Pesticides – Advances in Chemical and Botanical Pesticides

tested concentrations (Figure **6**).

*Concentrations in µg/ml*

**-20**

**0**

**In creasin g % of d ry w eigh t from**

**20**

**40**

**zero tim e** **60**

**80**

**100**

dichlorobenzotriazole inhibited both fresh and dry weights of wheat seedlings. Fresh weight of the emerged wheat seedlings was reduced with 45.1 - 94.7% at a concentration of 2 - 30 g/ml with EC50 values equaled 2.9 g/ml, while their dry weight was reduced increasingly with increasing the concentration with 30.6 - 94.2% reduction with EC50 value equaled 3.6 g/ml. At 50 g/ml, it completely prevented seeds emergence. Post-emergence treatment of wheat seedlings with 5,6-dichlorobenzotriazole and 3-methyl-1-(2,4-dinitrophenyl)pyrazol-5-one affected their dry weight increase depending on concentration and time after treatment. Both two compounds reduced this increasing rate in comparison to control at all times. The highest effect was obtained during the first three days after treatment at all concentrations. 3-Methyl-1-(2,4-dinitrophenyl)pyrazol-5-one highly affected it during seven days after treatment. It was more potent than 5,6-dichlorobenzotriazole nearly at all the

**In cr e a sin g % o f d ry w e ig h t fro m**

**-20**

**0**

**0 3 7 15 25**

0

10

50

100

200

**Time after treatment (Days)**

**20**

**40**

**zero tim e**

**60**

**80**

**100**

Single post-emergence treatment with both 5,6-dichlorobenzotriazole and 3-methyl-1-(2,4 dinitro-phenyl)pyrazol-5-one affected the total soluble sugars contents in a function of concentration and time after treatment (Figure **7**). Both reduced and non-reduced sugars alternatively changed regarding the time after treatment. 100 *µ*g/ml was the most effective concentration reducing TSS increasingly with time after treatment. Low activity at 200 *µ*g/ml might be referred to its difficult penetration. However 3-methyl-1-(2,4-dinitrophenyl)pyrazol-5-one showed its maximum activity after 7 days at 100 *µ*g/ml. The highest effect of 5,6-dichlorobenzotriazole on chlorophyll was after 3 days. At 15 days after treatment, chlorophyll contents were enhanced at all concentrations. Chlorophyll **a** was more sensitive than **b** Vice versa, 3-methyl-1-(2,4-dinitrophenyl)pyr-azol-5-one affected chlorophyll **a** less than **b.** Enhancement was noticed at low concentrations at all the tested periods. The most reducing concentration was 100 µg/ml. Treatment with 5,6-dichlorobenzotriazole reduced the soluble phenolics content mostly until 3 days after treatment at all concentrations systematically with increasing the tested concentration. This effect was fluctuated

**Figure 6.** Effect of post emergence treatment on wheat seedlings dry weight. **Left**, 5,6-dichlorobenzotriazole; **Right**, 3-methyl-1-(2,4-dinitrophenyl)pyrazol-5-one

**0 3 7 15 25**

**Time after treatment (Days)**

Several prepared organic compounds are tested for their pesticidal actions. Indole derivatives inhibited hyphal growth of several plant pathogenic fungi based on treated fungus and structure affecting sugars, RNA and DNA contents as well as enzymes disturbing cell physiology. They caused lethality, larval weight reduction, inhibition of pupation and adult emergence with inhibiting egg hatchability of *S. littoralis* Boisd. Tested pyrazole, imidazole and oxazole derivatives exhibited weak lethality with inhibition of insect palatability and moderate to high fungitoxic and phytotoxic effects according to structure, fungus and plant seedlings. Imidazolidine and oxazolone derivatives were antifeedants more than killers against *S. littoralis* and 5,5 diphenylimidazol-idin-2,4-dione was the most effective structure. Their phytocidal and fungicidal activities were higher than insecticidal effects and 5,5-diphenylimidazolidin-2-thione-4 one was the most useful structure. Benzotriazoles changed in their fungicidal effects and 5,6 dichlorobenzotriazole was highly to moderately toxic against the treated fungi affecting both polyphenoloxidase, peroxidase and DNA & RNA contents. They caused lower effects on haemoglobin and haematocrit of rats, whereas dicoumarol and furopyrone highly reduced them. However dimethyl- and dichloro- substituent increased the activity of non-substituted benzotriazole on RBC's, WBC's and sAST, aceylation of 5,6-dimethylbenzotriazole decreased its effect on both male and female RBC's, sAST. Benzylidine derivatives caused moderately phytotoxic effects. Dibenzylidineacetone caused nearly the same effect as methomyl against cotton leaf worm. They differed in their mortality on *E. vermiculata* and *T. pisana* snails. Significantly *C. versicolor* was more sensitive than *G. trabeum* to benzylidine and pyrazole derivatives. 3- Methyl-1-(2,4-dinitro-phenyl)-pyrazol-5-one was the most effective followed by 1,3 diphenylpropen-3-one, exceeding boric acid, as a standard in case of un-leached poplar samples, while no differences were observed between them in leached samples. In Scots pine sapwood, significance appeared in both samples. 1,3-Diphenylpropen-3-one was approximately retained in the same amount in both wood specimens. Although 3-methyl-1-(2,4-dinitrophenyl)-pyrazol-5-one was retained in one fifth of 1,3-diphenylpropen-3-one in *P. nigra*, it was more effective. Benzotriazole, indole and pyrazole derivatives inhibited wheat seedlings growth more than seed germination process. Pyrazoles were less than others inhibiting seed germination, effects on vegetation depended on structure. Some of derivatives exceeded the standard herbicides in their effects. 5,6-dichlorolbenzoriazole was the most effective inhibiting monocotyledons and dicotyledonous seedlings growth. Its pre emergence treatment inhibited wheat seedlings fresh and dry weights. They also affected the total soluble sugars, chlorophyll and soluble phenolic contents in plants. The configuration of 5,6-dichlorolbenzotriazole and 5,6-dichloro- substituent may be required to get good results. These results may exhibit 5,6 dichlorobenzotriazole as pre-emergent phytocidal compound, while 3-methyl-1-(2,4-dinitrophenyl)pyrazol-5-one as post-emergent. These results proveed 5,6-dichlorobenzotriazole to inhibit the chlorophyll content, cell division leading to membrane disruption, inhibiting photosynthesis, growth abnormalities leading to desiccation, tissue necrosis and decay and decrease the plant size emphasizing our obtained results on fresh and dry weight. In conclusion, this research might help finding active molecules are not famous as pesticides to be useful in integrated management programs.

#### **Author details**

Ahmed S. Abdel-Aty *Department of Pesticide Chemistry &Technology, Faculty of Agriculture, El-Shatby, Alexandria University, Alexandria, Egypt*

#### **5. References**

[4] Abdel-Aty A S (2004) Pesticidal effects of some benzylidine derivatives. Alex. J. of Agricultural Res. 49 (1): 99- 105.

94 Pesticides – Advances in Chemical and Botanical Pesticides

integrated management programs.

*Alexandria University, Alexandria, Egypt*

derivatives. J. Am. Chem. Soc. 74: 4719.

Thames Polytechnic London, S.E. 16 6PF 886p.

*Department of Pesticide Chemistry &Technology, Faculty of Agriculture, El-Shatby,*

[1] Benson F R, Hartizel LW, Savell WL(1952) 5,6-Dimethyl benzotriazole and its acyl

[2] Vogel A I (1976) A text book of practical organic chemistry 4th edition. BNNO 582443946

[3] Abdel-Aty A S (1996) Rodenticidal activity of certain organic molecules "Chemistry and rodenticidal activity of some benzotriazole and coumarin derivatives". MSc, Pesticide

Chem. Department, Faculty of Agriculture, Alexandria University, Egypt.

**Author details**

**5. References**

Ahmed S. Abdel-Aty

moglobin and haematocrit of rats, whereas dicoumarol and furopyrone highly reduced them. However dimethyl- and dichloro- substituent increased the activity of non-substituted benzotriazole on RBC's, WBC's and sAST, aceylation of 5,6-dimethylbenzotriazole decreased its effect on both male and female RBC's, sAST. Benzylidine derivatives caused moderately phytotoxic effects. Dibenzylidineacetone caused nearly the same effect as methomyl against cotton leaf worm. They differed in their mortality on *E. vermiculata* and *T. pisana* snails. Significantly *C. versicolor* was more sensitive than *G. trabeum* to benzylidine and pyrazole derivatives. 3- Methyl-1-(2,4-dinitro-phenyl)-pyrazol-5-one was the most effective followed by 1,3 diphenylpropen-3-one, exceeding boric acid, as a standard in case of un-leached poplar samples, while no differences were observed between them in leached samples. In Scots pine sapwood, significance appeared in both samples. 1,3-Diphenylpropen-3-one was approximately retained in the same amount in both wood specimens. Although 3-methyl-1-(2,4-dinitrophenyl)-pyrazol-5-one was retained in one fifth of 1,3-diphenylpropen-3-one in *P. nigra*, it was more effective. Benzotriazole, indole and pyrazole derivatives inhibited wheat seedlings growth more than seed germination process. Pyrazoles were less than others inhibiting seed germination, effects on vegetation depended on structure. Some of derivatives exceeded the standard herbicides in their effects. 5,6-dichlorolbenzoriazole was the most effective inhibiting monocotyledons and dicotyledonous seedlings growth. Its pre emergence treatment inhibited wheat seedlings fresh and dry weights. They also affected the total soluble sugars, chlorophyll and soluble phenolic contents in plants. The configuration of 5,6-dichlorolbenzotriazole and 5,6-dichloro- substituent may be required to get good results. These results may exhibit 5,6 dichlorobenzotriazole as pre-emergent phytocidal compound, while 3-methyl-1-(2,4-dinitrophenyl)pyrazol-5-one as post-emergent. These results proveed 5,6-dichlorobenzotriazole to inhibit the chlorophyll content, cell division leading to membrane disruption, inhibiting photosynthesis, growth abnormalities leading to desiccation, tissue necrosis and decay and decrease the plant size emphasizing our obtained results on fresh and dry weight. In conclusion, this research might help finding active molecules are not famous as pesticides to be useful in

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[42] Olgen S, Kiliç Z, Ada A O, Coban T (2007) Synthesis and evaluation of novel N-H and N-substituted indole-2- and 3-carboxamide derivatives as antioxidants agents. J Enzyme Inhib Med Chem. 22 (4): 457–462.

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(3): 459-64.

129–136.

158: 373–377.

1725.

Biotechnol. 31 (11): 517-24.

*phaseolina*. J Environ Biol. 28 (1): 15-20.

*nidulans*. *Carcinogenesis* 4 (11): 1409-11.

*Arabidopis thaliana.* Plant J. 50: 886–901.

[25] Wintrobe MM (1965) Clinical hematology, 4th ed. Lea & Febiger philadelphia.

[27] Reitman S, Frankel S (1957) Acoduimetric method for the determination of serum glutamic oxaloacetate and glutamic pyruvate trans aminase. Am. J. Clin Path. 28:56. [28] Yu T, Zheng X D (2007) Indole-3-acetic acid enhances the biocontrol of *Penicillium expansum* and *Botrytis cinerea* on pear fruit by *Cryptococcus laurentii*.  J Ind Microbiol

[30] Wang H X, Ng T B (2002) Demonstration of antifungal and anti-human immunodeficiency virus reverse transcriptase activities of 6-methoxy-2 benzoxazolinone and antibacterial activity of the pineal indole 5-methoxyindole-3-acetic

[31] Ryu C K, Lee J Y, Park R E, Ma M Y, Nho J H (2007) Synthesis and antifungal activity of

[32] Sharaf E F, Farrag A A (2004) Induced resistance in tomato plants by IAA against

[33] Kumar V, Kumar A, Kharwar R N (2007) Antagonistic potential of fluorescent pseudomonads and control of charcoal rot of chickpea caused by *Macrophomina*

[34] Folkes L K, wardman P (2001) Oxidative activation of indole-3-acetic acid to cytotoxic species a potential new role for plant auxins in cancer therapy. Biochem. Pharmacol. 61:

[35] Kappas A (1983) Genotoxic activity of plant growth-regulating hormones in *Aspergillus*

[36] Ali S E, Abdel-Aty A S (2010) Insecticidal activity of some indole derivatives. against

[37] Ben Jannet H, Harzallah-Skhiri F, Mighri Z, Simmonds M S, Blaney W M (2000) Responses of *Spodoptera littoralis* larvae to Tunisian plant extracts and to neo-clerodane

[39] Gigolashvili T, Berger B, Mock H P, Muller C, Weisshaar B, Flugge U I (2007) The transcription factor HIG1/MYB51 regulates indlic glucosinolate biosynthesis in

[40] Bodur E, Cokugras A N (2005) The effects of indole-3-acetic acid on human and horse serum butyryl cholinesterase. Extended Abstracts/Chemico-Biological Interactions 157-

[41] De Melo M P, Pithon-Curi T C, Curi R (2004) Indole-3-acetic acid increases glutamine utilization by high peroxidase activity-presenting leukocytes. Life Sci. 75 (14): 1713–

diterpenoids isolated from *Ajuga pseudoiva* leaves. Fitoterapia 71 (2): 105–112. [38] Glazebrook J (2005) Contrasting mechanisms of defense against biotrophic and

acid. Comp Biochem. Physiol. C Toxicol. Pharmacol. 132 (2): 261-268.

1H-indole-4,7-diones. Bioorg. Med. Chem. Lett*.* 17 (1): 127-131.

*Fusarium oxysporum lycopersici*. Pol. J. Microbiol. 53 (2): 111-116.

*Spodoptera littoralis* (Boisd.). Alex. J. Agric. Res. 55 (1): 1 - 11.

necrotrophic pathogens. Annu. Rev. Phytopathol. 43: 205–227.

[26] Dacie J V, Lewis S M (1991) Practical hematology, 7th ed. 624p.

## **Endocrine Disrupting Pesticides**

Svetlana Hrouzková and Eva Matisová

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/46226

### **1. Introduction**

98 Pesticides – Advances in Chemical and Botanical Pesticides

16 (2): 111-125.

395.

96.

[58] Abdel-Aty A S, Mohareb A S O (2008) Preliminary evaluation of certain benzylidine and pyrazole derivatives against wood decay fungi. Journal of Pest Cont. and Environ. Sci.

[59] Kramer C, Kreisel G, Fahr K, Kässbohrer J, Schlosser D (2004) Degradation of 2 fluorophenol by the brown-rot fungus *Gloeophyllum striatum*: evidence for the involvement of extracellular Fenton chemistry. Appl Microbiol Biotechnol. 64 (3): 387-

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Food and environmental samples represent nowadays an enormous challenge to analytical chemists in their efforts to determine residues of pesticides at trace levels, as pesticides can represent a risk for consumer and also safeguard the biodiversity in the environment. The concern has increased as certain pesticides and other synthetic chemicals may act as pseudo hormones which disrupt the normal function of the endocrine system in humans and wildlife (Colborn et al., 1993; Lintelmann et al., 2003). This specific category of pollutants comprises the compounds that may affect the normal hormonal function or possess endocrine-related functions, known as endocrine disrupting chemicals (EDCs) or endocrine disrupters. During the last decades the interest and concern related to endocrine disrupters among scientists, regulators and public has increased. In the last years a great deal of concern has been expressed worldwide over the increasing levels of EDCs found in the environment. This anxiety is caused by the adverse effects of these pollutants on the hormone systems of humans and wildlife even when present at levels under ppb (Jobling, 2004).

Known and potential EDCs in food and the environment originate from many different sources. Endocrine disrupting pesticides (EDPs) are the largest group of EDCs in numbers compared to other chemical groups. They are active at low concentrations in food daily consuming by adult population and in agricultural commodities consumed in large quantities especially by infants and children. Organisms under development are very sensitive to negative effects of EDPs. Understanding in which and how much biologically active compounds are in the environmental samples or products of human consumption is important not just to scientists and environmentalists, but also to governments, pediatricians, genetics, and the general public (LaFleur & Schug, 2011).

This contribution is devoted to pesticides that exhibit or are supposed to exhibit endocrine disrupting properties. First, the terms, definition and current state of EDCs list creation are discussed. Then the selected EDPs and their categories are presented. Next the common

© 2012 Hrouzková and Matisová, licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 Hrouzková and Matisová, licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

analytical methods including sample preparation for the identification and quantification of EDPs by chromatographic analytical methods at ultratrace concentration level are briefly covered. The combinations of fast and effective sample preparation methods with conventional and fast capillary gas chromatography (GC) are presented. Selective mass spectrometric (MS) detection with negative chemical ionization (NCI) is discussed and compared to electron ionization (EI). The results leading to selectivity enhancement and decrease of the limits of quantification of selected EDPs using mass spectrometer operated in NCI mode are shown. Real-life analysis demonstrates the potential of studied sample preparation followed by fast gas chromatography.

#### **2. Terms, definitions and background**

The endocrine system is a complex integrative network of glands, hormones and receptors. It provides the key communication and control link between the nervous system and bodily functions such as reproduction, immunity, metabolism and behaviour. The endocrine system uses hormones to act as messengers that regulate reproduction, metabolism, growth, development, natural defences to stress, as well as water, electrolyte, and nutritional balance of the blood. Homeostasis is the balance of functions or levels in the body, returning biological variables to their biochemical baseline when perturbed and keeping them there. Maintaining homeostasis is one of the most important functions of the endocrine system. Therefore, the endocrine system includes number of central nervous system-pituitary-target organ feedback mechanisms that enable the body to react very flexibly on internal or external changes of hormone status (Lintelmann et al., 2003). This complex system is very sensitive toward disturbing influences that can severely impair the whole development of the organisms. A number of naturally occurring and synthetic chemicals have been shown to exert these adverse effects upon the endocrine system across animal classes including mammals. Concern for these chemicals initially focused on chemicals with estrogenic activity, and thus they were commonly referred to as environmental estrogens, or xenoestrogens (Rhomberg & Seeley, 2005). The initial focus has expanded to include compounds with androgenic activity, as well as thyroid-active chemicals (Rhomberg & Seeley, 2005). Consequently, also different variable terms appeared, e. g. endocrine disrupter (mainly used in Europe)/endocrine disruptor (in America), hormone mimics, hormone inhibitors, hormonally active chemicals, endocrine modulators (Jobling, 1998). Today, these compounds are commonly referred to as endocrine disrupting chemicals.

By definition adopted by European Commission (EC), "an endocrine disrupter is an exogenous substance or mixture that alters function(s) of the endocrine system and consequently causes adverse health effects in an intact organism, or its progeny, or (sub) populations" (European Commission [EC], Endocrine disrupters website, 2011). EDCs were defined by Unites States Environmental Protection Agency (US EPA) as "an exogenous agent that interferes with synthesis, secretion, transport, metabolism, binding action, or elimination of natural blood-borne hormones that are present in the body and are responsible for homeostasis, reproduction, and developmental process". According to Diamanti-Kandarakis et al., 2009, it is necessary to broaden the term - the EDCs is a compound, which through environmental or inappropriate developmental exposures, alters the hormonal and homeostatic systems that enable the organism to communicate and respond to environment.

100 Pesticides – Advances in Chemical and Botanical Pesticides

preparation followed by fast gas chromatography.

**2. Terms, definitions and background**

analytical methods including sample preparation for the identification and quantification of EDPs by chromatographic analytical methods at ultratrace concentration level are briefly covered. The combinations of fast and effective sample preparation methods with conventional and fast capillary gas chromatography (GC) are presented. Selective mass spectrometric (MS) detection with negative chemical ionization (NCI) is discussed and compared to electron ionization (EI). The results leading to selectivity enhancement and decrease of the limits of quantification of selected EDPs using mass spectrometer operated in NCI mode are shown. Real-life analysis demonstrates the potential of studied sample

The endocrine system is a complex integrative network of glands, hormones and receptors. It provides the key communication and control link between the nervous system and bodily functions such as reproduction, immunity, metabolism and behaviour. The endocrine system uses hormones to act as messengers that regulate reproduction, metabolism, growth, development, natural defences to stress, as well as water, electrolyte, and nutritional balance of the blood. Homeostasis is the balance of functions or levels in the body, returning biological variables to their biochemical baseline when perturbed and keeping them there. Maintaining homeostasis is one of the most important functions of the endocrine system. Therefore, the endocrine system includes number of central nervous system-pituitary-target organ feedback mechanisms that enable the body to react very flexibly on internal or external changes of hormone status (Lintelmann et al., 2003). This complex system is very sensitive toward disturbing influences that can severely impair the whole development of the organisms. A number of naturally occurring and synthetic chemicals have been shown to exert these adverse effects upon the endocrine system across animal classes including mammals. Concern for these chemicals initially focused on chemicals with estrogenic activity, and thus they were commonly referred to as environmental estrogens, or xenoestrogens (Rhomberg & Seeley, 2005). The initial focus has expanded to include compounds with androgenic activity, as well as thyroid-active chemicals (Rhomberg & Seeley, 2005). Consequently, also different variable terms appeared, e. g. endocrine disrupter (mainly used in Europe)/endocrine disruptor (in America), hormone mimics, hormone inhibitors, hormonally active chemicals, endocrine modulators (Jobling, 1998). Today, these

compounds are commonly referred to as endocrine disrupting chemicals.

By definition adopted by European Commission (EC), "an endocrine disrupter is an exogenous substance or mixture that alters function(s) of the endocrine system and consequently causes adverse health effects in an intact organism, or its progeny, or (sub) populations" (European Commission [EC], Endocrine disrupters website, 2011). EDCs were defined by Unites States Environmental Protection Agency (US EPA) as "an exogenous agent that interferes with synthesis, secretion, transport, metabolism, binding action, or elimination of natural blood-borne hormones that are present in the body and are responsible for homeostasis, reproduction, and developmental process". According to Diamanti-Kandarakis et al., 2009, it is necessary to broaden the term - the EDCs is a Exposure to EDCs may cause disorder of endocrine system in a number of ways (Mendes, 2002). Many disrupters interact directly with hormone receptors, whereas some cause indirect activation of hormone receptors. They interfere by mimicking the action of a naturally-produced hormone, such as estrogen or testosterone, and thereby setting off similar chemical reactions in the body. EDCs can interfere by blocking the receptors in cells receiving the hormones (hormone receptors), thereby preventing the action of normal hormones. Some receptors interact with each other, such as through "cross-talk" between the estrogen and the growth factor receptors (Dybing, 2006). In other situations, EDCs may interact with multiple receptors. It is well-known, that inhibition of hormone synthesis and hormone transport, as well as alteration in hormone metabolism can affect endocrine system as the concentration of natural hormones alters. An example of how EDCs can interfere with receptor sites is shown in Fig. 1. The important role of well-working endocrine system functioning is the proper hormone-receptor binding at the appropriate level and time (Fig. 1. A).  B) at inappropriate times compared to natural body's hormones (LaFleur & Schug, 2011). At the environmental level, wildlife is particularly vulnerable to the endocrine disrupting effects of pesticides, effects noted in invertebrates, reptiles, fish, birds and mammals were reviewed by Mnif et al., 2007. Many pesticides and industrial chemicals are capable of interfering with the proper function of estrogen, androgen and thyroid hormones at the human level. For example, during pregnancy, lipophilic xenobiotics stored in maternal adipose tissue can be mobilized and enter the blood circulation and reach the placenta. As it was searched by Lopez-Espinosa et al., 2007, the presence of more pesticides in placenta was significantly associated with lower birth weight.

**Figure 1.** Outline of normal hormonal response (A) and EDCs interference with hormone receptors (B).

Changes in hormone levels affect developing organisms more than adults and can result in abnormalities in reproduction, growth, development and can disorder the immune system, as it was discussed by Mnif et al., 2011. A central feature of endocrine disruption is that may cause detrimental effects on organisms at very low chemical concentrations (Fang et al., 2001). Effects of EDCs at very low concentrations can be different from effects of the same chemical at higher concentrations (Colborn, 2012). Traditional approaches to determining safe exposure levels (for example, chemical risk assessments) do not work with EDCs.

## **3. Compounds of interest**

The groups of molecules identified as EDCs are highly heterogeneous and include natural chemicals found in human and animal food (phytoestrogenes), synthetic chemicals used as industrial solvents/lubricants and their by-products, plastics, plasticizers, pesticides, pharmaceuticals, etc. The scope of EDCs here has been narrowed specifically to known and potential endocrine disrupting pesticides.

The first list of suspected EDCs was published in scientific literature in 1993 by Theo Colborn (Colborn et al., 1993), followed by popular book for the layperson "Our stolen future" (Colborn et al., 1996). This book was instrumental in public awareness of the need to find out more.

In United States, the US EPA has been authorized to screen all manufacturing or processing chemicals and formulations for potential endocrine activity. The Endocrine Disruption Screening Program (EDSP) of EPA is mandated to use validated methods for screening and testing chemicals to identify potential endocrine disruptors, determine adverse effects, doseresponse, assess risk and ultimately manage risk under laws. It is realized in two-tiered screening and testing process. In Tier 1, EPA hopes to identify chemicals that have potential to interact with the endocrine system. In Tier 2, EPA determines the specific effect caused by each disruptor and establishes the dose at which the effect occurs. In 2009, EPA released the Final list of Chemicals for Tier 1 Screening in the EDSP (United States Environmental Protection Agency [US EPA] Document, 2009), which is an update of Initial list from 2007 (some chemicals were removed). On November 2010 the US EPA published the second list of chemicals for further testing. This list of 134 chemicals includes a large number of pesticides (US EPA Document, 2010). The selection showing pesticides for EDSP screening is summarized in Table 1.

The European Union (EU) has done extensive work towards official designation of endocrine disrupting substances, collecting literature studies on many chemicals. In December 1999, the European Commission adopted a document entitled "Community Strategy for Endocrine Disrupters" to address the problem of EDCs. A part of this strategy was to establish a priority list that are presumably responsible for damaging human health by interference with hormones and to require the further evaluation of their role in endocrine disruption (EC document, 1999). The creation of the list was based on the published studies of these chemicals and was divided into categories according to

with EDCs.

find out more.

summarized in Table 1.

**3. Compounds of interest**

potential endocrine disrupting pesticides.

Changes in hormone levels affect developing organisms more than adults and can result in abnormalities in reproduction, growth, development and can disorder the immune system, as it was discussed by Mnif et al., 2011. A central feature of endocrine disruption is that may cause detrimental effects on organisms at very low chemical concentrations (Fang et al., 2001). Effects of EDCs at very low concentrations can be different from effects of the same chemical at higher concentrations (Colborn, 2012). Traditional approaches to determining safe exposure levels (for example, chemical risk assessments) do not work

The groups of molecules identified as EDCs are highly heterogeneous and include natural chemicals found in human and animal food (phytoestrogenes), synthetic chemicals used as industrial solvents/lubricants and their by-products, plastics, plasticizers, pesticides, pharmaceuticals, etc. The scope of EDCs here has been narrowed specifically to known and

The first list of suspected EDCs was published in scientific literature in 1993 by Theo Colborn (Colborn et al., 1993), followed by popular book for the layperson "Our stolen future" (Colborn et al., 1996). This book was instrumental in public awareness of the need to

In United States, the US EPA has been authorized to screen all manufacturing or processing chemicals and formulations for potential endocrine activity. The Endocrine Disruption Screening Program (EDSP) of EPA is mandated to use validated methods for screening and testing chemicals to identify potential endocrine disruptors, determine adverse effects, doseresponse, assess risk and ultimately manage risk under laws. It is realized in two-tiered screening and testing process. In Tier 1, EPA hopes to identify chemicals that have potential to interact with the endocrine system. In Tier 2, EPA determines the specific effect caused by each disruptor and establishes the dose at which the effect occurs. In 2009, EPA released the Final list of Chemicals for Tier 1 Screening in the EDSP (United States Environmental Protection Agency [US EPA] Document, 2009), which is an update of Initial list from 2007 (some chemicals were removed). On November 2010 the US EPA published the second list of chemicals for further testing. This list of 134 chemicals includes a large number of pesticides (US EPA Document, 2010). The selection showing pesticides for EDSP screening is

The European Union (EU) has done extensive work towards official designation of endocrine disrupting substances, collecting literature studies on many chemicals. In December 1999, the European Commission adopted a document entitled "Community Strategy for Endocrine Disrupters" to address the problem of EDCs. A part of this strategy was to establish a priority list that are presumably responsible for damaging human health by interference with hormones and to require the further evaluation of their role in endocrine disruption (EC document, 1999). The creation of the list was based on the published studies of these chemicals and was divided into categories according to **Table 1.** The selection of pesticide active ingredients (compound name/ chemical abstract number CAS) from Initial and Second List of Chemicals according to US EPA studied in Tier 1 in the frame of US EDSP.

documented/potential endocrinal effect. This list of chemicals divides compounds into the following categories according to their impact on endocrine system:

**Category 1**  Acetochlor / 34256-82-1; Alachlor / 15972-60-8; Amitrol = Aminotriazol / 61-82-5; Atrazine / 1912-24-9; Beta-HCH / 319-85-7; Bifenthrin / 82657-04-3; Bis-OH-Methoxychlor = 1,1,1-Trichloro-2,2-bis(4-hydroxyphenyl)ethane (HTPE) / 63-25-2; Carbaryl / 63-25-2; Cis-Nonachlor / 5103-73-1; Cyhalothrin / 91465-08-6; DDT (technical) = clofenotane / 50-29-3; Deltamethrin / 52918-63-5; Dibromoethane (EDB) / 106-93-4; Dibromochloropropane (DBCP) / 96-12-8; 1,3-Dichloro-2,2-bis(4-methoxy-3-methylphenyl)propane/ 30668-06-5; 2,4- Dichlorophenoxybutyric acid = 2,4-DB / 326354-18-7; Ethyl-4-hydroxybenzoate / 120-47-8; Ethylene thiourea (ETU) / 96-45-7; Fenarimol / 60168-88-9; Fenitrothion / 122-14-5; Fentin acetate = triphenyltin acetate / 900-95-8; Gamma-HCH (Lindane) / 58-89-9; Hexachlorobenzene (HCB) / 118-74-1; Hexachlorocyclohexane / 608-73-1; Chlordane (technical)/ 12789-03-6; Chlordane (cis- and trans-) / 57-74-9; Chlordimeform / 6164-98-3; Ioxynil / 1689-83-4; Kepone (Chlordecone) / 143-50-0; Ketoconazol / 65277-42-1; Linuron (Lorox) / 330-55-2; 4-MeO-o,p'- DDE / 65148-81-4; 4-MeO-o,p'-DDT / 65148-72-3; 5-MeO-o,p'-DDD / 65148-75-6; 5-MeO-o,p'-DDE / 65148-82-5; 5- MeO-o,p'-DDT / 65148-74-5; m,p'-DDD / 4329-12-8; Mancozeb / 8018-01-7; Maneb / 12427-38-2; 3-MeO-o,p'-DDE / 65148-80-3; Metam Natrium / 137-42-8; Methoxychlor / 72-43-5; Methyl p-Hydroxybenzoate / 99-76-3; Metiram (Metiram-complex) / 9006-42-2; Metribuzin / 21087-64-9; Mirex / 2385-85-5; Nitrofen / 1836-75-5; Propylparaben (n-propyl p-hydroxybenzoate) / 94-13-3; 3-OH-o,p'-DDT / 43216-70-2; 5-OH-o,p'-DDT / 65148-73-4; o,p'-DDAglycinat = N-[(2-chlorophenyl)(4-chlorophenyl) acettyl]glycin / 65148-83-6; o,p'-DDD / 53-19-0; o,p'-DDE / 3424- 82-6; o,p'-DDMU / 14835-94-0; o,p'-DDT / 789-02-6; Omethoate / 1113-02-6; p,p'-DDD / 72-54-8; p,p'-DDE / 72-55- 9; p,p'-DDT = clofenotane / 50-29-3; p,p'-Methoxychlor / 72-43-5; Pentachlorophenol (PCP) / 87-86-5; Phenol, 2- [[(tributylstannyl)oxy]carbonyl]- / 4342-30-7; Picloram / 1918-02-1; Procymidon / 32809-16-8; 2-Propenoic acid, 2 methyl-, methyl ester = Stannane, tributylmeacrylate / 326354-18-7; Quinalphos = Chinalphos / 13593-03-8; Resmethrin / 10453-86-8; Stannane, (benzoyloxy)tributyl- / 4342-36-3; Stannane, tributyl[(1-oxo-9,12 octadecadienyl)oxy]-, (Z,Z)- / 24124-25-2; Stannane, tributyl[[[1,2,3,4,4a,4b,5,6,10,10a-decahydro-1,4a-dimethyl-7-(1-methylethyl)-1-phenanthrenyl]carbonyl]oxy]-,[1R-(1a,4ab,4ba,10aa)]- / 26239-64-5; Stannane, tributylfluoro- / 1983-10-4; Terbutryn / 886-50-0; Thiram / 137-26-8; 1,1,1,2-Tetrachloro-2,2-bis(4-chlorophenyl) ethane (tetrachloro DDT) / 3563-45-9; Toxaphene = Camphechlor / 8001-35-2; Trans-Nonachlor / 39765-80-5; Tributyl[(2 methyl-1-oxo-2-propenyl)oxy]stannane / 2155-70-6; 1,1,1-Trichloro-2,2-bis(4-chlorophenyl) ethane / 2971-22-4; Trifluralin / 1582-09-8; Vinclozolin / 50471-44-8; Zineb / 12122-67-7; **Category 2**  Acephate / 30560-19-1; Aldicarb / 116-06-3; Aldrin / 309-00-2; Allethrin (d- trans allethrin) / 584-79-2; Bromoxynil / 1689-84-5; Carbendazim / 10605-21-7; Carbofuran / 1563-66-2; 4-Chloro-2-methylphenol / 1570-64-5; 4-Chloro-3-methylphenol / 59-50-7; p-Cresol /106-44-5; Cyanazine / 21725-46-2; Cypermethrin / 52315-07-8; Delta-HCH / 319-86-8; p,p'-DDA / 83-05-6; Diazinon / 333-41-5; 2,4-Dichlorophenoxy acetic acid (2,4-D) / 94-75-7; Dicofol = Kelthane / 115-32-2; Dieldrin / 60-57-1; Diisobutylphthalate / 84-69-5; Dimethoate / 60-51-5; Diuron / 330-54-1; Elsan = Dimephenthoate / 2597-03-7; Endosulfan / 115-29-7; Endosulfan (alpha) / 959-98-8; Endosulfan (beta) / 33213-65-9; Endrin / 72-20-8; Etridiazole / 2593-15-9; Fenothrin = sumithrin / 26002-80-2; Fenoxycarb / 72490-01-8; Fenvalerate / 51630-58-1; Fluvalinate / 69409-94-5; Heptachlor / 76-44-8; Chlorfenvinphos / 470-90-6; Iprodione / 36734-19-7; Malathion / 121-75-5; Methomyl /16752-77-5; Methylbromide (bromomethane) / 74-83-9; Methylparathion /298-00-0; Mevinphos = Phosdrin /7786-34-7; 4-Nitrophenol / 100-02-7; Oxychlordane /27304- 13-8; Parathion = Parathion(-ethyl) / 56-38-2; Permethrin / 52645-53-1; o-Phenylphenol / 90-43-7; Phosophamidon / 13171-21-6; Photomirex / 39801-14-4; Piperonyl butoxide / 51-03-6; Prochloraz / 67747-09-5; Prometryn /7287- 19-6; Propanil / 709-98-8; Pyrethrin / 121-29-9; Simazine / 122-34-9; Triadimefon / 43121-43-3; Triadimenol / 123- 88-6; Trichlorfon = Dipterex / 52-68-6; 2,4,5-Trichlorophenoxy acetic acid (2,4,5-T) / 93-76-5; Ziram / 137-30-4; **Category 3**  Abamectin / 71751-41-2; Amitraz / 33089-61-1; Azadirachtin / 11141-17-6; Benomyl / 17804-35-2; Bitertanol / 55179-31-2; Bromacil / 314-40-9; Clofentezine = chlorfentezine / 74115-24-5; Cyproconazole / 94361-07-6; Demefion / 682-80-4; Demeton-s-methyl / 919-86-8; Difenoconazole / 119446-68-3; Dichlorvos /62-73-7; Dimethylformamide (DMFA) / 68-12-2; Dinitrophenol / 25550-58-7; Dinoseb / 88-85-7; Diphenyl / 92-52-4; Epiconazole / 133855-98-8; Epoxiconazole / 135319-73-2; Esfenvalerate / 66230-04-4; Ethofenprox / 80844-07-1; Fenbuconazole / 114369-43-6; Fipronil / 120068-37-3; Fluazifop-butyl / 69806-50-4; Flutriafol / 76674-21-0; Formothion / 682-80-4; Glufosinate / 51276-47-2; Glufosinate-ammonium / 70393-85-0; Glyphosate / 1071-83-6; Heptachlor-epoxide / 1024-57-3; Chlordene / 3734-48-3; Chlorpyrifos / 2921-88-2; Imazalil / 3554-44-0; Molinate / 2212-67-1; Myclobutanil / 88671-89-0; Nabam / 142-59-6; Octachlorostyrene / 29082-74-4; Oryzalin / 19044-88-3; Oxydemeton-methyl / 301-12-2; Paraquat / 4685-14-7; Penconazole / 66246-88-6; Pendimethalin / 40487-42-1; Pentachloronitrobenzene (Quintozene) / 82-68-8; Prodiamine / 29091-21-2; Pronamide / 23950-58-5; Propiconazole / 60207-90-1; Ronnel = Fenchlorfos / 299-84-3; Tebuconazole / 107534-96-3; Tetrachlorvinphos = Gardona / 22248-79-9; Thiazopyr / 117718-60-2;

**Table 2.** The selection of endocrine disrupting pesticides (compound name/ chemical abstract number CAS) according to EU prioritization of EDCs into 3 categories.

For some chemicals the production and usage was already forbidden and others are still under testing. Majority of substances registered in this list of chemicals are pesticides (EC document, 2007). The selection of pesticides is summarized in Table. 2 according to their Category. From a total of 564 chemicals that had been suggested by various organizations or in published papers or reports as being suspected EDCs, 147 were considered likely to be either persistent in the environment or produced at high volumes. Of these, however, in a first assessment clear evidence of endocrine disrupting activity was noted for only 66 (assigned Category 1 using the criteria adopted in the study). A further 52 chemicals showed some evidence suggesting potential activity (Category 2). In total 118 substances were categorized in the first exercise of priority setting. Of the 66 chemicals in Category 1, humans were considered likely to be exposed to 60.

Selected substances have been included as persistent organic pollutants in the Stockholm Convention, which is a global treaty to protect human health and the environment from these compounds (EC document, 2006).

In June 2007, the new EU policy on chemicals, REACH - Registration, Evaluation, Authorization and Restriction of Chemicals, entered into force. The goal of REACH is a prompt, effective process for identifying the most hazardous chemicals on the European market and replacing them with safer alternatives. At the heart of the Authorization process is a "candidate list" of chemicals that meet the criteria of "Substances of Very High Concern" (SVHC) defined in the legislation, such as those that may cause cancer or persist in our bodies and the environment for long periods of time. Under REACH, SVHC are subject to the greatest scrutiny. The EU creates a specific list of these undesirable substances which will oblige importers, producers and downstream users to seek special authorization for continued use. Authorization may be denied, because REACH contains a provision that could replace some of these dangerous substances with safer alternatives. Under this activity, the International Chemical Secretariat (ChemSec) is a non-profit organization working for a toxic free world and publishing the SIN List (Substitute It Now). The SIN List applies REACH's own criteria to identify SVHC, and with the SIN 2.0 List update encompasses 378 chemicals. It contains 22 substances identified solely due to their endocrine disrupting properties. The following pesticides are included: Thiram (CAS 137-26-8), Zineb (CA S 12122-67-7).

### **4. EDPs analysis**

104 Pesticides – Advances in Chemical and Botanical Pesticides

Acetochlor / 34256-82-1; Alachlor / 15972-60-8; Amitrol = Aminotriazol / 61-82-5; Atrazine / 1912-24-9; Beta-HCH / 319-85-7; Bifenthrin / 82657-04-3; Bis-OH-Methoxychlor = 1,1,1-Trichloro-2,2-bis(4-hydroxyphenyl)ethane (HTPE) / 63-25-2; Carbaryl / 63-25-2; Cis-Nonachlor / 5103-73-1; Cyhalothrin / 91465-08-6; DDT (technical) = clofenotane / 50-29-3; Deltamethrin / 52918-63-5; Dibromoethane (EDB) / 106-93-4; Dibromochloropropane

Dichlorophenoxybutyric acid = 2,4-DB / 326354-18-7; Ethyl-4-hydroxybenzoate / 120-47-8; Ethylene thiourea (ETU) / 96-45-7; Fenarimol / 60168-88-9; Fenitrothion / 122-14-5; Fentin acetate = triphenyltin acetate / 900-95-8; Gamma-HCH (Lindane) / 58-89-9; Hexachlorobenzene (HCB) / 118-74-1; Hexachlorocyclohexane / 608-73-1; Chlordane (technical)/ 12789-03-6; Chlordane (cis- and trans-) / 57-74-9; Chlordimeform / 6164-98-3; Ioxynil / 1689-83-4; Kepone (Chlordecone) / 143-50-0; Ketoconazol / 65277-42-1; Linuron (Lorox) / 330-55-2; 4-MeO-o,p'- DDE / 65148-81-4; 4-MeO-o,p'-DDT / 65148-72-3; 5-MeO-o,p'-DDD / 65148-75-6; 5-MeO-o,p'-DDE / 65148-82-5; 5- MeO-o,p'-DDT / 65148-74-5; m,p'-DDD / 4329-12-8; Mancozeb / 8018-01-7; Maneb / 12427-38-2; 3-MeO-o,p'-DDE / 65148-80-3; Metam Natrium / 137-42-8; Methoxychlor / 72-43-5; Methyl p-Hydroxybenzoate / 99-76-3; Metiram (Metiram-complex) / 9006-42-2; Metribuzin / 21087-64-9; Mirex / 2385-85-5; Nitrofen / 1836-75-5; Propylparaben (n-propyl p-hydroxybenzoate) / 94-13-3; 3-OH-o,p'-DDT / 43216-70-2; 5-OH-o,p'-DDT / 65148-73-4; o,p'-DDAglycinat = N-[(2-chlorophenyl)(4-chlorophenyl) acettyl]glycin / 65148-83-6; o,p'-DDD / 53-19-0; o,p'-DDE / 3424- 82-6; o,p'-DDMU / 14835-94-0; o,p'-DDT / 789-02-6; Omethoate / 1113-02-6; p,p'-DDD / 72-54-8; p,p'-DDE / 72-55- 9; p,p'-DDT = clofenotane / 50-29-3; p,p'-Methoxychlor / 72-43-5; Pentachlorophenol (PCP) / 87-86-5; Phenol, 2- [[(tributylstannyl)oxy]carbonyl]- / 4342-30-7; Picloram / 1918-02-1; Procymidon / 32809-16-8; 2-Propenoic acid, 2 methyl-, methyl ester = Stannane, tributylmeacrylate / 326354-18-7; Quinalphos = Chinalphos / 13593-03-8; Resmethrin / 10453-86-8; Stannane, (benzoyloxy)tributyl- / 4342-36-3; Stannane, tributyl[(1-oxo-9,12-

octadecadienyl)oxy]-, (Z,Z)- / 24124-25-2; Stannane, tributyl[[[1,2,3,4,4a,4b,5,6,10,10a-decahydro-1,4a-dimethyl-7-(1-methylethyl)-1-phenanthrenyl]carbonyl]oxy]-,[1R-(1a,4ab,4ba,10aa)]- / 26239-64-5; Stannane, tributylfluoro- / 1983-10-4; Terbutryn / 886-50-0; Thiram / 137-26-8; 1,1,1,2-Tetrachloro-2,2-bis(4-chlorophenyl) ethane (tetrachloro DDT) / 3563-45-9; Toxaphene = Camphechlor / 8001-35-2; Trans-Nonachlor / 39765-80-5; Tributyl[(2 methyl-1-oxo-2-propenyl)oxy]stannane / 2155-70-6; 1,1,1-Trichloro-2,2-bis(4-chlorophenyl) ethane / 2971-22-4;

Acephate / 30560-19-1; Aldicarb / 116-06-3; Aldrin / 309-00-2; Allethrin (d- trans allethrin) / 584-79-2; Bromoxynil / 1689-84-5; Carbendazim / 10605-21-7; Carbofuran / 1563-66-2; 4-Chloro-2-methylphenol / 1570-64-5; 4-Chloro-3-methylphenol / 59-50-7; p-Cresol /106-44-5; Cyanazine / 21725-46-2; Cypermethrin / 52315-07-8; Delta-HCH / 319-86-8; p,p'-DDA / 83-05-6; Diazinon / 333-41-5; 2,4-Dichlorophenoxy acetic acid (2,4-D) / 94-75-7; Dicofol = Kelthane / 115-32-2; Dieldrin / 60-57-1; Diisobutylphthalate / 84-69-5; Dimethoate / 60-51-5; Diuron / 330-54-1; Elsan = Dimephenthoate / 2597-03-7; Endosulfan / 115-29-7; Endosulfan (alpha) / 959-98-8; Endosulfan (beta) / 33213-65-9; Endrin / 72-20-8; Etridiazole / 2593-15-9; Fenothrin = sumithrin / 26002-80-2; Fenoxycarb / 72490-01-8; Fenvalerate / 51630-58-1; Fluvalinate / 69409-94-5; Heptachlor / 76-44-8; Chlorfenvinphos / 470-90-6; Iprodione /

36734-19-7; Malathion / 121-75-5; Methomyl /16752-77-5; Methylbromide (bromomethane) / 74-83-9; Methylparathion /298-00-0; Mevinphos = Phosdrin /7786-34-7; 4-Nitrophenol / 100-02-7; Oxychlordane /27304- 13-8; Parathion = Parathion(-ethyl) / 56-38-2; Permethrin / 52645-53-1; o-Phenylphenol / 90-43-7; Phosophamidon / 13171-21-6; Photomirex / 39801-14-4; Piperonyl butoxide / 51-03-6; Prochloraz / 67747-09-5; Prometryn /7287- 19-6; Propanil / 709-98-8; Pyrethrin / 121-29-9; Simazine / 122-34-9; Triadimefon / 43121-43-3; Triadimenol / 123- 88-6; Trichlorfon = Dipterex / 52-68-6; 2,4,5-Trichlorophenoxy acetic acid (2,4,5-T) / 93-76-5; Ziram / 137-30-4;

Abamectin / 71751-41-2; Amitraz / 33089-61-1; Azadirachtin / 11141-17-6; Benomyl / 17804-35-2; Bitertanol / 55179-31-2; Bromacil / 314-40-9; Clofentezine = chlorfentezine / 74115-24-5; Cyproconazole / 94361-07-6; Demefion / 682-80-4; Demeton-s-methyl / 919-86-8; Difenoconazole / 119446-68-3; Dichlorvos /62-73-7; Dimethylformamide (DMFA) / 68-12-2; Dinitrophenol / 25550-58-7; Dinoseb / 88-85-7; Diphenyl / 92-52-4; Epiconazole / 133855-98-8; Epoxiconazole / 135319-73-2; Esfenvalerate / 66230-04-4; Ethofenprox / 80844-07-1; Fenbuconazole / 114369-43-6; Fipronil / 120068-37-3; Fluazifop-butyl / 69806-50-4; Flutriafol / 76674-21-0; Formothion / 682-80-4; Glufosinate / 51276-47-2; Glufosinate-ammonium / 70393-85-0; Glyphosate / 1071-83-6; Heptachlor-epoxide / 1024-57-3; Chlordene / 3734-48-3; Chlorpyrifos / 2921-88-2; Imazalil / 3554-44-0; Molinate / 2212-67-1; Myclobutanil / 88671-89-0; Nabam / 142-59-6; Octachlorostyrene / 29082-74-4; Oryzalin / 19044-88-3; Oxydemeton-methyl / 301-12-2; Paraquat / 4685-14-7; Penconazole / 66246-88-6; Pendimethalin / 40487-42-1; Pentachloronitrobenzene (Quintozene) / 82-68-8; Prodiamine / 29091-21-2; Pronamide / 23950-58-5;

Propiconazole / 60207-90-1; Ronnel = Fenchlorfos / 299-84-3; Tebuconazole / 107534-96-3; Tetrachlorvinphos =

**Table 2.** The selection of endocrine disrupting pesticides (compound name/ chemical abstract number

Trifluralin / 1582-09-8; Vinclozolin / 50471-44-8; Zineb / 12122-67-7;

Gardona / 22248-79-9; Thiazopyr / 117718-60-2;

CAS) according to EU prioritization of EDCs into 3 categories.

(DBCP) / 96-12-8; 1,3-Dichloro-2,2-bis(4-methoxy-3-methylphenyl)propane/ 30668-06-5; 2,4-

**Category 1**

**Category 2**

**Category 3**

Detection of EDPs and subsequent screening require sensitive and selective instrumental analytical techniques with sufficiently low limits of detection and quantification. Analyzing the EDPs at low concentration levels requires multistep sample preparation including cleaning and preconcentration of the resulting extract. As EDPs represent structurally diverse classes of substances, plentiful analytical methods could be applied for the identification and quantification of these compounds (Lagana et al., 2004; Petrovič et al., 2002). The most efficient approach to EDPs residues analysis involves the use of chromatographic methods (Comerton et al., 2009). Recently, methods based on biosensors have also been used (Bezbaruah & Kalita, 2010; Dostálek et al., 2007). Analytical techniques

as GC and liquid chromatography (LC) combined with MS or tandem MS are the techniques most frequently used and can reach satisfactory selectivity and sensitivity analyzing EDPs in complex food matrices mainly of food and environmental origin (Alder et al., 2006). Comparing mass-based methods with other analytical methods, such as Estrogen responsive chemically activated luciferase expression, Yeast estrogen screen, Enzyme-linked immunosorbent assay, it was shown that methods with MS detection (GC-MS, LC-MS, GC-MS/MS , LC-MS/MS) show lower detection limits (Chang et al., 2009). Comparing detection limits of enzymatic methods for the detection of organochlorine, organophosphate and carbamate pesticides with chromatographic methods it was concluded, that enzymatic methods achieve limits of detection in µg/l, whereas traditional chromatographic methods are often able to detect pesticides in ng/l (Van Dyk & Pletschke, 2011).

In the next part we will focus on analytical methods with limit of detection/quantification in the trace concentration level or ultratrace region and we will cite methods well-suited for analytical tests of low-level EDPs in food, environmental and biological samples.

Capillary gas chromatography coupled to MS detection has developed into a primary technique for identification and quantification of many EDCs using small bench-top instruments with sophisticated data systems (Holland, 2003). Electron ionization is the ionization technique of the first choice. In cases requiring enhanced sensitivity and selectivity the negative/positive chemical ionization is employed (Húšková et al., 2009a, 2009b, 2010a).

Within gas chromatographic techniques, fast GC technique satisfies the present day demands on faster and cost-effective analysis. Nowadays, fast GC can be performed on commercial gas chromatographs, which are standardly equipped with high-speed injection systems, electronic gas pressure control, rapid oven heating/cooling and fast detection (Dömötörová & Matisová, 2008). Advances in LC-MS interfacing, namely introduction of electrospray (ESI) and atmospheric pressure chemical ionization (APCI) have enabled sensitivity and reliability that are suitable for routine determinations of EDCs, particularly for more polar compounds that would require derivatization for GC-MS. LC-MS can reduce clean-up requirements over HPLC-UV (high performance liquid chromatography with ultraviolet detection), although care must be taken with matrix effects on ESI responses that may affect quantitation (Holland, 2003).

Signal enhancement and suppression due to matrix effects are reduced by the use of isotopelabelled internal standards or by application of matrix-matched standards. Tandem MS available on triple quadrupole, ion trap and hybrid analyzers are valuable for confirmation of identity, reduction of high background signals. They provide low limits of detection without the need for derivatization and sometimes also without the need of complicated sample preparation.

The overview of latest analytical methods combining preconcentration and chromatographic analytical methods for analysis of EDPs in food, environmental, and biological samples are summarized in Table 3. Various groups of EDPs were investigated by GC as carbamates, organochlorines, organophosphorous, organothiophosphates, organotins, triazines and others. Analysed samples varied from indoor air, water, sediments, food, to biological

may affect quantitation (Holland, 2003).

sample preparation.

as GC and liquid chromatography (LC) combined with MS or tandem MS are the techniques most frequently used and can reach satisfactory selectivity and sensitivity analyzing EDPs in complex food matrices mainly of food and environmental origin (Alder et al., 2006). Comparing mass-based methods with other analytical methods, such as Estrogen responsive chemically activated luciferase expression, Yeast estrogen screen, Enzyme-linked immunosorbent assay, it was shown that methods with MS detection (GC-MS, LC-MS, GC-MS/MS , LC-MS/MS) show lower detection limits (Chang et al., 2009). Comparing detection limits of enzymatic methods for the detection of organochlorine, organophosphate and carbamate pesticides with chromatographic methods it was concluded, that enzymatic methods achieve limits of detection in µg/l, whereas traditional chromatographic methods

In the next part we will focus on analytical methods with limit of detection/quantification in the trace concentration level or ultratrace region and we will cite methods well-suited for