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## **Meet the editors**

Sonia Soloneski holds a PhD in Natural Sciences and is an Assistant Professor of Molecular Cell Biology at the Faculty of Natural Sciences and Museum of La Plata, National University of La Plata, Argentina. Her graduate studies in Finland helped her in developing her doctoral thesis at the Department of Medical Genetics, University of Finland, Finland. She is a member of the National Sci-

entific and Technical Research Council (CONICET) of Argentina, the Latin American Association of Environmental Mutagenesis, Carcinogenesis and Teratogenesis (ALAMCTA), the Argentinean Society of Toxicology (ATA) and the Argentine Society of Genetics (SAG). Dr Soloneski has co-authored more than 40 scientific publications in reviewed scientific journals, and 100 abstracts of research papers as a first author. She is a regular lecturer at the International A. Hollaender courses, held by the International Association of Environmental Mutagen Societies (IAEMS), and is a referent for subjects related to genetic toxicology and the ecotoxicology field.

Marcelo L. Larramendy, PhD, is a Professor of Molecular Cell Biology at La Plata National University (UNLP), Argentina, and has been a researcher at the National Scientific and Technical Research Council (CONICET) of Argentina since 1981. He is also a member of the Executive Committee of the Latin American Association of Environmental Mutagenesis, Carcinogenesis and

Teratogenesis (ALAMCTA). Dr Larramendy has written more than 365 scientific papers in the field and has received several national and international awards. He is a Regular Lecturer at the International A. Hollaender Courses, held by the International Association of Environmental Mutagen Societies (IAEMS), and among others, a former guest scientist at NIH, Bethesda, USA, and University of Helsinki, Finland. Dr Larramendy is an expert in Molecular Cytogenetics and Genetic Toxicology.

Contents

**Preface IX** 

Chapter 1 **Insecticide Thiamethoxam:** 

Chapter 4 **Tree Injection as an Alternative** 

Chapter 6 **Adverse Effect of Insecticides** 

Mahdi Banaee

Beverly A. Wiltz

**Part 1 Basic and Alternative Control of Insect Pests 1** 

Andréia da Silva Almeida, Francisco Amaral Villela,

Chapter 3 **Photoremediation of Carbamate Residues in Water 39**  Anđelka V. Tomašević and Slavica M. Gašić

> **Method of Insecticide Application 61**  Joseph J. Doccola and Peter M. Wild

Chapter 5 **Development of a Prophylactic Butyrylcholinesterase Bioscavenger to Protect Against Insecticide** 

Chapter 7 **Production of Insecticidal Baculoviruses in Insect Cell Cultures: Potential and Limitations 127** 

Gabriela A. Micheloud and Gabriel Visnovsky

Chapter 8 **Factors Affecting Performance of Soil Termiticides 153** 

Juan D. Claus, Verónica V. Gioria,

**Toxicity Using a Homologous Macaque Model 79**  Yvonne Rosenberg, Xiaoming Jiang, Lingjun Mao, Segundo Hernandez Abanto, Keunmyoung Lee

**on Various Aspects of Fish's Biology and Physiology 101** 

Maria Ângela André Tillmann and Geri Eduardo Meneghello

**Daucus carota**

 **L.) 3** 

**A Bioactive Action on Carrot Seeds (**

Chapter 2 **The Pyrethroid Knockdown Resistance 17**  Ademir Jesus Martins and Denise Valle

### Contents

#### **Preface XI**



#### **Part 2 Further Applications 197**


### Preface

Following *Herbicides, Theory and Applications* (InTech, 2011), this new addition aims to shed more light on matters of scientific interest in pesticide and crop management.

Insects have played a role in shaping the history of mankind since the dawn of time. Reference to them can be found in ancient books, for example in three (or four) of the ten plagues of Egypt, to persuade Pharaoh to release the people of Israel from slavery.

The purpose of pest control is to be able to produce more and better quality food as well as decrease costs. Traditional undernourished nations now export foodstuffs, but also suffer from the epidemics of obesity and diabetes among others. The benefits of insecticides speak for themselves. The pitfalls and dangers of their excessive use to animal and human health, as well as the environment, do not.

We cannot disregard the interplay that exists between science, national governments, international agencies and economy, to name but a few. While many agents have very strict or forbidden indications of use in many countries, the truth is that these criteria are not seriously enforced or, even worse, are simply disregarded in some. A growing trend is for countries to rent their lands for cultivation to others that are less favored by geographical conditions and who have an urgent need to feed those populations. These "host" countries are usually in need of cash and, in some cases, without rule of law.

Standardization of agricultural practices is another matter that should not be disregarded in the overall equation. There is a general aversion to rely less on the old farmer's eye and apply the same rule(s), whether they are appropriate or not. It has been estimated that an apple tree receives no less than 26 "treatments". While minimum levels of insecticides are set, the equation sometimes does not consider the overall sum of those minimum values, which reaches alarming proportions in some cases.

Scientists have the right and obligation to raise their voice and air concerns about double standards. Active principles that are forbidden to be used in most European and North American countries are still being produced for sale in far away destinations. In other cases, local production is achieved by means of subsidiaries, sale of licenses and local reformulations. Two of the BRIC countries are the main world

#### XII Preface

producers of agrochemicals today. There are many reasons for this, and the list would be too extensive and not inclusive but, among other things, we have to consider the means governments have at their disposal to sanction proper legislation, enforce bans, lobbying groups, struggle against smuggling or parallel import, higher cost of alternatives, attractiveness to foreign investments and the price these commodities reach in international markets. As a director of corporate communications of one of the largest agrochemical companies once mentioned in an interview, "his company does not have to guarantee the safety of biotech food (goes without saying that other products by this conglomerate fall into this category by default). Assuring safety is FDA´s job"(Food and Drug Administration of USA). Companies change names and use green colored logos with plants or flowers to convey an image that cannot be further from the truth.

Few people do not recognize that this is actually a boomerang. A large portion of these crops will be shipped to far away destinations, thus finding a way (directly or indirectly) to the consumer's tables or to be used as animal feed. The "green credentials" of foodstuffs and their packaging are another misleading factor. Several international food crises that have taken place in the last decades have demonstrated this. Usually, appropriate measures are taken after a serious incident has taken place but, in general terms, too late for the victims involved. Scientists should also bear responsibilities vis à vis consumers and not only governments and companies in order to avoid repeating the mistakes from the past. Not only human beings, but also biomes, land and riparian organisms (fauna and flora) have to be considered. We all share one land, one air; political or legal borders are totally meaningless in this question.

It is our hope that this book will be of interest and use not only to scientists, but also to the food‐producing industry, governments, politicians and consumers as well. If we are able to stimulate this interest, albeit in a small way, we have achieved our goal.

> **Dr Guillermo Eli Liwszyc,** Physician, Specialist in Internal Medicine, former Guest Scientist at the University of Helsinki, Finland

## **Part 1**

**Basic and Alternative Control of Insect Pests** 

**1** 

*Brazil* 

**Insecticide Thiamethoxam: A Bioactive Action** 

Explorations of the substances potential that can act as promoters of growth-inducing activities are in substantial contribution of research to agriculture. Many of these activities are related to the activation capacity of the plants resistance mechanisms, thus enabling seek

With the modernization of agriculture, marked advances in farming techniques have been obtained, allowing mitigate the limiting factors with weather conditions such diseases, pests, among others. The plant physiology has promoted great advances in recent years with the advent of modern techniques such as the production of plants by tissue culture, genetic engineering and biotechnology. Among these modern techniques, the use of bioactive, capable of increasing the productive potential of plants, is an increasing use in the

In Brazil, the use of bioactive beginning to be explored and the results of several studies have shown that these substances provide significant increases in productivity and, in quality, as observed, for example, significant increases in the amount of larger fruits. Bioactivators are natural substances of plant origin that have actions similar to the main plant growth regulators, aimed at growth and development of the plant. Provide better

Moreover, they are complex organic substances, not bioregulators, growth modifiers, capable of working in the plant transcription factors and gene expression in membrane proteins by altering the ion transport. They also act in metabolic enzymes could affect the secondary metabolism and may alter the mineral nutrition, induce the production of precursors of plant hormones, leading to hormone synthesis and more intense response to

Applied to plants, bioactivators cause modification or alteration of specific metabolic and physiological processes, such as increasing the division and cell elongation, stimulation of chlorophyll synthesis and photosynthesis, flower bud differentiation, increasing the life of plants, softening the effects of adverse weather conditions and increasing the absorption of

practice of modern agriculture and widespread in countries highly technical.

physiological balance, favoring closer ties to the genetic potential of culture.

**1. Introduction** 

control through integrated management.

nutrients and plant hormones.

nutrients and setting their roots.

**on Carrot Seeds (***Daucus carota* **L.)** 

Andréia da Silva Almeida1, Francisco Amaral Villela2,

*1Ciência e Tecnologia de Sementes; 2Ciência e Tecnologia de Sementes; University Federal de Pelotas* 

Maria Ângela André Tillmann2 and Geri Eduardo Meneghello2

### **Insecticide Thiamethoxam: A Bioactive Action on Carrot Seeds (***Daucus carota* **L.)**

Andréia da Silva Almeida1, Francisco Amaral Villela2, Maria Ângela André Tillmann2 and Geri Eduardo Meneghello2

*1Ciência e Tecnologia de Sementes; 2Ciência e Tecnologia de Sementes; University Federal de Pelotas Brazil* 

#### **1. Introduction**

Explorations of the substances potential that can act as promoters of growth-inducing activities are in substantial contribution of research to agriculture. Many of these activities are related to the activation capacity of the plants resistance mechanisms, thus enabling seek control through integrated management.

With the modernization of agriculture, marked advances in farming techniques have been obtained, allowing mitigate the limiting factors with weather conditions such diseases, pests, among others. The plant physiology has promoted great advances in recent years with the advent of modern techniques such as the production of plants by tissue culture, genetic engineering and biotechnology. Among these modern techniques, the use of bioactive, capable of increasing the productive potential of plants, is an increasing use in the practice of modern agriculture and widespread in countries highly technical.

In Brazil, the use of bioactive beginning to be explored and the results of several studies have shown that these substances provide significant increases in productivity and, in quality, as observed, for example, significant increases in the amount of larger fruits. Bioactivators are natural substances of plant origin that have actions similar to the main plant growth regulators, aimed at growth and development of the plant. Provide better physiological balance, favoring closer ties to the genetic potential of culture.

Moreover, they are complex organic substances, not bioregulators, growth modifiers, capable of working in the plant transcription factors and gene expression in membrane proteins by altering the ion transport. They also act in metabolic enzymes could affect the secondary metabolism and may alter the mineral nutrition, induce the production of precursors of plant hormones, leading to hormone synthesis and more intense response to nutrients and plant hormones.

Applied to plants, bioactivators cause modification or alteration of specific metabolic and physiological processes, such as increasing the division and cell elongation, stimulation of chlorophyll synthesis and photosynthesis, flower bud differentiation, increasing the life of plants, softening the effects of adverse weather conditions and increasing the absorption of nutrients and setting their roots.

Insecticide Thiamethoxam: A Bioactive Action on Carrot Seeds (*Daucus carota* L.) 5

sources of provitamin A (Spinola et al, 1998). Careful selection of cultivars allows sowing of

The success of horticulture generally depends on the establishment of suitable stand for each crop; otherwise reductions may occur in the quantity and quality of final product (Silva

Under field conditions, carrot seed germination may have low, slow and irregular, resulting in uneven emergence and a heterogeneous population of plants (Corbineau et al., 1994). With increasing mechanization in vegetable production, establish rapid and uniform culture becomes increasingly important, and it is desirable that the evaluation of seed quality to

Establishment of appropriate stand depends on the use of seeds with high physiological potential, able to germinate rapidly and uniformly under a large variation of the environment. Speed and timing are very important because they allow reducing the degree

Reduced or uneven emergence can lead to developmental delays, problems with weed control, non-uniformity of culture in different phenological stages, interference on product quality and characteristics related to the efficiency of harvesting (Marcos Filho, 2005). In vegetables, backwardness and uneven development may be reflected in product quality and reducing the commercial value, such as lettuce, cabbage, carrots, cauliflower, eggplant

A survey on physiological quality of seeds of different kinds of vegetables such as carrots, peas, beets, tomato and watermelon industry, marketed and / or used by farmers, it was observed that germination of these seeds do not always fit the minimum standard of

Therefore, failure to stand and seedling vigor at low field level are frequent, with the need

In this context, considering the lack of information concerning the effect of thiamethoxam and the potential benefits that treatment can provide, the present study was to evaluate the

This work was conducted at the Laboratory of Seed Analysis Textbook and greenhouse, Faculty of Agronomy Eliseu Maciel, Universidade Federal de Pelotas (UFPel), Pelotas /

To establishment concentrations of thiamethoxam, it was used the following concentrations: 0.0, 0.05, 0.1, 0.2, 0.4, 0.8 mL / L, based on germination test three were selected. Tests conducted to evaluate the quality of seeds were performed with and without water stress. The seeds were treated in a plastic bag containing distilled water at 0.1 mL of water to 0.05 mL and 0.4 mL of product thiamethoxam to 3g of carrot seeds. The product was applied directly to the bottom of the plastic bag before putting the seeds. Then the seeds were placed in plastic bag and mixed until uniform coating of the seeds. It was used a volume of mixture (product + water) sufficient to promote a more even distribution of product on the seeds.

Water stress was achieved by the water potential of -0.4 MPa, using aqueous solutions of polyethylene glycol (PEG 6000). The calculation of the solute quantities was performed

for appropriate and sensitive methods to detect these differences in seed quality.

influence of thiamethoxam in physiological performance of carrot seeds.

For measurement of product and distilled water were used micropipettes.

carrots over the years in many regions (Filgueira, 2000).

provide information on their performance on the field.

and onion (Kikuta and Marcos Filho, 2007).

marketing required for each species (Nascimento, 1994).

Seed lots of carrot cultivar Brasilia represented by four lots.

of exposure of seeds and plants to adverse factors (Marcos Filho, 2005).

and Vieira, 2006).

**3. Methodology** 

Brazil.

The bioactivator acts in the expression of genes responsible for synthesis and activation of metabolic enzymes related to plant growth by altering the production of amino acid precursors of plant hormones. With the increased production of hormones, plant expressed greater vigor, germination and root development. With a greater number of roots, increases the absorption and resistance of plant stomata to water loss, which benefits the metabolism and increases resistance to stress.

Thiamethoxam, 3-(2 – chloro – thiazole - 5 - ylmethyl) - 5 - methyl (1,3,5) oxadiazinan - 4 ylidene-N- nitroamine, whose chemical structure is shown in Figure 1, is a systemic insecticide neonicotinoid group, family nitroguanidine, which acts on acetylcholine nicotinic receptor in membrane of insects, damaging the nervous system and causing them to death. It is used successfully in pest control in beginning cycle from different cultures. Due to numerous reports of field observations describing increases in vigor development and productivity, even in the absence of pests, has been considered a product that has phytotonic effect.

Fig. 1. Chemical structure of thiamethoxam. Source: Oliveira, et al., (2009).

With reference to the mechanism of action of thiamethoxam, the molecule has the ability to induce physiological changes in plants. Due to results obtained, it is concluded that this insecticide acts as bioactive two ways. The first, activating carrier proteins of cell membranes allowing greater ion transport by enhancing plant mineral nutrition. This increase in the availability of minerals promotes positive responses in the development and plant productivity. The second is related to increased enzyme activation caused by thiamethoxam at both the seed and the plant, thereby increasing both primary and secondary metabolism. It also increases the synthesis of amino acids, precursors of new proteins and endogenous synthesis of plant hormones. The responses of plants to these proteins and hormone biosynthesis may be related to significant increases in production.

Results of research related with soybean (*Glycine max*) (Castro et al., 2008; Cataneo, 2008), rice (*Oryza sativa*) (Clavijo, 2008; Almeida et al.; 2010), cotton (*Gossypium hirsutum* ) (Lauxen et al., 2010), bean (*Phaseolus vulgaris*) (Almeida et al., 2010) e vegetables like, lettuce (*Lactuca sativa*), tomato (*Lycopersicon esculentum*), pumpkin (*Cucurbita pepo* L.) and carrot (*Daucus carota*) indicate benefit effect of thiamethoxam uses.

#### **2. Bioactivator in physiological performance of carrot seeds**

Carrot (*Daucus carota* L.) is the most economical expression of vegetables among those whose edible portion is the root, and to highlight the nutritional value, as a major vegetable sources of provitamin A (Spinola et al, 1998). Careful selection of cultivars allows sowing of carrots over the years in many regions (Filgueira, 2000).

The success of horticulture generally depends on the establishment of suitable stand for each crop; otherwise reductions may occur in the quantity and quality of final product (Silva and Vieira, 2006).

Under field conditions, carrot seed germination may have low, slow and irregular, resulting in uneven emergence and a heterogeneous population of plants (Corbineau et al., 1994).

With increasing mechanization in vegetable production, establish rapid and uniform culture becomes increasingly important, and it is desirable that the evaluation of seed quality to provide information on their performance on the field.

Establishment of appropriate stand depends on the use of seeds with high physiological potential, able to germinate rapidly and uniformly under a large variation of the environment. Speed and timing are very important because they allow reducing the degree of exposure of seeds and plants to adverse factors (Marcos Filho, 2005).

Reduced or uneven emergence can lead to developmental delays, problems with weed control, non-uniformity of culture in different phenological stages, interference on product quality and characteristics related to the efficiency of harvesting (Marcos Filho, 2005).

In vegetables, backwardness and uneven development may be reflected in product quality and reducing the commercial value, such as lettuce, cabbage, carrots, cauliflower, eggplant and onion (Kikuta and Marcos Filho, 2007).

A survey on physiological quality of seeds of different kinds of vegetables such as carrots, peas, beets, tomato and watermelon industry, marketed and / or used by farmers, it was observed that germination of these seeds do not always fit the minimum standard of marketing required for each species (Nascimento, 1994).

Therefore, failure to stand and seedling vigor at low field level are frequent, with the need for appropriate and sensitive methods to detect these differences in seed quality.

In this context, considering the lack of information concerning the effect of thiamethoxam and the potential benefits that treatment can provide, the present study was to evaluate the influence of thiamethoxam in physiological performance of carrot seeds.

#### **3. Methodology**

4 Insecticides – Basic and Other Applications

The bioactivator acts in the expression of genes responsible for synthesis and activation of metabolic enzymes related to plant growth by altering the production of amino acid precursors of plant hormones. With the increased production of hormones, plant expressed greater vigor, germination and root development. With a greater number of roots, increases the absorption and resistance of plant stomata to water loss, which benefits the metabolism

Thiamethoxam, 3-(2 – chloro – thiazole - 5 - ylmethyl) - 5 - methyl (1,3,5) oxadiazinan - 4 ylidene-N- nitroamine, whose chemical structure is shown in Figure 1, is a systemic insecticide neonicotinoid group, family nitroguanidine, which acts on acetylcholine nicotinic receptor in membrane of insects, damaging the nervous system and causing them to death. It is used successfully in pest control in beginning cycle from different cultures. Due to numerous reports of field observations describing increases in vigor development and productivity, even in the absence of pests, has been considered a product that has

Fig. 1. Chemical structure of thiamethoxam. Source: Oliveira, et al., (2009).

*carota*) indicate benefit effect of thiamethoxam uses.

**2. Bioactivator in physiological performance of carrot seeds** 

With reference to the mechanism of action of thiamethoxam, the molecule has the ability to induce physiological changes in plants. Due to results obtained, it is concluded that this insecticide acts as bioactive two ways. The first, activating carrier proteins of cell membranes allowing greater ion transport by enhancing plant mineral nutrition. This increase in the availability of minerals promotes positive responses in the development and plant productivity. The second is related to increased enzyme activation caused by thiamethoxam at both the seed and the plant, thereby increasing both primary and secondary metabolism. It also increases the synthesis of amino acids, precursors of new proteins and endogenous synthesis of plant hormones. The responses of plants to these proteins and hormone biosynthesis may be related to significant increases in production. Results of research related with soybean (*Glycine max*) (Castro et al., 2008; Cataneo, 2008), rice (*Oryza sativa*) (Clavijo, 2008; Almeida et al.; 2010), cotton (*Gossypium hirsutum* ) (Lauxen et al., 2010), bean (*Phaseolus vulgaris*) (Almeida et al., 2010) e vegetables like, lettuce (*Lactuca sativa*), tomato (*Lycopersicon esculentum*), pumpkin (*Cucurbita pepo* L.) and carrot (*Daucus* 

Carrot (*Daucus carota* L.) is the most economical expression of vegetables among those whose edible portion is the root, and to highlight the nutritional value, as a major vegetable

and increases resistance to stress.

phytotonic effect.

This work was conducted at the Laboratory of Seed Analysis Textbook and greenhouse, Faculty of Agronomy Eliseu Maciel, Universidade Federal de Pelotas (UFPel), Pelotas / Brazil.

Seed lots of carrot cultivar Brasilia represented by four lots.

To establishment concentrations of thiamethoxam, it was used the following concentrations: 0.0, 0.05, 0.1, 0.2, 0.4, 0.8 mL / L, based on germination test three were selected. Tests conducted to evaluate the quality of seeds were performed with and without water stress.

The seeds were treated in a plastic bag containing distilled water at 0.1 mL of water to 0.05 mL and 0.4 mL of product thiamethoxam to 3g of carrot seeds. The product was applied directly to the bottom of the plastic bag before putting the seeds. Then the seeds were placed in plastic bag and mixed until uniform coating of the seeds. It was used a volume of mixture (product + water) sufficient to promote a more even distribution of product on the seeds. For measurement of product and distilled water were used micropipettes.

Water stress was achieved by the water potential of -0.4 MPa, using aqueous solutions of polyethylene glycol (PEG 6000). The calculation of the solute quantities was performed

Insecticide Thiamethoxam: A Bioactive Action on Carrot Seeds (*Daucus carota* L.) 7

It is noted in Figure 1 that germination of four seed lots, without (Figure 1A) and with (Figure 1B) water stress, treated with thiamethoxam showed significant difference compared to control. Increases in germination were marked and varied according to the lots from 5 to 23 percentage points if the seeds have not been subjected to water stress and 4 to

In Figure 1B it appears that water stress reduced the percentage germination of seed lots. Lots 1 and 3 not treated, after the water stress reached below the standard of marketing, however, treatment stimulated germination of seeds and lots reached the minimum germination (70%) of the standard marketing. In soybean seeds was also observed that thiamethoxam accelerates germination, induces more growth of the embryonic axis and minimize the negative effects in situations of presence of aluminum, salinity and water

There is a trend of germination of treated lots with different concentrations of the product showed similar results, with the exception of Lot 3, in which the concentration of 0.4 mL / L

\* \*

\* \* \*

1234 **Lotes**

Lots

\* \* \* \* \*

1234 **Lotes**

Lots

(B) Fig. 1. Germination (%) of four seed lots of carrot, cultivar Brasilia, submitted (A) or not (B) to water stress. \* Different from the control by Dunnet test at probability level of 5%.

(A)

\*

\* \*

0,4mL/l 0,05mL/l 0,0mL/l

0,4mL/l 0,05mL/l 0,0mL/l

\*

\* \*

40

50

60

70

**Germinacão (%)**

Germination (%)

80

90

100

**Germinacão (%)**

Germination (%)

15 to be subjected to stress.

deficiency (Cataneo et al., 2006).

was more efficient.

according to Villela et al. (1991). Polyethylene glycol solutions, thus obtained were applied to the paper substrate, in an amount equivalent to 2.5 times its dry weight in all parameters evaluated in the laboratory involving the germination test.

To evaluate the physiological quality of seeds were conducted the following tests:

Germination: it was used four replications of 50 seeds of each batch distributed in transparent plastic boxes (gerbox) on two sheets of white blotter paper, moistened 2.5 times the weight of paper, placed in a germination chamber set to maintain the temperature constant 25 º C. Counts were made in the seventh and fourteenth days after sowing, and assessments, carried out according to ISTA (2010) by computing the percentages of normal seedlings.

Accelerated aging: conducted with 4.0 g of seeds distributed in wire screen suspended and placed inside plastic boxes, type gerbox (mini-camera). Inside the germination boxes were placed 40 mL of water and then the boxes were taken to an incubator set at a constant temperature of 41° C for 48 hours and subsequently subjected to germination tests, as described above. The evaluation was performed seven days after sowing, by computing the percentage of normal seedlings.

Root length: four replicates of 50 seeds were sown on a line drawn in the upper third part of paper substrate. The rolls containing the seeds remained at 25 ° C for seven days, after being evaluated, the root length of normal seedlings, with the aid of a millimeter ruler. The root length was obtained by adding the measurements of each replicate and dividing by the number of seedlings, with results expressed in centimeters.

Speed of germination: performed according to the methodology of the germination test, determined by daily counts to stabilize the number of seedlings in the test and the speed calculation made according to Maguire (1962).

Emergence of seedlings in the greenhouse: four replications of 50 seeds were distributed in individual cells of polystyrene trays (Styrofoam), containing commercial substrate Plantimax ®. The trays were kept in the greenhouse and evaluations were performed at 16 days after sowing, counting seedlings in length and more than 1.0 cm. The results were expressed as a percentage of emergence.

Statistical procedure: completely randomized factorial 4x3 (four lots and three concentrations of the product) separately in the evaluation with and without water stress, with three replications. For comparison of means between control and concentrations, it was used Dunnet test, probability level of 5%.

#### **4. Results establishment of concentrations**

The concentrations selected based on the result of germination test of treated seeds with different concentrations of the product thiamethoxam, beyond control were 0.05 and 0.4 mL.

Germination of treated seeds in accordance with product concentrations were 70% (zero), 75% (0.05 mL / L), 72% (0.1 mL / L), 72% (0.2 mL / L), 75% (0.4 mL / L) and 70% (0.8 mL / L) without stress. The choice of the concentration of 0.05 mL / L was based on the fact that seeds showed germination similar to other concentrations and spend less of the product. On the other hand, the concentration of 0.4 mL / L was selected because germination test were seedlings showed well developed, open cotyledons and normal roots. At the concentration 0.8 mL / L was found that the seedlings were developed, but their roots had necrosis.

Statistical analysis performed by Dunnet's test showed significant results for comparison of means between control and concentrations of all parameters.

according to Villela et al. (1991). Polyethylene glycol solutions, thus obtained were applied to the paper substrate, in an amount equivalent to 2.5 times its dry weight in all parameters

Germination: it was used four replications of 50 seeds of each batch distributed in transparent plastic boxes (gerbox) on two sheets of white blotter paper, moistened 2.5 times the weight of paper, placed in a germination chamber set to maintain the temperature constant 25 º C. Counts were made in the seventh and fourteenth days after sowing, and assessments, carried

Accelerated aging: conducted with 4.0 g of seeds distributed in wire screen suspended and placed inside plastic boxes, type gerbox (mini-camera). Inside the germination boxes were placed 40 mL of water and then the boxes were taken to an incubator set at a constant temperature of 41° C for 48 hours and subsequently subjected to germination tests, as described above. The evaluation was performed seven days after sowing, by computing the

Root length: four replicates of 50 seeds were sown on a line drawn in the upper third part of paper substrate. The rolls containing the seeds remained at 25 ° C for seven days, after being evaluated, the root length of normal seedlings, with the aid of a millimeter ruler. The root length was obtained by adding the measurements of each replicate and dividing by the

Speed of germination: performed according to the methodology of the germination test, determined by daily counts to stabilize the number of seedlings in the test and the speed

Emergence of seedlings in the greenhouse: four replications of 50 seeds were distributed in individual cells of polystyrene trays (Styrofoam), containing commercial substrate Plantimax ®. The trays were kept in the greenhouse and evaluations were performed at 16 days after sowing, counting seedlings in length and more than 1.0 cm. The results were

Statistical procedure: completely randomized factorial 4x3 (four lots and three concentrations of the product) separately in the evaluation with and without water stress, with three replications. For comparison of means between control and concentrations, it was

The concentrations selected based on the result of germination test of treated seeds with different concentrations of the product thiamethoxam, beyond control were 0.05 and 0.4

Germination of treated seeds in accordance with product concentrations were 70% (zero), 75% (0.05 mL / L), 72% (0.1 mL / L), 72% (0.2 mL / L), 75% (0.4 mL / L) and 70% (0.8 mL / L) without stress. The choice of the concentration of 0.05 mL / L was based on the fact that seeds showed germination similar to other concentrations and spend less of the product. On the other hand, the concentration of 0.4 mL / L was selected because germination test were seedlings showed well developed, open cotyledons and normal roots. At the concentration 0.8 mL / L was found that the seedlings were developed, but their roots had necrosis. Statistical analysis performed by Dunnet's test showed significant results for comparison of

To evaluate the physiological quality of seeds were conducted the following tests:

out according to ISTA (2010) by computing the percentages of normal seedlings.

evaluated in the laboratory involving the germination test.

number of seedlings, with results expressed in centimeters.

calculation made according to Maguire (1962).

expressed as a percentage of emergence.

used Dunnet test, probability level of 5%.

mL.

**4. Results establishment of concentrations** 

means between control and concentrations of all parameters.

percentage of normal seedlings.

It is noted in Figure 1 that germination of four seed lots, without (Figure 1A) and with (Figure 1B) water stress, treated with thiamethoxam showed significant difference compared to control. Increases in germination were marked and varied according to the lots from 5 to 23 percentage points if the seeds have not been subjected to water stress and 4 to 15 to be subjected to stress.

In Figure 1B it appears that water stress reduced the percentage germination of seed lots. Lots 1 and 3 not treated, after the water stress reached below the standard of marketing, however, treatment stimulated germination of seeds and lots reached the minimum germination (70%) of the standard marketing. In soybean seeds was also observed that thiamethoxam accelerates germination, induces more growth of the embryonic axis and minimize the negative effects in situations of presence of aluminum, salinity and water deficiency (Cataneo et al., 2006).

There is a trend of germination of treated lots with different concentrations of the product showed similar results, with the exception of Lot 3, in which the concentration of 0.4 mL / L was more efficient.

Fig. 1. Germination (%) of four seed lots of carrot, cultivar Brasilia, submitted (A) or not (B) to water stress. \* Different from the control by Dunnet test at probability level of 5%.

Insecticide Thiamethoxam: A Bioactive Action on Carrot Seeds (*Daucus carota* L.) 9

balance of the plant, tolerating water deficit better (Castro, 2006). As observed in soybean root development increases the absorption of nutrients, increases the expression of leaf area

The data speed of germination, without (Figure 5A) and with (Figure 5B) stress show that the treated seeds had a higher rate compared to control. The concentrations used had similar results. Treated seeds germinated on average one day soon if they have not been subjected to water stress and two days are subject to stress. This effect is very promising because carrot seeds in field conditions have poor germination, slow and irregular resulting in uneven emergence (Corbineau et al., 1994). This increased speed of germination is caused by physiological changes that occur in the plant indirectly stimulating the production of hormones, resulting in increased vigor, root growth, water absorption and primary and

secondary metabolism, as observed in the sugarcane crop (Castro, 2007).

\* \* \* \* \* \* \* \*

0,4mL/l 0,05mL/l 0,0mL/l

> 0,4mL/l 0,05mL/l 0,0mL/l

1234

**Lotes**

Lots

(A)

\* \* \* \* \* \* \* \*

1234

**Lotes**

Lots

(B) Fig. 3. Root length (cm) of seedlings of four seed lots of carrot, cultivar Brasilia, without (A) and with (B) water stress. \* Different from the control by Dunnet test at probability level

and plant vigor (Tavares and Castro, 2005).

**Comprimento** 

**Comprimento** 

Root length (cm) Root length (cm)

of 5%.

**radicular (cm)**

**radicular (cm)**

According to Figure 2, germination after accelerated aging of treated seeds without (Figure 2A) and with (Figure 2B) water stress showed significant differences related to control. Positive difference varied according of lots, 2 to 11 percentage points in seed not submitted to stress and 2 to 9 in submitted to water stress. This superiority resistance occurs because thiamethoxam move through plant cells and actives several physiological reactions, such as functional protein expression related with plant defense mechanism avoid stress factors like drought, high temperatures, toxic effects, among others, improving productivity, leaf and radicular area, as found in soybean seed (Tavares e Castro, 2005).

Concentrations showed positive results in situations with and without water stress, but the concentration of 0.4 mL / L performed better for lots 2 and 3, without stress and 2, 3 and 4 with stress.

(B)

Fig. 2. Accelerated aging (%) of four seed lots of carrot, cultivar Brasilia, without (A) and with (B) water stress.\* It differs from the control by Dunnet test at probability level of 5%.

According to data presented in Figures 3 and 4, treated seeds showed marked differences in root length compared to untreated, on average 4 cm, in both cases without (Figure 3 and 4 A) and with (Figures 3 and 4 B) water stress. This effect of thiamethoxam of supporting the growth of the root system, confirming the effect of rooting observed by Pereira et al. (2007) in sugar cane and potatoes; and also by Tavares et al. (2007) in soybean. It is believed that the thiamethoxam increase water uptake and stomatal resistance, improving the water

According to Figure 2, germination after accelerated aging of treated seeds without (Figure 2A) and with (Figure 2B) water stress showed significant differences related to control. Positive difference varied according of lots, 2 to 11 percentage points in seed not submitted to stress and 2 to 9 in submitted to water stress. This superiority resistance occurs because thiamethoxam move through plant cells and actives several physiological reactions, such as functional protein expression related with plant defense mechanism avoid stress factors like drought, high temperatures, toxic effects, among others, improving productivity, leaf and

Concentrations showed positive results in situations with and without water stress, but the concentration of 0.4 mL / L performed better for lots 2 and 3, without stress and 2, 3 and 4

\* \* \* \* \* \* \* \*

0,4mL/l 0,05mL/l 0,0mL/l

0,4mL/l 0,05mL/l 0,0mL/l

1234

**Lotes**

Lots

(A)

\* \* \* \* \* \* \* \*

1234

**Lotes**

Lots

(B) Fig. 2. Accelerated aging (%) of four seed lots of carrot, cultivar Brasilia, without (A) and with (B) water stress.\* It differs from the control by Dunnet test at probability level of 5%. According to data presented in Figures 3 and 4, treated seeds showed marked differences in root length compared to untreated, on average 4 cm, in both cases without (Figure 3 and 4 A) and with (Figures 3 and 4 B) water stress. This effect of thiamethoxam of supporting the growth of the root system, confirming the effect of rooting observed by Pereira et al. (2007) in sugar cane and potatoes; and also by Tavares et al. (2007) in soybean. It is believed that the thiamethoxam increase water uptake and stomatal resistance, improving the water

radicular area, as found in soybean seed (Tavares e Castro, 2005).

**Envelhecimento** 

**Envelhecimento** 

**acelerado (%)**

Accelerated aging (%)

**acelerado (%)**

Accelerated aging (%)

with stress.

balance of the plant, tolerating water deficit better (Castro, 2006). As observed in soybean root development increases the absorption of nutrients, increases the expression of leaf area and plant vigor (Tavares and Castro, 2005).

The data speed of germination, without (Figure 5A) and with (Figure 5B) stress show that the treated seeds had a higher rate compared to control. The concentrations used had similar results. Treated seeds germinated on average one day soon if they have not been subjected to water stress and two days are subject to stress. This effect is very promising because carrot seeds in field conditions have poor germination, slow and irregular resulting in uneven emergence (Corbineau et al., 1994). This increased speed of germination is caused by physiological changes that occur in the plant indirectly stimulating the production of hormones, resulting in increased vigor, root growth, water absorption and primary and secondary metabolism, as observed in the sugarcane crop (Castro, 2007).

Fig. 3. Root length (cm) of seedlings of four seed lots of carrot, cultivar Brasilia, without (A) and with (B) water stress. \* Different from the control by Dunnet test at probability level of 5%.

Insecticide Thiamethoxam: A Bioactive Action on Carrot Seeds (*Daucus carota* L.) 11

\*

1234

**Lotes**

\* \* \* \* \* \* \* \*

1234

**Lotes**

(B)

Fig. 5. Speed of germination (days) of four seed lots of carrot cultivar Brasilia, without (A) and with (B) water stress.\* It differs from the control by Dunnet test at probability level of

(A)

\* \* \*

\*

\*

0,4mL/l 0,05mL/l 0,0mL/l

0,4mL/l 0,05mL/l 0,0mL/l

\* \*

**Velocidade de** 

5%.

**germinacão (dias)**

**Velocidade de** 

**germinacão (dias)**

(A)

Fig. 4. Root length (cm) of seedlings of four seed lots of carrot, cultivar Brasilia, without (A) and with (B) water stress.

(A)

Fig. 4. Root length (cm) of seedlings of four seed lots of carrot, cultivar Brasilia, without (A)

mL of product/ 3g of seed

and with (B) water stress.

(A)

Fig. 5. Speed of germination (days) of four seed lots of carrot cultivar Brasilia, without (A) and with (B) water stress.\* It differs from the control by Dunnet test at probability level of 5%.

Insecticide Thiamethoxam: A Bioactive Action on Carrot Seeds (*Daucus carota* L.) 13

In Figure 6, without (Figure 6A) and with (Figure 6B) water stress, it was observed that the emergence of seedlings in the greenhouse was stimulated, and the seeds treated with thiamethoxam showed significant differences compared to control. The positive differences compared to control vary according to lots, 9 to 17 percentage points if the seeds have not been subjected to water stress and 20 to 10 percentage points when subjected to stress. The two concentrations showed similar responses. These results confirm those found in soybean, to be seen increase in the root system and the percentage of seedling emergence also in water deficit conditions (Castro et al., 2006). According to the literature, soybean seeds treated with thiamethoxam have higher levels of amino acids, enzyme activity and synthesis of plant hormones that increase the plant responses to these proteins and these events provide significant increases in production and reducing the time of establishment of culture in the field, making it more tolerant to stress

The results obtained can be described that the product stimulated the performance of carrot seeds in all parameters evaluated, both in seeds subjected to water stress or not. Carrot seeds treated with the product thiamethoxam showed significant increases in germination and vigor for all lots. Among the aspects of vigor, the product stimulated the growth of the root length, which is of great importance to the culture of carrots and this result was

The product was more effective in stimulating the quality of seeds not subjected to water stress, with the exception of root length which positive change was similar for seeds subjected to stress or not. In all parameters evaluated, increases in the quality varied according to the lot. Concentrations of the product for most tests evaluated did not differ,

The application of thiamethoxam has strong interest for the culture of carrot, whose edible portion is the root and, moreover, by presenting, in field conditions, poor germination, slow, irregular with uneven emergence, the product acts as an enhancer, by allowing the expression of seed germination potential, accelerate the growth of roots and increase the absorption of nutrients by the plant. These features of thiamethoxam combined with the use of genetics and physiological high-quality seed powers the productive capacity of the

Thiamethoxam product stimulates the physiological performance of carrot seeds subjected

Concentrations of 0.05 and 0.4 mL of the product is effective, however there is a tendency of

ALMEIDA, A.S.; TILLMANN, M.A.A.; VILLELA, F. A.; PINHO, M.S. Bioativador no

desempenho fisiológico de sementes de cenoura. Revista Brasileira de Sementes,

factors (Castro, 2006).

culture.

**5. Conclusions** 

**6. References** 

obtained in the laboratory confirmed in the greenhouse.

however there was a trend of higher concentration to the higher values.

to water stress or not, with variable intensity according to lot.

higher concentration to the higher increases in quality.

Brasília,v.31, n. 3, p. 87-95, 2009.

(A)

Fig. 6. Emergence of seedlings in the greenhouse for four seed lots of carrot, cultivar Brasilia without (A) and with (B) water stress. \* Different from the control by Dunnet test at probability level of 5%.

In Figure 6, without (Figure 6A) and with (Figure 6B) water stress, it was observed that the emergence of seedlings in the greenhouse was stimulated, and the seeds treated with thiamethoxam showed significant differences compared to control. The positive differences compared to control vary according to lots, 9 to 17 percentage points if the seeds have not been subjected to water stress and 20 to 10 percentage points when subjected to stress. The two concentrations showed similar responses. These results confirm those found in soybean, to be seen increase in the root system and the percentage of seedling emergence also in water deficit conditions (Castro et al., 2006). According to the literature, soybean seeds treated with thiamethoxam have higher levels of amino acids, enzyme activity and synthesis of plant hormones that increase the plant responses to these proteins and these events provide significant increases in production and reducing the time of establishment of culture in the field, making it more tolerant to stress factors (Castro, 2006).

The results obtained can be described that the product stimulated the performance of carrot seeds in all parameters evaluated, both in seeds subjected to water stress or not. Carrot seeds treated with the product thiamethoxam showed significant increases in germination and vigor for all lots. Among the aspects of vigor, the product stimulated the growth of the root length, which is of great importance to the culture of carrots and this result was obtained in the laboratory confirmed in the greenhouse.

The product was more effective in stimulating the quality of seeds not subjected to water stress, with the exception of root length which positive change was similar for seeds subjected to stress or not. In all parameters evaluated, increases in the quality varied according to the lot. Concentrations of the product for most tests evaluated did not differ, however there was a trend of higher concentration to the higher values.

The application of thiamethoxam has strong interest for the culture of carrot, whose edible portion is the root and, moreover, by presenting, in field conditions, poor germination, slow, irregular with uneven emergence, the product acts as an enhancer, by allowing the expression of seed germination potential, accelerate the growth of roots and increase the absorption of nutrients by the plant. These features of thiamethoxam combined with the use of genetics and physiological high-quality seed powers the productive capacity of the culture.

#### **5. Conclusions**

12 Insecticides – Basic and Other Applications

\* \* \* \* \* \* \* \*

1234

0,4mL/l 0,05mL/l 0,0mL/l

0,4mL/l 0,05mL/l 0,0mL/l

**Lotes**

Lots

(A)

\* \* \* \* \* \* \*

1234

**Lotes**

Lots

Fig. 6. Emergence of seedlings in the greenhouse for four seed lots of carrot, cultivar Brasilia

without (A) and with (B) water stress. \* Different from the control by Dunnet test at

\*

**Emergência em casa** 

**Emergência em casa** 

Emergence of seedlings in

probability level of 5%.

**de vegetacão (%)** 

the greenhouse (%)

**de vegetacão (%)** 

Emergence of seedlings in the greenhouse (%)

Thiamethoxam product stimulates the physiological performance of carrot seeds subjected to water stress or not, with variable intensity according to lot.

Concentrations of 0.05 and 0.4 mL of the product is effective, however there is a tendency of higher concentration to the higher increases in quality.

#### **6. References**

ALMEIDA, A.S.; TILLMANN, M.A.A.; VILLELA, F. A.; PINHO, M.S. Bioativador no desempenho fisiológico de sementes de cenoura. Revista Brasileira de Sementes, Brasília,v.31, n. 3, p. 87-95, 2009.

Insecticide Thiamethoxam: A Bioactive Action on Carrot Seeds (*Daucus carota* L.) 15

LAUXEN, L.R.; VILLELA, F. A.; SOARES, R. C. Desempenho fisiológico de sementes de

LUBUS, C.A.F.; FERRAZ, J.A.D.P.; CALAFIORI, M.H.; ZAMBON, S.; BUENO, B.F. Ensaio

MAGUIRE, J.D Speed of germination and in selection and evaluation for

NUNES, J.C. Bioativador de plantas: uma utilidade adicional para um produto

OLIVEIRA, V.S.; LIMA, J.M.; CARVALHO, R.F.; RIGITANO, R.L.O. Absorção do inseticida

PEREIRA, M.A.; CASTRO, P.R.C.; GARCIA, E.O; REIS, A. R. Efeitos fisiológicos de

REDDY, K.R.; REDDY, V.R.; BAKER, D.N.; McKINION, J.M. Effects of aldicarb on

REDDY, K.R.; REDDY, V.R.; BAKER, D.N.; McKINION, J.M. Is aldicarb a plant growth

TAVARES, S.; CASTRO, P.R.C.; RIBEIRO, R.V.; ARAMAKI, P.H. Avaliação dos efeitos

TAVARES, S.; CASTRO, P.R.C. Avaliação dos efeitos fisiológicos de Cruiser 35FS após

TEIXEIRA, N.T.; ZAMBON, S.; BOLLELA, E.R,; NAKANO; OLIVEIRA, D.A; CALAFIORI,

VILLELA, F.A; DONI-FILHO,L,; SEQUEIRA,E.L. Tabela de potencial osmótico em função

Brasileira, Brasília, v.26,n.11/12,p.1957-1968, 1991.

3, p. 61-68 , 2010.

10, p. 64-66, 1985.

p.30-31, 2006.

Vegetal, 2007.

American, p.79-80, 1990.

v.16, p.120-125, 1991.

Agricultura, Piracicaba, 2007.

1989.

2005.

Lavras, v. 32, n. 6, p. 1432-1435, 2009.

1962.

algodão tratadas com tiametoxam. Revista Brasileira de Sementes. Brasília, v. 32, n.

com diferentes dosagens de aldicard e de adubo visando a produtividade na cultura da batata (*Solarium tuberosum* L.), Ecossistema, Espírito Santo do Pinhal, v.

seedling emergence and vigor. Crop Science, Madison, v.2, n.2, p.176-177,

desenvolvido originalmente como inseticida. Revista SEEDNews, Pelotas, v.10, n.5,

tiametoxam em latossolos sob efeito de fosfato e vinhaça. Revista Química Nova,

Thiametoxan em plantas de feijoeiro. In: XI CONGRESSO BRASILEIRO DE FISIOLOGIA VEGETAL, Resumos, Gramado: Sociedade Brasileira de Fisiologia

photosynthesis, root growth and flowering of cotton. In: PLANT GROWTH REGULATION SOCIETY OF AMERICAN ANNUAL MEETING, 16., Arlington. Proceedings… Arligton: Plant Regulation Society of American, p.168-169,

regulator. In PLANT GROWTH REGULATION SOCIETY OF AMERICAN ANNUAL MEETING, 17., Proceedings… Saint Paul: Plant Regulation Society of

fisiológicos do tiametoxam no tratamento de sementes de soja. Revista da

tratamento de sementes de soja. In: ESCOLA SUPERIOR DE AGRICULTURA "LUIZ DE QUEIROZ". Relatório técnico ESALQ/Syngenta Piracicaba, p. 1-13,

M.H. Adubação e aldicarb influenciando os teores de N, P e K, nas folhas da cultura da batata (*Solarium tuberosum* L). Ecossistema, Espírito Santo do Pinhal,

da concentração de polietileno glicol 6000 e da temperatura. Pesquisa Agropecuária


ANANIA, F.R.; TEIXEIRA, N.T.; CALAFIORI, M.H.; ZAMBON,S. Influência de inseticidas

*hypogaea* L.) Ecossistema, Espírito Santo do Pinhal, v. 13, p. 121-124, 1988a. ANANIA, P,F.R.; TEIXEIRA, N.T.; CALAFIORI, M.H.; ZAMBON,S. Influência de inseticidas

CALAFIORI, M.H; TEIXEIRA, N.T; SCHMIDT, H A P.; ANANIA, P.F.R.; GRANDO, F.I.;

CASTRO, P.R.C.; PITELLI, A M.C.M.; PERES, L.E.P.; ARAMAKI, P.H. Análise da atividade hormonal de thiametoxam através de biotestes. Publicatio, UEPG, 2007 CASTRO, P.R.C. Agroquimicos de controle hormonal na agricultura tropical. Boletim, n.32,

CASTRO, P.R.C.; PITELLI, AM.C.M.; PERES, L.E.P. Avaliação do crescimento da raiz e parte

CASTRO, P.R.C.; SOARES, F.C.; ZAMBON, S.; MARTINS, A N.; Efeito do aldicarb no

CATANEO, A C.; ANDRÉO, Y.; SEIFFERT, M.; BÚFALO,J.; FERREIRA,L.C. Ação do

CORBINEAU, F.; PICARDE, M.A.; CÔME, D. Effects of temperature, oxigen and osmotic

De GRANDE, P.E. Influência de aldicarb e carbofuran na soja (*Glycine max* L.) Merrill. 137f.

DENARDIN, N.D. Ação do thiametoxan sobre a fixação biológica do nitrogênio e na

HORII, A; McCUE, P.; SHETTY, K. Enhancement of seed vigour following and phenolic

JUNQUEIRA, F.M.A; FORNER, M.A; CALAFIORI, M.H.; TEIXEIRA, N.T.; ZAMBON, S.;

Série Produtor Rural, USP/ ESALQ/ DIBD, Piracicaba, 46p., 2006.

Espírito Santo do Pinhal, v.14.p. 132-14, 1989.

Horticulturae, The Hague, v.354, p.9-15, 1994.

Relatório técnico, Passo Fundo, 2005.

Santo do Pinhal, v. 13, p. 101-107, 1988.

Queiroz", Universidade de São Paulo, Piracicaba, 1992.

Piracicaba, p.14-25, 2005.

Pinhal, v.20, p. 63-68, 1995.

1988b.

2006.

2007.

granulados sistêmicos nos teores de N-P-K nas folhas de amendoim (*Arachis* 

granulados sistêmicos nos teores de N-P-K nas folhas de limoeiro Taiti (*Citrus aurantifolia*.) cv. Peruano. Ecossistema, Espírito Santo do Pinhal, v. 13, p. 121-124,

PALAZZINI, R.; MARTINS, R.C.; OLIVEIRA, C.L.; ZAMBON, S. Efeitos nutricionais de inseticidas sistêmicos granulados sobre cafeeiros. Ecossistema.

aérea de plântulas de tomateiro MT, DGT E BRT germinadas em diferentes concentrações do inseticida thiametoxan. In ESCOLA SUPERIOR DE AGRICULTURA "LUIZ DE QUEIROZ". Relatório técnico ESALQ/Syngenta.

desenvolvimento do feijoeiro cultivar Carioca. Ecossistema. Espírito Santo do

inseticida Cruiser sobre a germinação do soja em condições de estresse. In: IVCONGRESSO BRASILEIRO DE SOJA, Resumos, Londrina, p.90,

pressure on germination of carrot seeds: evaluation of seed quality. Acta

Dissertação (Mestrado em Entomologia) - Escola Superior de Agricultura "Luiz de

promoção de ativadores de crescimento vegetal. In: Universidade de Passo Fundo.

elicitor treatment. Bioresource Technology, United States, v.98, n.3, p.623-632,

Aplicação de aldicarb em diferentes dosagens e tipos de adubação influenciando a produtividade na cultura da batata (*Solarium tuberosum* L.). Ecossistema, Espírito


**2** 

*Brazil* 

**The Pyrethroid Knockdown Resistance** 

New promising insect control efforts are now being evaluated such as biological alternatives or even transgenic insects and *Wolbachia* based strategies. Although it is increasingly clear that successful approaches must involve integrated actions, chemical insecticides unfortunately still play a central role in pest and vector control (Raghavendra et al., 2011). Development of new safe and effective compounds in conjunction with preservation of those currently being utilized are important measures to insure insecticide availability and efficiency for arthropod control. In this sense, understanding the interaction of insecticides with the insect organism (at physiological and molecular levels), the selected resistance

Pyrethroids are synthetic compounds derived from pyrethrum, present in *Chrysanthemum* flowers. Currently, pyrethroids are the most used insecticides against arthropod plagues in agriculture and livestock as well as in the control of vectors of veterinary and human health importance. They are chemically distinguished as type I (such as permethrin, compounds that lack an alpha-ciano group) and type II (with an alpha-ciano group, like deltamethrin) (T. G. Davies et al., 2007b). Pyrethroid insecticides have been largely adopted against vector mosquitoes through indoor, perifocal or ultra-low volume (ULV) applications. As of yet pyrethroids are the only class of insecticides approved for insecticide treated nets (ITNs), an important tool under expansion against malaria, mainly in the African continent (Ranson et al., 2011). The consequence of intense and uncontrolled pyrethroid use is the extremely

Just like DDT, pyrethroids act very fast in the central nervous system of the insects, leading to convulsions, paralysis and eventually death, an effect known as *knockdown*. However, unlike DDT, pyrethroids are not claimed to cause severe risks to the environment or to animal or human health, hence its widespread use. The main pyrethroid resistance mechanism (the knockdown resistance phenotype, *kdr*) occurs due to a point mutation in the voltage gated

Herein we aim to discuss the main mechanism of pyrethroid resistance, the knockdown resistance (*kdr*) mutation, its effect and its particularities among arthropods. The most common methods presently employed to detect the *kdr* mutation are also discussed. Some aspects regarding the other main pyrethroid resistance mechanisms, like alterations in behaviour, cuticle and detoxifying enzymes will be only briefly addressed. The proposal of this chapter is to review knockdown resistance to pyrethroids, nowadays the preferred insecticide class worldwide. This topic discusses aspects of general biology, physiology,

sodium channel in the central nervous system, the target of pyrethroids and DDT.

mechanisms and their dynamics in and among natural populations is obligatory.

rapid selection of resistant populations throughout the world.

**1. Introduction** 

Ademir Jesus Martins and Denise Valle *Fundação Oswaldo Cruz/ Instituto Oswaldo Cruz/* 

*Laboratório de Fisiologia e Controle de Artrópodes Vetores* 

WHEATON, T. A; CHILDERS, C.C.; TIMMER, L.W.; DUNCAN, L.W.; NIKDEL, S. Effects of aldicarb on the production, quality of fruits and situation of citrus plants in Florida. Proceedings of the Florida State for Horticultural Society, Tallahasse, v. 98, p. 6-10, 1985.

### **The Pyrethroid Knockdown Resistance**

Ademir Jesus Martins and Denise Valle

*Fundação Oswaldo Cruz/ Instituto Oswaldo Cruz/ Laboratório de Fisiologia e Controle de Artrópodes Vetores Brazil* 

#### **1. Introduction**

16 Insecticides – Basic and Other Applications

WHEATON, T. A; CHILDERS, C.C.; TIMMER, L.W.; DUNCAN, L.W.; NIKDEL, S. Effects of

1985.

aldicarb on the production, quality of fruits and situation of citrus plants in Florida. Proceedings of the Florida State for Horticultural Society, Tallahasse, v. 98, p. 6-10,

> New promising insect control efforts are now being evaluated such as biological alternatives or even transgenic insects and *Wolbachia* based strategies. Although it is increasingly clear that successful approaches must involve integrated actions, chemical insecticides unfortunately still play a central role in pest and vector control (Raghavendra et al., 2011). Development of new safe and effective compounds in conjunction with preservation of those currently being utilized are important measures to insure insecticide availability and efficiency for arthropod control. In this sense, understanding the interaction of insecticides with the insect organism (at physiological and molecular levels), the selected resistance mechanisms and their dynamics in and among natural populations is obligatory.

> Pyrethroids are synthetic compounds derived from pyrethrum, present in *Chrysanthemum* flowers. Currently, pyrethroids are the most used insecticides against arthropod plagues in agriculture and livestock as well as in the control of vectors of veterinary and human health importance. They are chemically distinguished as type I (such as permethrin, compounds that lack an alpha-ciano group) and type II (with an alpha-ciano group, like deltamethrin) (T. G. Davies et al., 2007b). Pyrethroid insecticides have been largely adopted against vector mosquitoes through indoor, perifocal or ultra-low volume (ULV) applications. As of yet pyrethroids are the only class of insecticides approved for insecticide treated nets (ITNs), an important tool under expansion against malaria, mainly in the African continent (Ranson et al., 2011). The consequence of intense and uncontrolled pyrethroid use is the extremely rapid selection of resistant populations throughout the world.

> Just like DDT, pyrethroids act very fast in the central nervous system of the insects, leading to convulsions, paralysis and eventually death, an effect known as *knockdown*. However, unlike DDT, pyrethroids are not claimed to cause severe risks to the environment or to animal or human health, hence its widespread use. The main pyrethroid resistance mechanism (the knockdown resistance phenotype, *kdr*) occurs due to a point mutation in the voltage gated sodium channel in the central nervous system, the target of pyrethroids and DDT.

> Herein we aim to discuss the main mechanism of pyrethroid resistance, the knockdown resistance (*kdr*) mutation, its effect and its particularities among arthropods. The most common methods presently employed to detect the *kdr* mutation are also discussed. Some aspects regarding the other main pyrethroid resistance mechanisms, like alterations in behaviour, cuticle and detoxifying enzymes will be only briefly addressed. The proposal of this chapter is to review knockdown resistance to pyrethroids, nowadays the preferred insecticide class worldwide. This topic discusses aspects of general biology, physiology,

The Pyrethroid Knockdown Resistance 19

resistance remain to be elucidated. With respect to pyrethroid resistance, recent evidences point to an increase in the levels of expression of two cuticle genes in populations of two

The increased ability to detoxify insecticides is one of the main types of resistance, commonly referred to as metabolic resistance. It takes place when the activity of naturally detoxifying enzymes is enhanced, impeding the insecticide to reach its target. Among these enzymes, Multi function Oxidases (or Monoxigenases P450), Esterases and Glutathion-S-Transferases (GST) (ffrench-Constant et al., 2004; Hemingway & Ranson, 2000) are the major representative families. Although the molecular basis of metabolic resistance has been extensively studied, only few reports have investigated the specific metabolic pathways involved or their location in the insect organism. Many different mutations may be attributed to metabolic resistance, such as those leading to production of more enzymes, via gene duplication events or either increases in gene transcription rates, alterations in the normal tissue/time specificity of expression, point mutations leading to a gain of function or changes in the substrate specificity (ffrench-Constant et al., 2004; Hemingway et al., 2004; Perry et al., 2011). Detoxifying enzymes belong to superfamilies composed of numerous genes (Ranson et al., 2002), and it is not unusual for different enzymes to produce the same metabolites. Additionally, an alteration in one type of enzyme may lead to cross-resistance among different classes of insecticides (Ranson et al., 2011). However, population genetic markers that make feasible a complete diagnostic of the resistance mechanisms or their distribution are not yet available. Current studies are generally based on biochemical assays (Valle et al., 2006) and, to a lesser extent, on *microarray detox chips* (David et al., 2005; Vontas et al., 2007). Due to technical limitations, the most common reports are hence oriented to single gene responses, such as punctual mutations that increase the ability of a specific

enzyme in detoxifying an insecticide (Lumjuan et al., 2011; Morin et al., 2008).

(Y. Y. Yang et al., 2010).

Constant et al., 1998; Raymond et al., 2001).

Multi function P450 Oxidases are the enzymes most commonly associated to metabolic resistance to pyrethroids. However, despite much indirect evidence of P450 total activity increase or even detection of higher expression of some related genes (*cyp*), little is known about their metabolic activity. For instance, 111 genes code for P450 in *Anopheles gambiae*, but only two (*cyp6p3* and *cyp6m2*) were described to be involved in pyrethroid metabolism (Muller et al., 2008). Surprisingly, metabolic resistance can still vary during the course of the day. This is the case of an *Ae. aegypti* population whose resistance to the pyrethroid permethrin is mediated by the *cyp9M9* gene. Expression of this gene is regulated by transcriptional factors enrolled in the circadian rhythm of the insect, varying along the day

Finally, phenotypic or target site resistance is designated by modification of the insect molecule where the insecticide binds, inhibiting its effects. Neurotoxic insecticides have as their ultimate target different molecules from the insect central nervous system: the enzyme Acetylcholinesterase (for organophosphates and carbamates), the gama-aminobutiric acid receptor (for ciclodienes), the nicotinic acetylcholine receptors (for spinosyns and neonicotinoids) and the voltage gated sodium channel (for DDT and pyrethroids). Although the mutated target molecule decreases or even abolishes its affinity for the insecticide, it is essential that this alteration does not result in loss of function regarding the insect physiological processes. Since the classical target molecules are much conserved among animals, few mutations are permissive to guarantee the viability of their carriers (ffrench-

*Anopheles* species (Awolola et al., 2009; Vontas et al., 2007).

biochemistry, genetics and evolution, with focus on disease vector mosquitoes. It is expected that the amount and diversity of material available on this subject may well illustrate insecticide resistance in a broader context.

#### **2. Insecticide resistance mechanisms**

Besides the resistance to chemical insecticides caused by modifications in the target site (also called phenotypic resistance), other mechanisms commonly associated are: metabolic resistance, behavioral modification and alterations in the integument. In the first case, endogenous detoxifying enzymes become more efficient in metabolizing the insecticide, preventing it from reaching its target in the nervous system. This occurs due to 1) increase in the number of available molecules (by gene amplification or expression activation) or 2) mutation in the enzyme coding portion of the gene, so that its product metabolizes the insecticide more efficiently. These processes can be very complex and involve three major enzyme superfamilies: Esterases, Multi function Oxidases P450 and Glutathion-S-Transferases (Hemingway & Ranson, 2000; Montella et al., 2007). In contrast, there are few examples in literature regarding insect behavioral changes and tegument alterations.

Resistance to insecticides may be functionally defined as the ability of an insect population to survive exposure to dosages of a given compound that are lethal to the majority of individuals of a susceptible lineage of the same species (Beaty & Marquardt, 1996). Resistance is based on the genetic variability of natural populations. Under insecticide selection pressure, specific phenotypes are selected and consequently increase in frequency. Resistance can result from the selection of one or more mechanisms. In order to elucidate the molecular nature of resistance, many studies report laboratory controlled selection of different species (Chang et al., 2009; Kumar et al., 2002; Paeoporn et al., 2003; Rodriguez et al., 2003; Saavedra-Rodriguez et al., 2007). With selected lineages, it becomes easier to separate the role of each distinct mechanism. In a more direct approach, the current availability of a series of molecular tools enables detection of expression of altered molecules in model organisms so that the effect of the insecticide can be evaluated under specific and controlled circumstances (Smith et al., 1997).

Regardless of the mono or multi-factorial character of resistance, this phenomenon may be didactically divided into four categories: behavioral, cuticular, metabolic and phenotypic resistance. In the first case the insect simply avoids contact with the insecticide through behavioral adaptations, which are presumably related to genetic inheritance (Sparks et al., 1989). Among arthropods, mosquitoes are by far the group most intensely investigated in relation to behavioral resistance (Lockwood et al., 1984). For instance, *Anopheles* malaria vector mosquitoes from the Amazon Region had the habit of resting in the walls after a blood meal. There are registers that some populations changed their behavior after a period of indoor residual application of DDT to the dwelling walls (Roberts & Alecrim, 1991). Behavioral changes that minimize contact between insect and insecticide may cause a severe impact in the insecticide application efficacy, especially if resistance is selected by physiological features (Ranson et al., 2011).

Certain alterations in the insect cuticle may reduce insecticide penetration. However, these effects are unspecific, leading to resistance to a series of xenobiotic compounds. This mechanism is known as reduced penetration or cuticle resistance. It is probably not related to high levels of resistance by itself, but it can interact synergistically with other mechanisms. The physiological processes or molecular pathways which describe this type of

biochemistry, genetics and evolution, with focus on disease vector mosquitoes. It is expected that the amount and diversity of material available on this subject may well illustrate

Besides the resistance to chemical insecticides caused by modifications in the target site (also called phenotypic resistance), other mechanisms commonly associated are: metabolic resistance, behavioral modification and alterations in the integument. In the first case, endogenous detoxifying enzymes become more efficient in metabolizing the insecticide, preventing it from reaching its target in the nervous system. This occurs due to 1) increase in the number of available molecules (by gene amplification or expression activation) or 2) mutation in the enzyme coding portion of the gene, so that its product metabolizes the insecticide more efficiently. These processes can be very complex and involve three major enzyme superfamilies: Esterases, Multi function Oxidases P450 and Glutathion-S-Transferases (Hemingway & Ranson, 2000; Montella et al., 2007). In contrast, there are few

examples in literature regarding insect behavioral changes and tegument alterations.

Resistance to insecticides may be functionally defined as the ability of an insect population to survive exposure to dosages of a given compound that are lethal to the majority of individuals of a susceptible lineage of the same species (Beaty & Marquardt, 1996). Resistance is based on the genetic variability of natural populations. Under insecticide selection pressure, specific phenotypes are selected and consequently increase in frequency. Resistance can result from the selection of one or more mechanisms. In order to elucidate the molecular nature of resistance, many studies report laboratory controlled selection of different species (Chang et al., 2009; Kumar et al., 2002; Paeoporn et al., 2003; Rodriguez et al., 2003; Saavedra-Rodriguez et al., 2007). With selected lineages, it becomes easier to separate the role of each distinct mechanism. In a more direct approach, the current availability of a series of molecular tools enables detection of expression of altered molecules in model organisms so that the effect of the insecticide can be evaluated under specific and

Regardless of the mono or multi-factorial character of resistance, this phenomenon may be didactically divided into four categories: behavioral, cuticular, metabolic and phenotypic resistance. In the first case the insect simply avoids contact with the insecticide through behavioral adaptations, which are presumably related to genetic inheritance (Sparks et al., 1989). Among arthropods, mosquitoes are by far the group most intensely investigated in relation to behavioral resistance (Lockwood et al., 1984). For instance, *Anopheles* malaria vector mosquitoes from the Amazon Region had the habit of resting in the walls after a blood meal. There are registers that some populations changed their behavior after a period of indoor residual application of DDT to the dwelling walls (Roberts & Alecrim, 1991). Behavioral changes that minimize contact between insect and insecticide may cause a severe impact in the insecticide application efficacy, especially if resistance is selected by

Certain alterations in the insect cuticle may reduce insecticide penetration. However, these effects are unspecific, leading to resistance to a series of xenobiotic compounds. This mechanism is known as reduced penetration or cuticle resistance. It is probably not related to high levels of resistance by itself, but it can interact synergistically with other mechanisms. The physiological processes or molecular pathways which describe this type of

insecticide resistance in a broader context.

**2. Insecticide resistance mechanisms** 

controlled circumstances (Smith et al., 1997).

physiological features (Ranson et al., 2011).

resistance remain to be elucidated. With respect to pyrethroid resistance, recent evidences point to an increase in the levels of expression of two cuticle genes in populations of two *Anopheles* species (Awolola et al., 2009; Vontas et al., 2007).

The increased ability to detoxify insecticides is one of the main types of resistance, commonly referred to as metabolic resistance. It takes place when the activity of naturally detoxifying enzymes is enhanced, impeding the insecticide to reach its target. Among these enzymes, Multi function Oxidases (or Monoxigenases P450), Esterases and Glutathion-S-Transferases (GST) (ffrench-Constant et al., 2004; Hemingway & Ranson, 2000) are the major representative families. Although the molecular basis of metabolic resistance has been extensively studied, only few reports have investigated the specific metabolic pathways involved or their location in the insect organism. Many different mutations may be attributed to metabolic resistance, such as those leading to production of more enzymes, via gene duplication events or either increases in gene transcription rates, alterations in the normal tissue/time specificity of expression, point mutations leading to a gain of function or changes in the substrate specificity (ffrench-Constant et al., 2004; Hemingway et al., 2004; Perry et al., 2011). Detoxifying enzymes belong to superfamilies composed of numerous genes (Ranson et al., 2002), and it is not unusual for different enzymes to produce the same metabolites. Additionally, an alteration in one type of enzyme may lead to cross-resistance among different classes of insecticides (Ranson et al., 2011). However, population genetic markers that make feasible a complete diagnostic of the resistance mechanisms or their distribution are not yet available. Current studies are generally based on biochemical assays (Valle et al., 2006) and, to a lesser extent, on *microarray detox chips* (David et al., 2005; Vontas et al., 2007). Due to technical limitations, the most common reports are hence oriented to single gene responses, such as punctual mutations that increase the ability of a specific enzyme in detoxifying an insecticide (Lumjuan et al., 2011; Morin et al., 2008).

Multi function P450 Oxidases are the enzymes most commonly associated to metabolic resistance to pyrethroids. However, despite much indirect evidence of P450 total activity increase or even detection of higher expression of some related genes (*cyp*), little is known about their metabolic activity. For instance, 111 genes code for P450 in *Anopheles gambiae*, but only two (*cyp6p3* and *cyp6m2*) were described to be involved in pyrethroid metabolism (Muller et al., 2008). Surprisingly, metabolic resistance can still vary during the course of the day. This is the case of an *Ae. aegypti* population whose resistance to the pyrethroid permethrin is mediated by the *cyp9M9* gene. Expression of this gene is regulated by transcriptional factors enrolled in the circadian rhythm of the insect, varying along the day (Y. Y. Yang et al., 2010).

Finally, phenotypic or target site resistance is designated by modification of the insect molecule where the insecticide binds, inhibiting its effects. Neurotoxic insecticides have as their ultimate target different molecules from the insect central nervous system: the enzyme Acetylcholinesterase (for organophosphates and carbamates), the gama-aminobutiric acid receptor (for ciclodienes), the nicotinic acetylcholine receptors (for spinosyns and neonicotinoids) and the voltage gated sodium channel (for DDT and pyrethroids). Although the mutated target molecule decreases or even abolishes its affinity for the insecticide, it is essential that this alteration does not result in loss of function regarding the insect physiological processes. Since the classical target molecules are much conserved among animals, few mutations are permissive to guarantee the viability of their carriers (ffrench-Constant et al., 1998; Raymond et al., 2001).

The Pyrethroid Knockdown Resistance 21

compare relative position of the Nav blue domains in the different pannels). The pore forming module is composed of the S5-S6 segments and the loop between them, the latter acting as an ion selective filter in the extracellular entrance of the pore (Catterall et al., 2003; Goldin, 2003; Narahashi, 1992). Additionally, the *P-loop* residues D, E, K and A, respectively

from domains I, II, III and IV, are critical for the Na+ sensitivity (Zhou et al., 2004).

Fig. 1. Propagation of the action potential through a neuronal axon - In the resting potential stage (A) the axon cytoplasm has Na+ and K+ respectively in low and high concentrations compared to the surrounding extracellular fluid. The Na/K pump is constantly expelling three Na+ from the cell for every two K+ it transfers in, which confers a positive charge to the outer part of the membrane. When there is a nervous stimulus, the NaV opens and the membrane becomes permeable affording the influx of Na+, depolarizing the membrane charge (B). This is

the rising phase of the action potential. Soon (~1 millisecond), the NaV is deactivated, precluding further Na+ entrance to the cell (C), whilst K+ exits the cell through KV which is now opened, characterizing the falling phase of the action potential (D). The Na/K pump helps to reestablish the initial membrane potential. The action potential generates a wave of sequential depolarization along the axon. Figure based on T. G. Davies et al. (2007b).

The voltage gated sodium channel (NaV) is the effective target for a number of neurotoxins produced by plants and animals, as components of their predation or defense strategies. Knowledge that mutations in the NaV gene can endow resistance to both the most popular insecticides of the past (DDT) and nowadays (pyrethroids) is leading to significant progress in the understanding of the physiology, pharmacology and evolution of this channel (ffrench-Constant et al., 1998; O'Reilly et al., 2006).

#### **3. The role of the voltage gated sodium channel (NaV) in the nerve impulse propagation in insects**

The membrane of all excitable cells (neurons, myocites, endocrinous and egg cells) have voltage gated ion channels responsible for the generation of action potential. These cells react to changes in the electric potential of the membrane, modifying their permeability status (Alberts et al., 2002; Randall et al., 2001). Voltage gated sodium channels (NaV) are transmembrane proteins responsible for the initial action potential in excitable cells (Catterall, 2000). They are members of the protein superfamily which also includes voltage gated calcium (CaV) and potassium (KV) channels (Jan & Jan, 1992). Both NaV and CaV channels are constituted of four homologous domains whilst KV is a tetramer with only one domain. A proposed evolution pathway assumes that CaV have evolved from Kv by gene duplication during the evolution of multicelular eukaryotes. NaV channels are supposed to have evolved from an ancestral CaV family (family CaV3) (Spafford et al., 1999). Accordingly, the four NaV domains are more similar to their CaV counterparts than among themselves (Strong et al., 1993). The sodium channel is completely functional by itself, unless the kinetics of opening and closure of the voltage gated channel can be modified by other proteins, sometimes referred to as complementary subunits (beta subunit in mammals and TipE in *Drosophila*) (Catterall et al., 2003).

Cell action potential starts with the depolarization of the membrane, with the internal side attaining a more positive state (compare Figure 1, pannels A and B). A stimulus that causes the depolarization in a given region of the cell membrane promotes activation (opening) of the NaV in the vicinity. This process results in the influx of Na+ to the cell, enhancing depolarization of the membrane. The action potential works in a positive feedback, that is, once started there is no need of additional stimuli to progress. However, one millisecond after the channel has been activated, the surrounding membrane reaches the Na+ equilibrium potential, and the channel is deactivated. In this state, the pore is still open, but it assumes a conformation that halts the ion influx into the cell (Figure 1, C). After some further milliseconds, the membrane is repolarized and the channel closes, finally returning to its resting configuration (Figure 1, D). This whole process occurs in consonance with other channels and pumps, such as KV and sodium/ potassium pumps that restore the original electric potential of the cell (Catterall et al., 2003; Randall et al., 2001). The correct operation of sodium channels is essential for nerve impulse propagation. Hence, if the regular propagation of an impulse is altered, as due to the interaction with an insecticide, the organism suffers paralysis and can eventually die.

The structure of NaV is organized in four homologous domains (I-IV), each containing six hydrophobic segments (S1-S6) and a *P-loop* between S5 and S6 (Figure 2). The segments S1- S4 work as a voltage sensitive module. Since S4 segments are positively charged and sensitive to voltage changes, they move across the membrane in order to initiate the channel activation in response to membrane depolarization (schematically represented in Figure 1,

The voltage gated sodium channel (NaV) is the effective target for a number of neurotoxins produced by plants and animals, as components of their predation or defense strategies. Knowledge that mutations in the NaV gene can endow resistance to both the most popular insecticides of the past (DDT) and nowadays (pyrethroids) is leading to significant progress in the understanding of the physiology, pharmacology and evolution of this channel

**3. The role of the voltage gated sodium channel (NaV) in the nerve impulse** 

The membrane of all excitable cells (neurons, myocites, endocrinous and egg cells) have voltage gated ion channels responsible for the generation of action potential. These cells react to changes in the electric potential of the membrane, modifying their permeability status (Alberts et al., 2002; Randall et al., 2001). Voltage gated sodium channels (NaV) are transmembrane proteins responsible for the initial action potential in excitable cells (Catterall, 2000). They are members of the protein superfamily which also includes voltage gated calcium (CaV) and potassium (KV) channels (Jan & Jan, 1992). Both NaV and CaV channels are constituted of four homologous domains whilst KV is a tetramer with only one domain. A proposed evolution pathway assumes that CaV have evolved from Kv by gene duplication during the evolution of multicelular eukaryotes. NaV channels are supposed to have evolved from an ancestral CaV family (family CaV3) (Spafford et al., 1999). Accordingly, the four NaV domains are more similar to their CaV counterparts than among themselves (Strong et al., 1993). The sodium channel is completely functional by itself, unless the kinetics of opening and closure of the voltage gated channel can be modified by other proteins, sometimes referred to as complementary subunits (beta subunit in mammals and

Cell action potential starts with the depolarization of the membrane, with the internal side attaining a more positive state (compare Figure 1, pannels A and B). A stimulus that causes the depolarization in a given region of the cell membrane promotes activation (opening) of the NaV in the vicinity. This process results in the influx of Na+ to the cell, enhancing depolarization of the membrane. The action potential works in a positive feedback, that is, once started there is no need of additional stimuli to progress. However, one millisecond after the channel has been activated, the surrounding membrane reaches the Na+ equilibrium potential, and the channel is deactivated. In this state, the pore is still open, but it assumes a conformation that halts the ion influx into the cell (Figure 1, C). After some further milliseconds, the membrane is repolarized and the channel closes, finally returning to its resting configuration (Figure 1, D). This whole process occurs in consonance with other channels and pumps, such as KV and sodium/ potassium pumps that restore the original electric potential of the cell (Catterall et al., 2003; Randall et al., 2001). The correct operation of sodium channels is essential for nerve impulse propagation. Hence, if the regular propagation of an impulse is altered, as due to the interaction with an insecticide,

The structure of NaV is organized in four homologous domains (I-IV), each containing six hydrophobic segments (S1-S6) and a *P-loop* between S5 and S6 (Figure 2). The segments S1- S4 work as a voltage sensitive module. Since S4 segments are positively charged and sensitive to voltage changes, they move across the membrane in order to initiate the channel activation in response to membrane depolarization (schematically represented in Figure 1,

(ffrench-Constant et al., 1998; O'Reilly et al., 2006).

TipE in *Drosophila*) (Catterall et al., 2003).

the organism suffers paralysis and can eventually die.

**propagation in insects** 

compare relative position of the Nav blue domains in the different pannels). The pore forming module is composed of the S5-S6 segments and the loop between them, the latter acting as an ion selective filter in the extracellular entrance of the pore (Catterall et al., 2003; Goldin, 2003; Narahashi, 1992). Additionally, the *P-loop* residues D, E, K and A, respectively from domains I, II, III and IV, are critical for the Na+ sensitivity (Zhou et al., 2004).

Fig. 1. Propagation of the action potential through a neuronal axon - In the resting potential stage (A) the axon cytoplasm has Na+ and K+ respectively in low and high concentrations compared to the surrounding extracellular fluid. The Na/K pump is constantly expelling three Na+ from the cell for every two K+ it transfers in, which confers a positive charge to the outer part of the membrane. When there is a nervous stimulus, the NaV opens and the membrane becomes permeable affording the influx of Na+, depolarizing the membrane charge (B). This is the rising phase of the action potential. Soon (~1 millisecond), the NaV is deactivated, precluding further Na+ entrance to the cell (C), whilst K+ exits the cell through KV which is now opened, characterizing the falling phase of the action potential (D). The Na/K pump helps to reestablish the initial membrane potential. The action potential generates a wave of sequential depolarization along the axon. Figure based on T. G. Davies et al. (2007b).

The Pyrethroid Knockdown Resistance 23

suffer paralysis followed by death (*knockdown*) but, at most, presented a momentary paralysis followed by complete locomotion recovery, this phenotype being named *kdr* (*knockdown* resistance) (Busvine, 1951; Harrison, 1951; Milani, 1954). Since the introduction of pyrethroids, plenty of insect species exhibiting the *kdr* phenotype have been observed, attributed to previous DDT selection pressure, characterizing cross-resistance between both insecticides (Hemingway & Ranson, 2000). *Kdr* resistance results in 10-20 fold decrease in the sensitivity to the insecticide. However, *kdr* lineages of some species can exhibit up to 100 X increased pyrethroid resistance, an effect denominated *super-kdr*. *Kdr* and *super-kdr* alleles act as recessive traits and hence may persist at low levels in the population in

Over three decades after the description of the *kdr* effect, electrophysiological studies based on neuronal cells and tissues suggested that NaV had to be the target site for pyrethroids. These reports also indicated that cross-resistance between pyrethroids and DDT must be related to that channel (Pauron et al., 1989). Concomitantly, the gene *paralytic* (*para)* from *Drosophila melanogaster* was cloned and sequenced. This gene is placed in the *locus* related to behavioral changes and paralysis after exposure to high temperatures, similar to the *knockdown* effect produced by DDT and pyrethroids (Loughney et al., 1989). Comparisons within vertebrate nucleotide sequences revealed that *para* is homologous to the voltage gated sodium channel gene (*NaV*) (Loughney & Ganetzky, 1989). It was then shown, with a DDT resistant housefly lineage, that the *locus* homologous to *para* was in strong linkage with the *kdr* phenotype (Williamson et al., 1993). This evidence was soon extended to other insect species plagues or vectors, such as the tobacco budworm *Heliothis virescens* (Taylor et al., 1993), the cockroach *Blatella germanica* (Dong & Scott, 1994) and the mosquito *Aedes aegypti*

Hitherto, NaV is the only molecule incriminated as the target site for DDT and pyrethroids. Although it has been implied that type II pyrethroids can interact with the GABA receptor (which is the target, for instance, of the insecticide dieldrin), this interaction has not been considered toxically important (Soderlund & Bloomquist, 1989). Research on the molecular interaction between pyrethroids and their target site presently guides a series of approaches towards the development of a great variety of natural and synthetic neurotoxicants acting

A great variety of sodium channels have been identified by electrophysiological assays, purification and cloning (Goldin, 2001). In mammals, nine NaV genes are known, with distinct electrophysiological properties as well as different expression profiles in the tissues and throughout development (Goldin, 2002; Yu & Catterall, 2003), phylogenetic analyses revealing that all are members of only one unique family, deriving from relatively recent gene duplications and chromosome rearrangements. On the other hand, CaV and KaV have little protein sequence identity and present diverse functions, indicative of more ancient

The *NaV* orthologous genes and cDNAs from *D. melanogaster* and *An. gambiae* share, respectively, 56-62% and 82% of nucleotide identity, evidencing a high degree of conservation between these species. The *NaV* C-terminal is the most variable region, but as in all dipterans, it is mainly composed of aminoacids of short (Gly, Ala, Ser, Pro) or negative (Asp, Glu) side chains, suggesting a conserved function in this domain (T. G. Davies et al.,

**5. Molecular biology of the insect NaV and the** *kdr* **mutation** 

segregation of their coding genes (Catterall et al., 2003).

heterozygous individuals (T. G. Davies et al., 2007a).

(Severson et al., 1997).

on the NaV (Soderlund, 2010).

Fig. 2. The voltage gated sodium channel - Scheme representative of the NaV inserted in a cell membrane, showing its four homologous domains (I-IV), each with six hydrophobic segments (S1-S6). In blue, the voltage sensor segments (S4); in green, the S6 segments, which form the channel pore together with the S5 segments and the link (*P-loop*, in red) between them. Figure adapted from Nelson & Cox (2000).

In the closed state, the putative insecticide contact sites are blocked, corroborating the assumption that pyrethroids and DDT have more affinity to the Nav channel in its open state when the insecticide stabilizes the open conformation (O'Reilly et al., 2006). These insecticides, therefore, inhibit the channel transition to the non-conducting and deactivated states (T. E. Davies et al., 2008). By interacting with the channel, they form a sort of wedge between segments IIS5 and IIIS6 that restricts displacement of the pore forming helices S5 and S6, preventing closure of the channel. Consequently, the influx of Na+ is prolonged, and the cell is led to work at an abnormal state of hyper-excitability. The amplitude of the Na+ current will not decrease unless the cell's level of hyper-excitability is overcome by its ability to keep the sodium-potassium pump under operation. This process is responsible for the pyrethroid sublethal effect in insects, known as *knockdown* effect, which may lead to paralysis and death if prolonged (T. E. Davies et al., 2008; T. G. Davies et al., 2007b).

Predictive models suggest that DDT and pyrethroids interact with a long and narrow cavity delimited by the IIS4-S5 linker and the IIS5 and IIIS6 helices, accessible to lipophilic insecticides. Moreover, some of the aminoacids belonging to the helices engaged in contact with these insecticides are not conserved among arthropods and other animals, and this could be responsible for the selectivity of pyrethroid effects against insects (O'Reilly et al., 2006). The crystal structure of a NaV has been recently published (Payandeh et al., 2011), pointing to a better understanding of the channel function and to its interaction with targeted compounds in a near future.

Besides pyrethroids and DDT, other insecticides act on the voltage gated sodium channel, like the sodium channel blocker insecticides (SCBIs) and N-alkylamide inseticides (like BTG 502). There are few reports about these compounds. However, it is known that SCBIs, such as indoxicarb, act by blocking the impulse conduction, an effect opposite to that of DDT and pyrethroids (Du et al., 2011).

#### **4. The knockdown effect and the** *kdr* **phenotype**

In the early 1950s, no sooner had DDT been introduced as an insecticide than resistant strains of houseflies were described. When exposed to DDT, these insects in general did not

Fig. 2. The voltage gated sodium channel - Scheme representative of the NaV inserted in a cell membrane, showing its four homologous domains (I-IV), each with six hydrophobic segments (S1-S6). In blue, the voltage sensor segments (S4); in green, the S6 segments, which form the channel pore together with the S5 segments and the link (*P-loop*, in red) between

In the closed state, the putative insecticide contact sites are blocked, corroborating the assumption that pyrethroids and DDT have more affinity to the Nav channel in its open state when the insecticide stabilizes the open conformation (O'Reilly et al., 2006). These insecticides, therefore, inhibit the channel transition to the non-conducting and deactivated states (T. E. Davies et al., 2008). By interacting with the channel, they form a sort of wedge between segments IIS5 and IIIS6 that restricts displacement of the pore forming helices S5 and S6, preventing closure of the channel. Consequently, the influx of Na+ is prolonged, and the cell is led to work at an abnormal state of hyper-excitability. The amplitude of the Na+ current will not decrease unless the cell's level of hyper-excitability is overcome by its ability to keep the sodium-potassium pump under operation. This process is responsible for the pyrethroid sublethal effect in insects, known as *knockdown* effect, which may lead to

paralysis and death if prolonged (T. E. Davies et al., 2008; T. G. Davies et al., 2007b).

Predictive models suggest that DDT and pyrethroids interact with a long and narrow cavity delimited by the IIS4-S5 linker and the IIS5 and IIIS6 helices, accessible to lipophilic insecticides. Moreover, some of the aminoacids belonging to the helices engaged in contact with these insecticides are not conserved among arthropods and other animals, and this could be responsible for the selectivity of pyrethroid effects against insects (O'Reilly et al., 2006). The crystal structure of a NaV has been recently published (Payandeh et al., 2011), pointing to a better understanding of the channel function and to its interaction with

Besides pyrethroids and DDT, other insecticides act on the voltage gated sodium channel, like the sodium channel blocker insecticides (SCBIs) and N-alkylamide inseticides (like BTG 502). There are few reports about these compounds. However, it is known that SCBIs, such as indoxicarb, act by blocking the impulse conduction, an effect opposite to that of DDT and

In the early 1950s, no sooner had DDT been introduced as an insecticide than resistant strains of houseflies were described. When exposed to DDT, these insects in general did not

them. Figure adapted from Nelson & Cox (2000).

targeted compounds in a near future.

**4. The knockdown effect and the** *kdr* **phenotype** 

pyrethroids (Du et al., 2011).

suffer paralysis followed by death (*knockdown*) but, at most, presented a momentary paralysis followed by complete locomotion recovery, this phenotype being named *kdr* (*knockdown* resistance) (Busvine, 1951; Harrison, 1951; Milani, 1954). Since the introduction of pyrethroids, plenty of insect species exhibiting the *kdr* phenotype have been observed, attributed to previous DDT selection pressure, characterizing cross-resistance between both insecticides (Hemingway & Ranson, 2000). *Kdr* resistance results in 10-20 fold decrease in the sensitivity to the insecticide. However, *kdr* lineages of some species can exhibit up to 100 X increased pyrethroid resistance, an effect denominated *super-kdr*. *Kdr* and *super-kdr* alleles act as recessive traits and hence may persist at low levels in the population in heterozygous individuals (T. G. Davies et al., 2007a).

Over three decades after the description of the *kdr* effect, electrophysiological studies based on neuronal cells and tissues suggested that NaV had to be the target site for pyrethroids. These reports also indicated that cross-resistance between pyrethroids and DDT must be related to that channel (Pauron et al., 1989). Concomitantly, the gene *paralytic* (*para)* from *Drosophila melanogaster* was cloned and sequenced. This gene is placed in the *locus* related to behavioral changes and paralysis after exposure to high temperatures, similar to the *knockdown* effect produced by DDT and pyrethroids (Loughney et al., 1989). Comparisons within vertebrate nucleotide sequences revealed that *para* is homologous to the voltage gated sodium channel gene (*NaV*) (Loughney & Ganetzky, 1989). It was then shown, with a DDT resistant housefly lineage, that the *locus* homologous to *para* was in strong linkage with the *kdr* phenotype (Williamson et al., 1993). This evidence was soon extended to other insect species plagues or vectors, such as the tobacco budworm *Heliothis virescens* (Taylor et al., 1993), the cockroach *Blatella germanica* (Dong & Scott, 1994) and the mosquito *Aedes aegypti* (Severson et al., 1997).

Hitherto, NaV is the only molecule incriminated as the target site for DDT and pyrethroids. Although it has been implied that type II pyrethroids can interact with the GABA receptor (which is the target, for instance, of the insecticide dieldrin), this interaction has not been considered toxically important (Soderlund & Bloomquist, 1989). Research on the molecular interaction between pyrethroids and their target site presently guides a series of approaches towards the development of a great variety of natural and synthetic neurotoxicants acting on the NaV (Soderlund, 2010).

#### **5. Molecular biology of the insect NaV and the** *kdr* **mutation**

A great variety of sodium channels have been identified by electrophysiological assays, purification and cloning (Goldin, 2001). In mammals, nine NaV genes are known, with distinct electrophysiological properties as well as different expression profiles in the tissues and throughout development (Goldin, 2002; Yu & Catterall, 2003), phylogenetic analyses revealing that all are members of only one unique family, deriving from relatively recent gene duplications and chromosome rearrangements. On the other hand, CaV and KaV have little protein sequence identity and present diverse functions, indicative of more ancient segregation of their coding genes (Catterall et al., 2003).

The *NaV* orthologous genes and cDNAs from *D. melanogaster* and *An. gambiae* share, respectively, 56-62% and 82% of nucleotide identity, evidencing a high degree of conservation between these species. The *NaV* C-terminal is the most variable region, but as in all dipterans, it is mainly composed of aminoacids of short (Gly, Ala, Ser, Pro) or negative (Asp, Glu) side chains, suggesting a conserved function in this domain (T. G. Davies et al.,

The Pyrethroid Knockdown Resistance 25

pairs of mutually exclusive exons (Figure 3) (Olson et al., 2008). These sites are conserved in *M. domestica* (Lee et al., 2002) generating, in both species, 512 potential *NaV* transcripts by alternative splicing. However, they are not all necessarily expressed as less than 10 were

Fig. 3. Alternative splice in the insect voltage gated sodium channel gene. Scheme of NaV with the sites of alternative exons of *DmNaV* indicated in dark color. Exons *a*, *b*, *i*, *j*, *e* and *f* are optional, while *d*/*c* and *l*/*k* are mutually exclusive. Figure adapted from Oslon et al.

The aminoacid sequences translated from optional exons are conserved and generally consist of intracellular domains of the channel, suggesting functional relevance to these events. *NaV* transcript diversity derived from alternative splicing has been investigated in insects of many orders, revealing a high level of conservation, as shown in the cockroach *B. germanica* (Liu et al., 2001; Song et al., 2004), the silk worm *Bombyx mori* (Shao et al., 2009), the moth *Plutella xylostella* (Sonoda et al., 2008) and the mosquitoes *An. gambiae* (T. G. Davies et al., 2007a) and *Ae. aegypti* (Chang et al., 2009). However, in some species not all exons

There are two mutually exclusive exons (called c/d) that code for a region between IIS4 and IIS5 segments (Figure 3). The absence of one of these exons might be important for pyrethroid resistance, since the *super-kdr* mutation (Met918Thr) is located in this region, as will be discussed further. In the cockroach *B. germanica*, the mutually exclusive exon pair *k*/*l* codes for the voltage sensitive region at domain III. The two varieties *BgNaV1.1a* and *BgNaV1.1b*1, which contain the exons *l* and *k* respectively, exhibit distinct electrophysiological properties. Furthermore, *BgNaV1.1b* is 100X more resistant to the

RNA editing has an important role in the regulation of gene expression and protein diversity. Recent studies implicate RNA editing in the removal of exons in alternative splicing sites, in the antagonism of interference RNA process (iRNA), in the modulation of mRNA processing and in the generation of new exons (for a review see Y. Yang et al., 2008). The basic mechanism of diversity generated by RNA editing includes nucleoside modifications such as C to U or A to I deaminations. Besides, it is possible that non-

1 The genes annotation is in accordance with the nomenclature suggested by Goldin (2000).

were observed nor their expression detected (see Davies et al., 2007a).

pyrethroid deltamethrin than *BgNaV1.1a* (Du et al., 2006).

**5.2 Sodium channel RNA editing** 

actually observed in mRNA pools (Soderlund, 2010).

(2008).

2007a). Concerning size, the voltage gated sodium channel of *Ae. aegypti* (*AaNaV*), for instance, presents 293 Kb of genomic DNA, with 33 exons. Its longer observed transcript has an ORF of 6.4 Kb, coding for 2,147 aminoacids for a protein estimated in 241 KDa (Chang et al., 2009).

The existence of two NaV evolutionary lines in invertebrates, represented by the genes *para* and *DSC1* in *D. melanogaster*, has been suggested (Spafford et al., 1999). These lines do not correspond to the different genes observed among vertebrates, and they are supposed to have arisen after vertebrate and invertebrate splitting (Goldin, 2002). DSC1 plays a role in the olfactory system (Kulkarni et al., 2002) as it has been found in the peripheral nervous system and also at high density in the synaptic regions. DSC1 is sensitive to tetradotoxin, a specific NaV blocker (Zhang et al., 2011), while BSC1, its homologous in *B. germanica*, has also been identified as a putative sodium channel, being expressed in the cockroach nerve cord, muscle, gut, fat body and ovary (Liu et al., 2001). Neither *DSC1* nor *BSC1*, however, mapped with any *locus* related to insecticide resistance (Loughney et al., 1989; Salkoff et al., 1987). Actually, these channels probably represent prototypes of a new CaV family, highly related to the known NaV and CaV (Zhang et al., 2011; Zhou et al., 2004). On the other hand, in invertebrates, the *D. melanogaster para* gene (or *DmNaV*) and its equivalent in other species actually code for sodium channels and are related to pyrethroid/DDT resistance and to behavioral changes, as aforementioned.

In his review, Goldin (2002) suggested that two to four genes coding for sodium channels should exist in insects and that differences among them would not result from distinct genes but from pos-transcriptional regulation. Accordingly, even after publication of many insect genome sequences, there has been no mention whatsoever of NaV gene duplication. Furthermore, recent reports attribute the diversity in NaV sequences to alternative splicing and RNA editing. These modifications seem to be tissue and stage specific and might also have some influence on pyrethroid resistance (Liu et al., 2004; Song et al., 2004; Sonoda et al., 2008).

#### **5.1 Alternative mRNA splicing in the NaV**

Briefly, alternative splicing is a post-transcriptional regulated event characterized when certain exons are removed together with introns. This is a common mechanism of gene expression regulation and increment of protein diversity in eukaryotes. The process may occur in different ways: complete exons can be included or excluded (optional exons), splicing sites can be altered and introns can be retained in the mature mRNA. There are also mutually exclusive pairs of exons, when two exons never unite in the same transcript. Alternative mRNA splicing introduces variability in both sequence and size of the NaV intracellular region, which by itself should have an impact on its operation (T. G. Davies et al., 2007a).

The regulation for excision of an exon, in detriment of others, may be tissue and development specific. In the context of pyrethroid resistance, it is important to know to what extent alternative splicing events compromise the interaction between the insecticide and the channel. It is also necessary to investigate the amount of alternative transcripts in the course of development and their distribution in the different tissues of the insect. The sodium channel genes have alternative exons that potentially synthesize a great number of different mRNAs (Figure 3). There are also mutually exclusive exons that occur in the transmembrane regions of domains II and III (T. G. Davies et al., 2007a). In *D. melanogaster*, many alternative splicing sites have been identified, with seven optional regions and two

2007a). Concerning size, the voltage gated sodium channel of *Ae. aegypti* (*AaNaV*), for instance, presents 293 Kb of genomic DNA, with 33 exons. Its longer observed transcript has an ORF of 6.4 Kb, coding for 2,147 aminoacids for a protein estimated in 241 KDa (Chang et

The existence of two NaV evolutionary lines in invertebrates, represented by the genes *para* and *DSC1* in *D. melanogaster*, has been suggested (Spafford et al., 1999). These lines do not correspond to the different genes observed among vertebrates, and they are supposed to have arisen after vertebrate and invertebrate splitting (Goldin, 2002). DSC1 plays a role in the olfactory system (Kulkarni et al., 2002) as it has been found in the peripheral nervous system and also at high density in the synaptic regions. DSC1 is sensitive to tetradotoxin, a specific NaV blocker (Zhang et al., 2011), while BSC1, its homologous in *B. germanica*, has also been identified as a putative sodium channel, being expressed in the cockroach nerve cord, muscle, gut, fat body and ovary (Liu et al., 2001). Neither *DSC1* nor *BSC1*, however, mapped with any *locus* related to insecticide resistance (Loughney et al., 1989; Salkoff et al., 1987). Actually, these channels probably represent prototypes of a new CaV family, highly related to the known NaV and CaV (Zhang et al., 2011; Zhou et al., 2004). On the other hand, in invertebrates, the *D. melanogaster para* gene (or *DmNaV*) and its equivalent in other species actually code for sodium channels and are related to pyrethroid/DDT resistance and to

In his review, Goldin (2002) suggested that two to four genes coding for sodium channels should exist in insects and that differences among them would not result from distinct genes but from pos-transcriptional regulation. Accordingly, even after publication of many insect genome sequences, there has been no mention whatsoever of NaV gene duplication. Furthermore, recent reports attribute the diversity in NaV sequences to alternative splicing and RNA editing. These modifications seem to be tissue and stage specific and might also have some influence on pyrethroid resistance (Liu et al., 2004; Song et al., 2004; Sonoda et al.,

Briefly, alternative splicing is a post-transcriptional regulated event characterized when certain exons are removed together with introns. This is a common mechanism of gene expression regulation and increment of protein diversity in eukaryotes. The process may occur in different ways: complete exons can be included or excluded (optional exons), splicing sites can be altered and introns can be retained in the mature mRNA. There are also mutually exclusive pairs of exons, when two exons never unite in the same transcript. Alternative mRNA splicing introduces variability in both sequence and size of the NaV intracellular region, which by itself should have an impact on its operation (T. G. Davies et

The regulation for excision of an exon, in detriment of others, may be tissue and development specific. In the context of pyrethroid resistance, it is important to know to what extent alternative splicing events compromise the interaction between the insecticide and the channel. It is also necessary to investigate the amount of alternative transcripts in the course of development and their distribution in the different tissues of the insect. The sodium channel genes have alternative exons that potentially synthesize a great number of different mRNAs (Figure 3). There are also mutually exclusive exons that occur in the transmembrane regions of domains II and III (T. G. Davies et al., 2007a). In *D. melanogaster*, many alternative splicing sites have been identified, with seven optional regions and two

al., 2009).

2008).

al., 2007a).

behavioral changes, as aforementioned.

**5.1 Alternative mRNA splicing in the NaV**

pairs of mutually exclusive exons (Figure 3) (Olson et al., 2008). These sites are conserved in *M. domestica* (Lee et al., 2002) generating, in both species, 512 potential *NaV* transcripts by alternative splicing. However, they are not all necessarily expressed as less than 10 were actually observed in mRNA pools (Soderlund, 2010).

Fig. 3. Alternative splice in the insect voltage gated sodium channel gene. Scheme of NaV with the sites of alternative exons of *DmNaV* indicated in dark color. Exons *a*, *b*, *i*, *j*, *e* and *f* are optional, while *d*/*c* and *l*/*k* are mutually exclusive. Figure adapted from Oslon et al. (2008).

The aminoacid sequences translated from optional exons are conserved and generally consist of intracellular domains of the channel, suggesting functional relevance to these events. *NaV* transcript diversity derived from alternative splicing has been investigated in insects of many orders, revealing a high level of conservation, as shown in the cockroach *B. germanica* (Liu et al., 2001; Song et al., 2004), the silk worm *Bombyx mori* (Shao et al., 2009), the moth *Plutella xylostella* (Sonoda et al., 2008) and the mosquitoes *An. gambiae* (T. G. Davies et al., 2007a) and *Ae. aegypti* (Chang et al., 2009). However, in some species not all exons were observed nor their expression detected (see Davies et al., 2007a).

There are two mutually exclusive exons (called c/d) that code for a region between IIS4 and IIS5 segments (Figure 3). The absence of one of these exons might be important for pyrethroid resistance, since the *super-kdr* mutation (Met918Thr) is located in this region, as will be discussed further. In the cockroach *B. germanica*, the mutually exclusive exon pair *k*/*l* codes for the voltage sensitive region at domain III. The two varieties *BgNaV1.1a* and *BgNaV1.1b*1, which contain the exons *l* and *k* respectively, exhibit distinct electrophysiological properties. Furthermore, *BgNaV1.1b* is 100X more resistant to the pyrethroid deltamethrin than *BgNaV1.1a* (Du et al., 2006).

#### **5.2 Sodium channel RNA editing**

RNA editing has an important role in the regulation of gene expression and protein diversity. Recent studies implicate RNA editing in the removal of exons in alternative splicing sites, in the antagonism of interference RNA process (iRNA), in the modulation of mRNA processing and in the generation of new exons (for a review see Y. Yang et al., 2008). The basic mechanism of diversity generated by RNA editing includes nucleoside modifications such as C to U or A to I deaminations. Besides, it is possible that non-

<sup>1</sup> The genes annotation is in accordance with the nomenclature suggested by Goldin (2000).

The Pyrethroid Knockdown Resistance 27

to as the *super-kdr* mutation (Jamroz et al., 1998). However, since it occurs only in association with the Leu1014Phe mutation, its isolated effects are as yet unknown. Although no *superkdr* mutation has so far been identified in mosquitoes, it was suggested that Leu932Phe, in association with Ile936Val (both also in the IIS4-S5 linker), in *Culex* might play this role, being the first example of *super-kdr* in this group (T. G. Davies et al., 2007a). Accordingly, these sites have proved to be important for the interaction between NaV, in the *D.* 

Substitutions in site 929 are also involved in enhanced pyrethroid resistance, as is the case with the Lepidoptera *Plutella xylostella* mutation Thr929Ile, detected in association with Leu1014Phe (Schuler et al., 1998). However, in the maize weevil *Sitophilus zeamais*, the Thr929Ile was found alone (Araujo et al., 2011). In the louse *Pediculus capitis*, in turn, the Thr929Ile mutation was together with Leu932Phe (Lee et al., 2000). There were other substitutions in the same site: Thr/Cys and/or Thr/Val in the diamondblack moth *Frankliniella occidentalis* (Forcioli et al., 2002) and in the cat flea *Ctenocephalides felis* (Bass et

*Ae. aegypti* mosquitoes do not present any substitution in the classic 1014 *kdr* site, unlike many other insects or even mosquitoes from other genera, such as *Anopheles* and *Culex*, very likely because the 1014 site of *Ae. aegypti* NaV is coded by a CTA, in place of the TTA codon present in the majority of other insects. For this reason, two simultaneous nucleotide substitutions would be necessary in order to change from Leu (CTA) to Phe (TTT) or Ser (TCA) (Martins et al., 2009a; Saavedra-Rodriguez et al., 2007). Instead, mutations in different positions have been observed in *Ae. aegypti* populations from Latin America and Southeast Asia, but at least two sites seem to be indeed related to pyrethroid resistance: 1016 (Val to Ile or Gly) and 1534 (Phe to Cys), respectively in the IIS6 and IIIS6 segments (Brengues et al., 2003; Harris et al., 2010; Martins et al., 2009a, b; Saavedra-Rodriguez et al., 2007). Mutations in the vicinity of this site in the IIIS6 segment were also encountered in the southern cattle tick *Rhipicephalus microplus* (He et al., 1999) and in the two-spotted spider mite *Tetranychus* 

Although different NaV site mutations are known to confer resistance to pyrethroids, their number is quite restricted; additionally, far related taxa present alterations in the same homologous sites. For instance, the Leu1014Phe *kdr* mutation must have arisen at least on four independent occasions in *An. gambiae* (Pinto et al., 2007). Alterations that do not interfere with the endogenous physiological functions of the Nav must be rare as it is much conserved among animals (ffrench-Constant et al., 1998). As a matter of fact, most of the species studied so far have the *kdr* mutation in the 1014 site, few species proving otherwise

due to codon constraints, like *Ae. aegypti* and some anopheline species.

**6. Molecular assays for monitoring frequency of** *kdr* **mutation in insect** 

Currently, there are many PCR based diagnostic methods for *kdr* mutation available with elevated sensitivity and specificity. For technique choice, one must consider mainly the laboratory resources, facilities and training of technical personnel, which is as important as establishing an defining localities and frequency of sampling. There is neither consensus nor strict rules suitable for all insect species or even for different populations of the same species. Resistance is a very dynamic process depending upon a series of external factors. Therefore, resistance level as well as the selected mechanisms may fluctuate in a short

*melanogaster* sodium channel and pyrethroids or DDT (Usherwood et al., 2007).

al., 2004).

*urticae* (Tsagkarakou et al., 2009).

**natural populations** 

templated nucleotides can be inserted in the edited mRNA. This process alters the protein aminoacid constitution so that it differs from the predicted genomic DNA sequence (Brennicke et al., 1999).

Liu et al. (2004) claimed that RNA editing should be the main regulatory mechanism to modulate the insect NaV function. For instance, no correlation was found between a variety of *DmNaV* originated by alternative splicing and the observed changes in gating properties. Therefore it was implied that RNA editing might play a primary role in determining the voltage dependence of activation and deactivation of *DmNaV* variants (Olson et al., 2008). At least 10 A/I RNA editing substitutions were observed in the *DmNaV* in different points of the Drosophila life cycle indicating developmental regulation (Palladino et al., 2000). These sites are highly conserved in various organisms. Type U/C editing, which is more usual in mitochondria and plastids from higher plants, was also observed in *DmNaV* and *BgNaV*, with electrophysiological alterations in both cases (Liu et al., 2004). Hence, RNA editing should play an important role in the generation of channels with distinct affinities to insecticides. Thus, it seems reasonable to infer that insecticide pressure selects for an adaptive mechanism which might spatially and temporally modulate NaV mRNA editing. Still, in *Cx. quinquefasciatus* mosquitoes, diversity based on U/A editing in the sodium channel mRNA was shown to be related to pyrethroid resistance (Xu et al., 2006). In *Ae. aegypti*, however, recent analysis of *AaNaV* transcripts from a pyrethroid resistant lineage did not identify any sign of RNA editing (Chang et al., 2009).

#### **5.3 The** *kdr* **mutation**

The very first mutation identified as responsible for the *kdr* trait was a leucine to phenylalanine substitution (Leu1014Phe)2 in the NaV IIS6 segment of *M. domestica* (Ingles et al., 1996). Since then, the genomic sequence spanning the region coding for the IIS6 segment has been explored in a vast number of insects, in most of which, the same substitution being found at homologous sites (1014). Besides Phe, Ser is also encountered replacing Leu at the 1014 site in *An. gambiae*. They were initially observed respectively in western and eastern African regions, being commonly referred to as *w-kdr* and *e-kdr* mutations (Pinto et al., 2006). However, nowadays it is known that none of these alleles is restricted to either part of the continent (Ranson et al., 2011). A different substitution in the same 1014 site, Leu1014His, was also associated to pyrethroid resistance in the tobacco budworm *Heliothis virescens* (Park et al., 1999). Many studies identified at least 20 additional substitutions in the NaV sequence, the majority being placed between segments S4 and S5, or internally to segments S5 or S6 of domain II. However, for most of them, the relationship with pyrethroid resistance is only speculative. Good compilations have recently been presented (T. G. Davies et al., 2007a; Dong, 2007; Du et al., 2009).

It is noteworthy that many of these mutations are not in the precise domain of interaction between insecticide and NaV (O'Reilly et al., 2006). On the other hand, it is likely that substitutions in these points of interaction could result in the *super-kdr* trait, which has a more pronounced resistance effect (T. G. Davies et al., 2007b). This phenotype was also first described in *M. domestica* (Williamson et al., 1996) and *Haematobia irritans* (Guerrero et al., 1997). In both species, beyond the Leu1014Phe substitution, a Met918Thr mutation (in the IIS4-S5 linker) was disclosed in flies with very high resistant ratios to pyrethroids, referred

<sup>2</sup> Number refers conventionally to the position in the voltage gated sodium channel primary sequence of *M. domestica Vssc1*, according to Soderlund & Knipple 2003.

templated nucleotides can be inserted in the edited mRNA. This process alters the protein aminoacid constitution so that it differs from the predicted genomic DNA sequence

Liu et al. (2004) claimed that RNA editing should be the main regulatory mechanism to modulate the insect NaV function. For instance, no correlation was found between a variety of *DmNaV* originated by alternative splicing and the observed changes in gating properties. Therefore it was implied that RNA editing might play a primary role in determining the voltage dependence of activation and deactivation of *DmNaV* variants (Olson et al., 2008). At least 10 A/I RNA editing substitutions were observed in the *DmNaV* in different points of the Drosophila life cycle indicating developmental regulation (Palladino et al., 2000). These sites are highly conserved in various organisms. Type U/C editing, which is more usual in mitochondria and plastids from higher plants, was also observed in *DmNaV* and *BgNaV*, with electrophysiological alterations in both cases (Liu et al., 2004). Hence, RNA editing should play an important role in the generation of channels with distinct affinities to insecticides. Thus, it seems reasonable to infer that insecticide pressure selects for an adaptive mechanism which might spatially and temporally modulate NaV mRNA editing. Still, in *Cx. quinquefasciatus* mosquitoes, diversity based on U/A editing in the sodium channel mRNA was shown to be related to pyrethroid resistance (Xu et al., 2006). In *Ae. aegypti*, however, recent analysis of *AaNaV* transcripts from a pyrethroid resistant lineage did not identify any

The very first mutation identified as responsible for the *kdr* trait was a leucine to phenylalanine substitution (Leu1014Phe)2 in the NaV IIS6 segment of *M. domestica* (Ingles et al., 1996). Since then, the genomic sequence spanning the region coding for the IIS6 segment has been explored in a vast number of insects, in most of which, the same substitution being found at homologous sites (1014). Besides Phe, Ser is also encountered replacing Leu at the 1014 site in *An. gambiae*. They were initially observed respectively in western and eastern African regions, being commonly referred to as *w-kdr* and *e-kdr* mutations (Pinto et al., 2006). However, nowadays it is known that none of these alleles is restricted to either part of the continent (Ranson et al., 2011). A different substitution in the same 1014 site, Leu1014His, was also associated to pyrethroid resistance in the tobacco budworm *Heliothis virescens* (Park et al., 1999). Many studies identified at least 20 additional substitutions in the NaV sequence, the majority being placed between segments S4 and S5, or internally to segments S5 or S6 of domain II. However, for most of them, the relationship with pyrethroid resistance is only speculative. Good compilations have recently been presented (T. G. Davies et al., 2007a;

It is noteworthy that many of these mutations are not in the precise domain of interaction between insecticide and NaV (O'Reilly et al., 2006). On the other hand, it is likely that substitutions in these points of interaction could result in the *super-kdr* trait, which has a more pronounced resistance effect (T. G. Davies et al., 2007b). This phenotype was also first described in *M. domestica* (Williamson et al., 1996) and *Haematobia irritans* (Guerrero et al., 1997). In both species, beyond the Leu1014Phe substitution, a Met918Thr mutation (in the IIS4-S5 linker) was disclosed in flies with very high resistant ratios to pyrethroids, referred

2 Number refers conventionally to the position in the voltage gated sodium channel primary sequence

of *M. domestica Vssc1*, according to Soderlund & Knipple 2003.

(Brennicke et al., 1999).

sign of RNA editing (Chang et al., 2009).

**5.3 The** *kdr* **mutation** 

Dong, 2007; Du et al., 2009).

to as the *super-kdr* mutation (Jamroz et al., 1998). However, since it occurs only in association with the Leu1014Phe mutation, its isolated effects are as yet unknown. Although no *superkdr* mutation has so far been identified in mosquitoes, it was suggested that Leu932Phe, in association with Ile936Val (both also in the IIS4-S5 linker), in *Culex* might play this role, being the first example of *super-kdr* in this group (T. G. Davies et al., 2007a). Accordingly, these sites have proved to be important for the interaction between NaV, in the *D. melanogaster* sodium channel and pyrethroids or DDT (Usherwood et al., 2007).

Substitutions in site 929 are also involved in enhanced pyrethroid resistance, as is the case with the Lepidoptera *Plutella xylostella* mutation Thr929Ile, detected in association with Leu1014Phe (Schuler et al., 1998). However, in the maize weevil *Sitophilus zeamais*, the Thr929Ile was found alone (Araujo et al., 2011). In the louse *Pediculus capitis*, in turn, the Thr929Ile mutation was together with Leu932Phe (Lee et al., 2000). There were other substitutions in the same site: Thr/Cys and/or Thr/Val in the diamondblack moth *Frankliniella occidentalis* (Forcioli et al., 2002) and in the cat flea *Ctenocephalides felis* (Bass et al., 2004).

*Ae. aegypti* mosquitoes do not present any substitution in the classic 1014 *kdr* site, unlike many other insects or even mosquitoes from other genera, such as *Anopheles* and *Culex*, very likely because the 1014 site of *Ae. aegypti* NaV is coded by a CTA, in place of the TTA codon present in the majority of other insects. For this reason, two simultaneous nucleotide substitutions would be necessary in order to change from Leu (CTA) to Phe (TTT) or Ser (TCA) (Martins et al., 2009a; Saavedra-Rodriguez et al., 2007). Instead, mutations in different positions have been observed in *Ae. aegypti* populations from Latin America and Southeast Asia, but at least two sites seem to be indeed related to pyrethroid resistance: 1016 (Val to Ile or Gly) and 1534 (Phe to Cys), respectively in the IIS6 and IIIS6 segments (Brengues et al., 2003; Harris et al., 2010; Martins et al., 2009a, b; Saavedra-Rodriguez et al., 2007). Mutations in the vicinity of this site in the IIIS6 segment were also encountered in the southern cattle tick *Rhipicephalus microplus* (He et al., 1999) and in the two-spotted spider mite *Tetranychus urticae* (Tsagkarakou et al., 2009).

Although different NaV site mutations are known to confer resistance to pyrethroids, their number is quite restricted; additionally, far related taxa present alterations in the same homologous sites. For instance, the Leu1014Phe *kdr* mutation must have arisen at least on four independent occasions in *An. gambiae* (Pinto et al., 2007). Alterations that do not interfere with the endogenous physiological functions of the Nav must be rare as it is much conserved among animals (ffrench-Constant et al., 1998). As a matter of fact, most of the species studied so far have the *kdr* mutation in the 1014 site, few species proving otherwise due to codon constraints, like *Ae. aegypti* and some anopheline species.

#### **6. Molecular assays for monitoring frequency of** *kdr* **mutation in insect natural populations**

Currently, there are many PCR based diagnostic methods for *kdr* mutation available with elevated sensitivity and specificity. For technique choice, one must consider mainly the laboratory resources, facilities and training of technical personnel, which is as important as establishing an defining localities and frequency of sampling. There is neither consensus nor strict rules suitable for all insect species or even for different populations of the same species. Resistance is a very dynamic process depending upon a series of external factors. Therefore, resistance level as well as the selected mechanisms may fluctuate in a short

The Pyrethroid Knockdown Resistance 29

Fig. 4. Examples for kdr genotyping based on PCR methods. A – Allelic specific PCR with specific primers in different orientations; B – Allelic specific PCR with specific primers in the same orientation but with additional and differently sized [GC]n tails, in addition to a mismatch in the 3rd base before the 3'-end; C – TaqMan assay based on specific probes with

a different luminescence for each allele. Figure adapted from Yanola et al. (2011).

period of time and space (Kelly-Hope et al., 2008). Moreover, one must be aware about the patterns of distribution and structure of the evaluated populations in order to determine an adequate frequency and sampling size (Ranson et al., 2011).

Allele-specific PCR assays (AS-PCR), as the name suggests, consists of amplification and detection of a specific allele from the DNA of an individual, who is further classified as hetero or homozygous for that allele. Many methodologies based on this strategy have been well succeeded in high-throughput individual diagnostic of *kdr* mutations. Herein, we highlight some PCR based amplifications by allele-specific primers and TaqMan genotyping.

There is ample variation for PCR methods based on allele specific primers. As a first example, one can use two primers (forward and reverse) common for both alleles that amplify a region spanning the mutation site. In this case, additional specific primers, bearing the SNP (single nucleotide polymorphism) at the 3'-end, have opposite orientations in relation to each other (Figure 2-A). The common primers will pair themselves giving rise to a bigger product (that can also be assumed as the positive control reaction) and shorter ones, the consequence of pairing with each allele-specific primer of contrary orientation. The common primers must anneal at sites that result in differently sized products when paring with the specific ones. If both alleles are present (cases when the individual is heterozygous) three products with distinct sizes will be produced (Chen et al., 2010; Harris et al., 2010).

Instead of amplifying a common region for both alleles, it is possible to directly obtain only the specific products (Figure 2-B). This can be accomplished by using only one common primer in one orientation and the two allele specific primers in the opposite sense. However, since the specific primers are at the same orientation and their specificity continues lying upon the 3'-end, something should be incremented in order to obtain distinguishable products. Germer & Higuchi, (1999), later improved by Wang et al. (2005), proposed attaching a GC-tail of different sizes to the 5'-end of the specific primers in a way that the products could be distinguishable by their Tm in a melting curve analysis. In this case the mix reaction contains a fluorescent dye, which lights up when bounded to double strand DNA, carried out in a fluorescence-detecting thermocycler ("Real time PCR"). Additionally, a different mismatch (pirimidine for purine or vice–versa) is added to the third site before the 3'-end of each allele specific primer, in order to strengthen their specificity (Okimoto & Dodgson, 1996). Alternatively, the products can also be distinguishable in a gel electrophoresis.

The second group of techniques is based on the amplification of a region spanning the *kdr* mutation site followed by the detection of the different alleles by specific hybridization with minor groove binding (MGB) DNA fluorescent probes, also known as TaqMan assay (Figure 2-C). Different alleles can be detected in the same reaction, since each probe is attached to a distinct fluorophore. The probe is constituted of an oligonucleotide specific for the SNP with a reporter fluorescent dye in the 5'-end and a non fluorescent quencher in the 3'-end (Araujo et al., 2011; Morgan et al., 2009; Yanola et al., 2011). Bass et al., (2007) concluded that TaqMan probes were the most accurate for *kdr* genotyping among six different evaluated methods.

Other techniques have also been applied. The *Hola* (Heated Oligonucleotide Ligation Assay, see details in Black et al., 2006) revealed high specificity in detecting different NaV alleles in the 1011 (Ile, Met and Val) and 1016 (Val, Ile and Gly) sites from Thai *Ae. aegypti* populations (Rajatileka et al., 2008) and in the 1014 site of *Cx. quinquefasciatus* from Sri Lanka (Wondji et

period of time and space (Kelly-Hope et al., 2008). Moreover, one must be aware about the patterns of distribution and structure of the evaluated populations in order to determine an

Allele-specific PCR assays (AS-PCR), as the name suggests, consists of amplification and detection of a specific allele from the DNA of an individual, who is further classified as hetero or homozygous for that allele. Many methodologies based on this strategy have been well succeeded in high-throughput individual diagnostic of *kdr* mutations. Herein, we highlight some PCR based amplifications by allele-specific primers and TaqMan

There is ample variation for PCR methods based on allele specific primers. As a first example, one can use two primers (forward and reverse) common for both alleles that amplify a region spanning the mutation site. In this case, additional specific primers, bearing the SNP (single nucleotide polymorphism) at the 3'-end, have opposite orientations in relation to each other (Figure 2-A). The common primers will pair themselves giving rise to a bigger product (that can also be assumed as the positive control reaction) and shorter ones, the consequence of pairing with each allele-specific primer of contrary orientation. The common primers must anneal at sites that result in differently sized products when paring with the specific ones. If both alleles are present (cases when the individual is heterozygous) three products with distinct sizes will be

Instead of amplifying a common region for both alleles, it is possible to directly obtain only the specific products (Figure 2-B). This can be accomplished by using only one common primer in one orientation and the two allele specific primers in the opposite sense. However, since the specific primers are at the same orientation and their specificity continues lying upon the 3'-end, something should be incremented in order to obtain distinguishable products. Germer & Higuchi, (1999), later improved by Wang et al. (2005), proposed attaching a GC-tail of different sizes to the 5'-end of the specific primers in a way that the products could be distinguishable by their Tm in a melting curve analysis. In this case the mix reaction contains a fluorescent dye, which lights up when bounded to double strand DNA, carried out in a fluorescence-detecting thermocycler ("Real time PCR"). Additionally, a different mismatch (pirimidine for purine or vice–versa) is added to the third site before the 3'-end of each allele specific primer, in order to strengthen their specificity (Okimoto & Dodgson, 1996). Alternatively, the products can also be distinguishable in a gel

The second group of techniques is based on the amplification of a region spanning the *kdr* mutation site followed by the detection of the different alleles by specific hybridization with minor groove binding (MGB) DNA fluorescent probes, also known as TaqMan assay (Figure 2-C). Different alleles can be detected in the same reaction, since each probe is attached to a distinct fluorophore. The probe is constituted of an oligonucleotide specific for the SNP with a reporter fluorescent dye in the 5'-end and a non fluorescent quencher in the 3'-end (Araujo et al., 2011; Morgan et al., 2009; Yanola et al., 2011). Bass et al., (2007) concluded that TaqMan probes were the most accurate for *kdr* genotyping among six

Other techniques have also been applied. The *Hola* (Heated Oligonucleotide Ligation Assay, see details in Black et al., 2006) revealed high specificity in detecting different NaV alleles in the 1011 (Ile, Met and Val) and 1016 (Val, Ile and Gly) sites from Thai *Ae. aegypti* populations (Rajatileka et al., 2008) and in the 1014 site of *Cx. quinquefasciatus* from Sri Lanka (Wondji et

adequate frequency and sampling size (Ranson et al., 2011).

produced (Chen et al., 2010; Harris et al., 2010).

genotyping.

electrophoresis.

different evaluated methods.

Fig. 4. Examples for kdr genotyping based on PCR methods. A – Allelic specific PCR with specific primers in different orientations; B – Allelic specific PCR with specific primers in the same orientation but with additional and differently sized [GC]n tails, in addition to a mismatch in the 3rd base before the 3'-end; C – TaqMan assay based on specific probes with a different luminescence for each allele. Figure adapted from Yanola et al. (2011).

The Pyrethroid Knockdown Resistance 31

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### **7. Conclusions**

New strategies for arthropod control based on the release of laboratory manipulated insects that would suppress or substitute natural populations are being tested in the field with great prospect. The release of transgenic insects carrying a dominant lethal gene (RIDL) (Black et al., 2011) or of mosquitoes with the intracellular *Wolbachia*, that lead to refractoriness to other parasites (Werren et al., 2008) are currently the most discussed strategies. However, the laboratory handling process has to consider specific and sometimes complex aspects for each insect species, and it may take many years until field control based on this kind of approach can be effectively accomplished. Moreover, field studies that guarantee the environmental safety of releasing manipulated insects may take even longer. Hence, even if these strategies prove to be efficient to reduce, extinguish, or substitute a target insect population, the use of insecticides may still indeed play an essential role for many years to come, especially during periods of high insect or disease incidence.

Pyrethroids are largely the most adopted insecticide class in agriculture and for public health purposes. Their use tends to increase, since pyrethroids are the only safe compound to impregnate insecticide treated nets (ITNs), a strategy under expansion against mosquitoes. Advances regarding knowledge of its target, the voltage gated sodium channel, can contribute to the design of new compounds as well as the rapid identification of resistance related mutations. The continuous monitoring of insecticide resistance status, and its mechanisms, in natural populations has proven to be an important tool in the preservation of these compounds.

#### **8. Acknowledgements**

We thank Andre Torres for his illustrations presented in this work, the Instituto de Biologia do Exército (IBEx) and Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular (INCT-EM). English review and revision by Mitchell Raymond Lishon, native of Chicago, Illinois, U.S.A – U.C.L.A, 1969. Financial support: Fiocruz, Pronex-dengue/CNPq, Faperj, SVS/MS and CAPES.

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New strategies for arthropod control based on the release of laboratory manipulated insects that would suppress or substitute natural populations are being tested in the field with great prospect. The release of transgenic insects carrying a dominant lethal gene (RIDL) (Black et al., 2011) or of mosquitoes with the intracellular *Wolbachia*, that lead to refractoriness to other parasites (Werren et al., 2008) are currently the most discussed strategies. However, the laboratory handling process has to consider specific and sometimes complex aspects for each insect species, and it may take many years until field control based on this kind of approach can be effectively accomplished. Moreover, field studies that guarantee the environmental safety of releasing manipulated insects may take even longer. Hence, even if these strategies prove to be efficient to reduce, extinguish, or substitute a target insect population, the use of insecticides may still indeed play an essential role for many years to

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**3** 

**Photoremediation of Carbamate** 

Pesticides are extensively world wide used for agriculture and for non-agricultural purposes. The major environmental concern of used pesticides is their ability to leach down to subsoil and contaminate the ground water, or, if they immobile, they could persist on the top soil and become harmful to microorganisms, plants, animal and people (Jha & Mishra 2005; Radivojević et al., 2008). Harmfull pesticide residues can contaminate the environment and accumulate in ecosystems than entering the human food chain (Đurović et al., 2010; Gašić et al., 2002a; Gevao et al., 2000). Pesticides have various characteristics that determine how act once in soil where it could accumulate to toxic level. Generally, soil and groundwater pollution are the major consequences environmental effects of pesticides application. Pesticides can reach water through surface runoff from treated plants and soil. Pesticide sprays usually directly hit non-target vegetation or can drift or volatilize from the treated areas that contaminate air, soil, and non-target plants. Finally, using of pesticides has resulted in acute and chronic ecological damage either by direct injury such as birds and fish

Carbamates are large group of pesticides which have been extensively used in almost sixty years. In this chapter an attempt is made to give the available data of the carbamates used as pesticides, their physico-chemical and toxicological characteristics, behaviour and fate in the environment, types of formulations which exist on the market as well as photochemical degradation for the certain members. Owing to widespread use in agriculture and relatively good solubility in water carbamate compounds can contaminate surface and ground waters

In this chapter we will also discuss some very important photocatalytic methods for remediation of water containing carbamate residues: direct photodegradation (photolysis), photosensitized degradation and photocatalytic degradation (including heterogeneous TiO2

Carbamates were developed into commercial pesticides in the 1950s. It is a very huge family which members are effective as insecticides, herbicides, and fungicides, but they are most commonly used as insecticides. More than 50 carbamates are known. The most often used

and therefore carries a risk to various consumers, as well as the environment.

and ZnO processes and photo-Fenton and Fenton-like processes).

**1. Introduction** 

or by indirect.

**2. Carbamates** 

Anđelka V. Tomašević and Slavica M. Gašić *Institute of Pesticides and Environmental Protection,* 

**Residues in Water** 

*Belgrade-Zemun,* 

*Serbia* 


### **Photoremediation of Carbamate Residues in Water**

Anđelka V. Tomašević and Slavica M. Gašić *Institute of Pesticides and Environmental Protection, Belgrade-Zemun,* 

*Serbia* 

#### **1. Introduction**

38 Insecticides – Basic and Other Applications

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Pesticides are extensively world wide used for agriculture and for non-agricultural purposes. The major environmental concern of used pesticides is their ability to leach down to subsoil and contaminate the ground water, or, if they immobile, they could persist on the top soil and become harmful to microorganisms, plants, animal and people (Jha & Mishra 2005; Radivojević et al., 2008). Harmfull pesticide residues can contaminate the environment and accumulate in ecosystems than entering the human food chain (Đurović et al., 2010; Gašić et al., 2002a; Gevao et al., 2000). Pesticides have various characteristics that determine how act once in soil where it could accumulate to toxic level. Generally, soil and groundwater pollution are the major consequences environmental effects of pesticides application. Pesticides can reach water through surface runoff from treated plants and soil. Pesticide sprays usually directly hit non-target vegetation or can drift or volatilize from the treated areas that contaminate air, soil, and non-target plants. Finally, using of pesticides has resulted in acute and chronic ecological damage either by direct injury such as birds and fish or by indirect.

Carbamates are large group of pesticides which have been extensively used in almost sixty years. In this chapter an attempt is made to give the available data of the carbamates used as pesticides, their physico-chemical and toxicological characteristics, behaviour and fate in the environment, types of formulations which exist on the market as well as photochemical degradation for the certain members. Owing to widespread use in agriculture and relatively good solubility in water carbamate compounds can contaminate surface and ground waters and therefore carries a risk to various consumers, as well as the environment.

In this chapter we will also discuss some very important photocatalytic methods for remediation of water containing carbamate residues: direct photodegradation (photolysis), photosensitized degradation and photocatalytic degradation (including heterogeneous TiO2 and ZnO processes and photo-Fenton and Fenton-like processes).

#### **2. Carbamates**

Carbamates were developed into commercial pesticides in the 1950s. It is a very huge family which members are effective as insecticides, herbicides, and fungicides, but they are most commonly used as insecticides. More than 50 carbamates are known. The most often used

Photoremediation of Carbamate Residues in Water 41

It is known that esters or N-substituted derivates of carbamic acid are unstable compounds, especially under alkaline conditions. Decomposition under this conditions takes place and

Derivates of carbamic acid as salts or esters are more stable than carbamic acid. This enhanced stability is the basis for synthesis of many derivates that are biologically active

Carbamate ester derivates are crystalline solids of low vapor pressure with variable, but usually low water solubility. They are moderately soluble in solvents such as benzene, toluene, xylene, chloroform, dichloromethane and 1,2-dichloromethane. Generally, they are poorly soluble in nonpolar organic solvents such as petroleum hydrocarbons but highly soluble in polar organic solvents such as methanol, ethanol, acetone, dimethylformamide,

Most carbamates are active inhibitors of acetylholinesteraze (AChE), but some carbamates as benzimidazole have no acetylcholinesterase activity. Carbamates toxicity to insects, nematodes, and mammals is based on inhibition of acetylcholinesterase, which is the enzyme responsible for the hydrolysis of acetycholine into choline and acetic acid. Acetylcholine (ACh) is a substance that transmits a nerve impulse from a nerve cell to a specific receptor such as another nerve cell or a muscle cell. Acetylcholine, in essence, acts as a chemical switch. When it is present (produced by nerve cell) it turns the nerve impulse on. When it is absent, the nerve impulse is discontinued. The nerve transmission ends when the enzyme aceylcholinesterase breaks down the acetylcholine into choline and acetic acid. Without the action of this enzyme acetylcholine builds up at the junction of nerve cell and the receptor site, and the nerve impulse continues. Carbamate insecticides block (or inhibit) the ability of this enzyme, acetylcholinesterase, to break down the acetylcholine and the

In mammals, cholinesterase inhibition caused by carbamates is labile, reversible process. Estimates of the recovery time in humans range from immediate up to four days, depending on the dose, the specific pesticide and the method of exposure. The breakdown of carbamate compounds within an organisms is a complex process and is depended on the specific pesticide structure. The rapid degradation of carbamates *in vivo* by mammals occurs by hydrolysis, oxidation and conjugation. The end products include amines, alcohols or phenol

Inhibition of acetylholinesteraze (AChE) by carbamates causes toxic effects in animals and human beings that result in variety of poisoning symptoms. Carbamates acute toxicity and poisoning are dose related. Acute poisoning occurs rapidly after exposure. Ingestion of carbamate insecticides at low doses can cause excessive salivation and an increase in the rate of breathing within 30 min. At higher doses this is followed by excessive tearing, urination, no control defecation, nausea and vomiting. At the highest doses, symptoms can include those listed above along with violent intestinal movements, muscle spasm and convulsions. Death has occurred in a few instances, usually due to respiratory failure resulting from

While the insecticidal carbamate produce the typical symptoms of cholinesterase inhibition, they don't appear to induce a delayed neurotoxic reaction similar to that seen with some organophosphourus compound. Chronic exposure to carbamate compounds may cause

derivates. The urinary route is the main excretory route (Machemer & Pickel, 1994).

the compounds as alcohol, phenol, ammonia, amine and carbon dioxide are formed.

**2.1 Carbamates physical and chemical properties** 

**2.2 Carbamates mode of action and toxicity** 

nerve impulse (Kamrin, 1997; Machemer & Pickel, 1994).

paralysis of the respiratory muscles (Kamrin, 1997; WHO, 1986).

pesticides.

etc (WHO, 1986).

members of carbamate group are: aldicarb, asulam, bendiocarb, carbaryl, carbetamid, carbofuran, carbosulfan, chlorpropham, desmedipham, ethiofencarb, formetanate, furatiocarb, fenoxycarb, isoprocarb, methiocarb, methomyl, oxamyl, phenmedipham, pirimicarb, promecarb, propamocarb and propoxur.

Carbamates are N-substituited esters of carbamic acid. Their general formula is:

$$\begin{array}{c} \text{O} \\ \text{R}\_{1}\text{NH} \longrightarrow \text{C} \longrightarrow \text{OR}\_{2} \end{array}$$

Fig. 1. General carbamate structure, where R2 is an aromatic or aliphatic moiety, if R1 is a methyl group it is carbamate insecticide, if R1 is an aromatic moiety it is carbamate herbicide and if R1 is a benzimidazole moiety it is carbamate fungicide (WHO, 1986).


Table 1. Relationship of chemical structure and pesticide activity of carbamates (WHO, 1986).

members of carbamate group are: aldicarb, asulam, bendiocarb, carbaryl, carbetamid, carbofuran, carbosulfan, chlorpropham, desmedipham, ethiofencarb, formetanate, furatiocarb, fenoxycarb, isoprocarb, methiocarb, methomyl, oxamyl, phenmedipham,

O

OR2

Carbamates are N-substituited esters of carbamic acid. Their general formula is:

R1NH C

and if R1 is a benzimidazole moiety it is carbamate fungicide (WHO, 1986).

O

O

C

C

alkyl NH O aryl

O

aryl NH O alkyl

O

C

Table 1. Relationship of chemical structure and pesticide activity of carbamates (WHO, 1986).

aryl NH O alkyl

O

aryl NH O alkyl

O

NH O aryl

NH O N alkyl

Fig. 1. General carbamate structure, where R2 is an aromatic or aliphatic moiety, if R1 is a methyl group it is carbamate insecticide, if R1 is an aromatic moiety it is carbamate herbicide

**activity Chemical structure Common or other names** 

aldoxycarb, allyxycarb, aminocarb, BPMC, bendiocarb, bufencarb, butacarb, carbanolate, carbaryl, carbofuran, cloethocarb, dimetilan, dioxacarb, ethiofencarb, formetanate, hoppcide, isoprocarb, trimethacarb, MPMC, methiocarb, metolcarb, mexacarbate, pirimicab, promacyl, promecarb, propoxur, MTMC, XMC, xylylcarb

aldicarb, methomyl, oxamyl, thiofanox, thiodicarb

asulam, barban, carbetamide, chlorbufam, desmedipham, phenmedipham, swep

dichlormate, karbutilate, terbucarb

propham, chlorpropham

benomyl, carbendazim, thiophanatemethyl, thiophanate-ethyl

pirimicarb, promecarb, propamocarb and propoxur.

CH3 C

CH3 C

**Pesticide** 

**Insecticide** 

**Herbicide** 

**Herbicide and sprout** 

**Fungicide** 

**inhibitors** C

#### **2.1 Carbamates physical and chemical properties**

It is known that esters or N-substituted derivates of carbamic acid are unstable compounds, especially under alkaline conditions. Decomposition under this conditions takes place and the compounds as alcohol, phenol, ammonia, amine and carbon dioxide are formed.

Derivates of carbamic acid as salts or esters are more stable than carbamic acid. This enhanced stability is the basis for synthesis of many derivates that are biologically active pesticides.

Carbamate ester derivates are crystalline solids of low vapor pressure with variable, but usually low water solubility. They are moderately soluble in solvents such as benzene, toluene, xylene, chloroform, dichloromethane and 1,2-dichloromethane. Generally, they are poorly soluble in nonpolar organic solvents such as petroleum hydrocarbons but highly soluble in polar organic solvents such as methanol, ethanol, acetone, dimethylformamide, etc (WHO, 1986).

#### **2.2 Carbamates mode of action and toxicity**

Most carbamates are active inhibitors of acetylholinesteraze (AChE), but some carbamates as benzimidazole have no acetylcholinesterase activity. Carbamates toxicity to insects, nematodes, and mammals is based on inhibition of acetylcholinesterase, which is the enzyme responsible for the hydrolysis of acetycholine into choline and acetic acid. Acetylcholine (ACh) is a substance that transmits a nerve impulse from a nerve cell to a specific receptor such as another nerve cell or a muscle cell. Acetylcholine, in essence, acts as a chemical switch. When it is present (produced by nerve cell) it turns the nerve impulse on. When it is absent, the nerve impulse is discontinued. The nerve transmission ends when the enzyme aceylcholinesterase breaks down the acetylcholine into choline and acetic acid. Without the action of this enzyme acetylcholine builds up at the junction of nerve cell and the receptor site, and the nerve impulse continues. Carbamate insecticides block (or inhibit) the ability of this enzyme, acetylcholinesterase, to break down the acetylcholine and the nerve impulse (Kamrin, 1997; Machemer & Pickel, 1994).

In mammals, cholinesterase inhibition caused by carbamates is labile, reversible process. Estimates of the recovery time in humans range from immediate up to four days, depending on the dose, the specific pesticide and the method of exposure. The breakdown of carbamate compounds within an organisms is a complex process and is depended on the specific pesticide structure. The rapid degradation of carbamates *in vivo* by mammals occurs by hydrolysis, oxidation and conjugation. The end products include amines, alcohols or phenol derivates. The urinary route is the main excretory route (Machemer & Pickel, 1994).

Inhibition of acetylholinesteraze (AChE) by carbamates causes toxic effects in animals and human beings that result in variety of poisoning symptoms. Carbamates acute toxicity and poisoning are dose related. Acute poisoning occurs rapidly after exposure. Ingestion of carbamate insecticides at low doses can cause excessive salivation and an increase in the rate of breathing within 30 min. At higher doses this is followed by excessive tearing, urination, no control defecation, nausea and vomiting. At the highest doses, symptoms can include those listed above along with violent intestinal movements, muscle spasm and convulsions. Death has occurred in a few instances, usually due to respiratory failure resulting from paralysis of the respiratory muscles (Kamrin, 1997; WHO, 1986).

While the insecticidal carbamate produce the typical symptoms of cholinesterase inhibition, they don't appear to induce a delayed neurotoxic reaction similar to that seen with some organophosphourus compound. Chronic exposure to carbamate compounds may cause

Photoremediation of Carbamate Residues in Water 43

steric and electronic properties of the carbamates. The carbamate derivates with herbicidal action are substantially more stable to alkaline hydrolysis than the methyl carbamate

Carbamate compounds degrade through chemical hydrolysis and this is the first step in the metabolic degradation. The hydrolysis products will be further metabolized in soil and plant. Chemical degradation does not appear to have much influence in the total degradation of pesticides in soil. Carbamate compounds are adsorbed and translocated through plants and treated crops. In most cases, carbamates will break down quickly in

Finally carbamates are metabolized by microorganisms, plants and animals or broken down in water or soil. In water carbamates degraded by chemical hydrolysis, but photodegradation and aquatic microbes may also contribute degradation. Generally, in alkaline water and under sunlight carbamate compounds will decompose more rapidly

Carbamate products come in variety of solid and liquid formulations on the market. They contain beside carbamate compounds inert ingredients which could be toxic, flammable or reactive. Examples of inert ingredients are wetting agents, spreaders, dispersing agents, solvents, solubilizers, carriers, ticker, surfactants and so on. A surfactant is a substance that reduced surface tension of a system, allowing oil-based and water-based substances to mix more readily. A common groups of non-ionic surfactants are the alkylphenol polyethoxylates or alcohol ethoxylates which may be used in pesticide formulations. Nonyl phenols, one of the members of above mention alkylphenol surfactant has been linked to endocrine-disrupting effects in aquatic animals and should be substituted by less hazardous alternatives. Commonly used formulation types include liquid and dry formulations as emulsifiable concentrates (EC), soluble concentrates (SL), suspension concentrates (SC), than wettable powders (WP), water dispersible granules (WG), granules (GR), etc, and they are

Pesticides are very often formulated as emulsifiable concentrates (EC) which produce emulsions when dissolved in water. The first problem in defining this formulation is the selection of an adequate surfactants (emulsifiers) for the intended purposes (Gašić et al., 1998a, 1998b, 2002b; Shinoda & Friberg, 1986). Recently there is increasing interest in the effect of emulsifiers on toxicity to mammals and fish. These effects can be due to inherent toxicity of the surfactant itself or to the enhancing effect that the emulsifiers may have on toxicity of active ingredient. So, the formulation type can have implications for product efficacy and exposure to humans and other non-target organisms (Knowles, 2005, 2006;

While the toxicity of the active ingredient of a pesticide is property which can not be changed, the acute toxicity effects of the formulation are strongly influenced by the way in which the active ingredient is formulated. While pesticide formulations are influenced by both the physical and chemical properties of the active ingredient and the economic pressures of the marketplace, there are formulation choices which will increase the safety of

The type of pesticide formulations and in, some cases, the choice of product of the same formulation type can significantly affect the results obtained in practical use. Safety, efficacy, residual life, cost, availability and ease of use must all be considered in selecting

derivatives, which have an insecticidal action (Kaufman, 1967).

plants and the residues in plants will last not very long .

signed by international coding system (CropLife, 2008).

pesticide formulations (Mollet & Grubenmann, 2001).

(WHO, 1986).

Sher, 1984).

**2.4 Formulations** 

adverse effects on organs or acetylcholinesterase levels. These effects are unlikely to occur in humans at expected exposure levels (Kamrin, 1997).

The acute toxicity of different member of carbamates ranges from highly toxic to only slightly toxic. The LD50 for the rats ranges from less than 1 mg/kg to over 5000 mg/kg body weight. The acute dermal toxicity of carbamates is generally low to moderate except aldicarb which is very toxic. The carbamates in short term and long term toxicity studies showed different toxicity. Some carbamates are very toxic and others less. Carbamate pesticides are transformed metabolically by variety of chemical reactions in more water soluble molecules which can be excreted via the urine. Rats eliminate carbamate compounds rapidly in that way. Most metabolites are excreted within 24 h of exposure and therefore carbamate residues don't accumulate in animals (Kamrin, 1997; WHO, 1986). In the study of carbofuran toxicity on rats during subhronic exposure the histopathological changes in liver and kidneys were observed but there was cell regeneration in all test groups as well (Brkić et al., 2008).

Aldicarb is the most toxic among the carbamates and establish acceptable daily intake (ADI) for humans is 0.001 mg/kg/body weight. The other carbamates have ADIs values in range of 0.001-0.1 mg/kg/ body weight (WHO, 1986). According to the European Food Safety Authority (2009), the lowest ADI has carbofuran 0.00015 mg/kg/body weight (EU Pesticide Database, 2011).

Many carbamates have been studied for reproductive, teratogenic, mutagenic and carcinogenic effects and the results of this is that a few members of this family has been banned by the regulatory bodies worldwide.

#### **2.3 Environmental fate**

Generally, carbamates remain active for a few hours to a few month in soils and crop, but they may leave residues in agricultural products (Takino et al., 2004). The rate of degradation in soil depends on soil type, soil moisture, adsorption, pH, soil temperature, concentration of pesticide, microbial activity and photodecomposition. The higher the organic content, the greater the binding to soil and thus the greater the persistence. Also, the higher the soil acidity, the longer it takes for carbamates to be degraded. Carbamate insecticides are mainly applied on the plants, but can reach the soil, while carbamate nematocides and herbicides are applied directly to the soil. Generally, in soil carbamates degraded by chemical hydrolysis and microbial processes. Microorganisms that have capability to degrade carbamate pesticides play a significant role in the break dawn and elimination of them from environment. Because the different carbamates have different properties, it is clear that each of them should be evaluated on its own merits, and no extrapolation of results can be made from one carbamate to another. One carbamate may be easily decomposed, while another may be strongly adsorbed on soil. Some leach out easily and may reach groundwater. In these processes, the soil type and water solubility are of great importance. Furthermore, it should be recognized that this not only concerns the parent compound but also the breakdown products or metabolites (Kamrin, 1997; WHO, 1986).

Persistence of carbamate herbicides is increased by application to dry soil surface or by soil incorporation. Environmental factors which increasing microbial activity in soil generally decrease the persistence of carbamate herbicides. In most of degradation reactions the initial cleavage of the molecule occurs at ester linkage. Enzymatic hydrolysis of some carbamates can be correlated to soil acidity, and rate differences explained by consideration of certain steric and electronic properties of the carbamates. The carbamate derivates with herbicidal action are substantially more stable to alkaline hydrolysis than the methyl carbamate derivatives, which have an insecticidal action (Kaufman, 1967).

Carbamate compounds degrade through chemical hydrolysis and this is the first step in the metabolic degradation. The hydrolysis products will be further metabolized in soil and plant. Chemical degradation does not appear to have much influence in the total degradation of pesticides in soil. Carbamate compounds are adsorbed and translocated through plants and treated crops. In most cases, carbamates will break down quickly in plants and the residues in plants will last not very long .

Finally carbamates are metabolized by microorganisms, plants and animals or broken down in water or soil. In water carbamates degraded by chemical hydrolysis, but photodegradation and aquatic microbes may also contribute degradation. Generally, in alkaline water and under sunlight carbamate compounds will decompose more rapidly (WHO, 1986).

#### **2.4 Formulations**

42 Insecticides – Basic and Other Applications

adverse effects on organs or acetylcholinesterase levels. These effects are unlikely to occur in

The acute toxicity of different member of carbamates ranges from highly toxic to only slightly toxic. The LD50 for the rats ranges from less than 1 mg/kg to over 5000 mg/kg body weight. The acute dermal toxicity of carbamates is generally low to moderate except aldicarb which is very toxic. The carbamates in short term and long term toxicity studies showed different toxicity. Some carbamates are very toxic and others less. Carbamate pesticides are transformed metabolically by variety of chemical reactions in more water soluble molecules which can be excreted via the urine. Rats eliminate carbamate compounds rapidly in that way. Most metabolites are excreted within 24 h of exposure and therefore carbamate residues don't accumulate in animals (Kamrin, 1997; WHO, 1986). In the study of carbofuran toxicity on rats during subhronic exposure the histopathological changes in liver and kidneys were observed but there was cell regeneration in all test groups as well (Brkić et

Aldicarb is the most toxic among the carbamates and establish acceptable daily intake (ADI) for humans is 0.001 mg/kg/body weight. The other carbamates have ADIs values in range of 0.001-0.1 mg/kg/ body weight (WHO, 1986). According to the European Food Safety Authority (2009), the lowest ADI has carbofuran 0.00015 mg/kg/body weight (EU Pesticide

Many carbamates have been studied for reproductive, teratogenic, mutagenic and carcinogenic effects and the results of this is that a few members of this family has been

Generally, carbamates remain active for a few hours to a few month in soils and crop, but they may leave residues in agricultural products (Takino et al., 2004). The rate of degradation in soil depends on soil type, soil moisture, adsorption, pH, soil temperature, concentration of pesticide, microbial activity and photodecomposition. The higher the organic content, the greater the binding to soil and thus the greater the persistence. Also, the higher the soil acidity, the longer it takes for carbamates to be degraded. Carbamate insecticides are mainly applied on the plants, but can reach the soil, while carbamate nematocides and herbicides are applied directly to the soil. Generally, in soil carbamates degraded by chemical hydrolysis and microbial processes. Microorganisms that have capability to degrade carbamate pesticides play a significant role in the break dawn and elimination of them from environment. Because the different carbamates have different properties, it is clear that each of them should be evaluated on its own merits, and no extrapolation of results can be made from one carbamate to another. One carbamate may be easily decomposed, while another may be strongly adsorbed on soil. Some leach out easily and may reach groundwater. In these processes, the soil type and water solubility are of great importance. Furthermore, it should be recognized that this not only concerns the parent compound but also the breakdown products or metabolites (Kamrin, 1997; WHO,

Persistence of carbamate herbicides is increased by application to dry soil surface or by soil incorporation. Environmental factors which increasing microbial activity in soil generally decrease the persistence of carbamate herbicides. In most of degradation reactions the initial cleavage of the molecule occurs at ester linkage. Enzymatic hydrolysis of some carbamates can be correlated to soil acidity, and rate differences explained by consideration of certain

humans at expected exposure levels (Kamrin, 1997).

banned by the regulatory bodies worldwide.

al., 2008).

1986).

Database, 2011).

**2.3 Environmental fate** 

Carbamate products come in variety of solid and liquid formulations on the market. They contain beside carbamate compounds inert ingredients which could be toxic, flammable or reactive. Examples of inert ingredients are wetting agents, spreaders, dispersing agents, solvents, solubilizers, carriers, ticker, surfactants and so on. A surfactant is a substance that reduced surface tension of a system, allowing oil-based and water-based substances to mix more readily. A common groups of non-ionic surfactants are the alkylphenol polyethoxylates or alcohol ethoxylates which may be used in pesticide formulations. Nonyl phenols, one of the members of above mention alkylphenol surfactant has been linked to endocrine-disrupting effects in aquatic animals and should be substituted by less hazardous alternatives. Commonly used formulation types include liquid and dry formulations as emulsifiable concentrates (EC), soluble concentrates (SL), suspension concentrates (SC), than wettable powders (WP), water dispersible granules (WG), granules (GR), etc, and they are signed by international coding system (CropLife, 2008).

Pesticides are very often formulated as emulsifiable concentrates (EC) which produce emulsions when dissolved in water. The first problem in defining this formulation is the selection of an adequate surfactants (emulsifiers) for the intended purposes (Gašić et al., 1998a, 1998b, 2002b; Shinoda & Friberg, 1986). Recently there is increasing interest in the effect of emulsifiers on toxicity to mammals and fish. These effects can be due to inherent toxicity of the surfactant itself or to the enhancing effect that the emulsifiers may have on toxicity of active ingredient. So, the formulation type can have implications for product efficacy and exposure to humans and other non-target organisms (Knowles, 2005, 2006; Sher, 1984).

While the toxicity of the active ingredient of a pesticide is property which can not be changed, the acute toxicity effects of the formulation are strongly influenced by the way in which the active ingredient is formulated. While pesticide formulations are influenced by both the physical and chemical properties of the active ingredient and the economic pressures of the marketplace, there are formulation choices which will increase the safety of pesticide formulations (Mollet & Grubenmann, 2001).

The type of pesticide formulations and in, some cases, the choice of product of the same formulation type can significantly affect the results obtained in practical use. Safety, efficacy, residual life, cost, availability and ease of use must all be considered in selecting

Photoremediation of Carbamate Residues in Water 45

Among AOPs, heterogeneous photocatalysis using TiO2 as photocatalyst appears as the most emerging destructive technology. The following mechanism of the TiO2 photocatalysis has been proposed (Daneshvar et al., 2003; Gomes da Silva Faria, 2003; Karkmaz et al.,

(UV) eCB-

(H2O ↔ H+ + OH-)ads + hVB+ H+ + •OH (6)

ZnO is also frequently used as a catalyst in heterogeneous photocatalytic reactions. The biggest advantage of ZnO in comparison to TiO2 is that it absorbs over a larger fraction of the UV spectrum and the corresponding threshold wavelength of ZnO is 387 nm. Upon irradiation, valence band electrons are promoted to the conduction band leaving a hole behind. These electron-hole pairs can either recombine or interact separately with other molecules. The holes at the ZnO valence band can oxidize adsorbed water or hydroxide ions to produce hydroxyl radicals. Electron in the conduction band at the catalyst surface can reduce molecular oxygen to superoxide anion. This radical may form organic peroxides or hydrogen peroxide in the presence of organic scavengers. The hydroxyl radical attacks organic compounds (R) and intermediates (Int) are formed. These intermediates react with hydroxyl radicals to produce the final products (P). The mechanism of heterogeneous photocatalysis in the presence of ZnO can be given by the following reactions (Behnajady et

al., 2006; Daneshvar et al., 2004, 2007; Pera-Titus at al,. 2004; Tomaševic at al., 2010a):

eCB- + hVB+ heat (10)

ZnO + *h*


hVB+ + H2Oads H+ +

hVB+ + –OHads

eCB- + O2 O2

O2

d) oxidation of the organic reactant via successive attacks by •OH radicals:

●-(3)

+ hVB+ (4)


R + •OH R'• + H2O (7)

(UV) eCB- + hVB+ (9)

+ H+ H2O2 + O2 (14)

OHads (11)

OHads (12)

– (13)

R + h+ R•+ degradation products (8)

e- + O2 → O2

a) absorption of efficient photons by titania (*hv*  Eg=3.2 eV):

(O2)ads + eCB- O2

c) neutralization of OH- groups into •OH by photoholes:

2004; Tomaševic at al., 2010a):

b) oxygen ionosorption:

TiO2 + *h*

e) or by direct reaction with holes:

formulation. The ways in which pesticides are formulated considerably influence their persistence. Formulations in order of increasing persistence on plants are prepared in the way that more readly adsorbed on the soil fractions and not appreciably degradated (Edwards, 1975).

#### **3. Photodegradation processes for carbamates wastewater treatments**

#### **3.1 Photolysis**

Photolysis (direct photodegradation reaction) is photodegradation process without any catalysts and use light only for degradation of different organic molecules, including pesticides and related compounds. Direct irradiation will lead to the promotion of the pesticides to their excited singlet states and such excited states can then undergo among homolysis, heterolysis or photoionization processes (Burrows et al., 2002). Direct photodegradation by solar light is limited and various lamps have been used for irradiation of contaminated water solutions. The photolysis of contaminants (including pesticides) in aqueos solution depends on the different reaction parameters such as type of light, lamp distance, temperature, initial concentration of pesticides, type of water, pH, the presence of humic and fulvic acids, the presence of O2, O3, O2/O3 and H2O2, the presence of inorganic ions and organic matter dissolved in water (Burrows et al., 2002; Tomaševic at al., 2010a).

#### **3.2 Photosensitized degradation**

The photosensitized reaction is based on the absorption of light by a molecule of the sensitizer and includes an energy transfer from molecul excited state to the pesticides. The most famous sensitizers are aceton, rose Bengal, methylene blue and humic and fulvic acids (Burrows et al., 2002).

#### **3.3 Advanced oxidation processes**

Advanced Oxidation Processes (AOPs) include catalytic and photochemical methods and have H2O2, O3 or O2 as oxidant. The principal active species in this system is the hydroxyl radical •OH, which is an extremely reactive and non-selective oxidant for organic contaminants (Legrini at al., 1993; Sun Pignatello, 1993). The main advantage of these processes is a complete mineralization of many organic pollutants (Andreozzi at al., 1999; Neyens Baeyens, 2003). Several of AOPs are currently employed for the elimination of pesticides from water: heterogeneous photocatalytic reactions with semiconductor oxides TiO2 (Malato et al., 2002a, 2002b; Tomaševic at al., 2010a) or ZnO (Tomaševic at al., 2010a) as photocatalysts, photo-Fenton (Malato et al., 2002a; Tamimi et al, 2008; Tomaševic at al., 2010b) and photo-assisted Fenton processes (Huston Pignatello, 1999). Electro-photo-Fenton (Kesraoui Abdessalem et al., 2010) and electrochemical oxidation processes (Tomašević et al., 2009a) have been seldom studied.

Heterogeneous photocatalysis is combination of semiconductor particles (TiO2, ZnO, Fe2O3, CdS, ZnS), UV/solar light and different oxidants (H2O2, K2S2O8, KIO4, KBrO3). The main equations of the heterogeneous photocatalysis are (Andreozzi et al., 1999; Daneshvar et al., 2003; Karkmaz et al., 2004; Legrini at al., 1993):

$$\mathbf{C} + h\nu \to \mathbf{C} \text{ (e} + \text{h}^\* \text{)}\tag{1}$$

$$\rm H^{\*} + H\_{2}O \rightarrow \rm \bullet OH + H^{\*} \tag{2}$$

$$\mathbf{e} \mathbf{\cdot} + \mathbf{O}\_{\mathbf{I}} \mathbf{\cdot} \to \mathbf{O}\_{\mathbf{I}} \mathbf{\bullet} \tag{3}$$

Among AOPs, heterogeneous photocatalysis using TiO2 as photocatalyst appears as the most emerging destructive technology. The following mechanism of the TiO2 photocatalysis has been proposed (Daneshvar et al., 2003; Gomes da Silva Faria, 2003; Karkmaz et al., 2004; Tomaševic at al., 2010a):

a) absorption of efficient photons by titania (*hv*  Eg=3.2 eV):

$$\text{TiO}\_2 + h\nu \text{(UV)} \rightarrow \text{e} \text{c} \text{x}^\* + \text{h} \text{v} \text{ $\!^\*$ }\tag{4}$$

b) oxygen ionosorption:

44 Insecticides – Basic and Other Applications

formulation. The ways in which pesticides are formulated considerably influence their persistence. Formulations in order of increasing persistence on plants are prepared in the way that more readly adsorbed on the soil fractions and not appreciably degradated

Photolysis (direct photodegradation reaction) is photodegradation process without any catalysts and use light only for degradation of different organic molecules, including pesticides and related compounds. Direct irradiation will lead to the promotion of the pesticides to their excited singlet states and such excited states can then undergo among homolysis, heterolysis or photoionization processes (Burrows et al., 2002). Direct photodegradation by solar light is limited and various lamps have been used for irradiation of contaminated water solutions. The photolysis of contaminants (including pesticides) in aqueos solution depends on the different reaction parameters such as type of light, lamp distance, temperature, initial concentration of pesticides, type of water, pH, the presence of humic and fulvic acids, the presence of O2, O3, O2/O3 and H2O2, the presence of inorganic ions and organic matter dissolved in water (Burrows et al., 2002; Tomaševic at al., 2010a).

The photosensitized reaction is based on the absorption of light by a molecule of the sensitizer and includes an energy transfer from molecul excited state to the pesticides. The most famous sensitizers are aceton, rose Bengal, methylene blue and humic and fulvic acids

Advanced Oxidation Processes (AOPs) include catalytic and photochemical methods and have H2O2, O3 or O2 as oxidant. The principal active species in this system is the hydroxyl radical •OH, which is an extremely reactive and non-selective oxidant for organic contaminants (Legrini at al., 1993; Sun Pignatello, 1993). The main advantage of these processes is a complete mineralization of many organic pollutants (Andreozzi at al., 1999; Neyens Baeyens, 2003). Several of AOPs are currently employed for the elimination of pesticides from water: heterogeneous photocatalytic reactions with semiconductor oxides TiO2 (Malato et al., 2002a, 2002b; Tomaševic at al., 2010a) or ZnO (Tomaševic at al., 2010a) as photocatalysts, photo-Fenton (Malato et al., 2002a; Tamimi et al, 2008; Tomaševic at al., 2010b) and photo-assisted Fenton processes (Huston Pignatello, 1999). Electro-photo-Fenton (Kesraoui Abdessalem et al., 2010) and electrochemical oxidation processes

Heterogeneous photocatalysis is combination of semiconductor particles (TiO2, ZnO, Fe2O3, CdS, ZnS), UV/solar light and different oxidants (H2O2, K2S2O8, KIO4, KBrO3). The main equations of the heterogeneous photocatalysis are (Andreozzi et al., 1999; Daneshvar et al.,

h+ + H2O → ●OH + H+ (2)

C + *hν* → C (e- + h+) (1)

**3. Photodegradation processes for carbamates wastewater treatments** 

(Edwards, 1975).

**3.1 Photolysis** 

**3.2 Photosensitized degradation** 

**3.3 Advanced oxidation processes** 

(Tomašević et al., 2009a) have been seldom studied.

2003; Karkmaz et al., 2004; Legrini at al., 1993):

(Burrows et al., 2002).

$$(\text{O}\_2)\_{\text{ads}} + \text{e}\_{\text{CN}'} \rightarrow \text{O}\_2\text{"{}'} \tag{5}$$

c) neutralization of OH- groups into •OH by photoholes:

$$\text{(H}\_{2}\text{O} \leftrightarrow \text{H}^{\*} + \text{OH}^{\*}\text{)}\_{\text{ads}} + \text{h}\_{\text{VB}}^{\*} \rightarrow \text{H}^{\*} + \text{\textasciic}{\text{OH}}\tag{6}$$

d) oxidation of the organic reactant via successive attacks by •OH radicals:

$$\text{R} + \text{"OH} \rightarrow \text{R}"\text{"} + \text{H}\_2\text{O} \tag{7}$$

e) or by direct reaction with holes:

$$\mathbf{R} + \mathbf{h}^\* \to \mathbf{R}^{\*\*} \to \text{degradation products} \tag{8}$$

ZnO is also frequently used as a catalyst in heterogeneous photocatalytic reactions. The biggest advantage of ZnO in comparison to TiO2 is that it absorbs over a larger fraction of the UV spectrum and the corresponding threshold wavelength of ZnO is 387 nm. Upon irradiation, valence band electrons are promoted to the conduction band leaving a hole behind. These electron-hole pairs can either recombine or interact separately with other molecules. The holes at the ZnO valence band can oxidize adsorbed water or hydroxide ions to produce hydroxyl radicals. Electron in the conduction band at the catalyst surface can reduce molecular oxygen to superoxide anion. This radical may form organic peroxides or hydrogen peroxide in the presence of organic scavengers. The hydroxyl radical attacks organic compounds (R) and intermediates (Int) are formed. These intermediates react with hydroxyl radicals to produce the final products (P). The mechanism of heterogeneous photocatalysis in the presence of ZnO can be given by the following reactions (Behnajady et al., 2006; Daneshvar et al., 2004, 2007; Pera-Titus at al,. 2004; Tomaševic at al., 2010a):

$$\rm ZnO + h\nu \text{(UV)} \rightarrow \rm e\_{CB}\* + h\_{VB}\* \tag{9}$$

$$\text{e}\_{\text{CB}^\ast} \text{} + \text{h}\_{\text{VB}^\ast} \text{} \rightarrow \text{heat} \tag{10}$$

$$\rm{H\_{VB}^\* + H\_2O\_{ads} \to H^\* + "OH\_{ads}}\tag{11}$$

 hVB+ + –OHads OHads (12)

$$\text{e}\_{\text{CF}} + \text{O}\_{2} \rightarrow \text{O}\_{2}"\text{-}\tag{13}$$

$$\rm O\_2^{\bullet-} + HO\_2^{\bullet} + H^+ \to H\_2O\_2 + O\_2 \tag{14}$$

Photoremediation of Carbamate Residues in Water 47

The degradation of asulam was studied in homogeneous aqueous solution in the presence of molecular oxygen at pH 3.0-3.4, by irradiation at 365 nm and by solar irradiation (Catastini et al., 2002a). When the iron(III) aquacomplexes was photoreduced to iron(II) ions and hydroxyl radicals the degradation of asulam in the presence of oxygen continud to completion. The Fe2+ ions are oxidized back to Fe3+ ions through various pathways such as photooxidation and oxidation by H2O2 generated within the system, where another

forms. Their experimental results indicate that the presence of Fe3+, Fe2+ and molecular oxygen accelerate the mineralization of asulam. Also, less than 10% conversion of asulam was observed when the irradiation was performeds in the presence of 0.01 M 2-propanol, used as hydroxyl radical scavenger. Complete conversion and nearly complete TOC reduction of 23 mg/L of asulam was achieved with 16.7 mg/L of Fe3+ ions, within 17 h (at 365 nm) and 28-30 h (under solar light). In this process intermediates or degradation byproducts of asulam were not identified. The photodegradation of the herbicide asulam in aqueous solution (1.0 x 10-4 M or 23 mg/L) has been investigated with and without Fe(III) (Catastini et al., 2002b).The asulam disappearance were monitored by photolysis at 254 nm as a functuion of pH and oxygen concentration and no complete transformation of organic carbon into CO2 was observed. In the presence of Fe(III) at 365 nm the complete

Bendiocarb (IUPAC name: 2,3-isopropyldenedioxyphenyl methylcarbamate, 2,2-dimethyl-1,3-benzodioxol-4-yl methylcarbamate) is systemic insecticide with contact and stomach action. It is active against many public health, industrial and storage pest. This active ingredient is especially useful inside buildings, due to its low odor and lack of corrosive and staining properties. It comes in variety formulations type as DP, FS, GR, SC, WP on the market. The current regulation status of this active ingredient under directive 91/414/EEC

Evaluation of different pathway (photolysis, photo-Fenton, H2O2/UV and electro-Fenton) of bendiocarb (112-188 mg/L) photodegradation have been proposed (Aaron & Oturan, 2001). The conversion of insecticide was apparently much faster in the H2O2/UV and photo-Fenton proces (λ = 254 nm, 68 mg/L of H2O2 and 55.8 mg/L of Fe3+) than in the other processes. Also, the degradation mechanism of bendiocarb has been proposed. The photolysis of aqueous bendiocarb (3.3 x 10-3 M, 4 h, room temperature, 125 W mediumpressure mercury lamp) has been examined by GC-MS (Climent & Miranda, 1996). Upon irradiation the only one photo-product (corresponding phenol) was detected and 30%

Carbaryl (IUPAC name: 1-naphthyl methylcarbamate) is insecticide with contact and stomach action and has slight systemic properties. It is used for control of chewing and sucking insects on more than 120 different crops, including vegetables, tree fruit (including citrus), mangoes, bananas, strawberries, nuts, vines, olives, okra, cucurbits, peanuts, soya beans, cotton, rice, tobacco, cereals, beet, maize, sorghum, alfalfa, potatoes, ornamentals, forestry, etc, than for control earthworms in turf and as a growth regulator for fruit thinning of apples. Also it is used against an animal ectoparasiticide. Carbaryl can be found formulated as DP, GR, OF, RB, SC, TK and WP. The current regulation status of this active

is not included in Annex 1 (EU Pesticide Database, 2011; Tomlin, 2009).

mineralization of asulam has been achieved.

conversion of bendiocarb was achieved.

**4.3 Bendiocarb** 

**4.4 Carbaryl** 

OH

$$\text{O}\_2\text{"} + \text{R} \rightarrow \text{R-CO"} \tag{15}$$

$$\text{"OH}\_{\text{ads}} + \text{R} \to \text{Int.} \to \text{P} \tag{16}$$

Fenton' s processes belong to AOPs and utilize H2O2 activation by iron salts. The classic Fenton' s reagent is a mixture of ferrous ion and H2O2 in acidic solution or suspension (Neyens & Baeyens, 2003; Tamimi at al.,2008):

$$\text{Fe}^{2+} + \text{H}\_2\text{O}\_2 \rightarrow \text{Fe}^{3+} + \text{OH} \cdot + \text{\textasciicant} \tag{17}$$

Equation (17) presents the most important steps of a Fenton reaction and involves electron transfer between H2O2 and Fe(II) with oxidation of Fe(II) to Fe(III) and the resulting production of highly reactive hydroxyl radical ●OH and potentially reactive ferryl species. The degradation of pesticides by Fenton' s reagent can be strongly accelerated upon UV or UV-visible light. This process is the photo-Fenton reaction (Malato et al., 2002a, 2002b; Tamimi et al, 2008; Tomaševic at al., 2010b). Equation (17) is the key of photo-Fenton processes. The obtained Fe3+ ion or its Fe(OH)2+ complexes act as light absorbing species, that produce another hydroxyl radical, while the initial Fe2+ ion is regained:

$$\text{Fe(OH)}^{2+} + lw \rightarrow \text{Fe}^{2+} + \text{OH} \tag{18}$$

The main advantage of the photo-Fenton process is light sensitivity up to a wavelength of 600 nm (Malato et al., 2002a).

#### **4. Photodegradation of carbamate pesticides**

#### **4.1 Aldicarb**

Aldicarb (IUPAC name: 2-methyl-2-(methylthio)propionaldehyde O-methylcarbamoyoxime) is a systemic oxime carbamate pesticide, effective against a variety of insects, mites, and nematodes. It is sold commercially only in granular form (GR). Aldicarb is applied on a variety of crops, including cotton, sugar beet, sugarcane, citrus fruits, potatoes, sweet potatoes, peanuts, beans (dried beans), soybeans, pecans, and ornamental plants. Home and garden use is not permitted in many countries. The current regulation status of this active ingredient under directive 91/414/EEC is not included in Annex 1 (EU Pesticide Database, 2011; Tomlin, 2009).

The complete conversion of 38 mg/L of aldicarb and 62% reduction in TOC content using the photo-Fenton reaction (Fe(III)/H2O2/UV) within 120 min in acidic aqueous solution (pH 2.8) at 25 C with fluorescent blacklight irradiation (300-400 nm) has been considered (Huston Pignatello, 1999). They also observed the formation of sulfate and nitrate ions during the photo-Fenton process.

#### **4.2 Asulam**

Asulam (IUPAC name: methyl sulanylcarbamate) is selective systemic herbicide, which is used for control of annual and perennial grasses and broad-leaved weeds in spinach, oilseed poppies, alfalfa, some ornamentals, sugar cane, bananas, coffee, tea, cocoa, coconuts, rubber, fruit trees and bushes, and forestry. It could be found only as soluble concentrate (SL) on the market. The current regulation status of this active ingredient under directive 91/414/EEC is not included in Annex 1 (EU Pesticide Database, 2011; Tomlin, 2009).

The degradation of asulam was studied in homogeneous aqueous solution in the presence of molecular oxygen at pH 3.0-3.4, by irradiation at 365 nm and by solar irradiation (Catastini et al., 2002a). When the iron(III) aquacomplexes was photoreduced to iron(II) ions and hydroxyl radicals the degradation of asulam in the presence of oxygen continud to completion. The Fe2+ ions are oxidized back to Fe3+ ions through various pathways such as photooxidation and oxidation by H2O2 generated within the system, where another OH forms. Their experimental results indicate that the presence of Fe3+, Fe2+ and molecular oxygen accelerate the mineralization of asulam. Also, less than 10% conversion of asulam was observed when the irradiation was performeds in the presence of 0.01 M 2-propanol, used as hydroxyl radical scavenger. Complete conversion and nearly complete TOC reduction of 23 mg/L of asulam was achieved with 16.7 mg/L of Fe3+ ions, within 17 h (at 365 nm) and 28-30 h (under solar light). In this process intermediates or degradation byproducts of asulam were not identified. The photodegradation of the herbicide asulam in aqueous solution (1.0 x 10-4 M or 23 mg/L) has been investigated with and without Fe(III) (Catastini et al., 2002b).The asulam disappearance were monitored by photolysis at 254 nm as a functuion of pH and oxygen concentration and no complete transformation of organic carbon into CO2 was observed. In the presence of Fe(III) at 365 nm the complete mineralization of asulam has been achieved.

#### **4.3 Bendiocarb**

46 Insecticides – Basic and Other Applications


 Fe2+ + H2O2 → Fe3+ + OH- + ●OH (17) Equation (17) presents the most important steps of a Fenton reaction and involves electron transfer between H2O2 and Fe(II) with oxidation of Fe(II) to Fe(III) and the resulting production of highly reactive hydroxyl radical ●OH and potentially reactive ferryl species.

UV-visible light. This process is the photo-Fenton reaction (Malato et al., 2002a, 2002b; Tamimi et al, 2008; Tomaševic at al., 2010b). Equation (17) is the key of photo-Fenton processes. The obtained Fe3+ ion or its Fe(OH)2+ complexes act as light absorbing species,

 Fe(OH)2+ + *hv* → Fe2+ + ●OH (18) The main advantage of the photo-Fenton process is light sensitivity up to a wavelength of

Aldicarb (IUPAC name: 2-methyl-2-(methylthio)propionaldehyde O-methylcarbamoyoxime) is a systemic oxime carbamate pesticide, effective against a variety of insects, mites, and nematodes. It is sold commercially only in granular form (GR). Aldicarb is applied on a variety of crops, including cotton, sugar beet, sugarcane, citrus fruits, potatoes, sweet potatoes, peanuts, beans (dried beans), soybeans, pecans, and ornamental plants. Home and garden use is not permitted in many countries. The current regulation status of this active ingredient under directive 91/414/EEC is not included in Annex 1 (EU Pesticide Database,

The complete conversion of 38 mg/L of aldicarb and 62% reduction in TOC content using the photo-Fenton reaction (Fe(III)/H2O2/UV) within 120 min in acidic aqueous solution (pH 2.8) at 25 C with fluorescent blacklight irradiation (300-400 nm) has been considered (Huston Pignatello, 1999). They also observed the formation of sulfate and nitrate ions

Asulam (IUPAC name: methyl sulanylcarbamate) is selective systemic herbicide, which is used for control of annual and perennial grasses and broad-leaved weeds in spinach, oilseed poppies, alfalfa, some ornamentals, sugar cane, bananas, coffee, tea, cocoa, coconuts, rubber, fruit trees and bushes, and forestry. It could be found only as soluble concentrate (SL) on the market. The current regulation status of this active ingredient under directive 91/414/EEC

is not included in Annex 1 (EU Pesticide Database, 2011; Tomlin, 2009).

that produce another hydroxyl radical, while the initial Fe2+ ion is regained:

s processes belong to AOPs and utilize H2O2 activation by iron salts. The classic

s reagent is a mixture of ferrous ion and H2O2 in acidic solution or suspension

(15)

OHads + R Int. P (16)

s reagent can be strongly accelerated upon UV or

O2

(Neyens & Baeyens, 2003; Tamimi at al.,2008):

The degradation of pesticides by Fenton'

**4. Photodegradation of carbamate pesticides** 

600 nm (Malato et al., 2002a).

**4.1 Aldicarb** 

2011; Tomlin, 2009).

**4.2 Asulam** 

during the photo-Fenton process.

Fenton'

Fenton'

Bendiocarb (IUPAC name: 2,3-isopropyldenedioxyphenyl methylcarbamate, 2,2-dimethyl-1,3-benzodioxol-4-yl methylcarbamate) is systemic insecticide with contact and stomach action. It is active against many public health, industrial and storage pest. This active ingredient is especially useful inside buildings, due to its low odor and lack of corrosive and staining properties. It comes in variety formulations type as DP, FS, GR, SC, WP on the market. The current regulation status of this active ingredient under directive 91/414/EEC is not included in Annex 1 (EU Pesticide Database, 2011; Tomlin, 2009).

Evaluation of different pathway (photolysis, photo-Fenton, H2O2/UV and electro-Fenton) of bendiocarb (112-188 mg/L) photodegradation have been proposed (Aaron & Oturan, 2001). The conversion of insecticide was apparently much faster in the H2O2/UV and photo-Fenton proces (λ = 254 nm, 68 mg/L of H2O2 and 55.8 mg/L of Fe3+) than in the other processes. Also, the degradation mechanism of bendiocarb has been proposed. The photolysis of aqueous bendiocarb (3.3 x 10-3 M, 4 h, room temperature, 125 W mediumpressure mercury lamp) has been examined by GC-MS (Climent & Miranda, 1996). Upon irradiation the only one photo-product (corresponding phenol) was detected and 30% conversion of bendiocarb was achieved.

#### **4.4 Carbaryl**

Carbaryl (IUPAC name: 1-naphthyl methylcarbamate) is insecticide with contact and stomach action and has slight systemic properties. It is used for control of chewing and sucking insects on more than 120 different crops, including vegetables, tree fruit (including citrus), mangoes, bananas, strawberries, nuts, vines, olives, okra, cucurbits, peanuts, soya beans, cotton, rice, tobacco, cereals, beet, maize, sorghum, alfalfa, potatoes, ornamentals, forestry, etc, than for control earthworms in turf and as a growth regulator for fruit thinning of apples. Also it is used against an animal ectoparasiticide. Carbaryl can be found formulated as DP, GR, OF, RB, SC, TK and WP. The current regulation status of this active

Photoremediation of Carbamate Residues in Water 49

Various carbofuran photodegradation processes (by ozon, UV photolysis, Fenton, O3 + UV, UV + H2O2 and photo-Fenton) upon polychromatic UV irradiation were evaluated (Benitez et al., 2002). For all these reactions, the apparent pseudo-first–order rate constants are evaluated in order to compare the efficiency of each process. The most effective process in removing carbofuran from water was the photo-Fenton system (UV + Fe2+ + H2O2) with rate constants k from 17.2 x 10-4/s to >200.0 x 10-4/s. The degradation of pure carbofuran and commercial product Furadan 4F in acidic aqueous solution upon polychromatic light (300-400 nm) by photo-assisted Fenton process has been studied (Huston Pignatello, 1999). The complete conversion of 2.0 x 10-4 M of pure carbofuran and more than 90% TOC reduction in the water solution within 120 min has been achieved. Nitrate and oxalate ions were detected as organic ionic species after the treatment. Also, the results show that the adjuvants in Furadan 4F have little or no influence on degradation of carbofuran nor of TOC mineralization. Two different Advanced oxidation processes (photo- and electro-Fenton) have been used for photodegradation of carbofuran in water (Kesraoui Abdessalem et al., 2010). For the photo-Fenton process TOC removal ratio was influenced by the initial concentration of the pesticides and the amout of Fe3+ and H2O2. The TOC measurement indicate an efficient mineralization of 93 and 94% respectively, for photo- and electro-Fenton processes after 480 min of treatment. Carbofuran could not be mineralized on AlFe-PILC and Fe-ZSM-5 zeolite catalysts in the heterogeneous photo-Fenton reactions at 575.6 nm, even in the catalytic reaction promoted at high temperature (Tomašević et al., 2007a,

Ethiofencarb (IUPAC name: -ethythio--tolyl methylcarbamate) is systemic insecticide with contact and stomach action. It is applied for control of aphids on pome fruit , stone fruit and soft fruit, than vegetables, ornamentals and sugar beet. Formulations types which can be found on the market are: emulsifieble concentrate (EC), emulsions oil in water (EW) and granules (GR). The current regulation status of this active ingredient under directive

Solar photodegradation of ethiofencarb was examined in pure water, natural water and in the pure water containing 10mg/L of humic acids (Vialaton Richard, 2002). Photosensitized reactions are main degradation pathway of pesticide in natural water and in the presence of humic acids. Photosensitized transformations were shown to be largely due to photoreactants other than singlet oxygen and hydroxyl radicals. A comparative photolysis reactions of ethiofencarb in water and non-water media were performed in the presence of simulated solar light (Sanz-Asensio et al., 1999). The studies showed that the photolysis reaction follows pseudo-first-order kinetics and that the degradation kinetics depend on the solvent polarity. In the water media the reaction of pesticide degradation was completed for 30 h. Also, the photoproducts are dependent on the solvent and the main photoproduct in water was 2-(methyl)phenyl-N-methylcarbamate. The photolysis of aqueous ethiofencarb (3.3 x 10-3 M, 4 h, room temperature, 125 W medium-pressure mercury lamp) has been examined by GC-MS (Climent Miranda, 1996). Upon irradiation three photoproducts were detected and 66% conversion of ethiofencarb was achieved. The main product was 2-methylphenyl methylcarbamate, and two corresponding phenols also were

91/414/EEC is not included in Annex 1 (EU Pesticide Database, 2011; Tomlin, 2009).

2007b).

registered.

**4.7 Ethiofenocarb** 

ingredient under directive 91/414/EEC is not included in Annex 1 (EU Pesticide Database, 2011; Tomlin, 2009).

The degradation of carbaryl under UV light using a continuous flow of TiO2 slurry shown that the degradation proceeds through a multi-step process involving the attack of the substrate by ●OH radicals (Peris at al., 1993). The studies on the degradation of carbaryl under simulated solar light in aqueous TiO2 dispersions showed that the reaction follows pseudo-first-order kinetics and the complete mineralization (to CO2, nitrate and ammonium ions ) is achieved in less 30 min (Pramauro et al., 1997). The effect of ionic and non-ionic aliphatic surfactants (constitute an important ingredient of pesticide formulations and can influence the degradation of pesticide) on the degradation of aqueous carbaryl solutions (20 mg/L) containing 500 mg/L of TiO2 (anatase) in the presence of simulated solar light (1500 W Xenon lamp with 340 nm cut-off filter) was investigated (Bianco Prevot at al., 1999). Depending on the surfactant and on the initial pH of the solution, an inhibition ot the photodegradation rate was observed. Also, mineralization of the carbaryl to CO2, nitrate and ammonium ions was evidence in the presence of added surfactants, suggesting the feasibility of photocatalytic treatment of aqueous pesticide wastes.

#### **4.5 Carbetamid**

Carbetamid (IUPAC name: (R)-1-(ethylcarbamoyl)ethyl carbamilate) is selective herbicide, absorbed by the roots, and also by the leaves. It is used for control of annualgrasses and some broad-leaved weeds, alfalfa, sainfoin, brassicas, field beans, peas, lentils, sugar beast, oilseed rape, chicory, endive, sunflowers, caraway, strawberries, wines, and fruit orchards. Formulations types for this active ingredient are EC and WP. The current regulation status of this active ingredient under directive 91/414/EEC is included in Annex 1, expiration of inclusion: 31/05/2021 (EU Pesticide Database, 2011; Tomlin, 2009).

Photodegradation of herbicide carbetamide with ultraviolet light (λ > 290 nm) in the presence of TiO2, H2O2 and ozone was studied in the aqueous solutions (Mansour et al., 1992). Using spectrometric methods several photoproducts were isolated and identified, suggesting that photodegradation pathways of carbetamide in the presence of TiO2 and H2O2 are hydroxylations of the aromatic ring. Also, UV-ozonation rapidly oxydized carbetamide to water, ammonia and CO2. The kinetics of photodegradation of carbetamide in water in the presence of TiO2 (Degussa P 25 grade, surface area 50.0 m2/g) or ZnO (surface area 9.5 m2/g) were examined upon λ 310 nm (Percherancier et al., 1995). The effects of various parameters, such as the kind of semiconductor, mass of TiO2, initial concentration of pesticide, radiation flux and quantum yield were studied. The degradation with ZnO is faster than that with TiO2 in spite of the lager surface area of the later catalyst. Also, the mechanism of the carbetamide photocatalytic degradation has been proposed.

#### **4.6 Carbofuran**

Carbofuran (IUPAC name: 2,3-dihydro-2,2-dimethylbenzofuran-7-yl methylcarbamate) is systemic insecticide with predominantly contact and stomach action. It is used for control of soil-dwelling and foliar-feeding insects and nematodes in vegetables, ornamentals, beet, maize, sorghum, sunflowers, oilseed rape, potatoes, alfalfa, peanuts, soya beans, sugar cane, rice, cotton, coffee, cucurbits, tobacco, lavender, citrus, wines, strawberries, bananas, mushrooms and other crops. This active ingredient is prepared as FS, GR, SC and WP formulation. The current regulation status of this active ingredient under directive 91/414/EEC is not included in Annex 1 (EU Pesticide Database, 2011; Tomlin, 2009).

ingredient under directive 91/414/EEC is not included in Annex 1 (EU Pesticide Database,

The degradation of carbaryl under UV light using a continuous flow of TiO2 slurry shown that the degradation proceeds through a multi-step process involving the attack of the substrate by ●OH radicals (Peris at al., 1993). The studies on the degradation of carbaryl under simulated solar light in aqueous TiO2 dispersions showed that the reaction follows pseudo-first-order kinetics and the complete mineralization (to CO2, nitrate and ammonium ions ) is achieved in less 30 min (Pramauro et al., 1997). The effect of ionic and non-ionic aliphatic surfactants (constitute an important ingredient of pesticide formulations and can influence the degradation of pesticide) on the degradation of aqueous carbaryl solutions (20 mg/L) containing 500 mg/L of TiO2 (anatase) in the presence of simulated solar light (1500 W Xenon lamp with 340 nm cut-off filter) was investigated (Bianco Prevot at al., 1999). Depending on the surfactant and on the initial pH of the solution, an inhibition ot the photodegradation rate was observed. Also, mineralization of the carbaryl to CO2, nitrate and ammonium ions was evidence in the presence of added surfactants, suggesting the

Carbetamid (IUPAC name: (R)-1-(ethylcarbamoyl)ethyl carbamilate) is selective herbicide, absorbed by the roots, and also by the leaves. It is used for control of annualgrasses and some broad-leaved weeds, alfalfa, sainfoin, brassicas, field beans, peas, lentils, sugar beast, oilseed rape, chicory, endive, sunflowers, caraway, strawberries, wines, and fruit orchards. Formulations types for this active ingredient are EC and WP. The current regulation status of this active ingredient under directive 91/414/EEC is included in Annex 1, expiration of

Photodegradation of herbicide carbetamide with ultraviolet light (λ > 290 nm) in the presence of TiO2, H2O2 and ozone was studied in the aqueous solutions (Mansour et al., 1992). Using spectrometric methods several photoproducts were isolated and identified, suggesting that photodegradation pathways of carbetamide in the presence of TiO2 and H2O2 are hydroxylations of the aromatic ring. Also, UV-ozonation rapidly oxydized carbetamide to water, ammonia and CO2. The kinetics of photodegradation of carbetamide in water in the presence of TiO2 (Degussa P 25 grade, surface area 50.0 m2/g) or ZnO (surface area 9.5 m2/g) were examined upon λ 310 nm (Percherancier et al., 1995). The effects of various parameters, such as the kind of semiconductor, mass of TiO2, initial concentration of pesticide, radiation flux and quantum yield were studied. The degradation with ZnO is faster than that with TiO2 in spite of the lager surface area of the later catalyst. Also, the mechanism of the carbetamide photocatalytic degradation has been proposed.

Carbofuran (IUPAC name: 2,3-dihydro-2,2-dimethylbenzofuran-7-yl methylcarbamate) is systemic insecticide with predominantly contact and stomach action. It is used for control of soil-dwelling and foliar-feeding insects and nematodes in vegetables, ornamentals, beet, maize, sorghum, sunflowers, oilseed rape, potatoes, alfalfa, peanuts, soya beans, sugar cane, rice, cotton, coffee, cucurbits, tobacco, lavender, citrus, wines, strawberries, bananas, mushrooms and other crops. This active ingredient is prepared as FS, GR, SC and WP formulation. The current regulation status of this active ingredient under directive

91/414/EEC is not included in Annex 1 (EU Pesticide Database, 2011; Tomlin, 2009).

feasibility of photocatalytic treatment of aqueous pesticide wastes.

inclusion: 31/05/2021 (EU Pesticide Database, 2011; Tomlin, 2009).

2011; Tomlin, 2009).

**4.5 Carbetamid** 

**4.6 Carbofuran** 

Various carbofuran photodegradation processes (by ozon, UV photolysis, Fenton, O3 + UV, UV + H2O2 and photo-Fenton) upon polychromatic UV irradiation were evaluated (Benitez et al., 2002). For all these reactions, the apparent pseudo-first–order rate constants are evaluated in order to compare the efficiency of each process. The most effective process in removing carbofuran from water was the photo-Fenton system (UV + Fe2+ + H2O2) with rate constants k from 17.2 x 10-4/s to >200.0 x 10-4/s. The degradation of pure carbofuran and commercial product Furadan 4F in acidic aqueous solution upon polychromatic light (300-400 nm) by photo-assisted Fenton process has been studied (Huston Pignatello, 1999). The complete conversion of 2.0 x 10-4 M of pure carbofuran and more than 90% TOC reduction in the water solution within 120 min has been achieved. Nitrate and oxalate ions were detected as organic ionic species after the treatment. Also, the results show that the adjuvants in Furadan 4F have little or no influence on degradation of carbofuran nor of TOC mineralization. Two different Advanced oxidation processes (photo- and electro-Fenton) have been used for photodegradation of carbofuran in water (Kesraoui Abdessalem et al., 2010). For the photo-Fenton process TOC removal ratio was influenced by the initial concentration of the pesticides and the amout of Fe3+ and H2O2. The TOC measurement indicate an efficient mineralization of 93 and 94% respectively, for photo- and electro-Fenton processes after 480 min of treatment. Carbofuran could not be mineralized on AlFe-PILC and Fe-ZSM-5 zeolite catalysts in the heterogeneous photo-Fenton reactions at 575.6 nm, even in the catalytic reaction promoted at high temperature (Tomašević et al., 2007a, 2007b).

#### **4.7 Ethiofenocarb**

Ethiofencarb (IUPAC name: -ethythio--tolyl methylcarbamate) is systemic insecticide with contact and stomach action. It is applied for control of aphids on pome fruit , stone fruit and soft fruit, than vegetables, ornamentals and sugar beet. Formulations types which can be found on the market are: emulsifieble concentrate (EC), emulsions oil in water (EW) and granules (GR). The current regulation status of this active ingredient under directive 91/414/EEC is not included in Annex 1 (EU Pesticide Database, 2011; Tomlin, 2009).

Solar photodegradation of ethiofencarb was examined in pure water, natural water and in the pure water containing 10mg/L of humic acids (Vialaton Richard, 2002). Photosensitized reactions are main degradation pathway of pesticide in natural water and in the presence of humic acids. Photosensitized transformations were shown to be largely due to photoreactants other than singlet oxygen and hydroxyl radicals. A comparative photolysis reactions of ethiofencarb in water and non-water media were performed in the presence of simulated solar light (Sanz-Asensio et al., 1999). The studies showed that the photolysis reaction follows pseudo-first-order kinetics and that the degradation kinetics depend on the solvent polarity. In the water media the reaction of pesticide degradation was completed for 30 h. Also, the photoproducts are dependent on the solvent and the main photoproduct in water was 2-(methyl)phenyl-N-methylcarbamate. The photolysis of aqueous ethiofencarb (3.3 x 10-3 M, 4 h, room temperature, 125 W medium-pressure mercury lamp) has been examined by GC-MS (Climent Miranda, 1996). Upon irradiation three photoproducts were detected and 66% conversion of ethiofencarb was achieved. The main product was 2-methylphenyl methylcarbamate, and two corresponding phenols also were registered.

Photoremediation of Carbamate Residues in Water 51

(Tomašević et al., 2009b, 2010a; Tomašević, 2011) and the influence of reaction conditions (initial concentration of methomyl, catalysts type and concentration, pH, presence of Clions) were studied. The results (Table 2) showed that the degradation of methomyl was much faster with ZnO than with TiO2. The IC results confirmed that mineralization of methomyl led to the formation of sulfate, nitrate, and ammonium ions during the all

> **AlFe-PILC FeZSM-5**

> > d = 20 mm d = 50 mm d = 75 mm d = 200 mm

0 60 120 180 240 300 **Illumination time (min)**

Fig. 2. Photodegradation of methomyl with 5 g/L of catalysts (Tomašević, 2011).

0 60 120 180 240 300 360 420 480 540 600 **Time (min)**

Fig. 3. The effect of lamp distance on the photolysis rate of methomyl (Tomašević, 2011).

investigated processes (Tomašević et al., 2010a, 2010b; Tomašević, 2011).

0.0

0.0000

0.5000

1.0000

1.5000

**ln (C0/C)**

2.0000

2.5000

3.0000

0.2

0.4

**C/C0**

0.6

0.8

1.0

#### **4.8 Formetanate**

Formetanate (IUPAC name: 3-dimethylaminomethyleneaminophenyl methylcarbamate) is acaricide and insecticide with contact and stomach action. It is used for control of spider mites and some insects on ornamentals, pome fruit, stone fruit, citrus fruit, vegetables and alfalfa. It is sold commercially only as soluble powder (SP). The current regulation status of this active ingredient under directive 91/414/EEC is included in Annex 1, expiration of inclusion: 30/09/2017 (EU Pesticide Database, 2011; Tomlin, 2009).

The solar driven photo-Fenton process using pilot-scale compound parabolic collector was applied to the degradation of formetanate in the form of AgrEvo formulated product Dicorzol (Fallman et al., 1999). The results shown that a good conversion of formetanate was achieved (about 25 min was a TOC half-life and about 70 min was the time necessary for degradation of 80% of TOC). The heterogeneous photocatalysis with TiO2 (200 mg/L) and homogeneous photocatalysis by photo-Fenton (0.05 mM of FeSO4 x 7H2O) of 50 mg/L of formetanate have been studied (Malato et al., 2002b). In the presence of 2.8 mg/L of Fe2+ complete conversion of formetanate and more than 90% TOC reduction was demonstrated in pilot-scale solar reactor. The kinetics of formetanate degradation by the TiO2 solar photocatalysis and by the solar photo-Fenton process were also investigated (Malato et al., 2002b, 2003).

#### **4.9 Methomyl**

Methomyl (IUPAC name: S-methyl N-(methylcarbamoyloxy)thioacetimidate) is systemic insecticide and acaricide with contact and stomach action. It is used for control of a wide range of insects and spider mites in fruit, vines, olives, hops, vegetables, ornamentals, field crops, cucurbits, flax, cotton, tobacco, soya beans, etc. Also it can be used for control of flies in animal and poultry houses and dairies. Formulations types for this active ingredient are SL, SP, WP. The current regulation status of this active ingredient under directive 91/414/EEC is included in Annex 1 expiration of inclusion: 31/08/2019 (EU Pesticide Database, 2011; Tomlin, 2009).

The solar driven homogeneous photo-Fenton and heterogeneous TiO2 processes for methomyl detoxification in water have been evaluated (Malato et al., 2002b, 2003). According to TOC removal, the photo-Fenton process was more efficient in degrading 50 mg/L of methomyl than was the TiO2 process. The both processes were capable of mineralizing more than 90% of the insecticide (Malato et al., 2002b). The photodegradation of methomyl by Fenton and photo-Fenton reactions were investigated (Tamimi et al., 2008). The degradation rate and the effect of reaction parameters (initial concentration of pesticide, pH, ferrous and H2O2 dosage, etc) were monitored. The photo-Fenton was more efficient than Fenton, both for methomyl degradation and TOC removal. The catalytic wet peroxide oxidation of methomyl at 575.6 nm (photo-Fenton reaction) with two types of heterogeneous iron catalysts (Fe-ZSM-5 zeolite and AlFe-pillared montmorillonite) were performed (Lazar et al., 2009; Tomašević et al., 2007c, 2009c, 2010a, 2010b; Tomašević, 2011). The effect of catalyst type on the reaction is shown in Fig. 2. The photolysis of 16.22 mg/L of methomyl in different types of water (deionized, disstiled and sea water) at 254 nm was performed (Tomašević et al., 2009c, 2010a; Tomašević, 2011) and the influence of reaction parameters to degradation of pesticide were investigated. The studies showed that the photolysis reactions depend on the lamp distance (Fig. 3), water type (Fig. 4), reaction temperature and pH. The photocatalytic removal of the methomyl from aqueous solutions upon UV/Vis (366 and 300- 400 nm) and natural solar light in the presence of TiO2 and ZnO has been examined

Formetanate (IUPAC name: 3-dimethylaminomethyleneaminophenyl methylcarbamate) is acaricide and insecticide with contact and stomach action. It is used for control of spider mites and some insects on ornamentals, pome fruit, stone fruit, citrus fruit, vegetables and alfalfa. It is sold commercially only as soluble powder (SP). The current regulation status of this active ingredient under directive 91/414/EEC is included in Annex 1, expiration of

The solar driven photo-Fenton process using pilot-scale compound parabolic collector was applied to the degradation of formetanate in the form of AgrEvo formulated product Dicorzol (Fallman et al., 1999). The results shown that a good conversion of formetanate was achieved (about 25 min was a TOC half-life and about 70 min was the time necessary for degradation of 80% of TOC). The heterogeneous photocatalysis with TiO2 (200 mg/L) and homogeneous photocatalysis by photo-Fenton (0.05 mM of FeSO4 x 7H2O) of 50 mg/L of formetanate have been studied (Malato et al., 2002b). In the presence of 2.8 mg/L of Fe2+ complete conversion of formetanate and more than 90% TOC reduction was demonstrated in pilot-scale solar reactor. The kinetics of formetanate degradation by the TiO2 solar photocatalysis and by the solar photo-Fenton process were also investigated (Malato et al.,

Methomyl (IUPAC name: S-methyl N-(methylcarbamoyloxy)thioacetimidate) is systemic insecticide and acaricide with contact and stomach action. It is used for control of a wide range of insects and spider mites in fruit, vines, olives, hops, vegetables, ornamentals, field crops, cucurbits, flax, cotton, tobacco, soya beans, etc. Also it can be used for control of flies in animal and poultry houses and dairies. Formulations types for this active ingredient are SL, SP, WP. The current regulation status of this active ingredient under directive 91/414/EEC is included in Annex 1 expiration of inclusion: 31/08/2019 (EU Pesticide

The solar driven homogeneous photo-Fenton and heterogeneous TiO2 processes for methomyl detoxification in water have been evaluated (Malato et al., 2002b, 2003). According to TOC removal, the photo-Fenton process was more efficient in degrading 50 mg/L of methomyl than was the TiO2 process. The both processes were capable of mineralizing more than 90% of the insecticide (Malato et al., 2002b). The photodegradation of methomyl by Fenton and photo-Fenton reactions were investigated (Tamimi et al., 2008). The degradation rate and the effect of reaction parameters (initial concentration of pesticide, pH, ferrous and H2O2 dosage, etc) were monitored. The photo-Fenton was more efficient than Fenton, both for methomyl degradation and TOC removal. The catalytic wet peroxide oxidation of methomyl at 575.6 nm (photo-Fenton reaction) with two types of heterogeneous iron catalysts (Fe-ZSM-5 zeolite and AlFe-pillared montmorillonite) were performed (Lazar et al., 2009; Tomašević et al., 2007c, 2009c, 2010a, 2010b; Tomašević, 2011). The effect of catalyst type on the reaction is shown in Fig. 2. The photolysis of 16.22 mg/L of methomyl in different types of water (deionized, disstiled and sea water) at 254 nm was performed (Tomašević et al., 2009c, 2010a; Tomašević, 2011) and the influence of reaction parameters to degradation of pesticide were investigated. The studies showed that the photolysis reactions depend on the lamp distance (Fig. 3), water type (Fig. 4), reaction temperature and pH. The photocatalytic removal of the methomyl from aqueous solutions upon UV/Vis (366 and 300- 400 nm) and natural solar light in the presence of TiO2 and ZnO has been examined

inclusion: 30/09/2017 (EU Pesticide Database, 2011; Tomlin, 2009).

**4.8 Formetanate** 

2002b, 2003).

**4.9 Methomyl** 

Database, 2011; Tomlin, 2009).

(Tomašević et al., 2009b, 2010a; Tomašević, 2011) and the influence of reaction conditions (initial concentration of methomyl, catalysts type and concentration, pH, presence of Clions) were studied. The results (Table 2) showed that the degradation of methomyl was much faster with ZnO than with TiO2. The IC results confirmed that mineralization of methomyl led to the formation of sulfate, nitrate, and ammonium ions during the all investigated processes (Tomašević et al., 2010a, 2010b; Tomašević, 2011).

Fig. 2. Photodegradation of methomyl with 5 g/L of catalysts (Tomašević, 2011).

Fig. 3. The effect of lamp distance on the photolysis rate of methomyl (Tomašević, 2011).

Photoremediation of Carbamate Residues in Water 53

For pure compounds TiO2 was a better catalyst than Na4W10O32, concerning the rate of photodegradation and mineralization. When the pesticide is used as formulation product, the decatungstate anion becomes as efficien or even more efficient than TiO2. This difference of reactivity is accounted for by the different nature of the active species during both photodegradation processes. The solar driven photo-Fenton process was applied to the degradation of oxamyl in the form of DuPont formulated product Vydate (Fallman et al., 1999). The obtained results shown that oxamyl was relatively recalcitrant (about 100 min was a TOC half-life and about 160 min was the time necessary for degradation of 80% of

Pirimicarb (IUPAC name: 2-dimethylamino-5,6-dimethylpyrimidin-4-yl dimethylcarbamate) is selective systemic insecticide with contact , stomach, and respiratory action. It is used as a selective aphicide for control a wide range of crops, including cereals, oil seeds, potatoes and other vegetables, ornamentals, and other non-food uses. Formulations types for this active ingredient are AE, DP, EC, FU, WG and WP. The current regulation status of this active ingredient under directive 91/414/EEC is included in Annex 1, expiration of

Photolysis of pirimicarb upon simulated solar light in natural water and in different aqueous solutions was investigated (Taboada et al., 1995). Aceton strongly increased degradation of pesticide, while methanol did not have any significant effect. The rate of pesticide degradation in the presence of river water was 4.5 times slower than in distilled water, and the half-life of pirimicarb in presence of dissolved humic and fulvic acids was 2- 10 times longer than in distilled water. In all studied solutions the degradation reaction followed a first-order kinetics. The solar light and simulated sunlight were used for the photolysis of pirimicarb in water (Romero et al., 1994). The photodegradation mechanism seemed to be similar under both conditions, but the half-life of pirimicarb was found to be about three times longer under natural than under simulated conditions. Also, four main products were isolated and identified by spectroscopic methods. The photolysis of aqueous pirimicarb (3.3 x 10-3 M, 4 h, room temperature) has been examined by GC-MS (Climent Miranda, 1996). Upon irradiation with 125 W medium-pressure mercury lamp three main

Promecarb (IUPAC name: 3-methyl-5-methylphenyl methylcarbamate) is an obsolete carbamate insecticide once used to combat foliage and fruit eating insects. It is systemic insecticide. Promecarb is highly toxic by ingestion and is adsorbed through the skin. Formulations type is EC. The current regulation status of this active ingredient under directive 91/414/EEC is not included in Annex 1 (EU Pesticide Database, 2011; Tomlin,

The photolysis of promecarb in water solution (3.3 x 10-3 M, 4 h, room temperature, 125 W medium-pressure mercury lamp) has been examined by GC-MS (Climent Miranda, 1996). Upon irradiation, 24% conversion of promecarb was achieved and photolysis of promecarb led to the phenol derivative (22%) as major product. Also, minor amounts of two

compounds (isomers arising from photo-Fries rearrangement) were also obtained.

inclusion: 31/07/2017 (EU Pesticide Database, 2011; Tomlin, 2009).

TOC).

**4.11 Pirimicarb** 

photoproducts were detected.

**4.12 Promecarb** 

2009).

Fig. 4. The effect of the type of water on the photolysis rate of methomyl (Tomašević, 2011).


Table 2. Kinetics of methomyl photodegradation at 366 nm (Tomašević, 2011).

#### **4.10 Oxamyl**

Oxamyl (IUPAC name: N,N-dimethyl-2-methylcarbamoyoxyimino-2-(methylthio) acetamide) is contact and systemic insecticide, acaricide and nematocide. It is used for control of chewing and sucking insects, spider mites and nematodes in ornamentals, frut trees, vegetables, cucurbits, beet, bananas, pineapples, peanuts, cotton, soya beans, tobacco, potatoes, and other crops. It could be found only as soluble concentrate (SL) on the market. The current regulation status of this active ingredient under directive 91/414/EEC is included in Annex 1, expiration of inclusion: 31/07/2016 (EU Pesticide Database, 2011; Tomlin, 2009).

An pre-industrial solar treatmen is used to prevent pollution of waters with commercial pesticide Vydate L, containing 24% oxamyl (Malato et al., 2000). Oxamyl is completely photodegraded, but mineralization is slow with illuminated TiO2 only. The use of additional oxidants such as peroxydisulphate enhanced the degradation rate by a factor of 7 compared to TiO2 alone. Solar photodegradation in aqueous solution of oxamyl in the presence of two photocatalysts TiO2 and sodium decatungstate Na4W10O32 is reported (Texier et al., 1999). For pure compounds TiO2 was a better catalyst than Na4W10O32, concerning the rate of photodegradation and mineralization. When the pesticide is used as formulation product, the decatungstate anion becomes as efficien or even more efficient than TiO2. This difference of reactivity is accounted for by the different nature of the active species during both photodegradation processes. The solar driven photo-Fenton process was applied to the degradation of oxamyl in the form of DuPont formulated product Vydate (Fallman et al., 1999). The obtained results shown that oxamyl was relatively recalcitrant (about 100 min was a TOC half-life and about 160 min was the time necessary for degradation of 80% of TOC).

#### **4.11 Pirimicarb**

52 Insecticides – Basic and Other Applications

0 60 120 180 240 300

Fig. 4. The effect of the type of water on the photolysis rate of methomyl (Tomašević, 2011).

Oxamyl (IUPAC name: N,N-dimethyl-2-methylcarbamoyoxyimino-2-(methylthio) acetamide) is contact and systemic insecticide, acaricide and nematocide. It is used for control of chewing and sucking insects, spider mites and nematodes in ornamentals, frut trees, vegetables, cucurbits, beet, bananas, pineapples, peanuts, cotton, soya beans, tobacco, potatoes, and other crops. It could be found only as soluble concentrate (SL) on the market. The current regulation status of this active ingredient under directive 91/414/EEC is included in Annex 1, expiration of inclusion: 31/07/2016 (EU Pesticide Database, 2011;

An pre-industrial solar treatmen is used to prevent pollution of waters with commercial pesticide Vydate L, containing 24% oxamyl (Malato et al., 2000). Oxamyl is completely photodegraded, but mineralization is slow with illuminated TiO2 only. The use of additional oxidants such as peroxydisulphate enhanced the degradation rate by a factor of 7 compared to TiO2 alone. Solar photodegradation in aqueous solution of oxamyl in the presence of two photocatalysts TiO2 and sodium decatungstate Na4W10O32 is reported (Texier et al., 1999).

Technical methomyl Parameters Water type

Table 2. Kinetics of methomyl photodegradation at 366 nm (Tomašević, 2011).

**Time (min)**

deionized water sea water distilled water

k (min-1) 0.0058 R 0.9880 t1/2 (min) 119.51

k (min-1) 0.0120 R 0.9915 t1/2 (min) 57.76

Deionized

0.0000 2.0000 4.0000 6.0000 8.0000 10.0000 12.0000 14.0000 16.0000

With 2.0 g/L of TiO2

With 2.0 g/ L of ZnO

**4.10 Oxamyl** 

Tomlin, 2009).

**Co/C**

Pirimicarb (IUPAC name: 2-dimethylamino-5,6-dimethylpyrimidin-4-yl dimethylcarbamate) is selective systemic insecticide with contact , stomach, and respiratory action. It is used as a selective aphicide for control a wide range of crops, including cereals, oil seeds, potatoes and other vegetables, ornamentals, and other non-food uses. Formulations types for this active ingredient are AE, DP, EC, FU, WG and WP. The current regulation status of this active ingredient under directive 91/414/EEC is included in Annex 1, expiration of inclusion: 31/07/2017 (EU Pesticide Database, 2011; Tomlin, 2009).

Photolysis of pirimicarb upon simulated solar light in natural water and in different aqueous solutions was investigated (Taboada et al., 1995). Aceton strongly increased degradation of pesticide, while methanol did not have any significant effect. The rate of pesticide degradation in the presence of river water was 4.5 times slower than in distilled water, and the half-life of pirimicarb in presence of dissolved humic and fulvic acids was 2- 10 times longer than in distilled water. In all studied solutions the degradation reaction followed a first-order kinetics. The solar light and simulated sunlight were used for the photolysis of pirimicarb in water (Romero et al., 1994). The photodegradation mechanism seemed to be similar under both conditions, but the half-life of pirimicarb was found to be about three times longer under natural than under simulated conditions. Also, four main products were isolated and identified by spectroscopic methods. The photolysis of aqueous pirimicarb (3.3 x 10-3 M, 4 h, room temperature) has been examined by GC-MS (Climent Miranda, 1996). Upon irradiation with 125 W medium-pressure mercury lamp three main photoproducts were detected.

#### **4.12 Promecarb**

Promecarb (IUPAC name: 3-methyl-5-methylphenyl methylcarbamate) is an obsolete carbamate insecticide once used to combat foliage and fruit eating insects. It is systemic insecticide. Promecarb is highly toxic by ingestion and is adsorbed through the skin. Formulations type is EC. The current regulation status of this active ingredient under directive 91/414/EEC is not included in Annex 1 (EU Pesticide Database, 2011; Tomlin, 2009).

The photolysis of promecarb in water solution (3.3 x 10-3 M, 4 h, room temperature, 125 W medium-pressure mercury lamp) has been examined by GC-MS (Climent Miranda, 1996). Upon irradiation, 24% conversion of promecarb was achieved and photolysis of promecarb led to the phenol derivative (22%) as major product. Also, minor amounts of two compounds (isomers arising from photo-Fries rearrangement) were also obtained.

Photoremediation of Carbamate Residues in Water 55

The authors are grateful to the Ministry of Education and Science of the Republic of Serbia for financial support (Project No. III 46008). The authors wish to thank also the DuPont de Nemours and FMC, USA companies for kindly support with the analytical standards. We

Andreozzi, R.; Caprio, V.; Insola, A. & Marotta, R. (1999). Advanced oxidation processes

Aaron, J.J. & Oturan, M.A. (2001). New Photochemical and Electrochemical Methods for the

Behnajady, M.A.; Modirshahla, N. & Hamzavi, R. (2006). Kinetic study on photocatalytic

Benitez, F.J.; Acero, J.L. Real. F.J. (2002). Degradation of Carbofuran by Using ozone, UV

Burrows, H.D.; Canle, M.L.; Santaballa, J.A. & Steenken, S. (2002). Reaction pathways and

Bianco Prevot, A.; Pramauro, E. & de la Guardia, M. (1999). Photocatalytic Degradation of

Brkić, D.; Vitorović, S.; Gašić, S. & Nešković, N. (2008). Carbofuran in Water: Subchronic

Catastini, C.; Sarakla, M.; Mailhot, G. & Bolte, M. (2002a). Iron (III) Aquacomplexes as

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Climent, M.H. & Miranda, M.A. (1996). Gas Chromatographic-Mass Spectrometric Study of

CropLife Tehnical Monograph No.2 (2008). http//www.croplife.org/en-us/technical\_

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would like to express thanks to Mr. Aleksandar F. Tomaši for technical assistance.

**6. Acknowledgment** 

**7. References** 

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#### **4.13 Propamocarb**

Propamocarb (IUPAC name: propyl 3-(dimethylamino)propylcarbamate) is systemic fungicide with protective action. It is used for specific control of Phycomycetes. Also it is used against of wide variety of pest on tomatoes and potatoes, lettuce, cucumber, cabbages, ornamentals, fruit, vegetables, and vegetable seedbeds. Formulations types on the market are SC and SL. The current regulation status of this active ingredient under directive 91/414/EEC is included in Annex 1 expiration of inclusion: 30/09/2017 (EU Pesticide Database, 2011; Tomlin, 2009).

The application of solar photo-Fenton process for degradation of DuPont commercial product Previcur (Fallman et al., 1999) confirmed that propamocarb was one of the hardest pesticides to degrade by process (106 min was a TOC half-life and more than 200 min was the time necessary for degradation of 80% of TOC).

#### **4.14 Propoxur**

Propoxur (IUPAC name: 2-isopropoxyphenyl methylcarbamate) is non-systemic insecticide with contact and stomach action. It is used for control of cockroaches, flies, fleas, mosquitoes, bugs, ants, millipedes and other insect pests in food storage areas, houses, animal houses, etc. Also it is used for control of sucking and chewing insects (including aphids) in fruit, vegetables, ornamentals, vines, maize, alfalfa, soya beans, cotton, sugar cane, rice, cocoa, forestry, etc, and against migratory locusts and grasshoppers. There are a lot of different formulations with this active ingredient as AE, DP, EC, FU, GR, RB, SL, UL, WP and Oil spray. The current regulation status of this active ingredient under directive 91/414/EEC is not included in Annex 1 (EU Pesticide Database, 2011; Tomlin, 2009).

An study of the photodegradation of aerated aqueous propoxur solution is given very interesting data (Sanjuan et al., 2000). Photolysis of 1.0 x 10-3 M solution (pH 6.8) with 125 W medium-pressure mercury lamp leads to an almost complete degradation of pesticide and the formation of photo-Fries rearrangement products, but with a relatively minor degree of mineralization. Photocatalyzed degradations in the presence of TiO2 (40 mg) or with 150 mg of triphenylpyrylium-Zeolite Y (TPY) were shown the same degree of propoxur mineralization. Laser flash photolysis (266 nm) has shown that the degradation could be initiated by a single electron transfer between excited 2,4,6-triphenylpyrylium cation and propoxur to form the corresponding 2,4,6-triphenylpyrylium radical and propoxur radical cation.

#### **5. Conclusion**

The reviewed literature reflects that in case of carbamate pesticides the most of the studies have been reported using photo-Fenton processes, photolysis and heterogeneous catalysis with TiO2 as a catalyst. This photodegradation processes have been proposed as an effective and attractive techniques for degradation of carbamate residues in water. The kinetics of all photodegradation processes depend on several main parameters such as the nature of pesticides, type of light, initial concentration of pesticides (and catalysts), pH of solution, temperature, and presence of oxidant. The AOPs provide an excellent opportunity to use solar light as an energy source. Photocatalytic processes can lead to the mineralization of toxic and hazardous carbamate pesticides into carbon dioxide, water and inorganic mineral salts.

#### **6. Acknowledgment**

54 Insecticides – Basic and Other Applications

Propamocarb (IUPAC name: propyl 3-(dimethylamino)propylcarbamate) is systemic fungicide with protective action. It is used for specific control of Phycomycetes. Also it is used against of wide variety of pest on tomatoes and potatoes, lettuce, cucumber, cabbages, ornamentals, fruit, vegetables, and vegetable seedbeds. Formulations types on the market are SC and SL. The current regulation status of this active ingredient under directive 91/414/EEC is included in Annex 1 expiration of inclusion: 30/09/2017 (EU Pesticide

The application of solar photo-Fenton process for degradation of DuPont commercial product Previcur (Fallman et al., 1999) confirmed that propamocarb was one of the hardest pesticides to degrade by process (106 min was a TOC half-life and more than 200 min was

Propoxur (IUPAC name: 2-isopropoxyphenyl methylcarbamate) is non-systemic insecticide with contact and stomach action. It is used for control of cockroaches, flies, fleas, mosquitoes, bugs, ants, millipedes and other insect pests in food storage areas, houses, animal houses, etc. Also it is used for control of sucking and chewing insects (including aphids) in fruit, vegetables, ornamentals, vines, maize, alfalfa, soya beans, cotton, sugar cane, rice, cocoa, forestry, etc, and against migratory locusts and grasshoppers. There are a lot of different formulations with this active ingredient as AE, DP, EC, FU, GR, RB, SL, UL, WP and Oil spray. The current regulation status of this active ingredient under directive

91/414/EEC is not included in Annex 1 (EU Pesticide Database, 2011; Tomlin, 2009).

An study of the photodegradation of aerated aqueous propoxur solution is given very interesting data (Sanjuan et al., 2000). Photolysis of 1.0 x 10-3 M solution (pH 6.8) with 125 W medium-pressure mercury lamp leads to an almost complete degradation of pesticide and the formation of photo-Fries rearrangement products, but with a relatively minor degree of mineralization. Photocatalyzed degradations in the presence of TiO2 (40 mg) or with 150 mg of triphenylpyrylium-Zeolite Y (TPY) were shown the same degree of propoxur mineralization. Laser flash photolysis (266 nm) has shown that the degradation could be initiated by a single electron transfer between excited 2,4,6-triphenylpyrylium cation and propoxur to form the corresponding 2,4,6-triphenylpyrylium radical and propoxur radical

The reviewed literature reflects that in case of carbamate pesticides the most of the studies have been reported using photo-Fenton processes, photolysis and heterogeneous catalysis with TiO2 as a catalyst. This photodegradation processes have been proposed as an effective and attractive techniques for degradation of carbamate residues in water. The kinetics of all photodegradation processes depend on several main parameters such as the nature of pesticides, type of light, initial concentration of pesticides (and catalysts), pH of solution, temperature, and presence of oxidant. The AOPs provide an excellent opportunity to use solar light as an energy source. Photocatalytic processes can lead to the mineralization of toxic and hazardous carbamate pesticides into carbon dioxide, water and inorganic mineral

**4.13 Propamocarb** 

**4.14 Propoxur** 

cation.

salts.

**5. Conclusion** 

Database, 2011; Tomlin, 2009).

the time necessary for degradation of 80% of TOC).

The authors are grateful to the Ministry of Education and Science of the Republic of Serbia for financial support (Project No. III 46008). The authors wish to thank also the DuPont de Nemours and FMC, USA companies for kindly support with the analytical standards. We would like to express thanks to Mr. Aleksandar F. Tomaši for technical assistance.

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*Part B*, Vol.45, No.7, pp. 626-632, ISSN 0360-1234

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Phase Microextraction Method for Determination of Triazine and Organophosphorus Pesticides in Soil. *Journal of Environmental Science and Health,* 

Photo-Fenton Method for Treating Water Containing Pesticides. *Catalysis Today,*

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Parameter for the Selection of an Appropriate Nonionic Emulsifier. *Journal of the* 

Areas of Serbia. *Journal of the Serbian Chemical Society,* Vol.67, No.12, pp. 887-892,

Additives. *Journal of the Serbian Chemical Society,* Vol.67, No.1, pp. 31-39, ISSN 0352-

Azo Dye in Aqueous Solution by UV Irradiation. *Journal of Photochemistry and* 

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Application/Residues in Soil Under Different Cropping Systems. *Bulletin of Environmental Contamination and Toxicology,* Vol.75, No.2, pp. 316-323, ISSN: 0007-


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Tamimi, M.; Qourzal, S.; Barka, N.; Assabbane, A. & Ait-Ichou, Y. (2008). Methomyl

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Tomašević, A.; Bošković, G.; Mijin, D. Kiss, E. (2007c). Decomposition of Methomyl Over

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Supported Iron Catalysts. *Reaction Kinetics and Catalysis Letters,* Vol.91, No.1, pp. 53-

A Study of the Electrochemical Behaviour of Methomyl on a Gold Electrode in a Neutral Electrolyte. *Journal of the Serbian Chemical Society,* Vol.74, No.5, pp. 573-579,

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**4** 

*USA* 

**Tree Injection as an Alternative Method of** 

Injection directly into the conductive tissues of trees was a method first investigated systematically by Leonardo da Vinci, but some of the most early tree injection experiments were not recorded until early in the 20th century (Roach, 1939, May, 1941, Costonis, 1981). Dutch elm disease, a destructive vascular wilt disease of elm renewed interest in tree injection in the 1970s (Jones and Gregory, 1971; McWain and Gregory, 1971; Jones et al., 1973; Gregory et al., 1973; Gregory and Jones, 1975; Shigo and Campana, 1979; Kielbaso et al. 1979; Shigo et al., 1980), when more common fungicide applications proved ineffective. During this time, several injection methods, including trunk infusion (Schreiber 1969), and pressurized trunk injections (Filer 1973; Helburg et al. 1973; Reil and Beutel 1976, Sachs et al., 1977; Kondo, 1978, Darvas et al., 1984, Navarro et al., 1992), were developed. Tree injection was also used for treatment of other tree pathogens (Guest et al., 1994; Fernández-Escobar et al.1994, 1999), insects, and physiological disorders (i.e., interveinal chlorosis) in the EU (Fernández-Escobar et al. 1993). Interest in tree injection technologies (McClure, 1992, Doccola et al., 2007; Smitley et al., 2010) in the US has also increased, with the introduction of several tree killing insects such as hemlock woolly adelgid (*Adelges tsugae*), Asian longhorned beetle (*Anoplophora glabripennis*) and emerald ash borer (*Agrilus planipennis*). In addition to new injection technology, formulations are being designed for injecting into trees that improve plant safety and reduce application time. Examples of the new technologies are the TREE I.V. micro-infusion system and Air/Hydraulic micro-injector (Arborjet, Inc. Woburn, MA, USA) and the Eco-ject® Microinjection System (Bioforest Technologies, Canada). Today, tree injection is an alternative method of chemical application with certain advantages: (1) efficient use of chemicals, (2) reduced potential environmental exposure, and (3) useful when soil and foliar applications are either ineffective or difficult to apply (Stipes, 1988; Sanchez-Zamora and Fernandez-Escobar, 2004). Tree injection into roots, trunks or limbs requires wounding of the tree, which has implications to the tree's health. The question often asked is, does the benefit gained by tree injection outweigh the risk of the wound caused by treatment? This question of cost-benefit is certainly valid. However, this concern must also be weighed against environmental (and off target) exposures when trees are sprayed or insecticides are applied to the soil. An underlying assumption is that the value of the tree and its treatment is greater than sustaining tree loss. Key factors weigh in to wound responses in trees that likewise demand consideration. These include (1) the tree species, (2) tree health, (3) the attributes of the

**1. Introduction** 

**Insecticide Application** 

Joseph J. Doccola and Peter M. Wild

*Arborjet, Inc. Woburn, MA* 

World Health Organization (1986). Carbamate pesticides: a general introduction. Environmental Health Criteria (EHC) No.64, World Health Organization (WHO), ISBN 9241542640, Geneva, Switzerland

### **Tree Injection as an Alternative Method of Insecticide Application**

Joseph J. Doccola and Peter M. Wild *Arborjet, Inc. Woburn, MA USA* 

#### **1. Introduction**

60 Insecticides – Basic and Other Applications

World Health Organization (1986). Carbamate pesticides: a general introduction.

ISBN 9241542640, Geneva, Switzerland

Environmental Health Criteria (EHC) No.64, World Health Organization (WHO),

Injection directly into the conductive tissues of trees was a method first investigated systematically by Leonardo da Vinci, but some of the most early tree injection experiments were not recorded until early in the 20th century (Roach, 1939, May, 1941, Costonis, 1981). Dutch elm disease, a destructive vascular wilt disease of elm renewed interest in tree injection in the 1970s (Jones and Gregory, 1971; McWain and Gregory, 1971; Jones et al., 1973; Gregory et al., 1973; Gregory and Jones, 1975; Shigo and Campana, 1979; Kielbaso et al. 1979; Shigo et al., 1980), when more common fungicide applications proved ineffective. During this time, several injection methods, including trunk infusion (Schreiber 1969), and pressurized trunk injections (Filer 1973; Helburg et al. 1973; Reil and Beutel 1976, Sachs et al., 1977; Kondo, 1978, Darvas et al., 1984, Navarro et al., 1992), were developed. Tree injection was also used for treatment of other tree pathogens (Guest et al., 1994; Fernández-Escobar et al.1994, 1999), insects, and physiological disorders (i.e., interveinal chlorosis) in the EU (Fernández-Escobar et al. 1993). Interest in tree injection technologies (McClure, 1992, Doccola et al., 2007; Smitley et al., 2010) in the US has also increased, with the introduction of several tree killing insects such as hemlock woolly adelgid (*Adelges tsugae*), Asian longhorned beetle (*Anoplophora glabripennis*) and emerald ash borer (*Agrilus planipennis*). In addition to new injection technology, formulations are being designed for injecting into trees that improve plant safety and reduce application time. Examples of the new technologies are the TREE I.V. micro-infusion system and Air/Hydraulic micro-injector (Arborjet, Inc. Woburn, MA, USA) and the Eco-ject® Microinjection System (Bioforest Technologies, Canada). Today, tree injection is an alternative method of chemical application with certain advantages: (1) efficient use of chemicals, (2) reduced potential environmental exposure, and (3) useful when soil and foliar applications are either ineffective or difficult to apply (Stipes, 1988; Sanchez-Zamora and Fernandez-Escobar, 2004). Tree injection into roots, trunks or limbs requires wounding of the tree, which has implications to the tree's health. The question often asked is, does the benefit gained by tree injection outweigh the risk of the wound caused by treatment? This question of cost-benefit is certainly valid. However, this concern must also be weighed against environmental (and off target) exposures when trees are sprayed or insecticides are applied to the soil. An underlying assumption is that the value of the tree and its treatment is greater than sustaining tree loss. Key factors weigh in to wound responses in trees that likewise demand consideration. These include (1) the tree species, (2) tree health, (3) the attributes of the

Tree Injection as an Alternative Method of Insecticide Application 63

is 44.4% carbon (Heukelekian, H. and S.A. Waksman. 1925). When mature, the xylem protoplast dies, leaving only cell wall. It is through the remaining lumen that water conduction occurs. The lumen simultaneously functions as a continuous and extensive

Water soluble insecticides are differentially absorbed by tree roots comparative to insoluble chemistries such as the avermectins (Wislocki, 1989). Imidacloprid and acephate are labeled in the US for soil application, but restricted in areas of ground water concern (for example, Long Island, N.Y., U.S.). In coarse textured, sandy soils and in areas with high precipitation, there is the potential for insecticide leaching. The insecticidal treatment of eastern hemlock (*Tsuga canadensis*) for hemlock woolly adelgid (*Adelges tsugae*) is an example. Eastern hemlock is a riparian species, which grows in moist soils, and near streams and rivers. In these environments, the use of trunk sprays increases the potential for exposure to off target organisms (e.g., aquatic invertebrates, fish). Tree injection of insecticides is an alternative method of application where these conditions exist. Tree injected imidacloprid applied directly to the vascular tissues is conducted upward within those tissues; the procedure

Canopy sprays are used to control defoliating insects, but drift and limited reach are issues in very tall (>15 meters) trees, where coverage from hydraulic sprayers is inadequate. Employing tree injections resolves these issues; the chemistries move within the vascular system into the canopy for systemic activity. Systemic injections are used to effectively control borers that feed under the bark, where active ingredients sprayed onto the surface of trees may not penetrate in biologically active concentrations. Soil applications are also used, but have a number of limitations. For example, they may be slower acting, require higher amounts of product or repeated applications, may migrate off-target, and be subject to microbial degradation. Finally, tree injections may be more economical to use. Although hydrolysis occurs within the plant, systemically injected chemistries may provide greater residual activity compared to other methods, (i.e., spray, drench) which are subject to drift, leaching, photolysis or microbial degradation. Repeated spray applications each season are necessary for adequate insect control. Aqueous photolysis and mean aerobic soil half-life of selected chemistries appear in Table 1. Soil applications of systemic insecticides are often made at significantly higher volumes (e.g., 5 to 10x) compared to tree injection in order to compensate for leaching, binding to soil particles, microbial degradation or the vagaries of pH and soil moisture. If there are good reasons to utilize tree injection, why are they not employed more often? The objection most often cited is that the application requires drilling into trees. This concern includes the physical wound, and the tree defenses triggered by the introduced formulation. Wounding in trees needs to be placed within context of other types of wounding against which trees evolved effective survival strategies. Trees are wounded in nature when insects bore into the bark and sapwood and when woodpeckers peck and bore into trees

after them. People also create wounds in trees for specific purposes.

**4. Soil and trunk spray applications compared to tree injection** 

conductive and adsorptive structure.

reduces the potential for unintended exposures.

**5. Pros of tree injection** 

chemistry applied and (4) the frequency that applications are made. Such issues present a broader and more complex paradigm and carry over into tree injection practices. In order to apply tree injections effectively, one needs a basic understanding of the (1) method of application, (2) the chemistry applied, and (3) tree condition. The aim of this paper is to recommend tree injection as an alternative application method for systemic insecticides to (1) protect trees against destructive insects, (2) to minimize potential environmental exposures, and (3) to manage tree wound responses.

#### **2. Tree anatomy and physiology**

The introduction and movement of liquid insecticides by injection is dependent upon tree vasculature. Anatomically, trees are highly connected systems (Shigo, 1989, 1991). Fibrous, non-woody roots absorb water and solutes (i.e., minerals in dissolved form) from the rhizosphere (root-soil environment). Hydraulic movement upward in the xylem is dependent upon transpiration from stomates, driven by the moisture lost from leaf surface to the ambient atmosphere (Greulach, 1973). Upward translocation of systemic insecticides also depends upon the rise of sap in trees.

Although movement of sap in the stem is generally upward (i.e., straight sectorial ascent), there is considerable variation in the path of water movement across species (Zanne et al., 2006). The ascent of water in trees follows two basic patterns, that of, spiral and vertical ascents. Systemic chemicals move upward in tree stems along the path of their respective ascents. Crown distribution of water is the most complete by spiral ascent (e.g., red oak), the least effective, by vertical ascent (e.g., white oak) (Rudinski and Vité, 1959). Spiral ascent occurs in a number of species, including conifer xylem (Kozlowski and Winget 1963, Kozlowski et al., 1967).

The size, pattern and distribution of vessels vary in trees. Hardwoods may be grouped as ring- or diffuse- porous; conifers are considered non-porous species (Chaney, 1988). Angiosperm trees have large, wide vessels associated with comparatively high flow rates, while gymnosperms rely solely on very small diameter tracheids to move water. The rate of water flow differs with tree species. Hagen-Poiseulle law describes the rate of flow as a function of the xylem radius to the 4th power (Kramer et al., 1996). Therefore hardwoods (e.g., oaks, elms) move injected liquid at a faster rate than conifers (e.g., pines, hemlocks). In feet per hour, ring porous hardwoods (red oak, ash, elm) move water at 92, 85 and 20; diffuse porous hardwoods (black walnut, maple, beech) move water at 13, 8 and 4; while conifers (pine, hemlock) move water at 6 and 3 (Coder, 1999). Conifers and diffuse porous hardwoods tend to use a larger proportion of sapwood than the ring porous hardwoods for water movement. Drilling more deeply (i.e., 30 rather than 15 mm) in these species serves to access a larger area of sapwood for the injection of systemic chemicals. Sinclair and Larsen investigated wood characteristics that correlated with ease of injection for deciduous trees and suggested the formula, relative frequency of vessels divided by specific gravity (1981).

#### **3. Sapwood composition**

Xylem (sapwood) is the conductive tissue of plants, made up of cellulose, lignin and other substances. Cellulose (C6H10O5)n is an organic polymer made up of glucose molecules linked together in long chains (Raven, Evert & Curtis, 1981). Lignin is a complex organic polymer that functions to strengthen wood. Cellulose makes up the cell wall of plants, and is 44.4% carbon (Heukelekian, H. and S.A. Waksman. 1925). When mature, the xylem protoplast dies, leaving only cell wall. It is through the remaining lumen that water conduction occurs. The lumen simultaneously functions as a continuous and extensive conductive and adsorptive structure.

#### **4. Soil and trunk spray applications compared to tree injection**

Water soluble insecticides are differentially absorbed by tree roots comparative to insoluble chemistries such as the avermectins (Wislocki, 1989). Imidacloprid and acephate are labeled in the US for soil application, but restricted in areas of ground water concern (for example, Long Island, N.Y., U.S.). In coarse textured, sandy soils and in areas with high precipitation, there is the potential for insecticide leaching. The insecticidal treatment of eastern hemlock (*Tsuga canadensis*) for hemlock woolly adelgid (*Adelges tsugae*) is an example. Eastern hemlock is a riparian species, which grows in moist soils, and near streams and rivers. In these environments, the use of trunk sprays increases the potential for exposure to off target organisms (e.g., aquatic invertebrates, fish). Tree injection of insecticides is an alternative method of application where these conditions exist. Tree injected imidacloprid applied directly to the vascular tissues is conducted upward within those tissues; the procedure reduces the potential for unintended exposures.

#### **5. Pros of tree injection**

62 Insecticides – Basic and Other Applications

chemistry applied and (4) the frequency that applications are made. Such issues present a broader and more complex paradigm and carry over into tree injection practices. In order to apply tree injections effectively, one needs a basic understanding of the (1) method of application, (2) the chemistry applied, and (3) tree condition. The aim of this paper is to recommend tree injection as an alternative application method for systemic insecticides to (1) protect trees against destructive insects, (2) to minimize potential environmental

The introduction and movement of liquid insecticides by injection is dependent upon tree vasculature. Anatomically, trees are highly connected systems (Shigo, 1989, 1991). Fibrous, non-woody roots absorb water and solutes (i.e., minerals in dissolved form) from the rhizosphere (root-soil environment). Hydraulic movement upward in the xylem is dependent upon transpiration from stomates, driven by the moisture lost from leaf surface to the ambient atmosphere (Greulach, 1973). Upward translocation of systemic insecticides

Although movement of sap in the stem is generally upward (i.e., straight sectorial ascent), there is considerable variation in the path of water movement across species (Zanne et al., 2006). The ascent of water in trees follows two basic patterns, that of, spiral and vertical ascents. Systemic chemicals move upward in tree stems along the path of their respective ascents. Crown distribution of water is the most complete by spiral ascent (e.g., red oak), the least effective, by vertical ascent (e.g., white oak) (Rudinski and Vité, 1959). Spiral ascent occurs in a number of species, including conifer xylem (Kozlowski and Winget 1963,

The size, pattern and distribution of vessels vary in trees. Hardwoods may be grouped as ring- or diffuse- porous; conifers are considered non-porous species (Chaney, 1988). Angiosperm trees have large, wide vessels associated with comparatively high flow rates, while gymnosperms rely solely on very small diameter tracheids to move water. The rate of water flow differs with tree species. Hagen-Poiseulle law describes the rate of flow as a function of the xylem radius to the 4th power (Kramer et al., 1996). Therefore hardwoods (e.g., oaks, elms) move injected liquid at a faster rate than conifers (e.g., pines, hemlocks). In feet per hour, ring porous hardwoods (red oak, ash, elm) move water at 92, 85 and 20; diffuse porous hardwoods (black walnut, maple, beech) move water at 13, 8 and 4; while conifers (pine, hemlock) move water at 6 and 3 (Coder, 1999). Conifers and diffuse porous hardwoods tend to use a larger proportion of sapwood than the ring porous hardwoods for water movement. Drilling more deeply (i.e., 30 rather than 15 mm) in these species serves to access a larger area of sapwood for the injection of systemic chemicals. Sinclair and Larsen investigated wood characteristics that correlated with ease of injection for deciduous trees and suggested the formula, relative frequency of vessels divided by specific gravity (1981).

Xylem (sapwood) is the conductive tissue of plants, made up of cellulose, lignin and other substances. Cellulose (C6H10O5)n is an organic polymer made up of glucose molecules linked together in long chains (Raven, Evert & Curtis, 1981). Lignin is a complex organic polymer that functions to strengthen wood. Cellulose makes up the cell wall of plants, and

exposures, and (3) to manage tree wound responses.

**2. Tree anatomy and physiology** 

also depends upon the rise of sap in trees.

Kozlowski et al., 1967).

**3. Sapwood composition** 

Canopy sprays are used to control defoliating insects, but drift and limited reach are issues in very tall (>15 meters) trees, where coverage from hydraulic sprayers is inadequate. Employing tree injections resolves these issues; the chemistries move within the vascular system into the canopy for systemic activity. Systemic injections are used to effectively control borers that feed under the bark, where active ingredients sprayed onto the surface of trees may not penetrate in biologically active concentrations. Soil applications are also used, but have a number of limitations. For example, they may be slower acting, require higher amounts of product or repeated applications, may migrate off-target, and be subject to microbial degradation. Finally, tree injections may be more economical to use. Although hydrolysis occurs within the plant, systemically injected chemistries may provide greater residual activity compared to other methods, (i.e., spray, drench) which are subject to drift, leaching, photolysis or microbial degradation. Repeated spray applications each season are necessary for adequate insect control. Aqueous photolysis and mean aerobic soil half-life of selected chemistries appear in Table 1. Soil applications of systemic insecticides are often made at significantly higher volumes (e.g., 5 to 10x) compared to tree injection in order to compensate for leaching, binding to soil particles, microbial degradation or the vagaries of pH and soil moisture. If there are good reasons to utilize tree injection, why are they not employed more often? The objection most often cited is that the application requires drilling into trees. This concern includes the physical wound, and the tree defenses triggered by the introduced formulation. Wounding in trees needs to be placed within context of other types of wounding against which trees evolved effective survival strategies. Trees are wounded in nature when insects bore into the bark and sapwood and when woodpeckers peck and bore into trees after them. People also create wounds in trees for specific purposes.

Tree Injection as an Alternative Method of Insecticide Application 65

**Buprestidae**: Emerald Ash Borer (*Agrilus planipennis*), an exotic introduced from Asia (China) was identified in Detroit, MI in 2002 (McCullough and Siegert, 2007; Anulewicz et al., 2008.). EAB attacks native ash (*Fraxinus*) species, preferentially Green (*F. pennsylvanica*) and Black (*F. nigra*), but also White (*F. americana*) and Blue (*F. quadrangulata*) ashes. EAB mines the phloem, cambium and scores the xylem as an actively developing larva. The vascular disruption reduces water movement upward into the canopy, and photosynthate transport through the phloem; unchecked infestations result in tree death. Unlike maple and birch attacked by ALB, ash trees do not bleed and EAB larvae do not remove frass from their galleries, so there are no visible signs of early infestation. Infestations often go undetected for several years, and symptoms in ash (epicormic sprouts, bark cracks, woodpecker flecks) and signs (d-shaped exit holes) do not occur until the damage has occurred. Goldspotted Oak Borer (*Agrilus coxalis*) is native to Southeastern Arizona, detected in San Diego County, California in 2004. It attacks coast live oak (*Quercus agrifolia*), canyon live oak (*Q. chrysolepis*) and California black oak (*Q. kelloggii*). Regarded as an invasive species in California, larval feeding kills phloem and cambium, which results in crown dieback and tree mortality (Coleman & Seybold, 2008). Other Buprestid borers include the two-lined chestnut borer (*A. bilineatus*) and the bronze birch borer (*A. anxius*). Adult two-lined chestnut borers attack stressed or declining oak trees. The bronze birch borer preferentially attacks European cutleaf birches such as *Betula jacquemontii, B.* 

The yellow-bellied sapsucker (*Sphyrapicus varius*) bores into the bark of trees to obtain sap. More than 250 species of woody plants are known to be attacked, but birch (*Betula* spp.), maple (*Acer* spp.) and hemlock (*Tsuga* spp.) are preferentially attacked (Ostry & Nicholls, 1978). Sapsucker damage is characterized by many closely spaced holes on the tree. The tree responds by proliferating new tissues at the wound sites. Woodpeckers feed primarily on wood boring insects. The Northern flicker (*Colaptes auratus*), Red-bellied woodpecker (*Melanerpes carolinus*), Downy woodpecker (*Picoides pubescens*), Hairy woodpecker (*Picoides*  villosus) and Red-headed woodpecker (*Melanerpes erthrocephalus*) drill holes into trees to extract insects or sap (Barnes, 1989). These woodpecker behaviors are generally not

People drill into trees for sap extraction and to apply treatments, including injection. In the northeastern US and Canada, Sugar maples (*Acer saccharum*) are tapped annually for maple syrup production. Healthy trees that are tapped according to established guidelines do not suffer adverse health effects and remain productive (Davenport & Staats, 1998), some for over 100 years. Arborists drill into trees to install cabling and lightning protection (ANSI A300 Part 3, 2006; ANSI A300 Part 4, 2008). Tree care specialists treat by injection to protect trees against destructive pests. In the US, destructive, exotic insects such as hemlock woolly adelgid (USDA/FS 2003), Asian longhorned beetle (USDA/FS 2008) and emerald ash borer (USDA/FS 2008a) have recently renewed interest in tree injection technology as an alternative method of insecticide application (McClure, 1992, Doccola et al., 2007; Smitley et al., 2010). To apply tree

*pendula and B. pendula* 'Youngii' (Dirr, 2009).

**7. Birds that drill into trees** 

regarded as detrimental to trees.

**8. People drill into trees** 


\*organic carbon adsorption coefficient

+mean aerobic

Table 1. Water solubility's, organic carbon adsorptions and half-lives of three chemistries systemically injected into trees.

#### **6. Wood boring insects**

Insect borers include species of Lepidoptera, Hymenoptera and Coleoptera. Borers may be further categorized as wood or cambium borers. Most native insects are opportunistic, attacking stressed and declining trees. When conditions favor epidemiology, trees are attacked and killed. Exotic insects are comparatively more aggressive and attack and kill healthy trees.

**Lepidoptera:** Clear-winged borers (Sessidae) include some serious pests including the ash borer (*Podesia syringae*). *Dioryctria* borers (Pyralidae) attack pines causing large masses of sap to exude. The Zimmerman pine moth (*Dioryctria zimmermani*) is a pest of Austrian and Scotch pines (*Pinus nigra, P. sylvestris*) in ornamental landscapes (Cranshaw & Leatherman, 2006).

**Hymenoptera:** Horntails (Siricidae) are sawflies that develop in damaged or stressed trees. A recent introduction in the US, the Sirex woodwasp (*Sirex noctilio*), a native of Europe, Asia and northern Africa has the potential to cause significant mortality in native pine stands (Haugen & Hoebeke, 2005).

**Coleoptera**: Several families of beetles bore into trees, which include the Scolytidae (bark beetles), Cerambycidae (Longhorned beetles or roundheaded borers), and Buprestidae (flatheaded borers). Some species vector spores of destructive pathogens.

**Scolytidae**: In Lodgepole pine (*Pinus contorta*) a native scolytid mountain pine beetle (*Dendroctonus ponderosae*) vectors *Ophiostoma clavigerum*, a blue staining fungus (Solheim and Krokene, 1998). MPB also infests ponderosa (*P. ponderosa*), sugar (*P. lambertiana*) and white (*P. monticola*) pines (Amman et al., 2002). An epidemic can cause widespread tree mortality. The Smaller European Elm bark beetle (*Scolytus multistriatus*) vectors spores of the bluestain fungus (*Ophiostoma novo-ulmi*) that cause Dutch elm disease, a vascular wilt disease that has devastated the American elm (*Ulmus americana*) in the United States.

**Cerambycidae**: Locust borer (*Megacyllene robiniae*) is a native that attacks, and can severely damage or kill stressed and healthy black locust *(Robinia pseudoacacia*) (Galford, 1984). The Asian longhorned beetle (*Anoplophora glabripennis*) was introduced from Asia (China) and identified in Brooklyn, New York in 1996. ALB has a broad host range in the US but preferentially infests maple (*Acer*), and birch (*Betula*) trees (Sawyer, 2010).

**Half-lives (days)** 

(Montgomery, 1993)

(Mushtaq et al. 1996)

Table 1. Water solubility's, organic carbon adsorptions and half-lives of three chemistries

Insect borers include species of Lepidoptera, Hymenoptera and Coleoptera. Borers may be further categorized as wood or cambium borers. Most native insects are opportunistic, attacking stressed and declining trees. When conditions favor epidemiology, trees are attacked and killed. Exotic insects are comparatively more aggressive and attack and kill

**Lepidoptera:** Clear-winged borers (Sessidae) include some serious pests including the ash borer (*Podesia syringae*). *Dioryctria* borers (Pyralidae) attack pines causing large masses of sap to exude. The Zimmerman pine moth (*Dioryctria zimmermani*) is a pest of Austrian and Scotch pines (*Pinus nigra, P. sylvestris*) in ornamental landscapes (Cranshaw & Leatherman,

**Hymenoptera:** Horntails (Siricidae) are sawflies that develop in damaged or stressed trees. A recent introduction in the US, the Sirex woodwasp (*Sirex noctilio*), a native of Europe, Asia and northern Africa has the potential to cause significant mortality in native pine stands

**Coleoptera**: Several families of beetles bore into trees, which include the Scolytidae (bark beetles), Cerambycidae (Longhorned beetles or roundheaded borers), and Buprestidae (flat-

**Scolytidae**: In Lodgepole pine (*Pinus contorta*) a native scolytid mountain pine beetle (*Dendroctonus ponderosae*) vectors *Ophiostoma clavigerum*, a blue staining fungus (Solheim and Krokene, 1998). MPB also infests ponderosa (*P. ponderosa*), sugar (*P. lambertiana*) and white (*P. monticola*) pines (Amman et al., 2002). An epidemic can cause widespread tree mortality. The Smaller European Elm bark beetle (*Scolytus multistriatus*) vectors spores of the bluestain fungus (*Ophiostoma novo-ulmi*) that cause Dutch elm disease, a vascular wilt disease that has

**Cerambycidae**: Locust borer (*Megacyllene robiniae*) is a native that attacks, and can severely damage or kill stressed and healthy black locust *(Robinia pseudoacacia*) (Galford, 1984). The Asian longhorned beetle (*Anoplophora glabripennis*) was introduced from Asia (China) and identified in Brooklyn, New York in 1996. ALB has a broad host range in the US but

headed borers). Some species vector spores of destructive pathogens.

devastated the American elm (*Ulmus americana*) in the United States.

preferentially infests maple (*Acer*), and birch (*Betula*) trees (Sawyer, 2010).

**Photolysis Soil+**

0.5

38.9

193.4

(Chevron, 1972g)

(Yoshida, 1990)

(Chukwudebe et al., 1997a)

stable

3.98x10-2 (Anderson, 1991)

(Chevron, 1972d)

3.6-10.9 (Mushtaq et al., 1998)

**Insecticide Water Sol (g/L) Ko/c\* Aqueous** 

0.48

300-400 (Cox et al., 1997)

>25000

(Worthing, 1987)

(Tomlin, 2004)

\*organic carbon adsorption coefficient

systemically injected into trees.

**6. Wood boring insects** 

(Haugen & Hoebeke, 2005).

(Yen & Wendt, 1993)

Acephate <sup>700</sup>

Imidacloprid 0.514

Emamectin 0.024

+mean aerobic

healthy trees.

2006).

**Buprestidae**: Emerald Ash Borer (*Agrilus planipennis*), an exotic introduced from Asia (China) was identified in Detroit, MI in 2002 (McCullough and Siegert, 2007; Anulewicz et al., 2008.). EAB attacks native ash (*Fraxinus*) species, preferentially Green (*F. pennsylvanica*) and Black (*F. nigra*), but also White (*F. americana*) and Blue (*F. quadrangulata*) ashes. EAB mines the phloem, cambium and scores the xylem as an actively developing larva. The vascular disruption reduces water movement upward into the canopy, and photosynthate transport through the phloem; unchecked infestations result in tree death. Unlike maple and birch attacked by ALB, ash trees do not bleed and EAB larvae do not remove frass from their galleries, so there are no visible signs of early infestation. Infestations often go undetected for several years, and symptoms in ash (epicormic sprouts, bark cracks, woodpecker flecks) and signs (d-shaped exit holes) do not occur until the damage has occurred. Goldspotted Oak Borer (*Agrilus coxalis*) is native to Southeastern Arizona, detected in San Diego County, California in 2004. It attacks coast live oak (*Quercus agrifolia*), canyon live oak (*Q. chrysolepis*) and California black oak (*Q. kelloggii*). Regarded as an invasive species in California, larval feeding kills phloem and cambium, which results in crown dieback and tree mortality (Coleman & Seybold, 2008). Other Buprestid borers include the two-lined chestnut borer (*A. bilineatus*) and the bronze birch borer (*A. anxius*). Adult two-lined chestnut borers attack stressed or declining oak trees. The bronze birch borer preferentially attacks European cutleaf birches such as *Betula jacquemontii, B. pendula and B. pendula* 'Youngii' (Dirr, 2009).

#### **7. Birds that drill into trees**

The yellow-bellied sapsucker (*Sphyrapicus varius*) bores into the bark of trees to obtain sap. More than 250 species of woody plants are known to be attacked, but birch (*Betula* spp.), maple (*Acer* spp.) and hemlock (*Tsuga* spp.) are preferentially attacked (Ostry & Nicholls, 1978). Sapsucker damage is characterized by many closely spaced holes on the tree. The tree responds by proliferating new tissues at the wound sites. Woodpeckers feed primarily on wood boring insects. The Northern flicker (*Colaptes auratus*), Red-bellied woodpecker (*Melanerpes carolinus*), Downy woodpecker (*Picoides pubescens*), Hairy woodpecker (*Picoides*  villosus) and Red-headed woodpecker (*Melanerpes erthrocephalus*) drill holes into trees to extract insects or sap (Barnes, 1989). These woodpecker behaviors are generally not regarded as detrimental to trees.

#### **8. People drill into trees**

People drill into trees for sap extraction and to apply treatments, including injection. In the northeastern US and Canada, Sugar maples (*Acer saccharum*) are tapped annually for maple syrup production. Healthy trees that are tapped according to established guidelines do not suffer adverse health effects and remain productive (Davenport & Staats, 1998), some for over 100 years. Arborists drill into trees to install cabling and lightning protection (ANSI A300 Part 3, 2006; ANSI A300 Part 4, 2008). Tree care specialists treat by injection to protect trees against destructive pests. In the US, destructive, exotic insects such as hemlock woolly adelgid (USDA/FS 2003), Asian longhorned beetle (USDA/FS 2008) and emerald ash borer (USDA/FS 2008a) have recently renewed interest in tree injection technology as an alternative method of insecticide application (McClure, 1992, Doccola et al., 2007; Smitley et al., 2010). To apply tree

Tree Injection as an Alternative Method of Insecticide Application 67

Emamectin benzoate is a semi-synthetic compound derived from the fermentation byproduct of a soil actinomycete, *Streptomyces avermitilis* (Jansson et al., 1996). Emamectin benzoate is a mixture of the benzoic acid salt of two structurally complex heterocyclic (glycoside) compounds. It occurs as a mixture of ≥90% benzoic acid salts of 4'-epimethylamino-4'-19 deoxyavermectin B1a and <10% 4'-epi-methylamino-4'-deoxyavermectin B1b (Wood, 2010). Emamectin benzoate is poorly (0.024 g/L) soluble in water (Tomlin, 2004). It has a Ko/c of >25,000 and is immobile in soils (Mushtaq et al. 1996). Emamectin benzoate has translaminar activity, but limited plant systemic activity when applied to the foliage (Copping, 2004). A novel micro-emulsion formulation (TREE-äge, Syngenta Crop Protection, LLC, Greensboro, NC) used for systemic tree injection is registered for use in the

Injected chemistries differ in their rate of movement in the vascular system, and in their residual activity. In Avocado (*Persea americana*), Acephate peaked in foliage 2 weeks following tree injection, whereas peak imidacloprid residues were not observed for 7-9 weeks following application (Morse et al., 2008). The slow upward movement of imidacloprid may be explained by its comparatively higher carbon adsorption, and may play a role in the extended activity observed in field studies (Doccola et al., 2007; Morse et al., 2008). Studies in green ash (*Fraxinus pennsylvanica* Marsh) and white ash (*F. americana* L.) have demonstrated that imidacloprid accumulates in the canopy, but tree injection could also provide a reservoir for continued systemic activity (Cregg et al., 2005; Tanis et al., 2006, 2007, 2009). Takai et al. (2003), reported 3 years of protection in pine trees against pine wilt nematode after injecting a liquid formulation of emamectin benzoate. In the US, emamectin benzoate was reported to provide 2 or more years of protection against Lepidopterous and Coleoptera pests, including Pine cone worm (*Dioryctria*)*,* Southern pine beetle (*Dendroctonus frontalis*) and Emerald ash borer (*Agrilus planipennis*) (Grosman et al., 2002, Grosman et al.,

Injection into plant tissues protects the chemistry from phytolysis and microbial degradation, mechanisms that breakdown the chemistry in the environment relatively quickly. Although hydrolysis occurs within the plant, some of the metabolites have insecticidal activity (for example, olefinic-, dihydroxy- and hydroxy-imidacloprid breakdown products of imidacloprid) (Sangha & Machemer, 1992; Suchail et al., 2001). Residual activity is based on the half-life of the chemistry, but carbon adsorption may also play a role in the activity observed in perennial tissues (such as in twig, branch and stem) over time. Injected formulations that provide multiple years of activity must move (spatially) from the original injection site in the xylem tissue into new vascular tissue in order to be effective against insects that perennially attack and feed in the lateral cambium. Residual activity of an injected insecticide provides protection against insect pests that have extended emergence periods, multiple generations per year, or are epidemic (i.e., increase

Apply treatments before damage (defoliation, vascular mining) occurs for optimum results. Oak trees defoliated by gypsy moth must use stored carbohydrates for recovery

**9.3 Emamectin benzoate** 

2009; Smitley et al., 2010).

exponentially over time).

**11. When to treat trees** 

US against specific Coleoptera and Lepidoptera pests.

**10. Behaviors of injected chemistries** 

injections effectively, one needs a basic understanding of the (1) method of application, (2) the chemistry applied, and (3) tree condition.

#### **9. Tree injection methodology**

Systemic tree injections effectively treat destructive insect pests of trees. Examples of the new technologies are the TREE I.V. micro-infusion system and the Air/Hydraulic microinjector (Arborjet, Inc. Woburn, MA, USA) and the Eco-ject® Micro-injection System (Bioforest Technologies, Inc., Canada). The TREE I.V. micro-infusion system and Air/Hydraulic micro-injector deliver 0.50 and 2.0 liters at injection pressures of 172 to 1379 kPa, respectively. These methods require the insertion of an interface into the sapwood (ArborplugTM) to inject a systemic insecticide. The Arborplug has an internal rubber septum which is pierced by an injector needle for liquid delivery. The Arborplug is 15 mm in length and has a diameter of either 7 or 9 mm. Drilling 15 mm deep provides a volumetric capacity of 0.6 to 1.1 cm3, respectively. The Eco-ject Micro-injection System loads re-usable microinjection capsules, but does not use a plug. Using such devices, one may deliver a number of systemic chemistries by tree injection. Here we discuss three insecticides which are, (1) acephate, (2) imidacloprid and (3) emamectin benzoate.

#### **9.1 Acephate**

Acephate (O,S-dimethyl acetylphosphoramidothioate) is water soluble (700 g/L) and readily absorbed by tree roots for systemic activity (Worthing, 1987; Kidd & James, 1991). It has a low Ko/c (organic carbon adsorption coefficient) of 0.48 (Montgomery, 1993); it is only weakly adsorbed in the soil. Acephate is an organo-phosphate insecticide designed for insecticidal activity and quick degradation. Acephate's stability is affected by pH. It has a comparatively shorter half-life (of 16-d, pH 9) in alkaline environments (Chevron, unpublished 1972b). Acephate is particularly mobile in coarse textured soils and has the potential to leach (Yen et al., 2000), but it is quickly degraded by microbial activity. In plants, acephate's half-life is approximately 5 to 10-d. Approximately 5 to 10% of acephate is degraded to methamidophos (which has insecticidal activity), the remainder to salts (of N, P and S) (Chevron, unpublished 1973). Acephate has both translaminar and systemic activity in plants. Acephate is a broad spectrum systemic, used for control of aphids, leaf miners, Lepidopterous larvae, sawflies, and thrips. 97.4% acephate is a soluble granular offered as an implant (Ace-Cap, Creative Sales, Fremont Nebraska) or tree injection formulation (ACEjet, Arborjet, Inc.).

#### **9.2 Imidacloprid**

Imidacloprid (1-[(6-chloropyridin-3-yl) methyl]-N-nitro-4, 5-dihydroimidazol-2-amine) is a chloronicotinyl (neonicotinoid) chemistry with a water solubility of 0.51 g/L (Yen and Wendt, 1993). Imidacloprid has moderate binding activity (Ko/c of 300 to 400) to clay and organic matter (Cox et al., 1997), however there is potential for the compound to move through porous, coarse textured soils (Jenkins, 1994). Imidacloprid has translaminar and systemic activity in plants (Buchholz and Nauen, 2002). Imidacloprid controls sucking insects such as adelgids, aphids, thrips, whiteflies, and some beetles, including Cerambycids. Examples of tree injection formulations of imidacloprid are Imicide (JJ Mauget, Arcadia, CA), Xytect (Rainbow Treecare Scientific Advancements, Minnetonka, MN) and IMA-jet (Arborjet, Inc.).

#### **9.3 Emamectin benzoate**

66 Insecticides – Basic and Other Applications

injections effectively, one needs a basic understanding of the (1) method of application, (2)

Systemic tree injections effectively treat destructive insect pests of trees. Examples of the new technologies are the TREE I.V. micro-infusion system and the Air/Hydraulic microinjector (Arborjet, Inc. Woburn, MA, USA) and the Eco-ject® Micro-injection System (Bioforest Technologies, Inc., Canada). The TREE I.V. micro-infusion system and Air/Hydraulic micro-injector deliver 0.50 and 2.0 liters at injection pressures of 172 to 1379 kPa, respectively. These methods require the insertion of an interface into the sapwood (ArborplugTM) to inject a systemic insecticide. The Arborplug has an internal rubber septum which is pierced by an injector needle for liquid delivery. The Arborplug is 15 mm in length and has a diameter of either 7 or 9 mm. Drilling 15 mm deep provides a volumetric capacity of 0.6 to 1.1 cm3, respectively. The Eco-ject Micro-injection System loads re-usable microinjection capsules, but does not use a plug. Using such devices, one may deliver a number of systemic chemistries by tree injection. Here we discuss three insecticides which are, (1)

Acephate (O,S-dimethyl acetylphosphoramidothioate) is water soluble (700 g/L) and readily absorbed by tree roots for systemic activity (Worthing, 1987; Kidd & James, 1991). It has a low Ko/c (organic carbon adsorption coefficient) of 0.48 (Montgomery, 1993); it is only weakly adsorbed in the soil. Acephate is an organo-phosphate insecticide designed for insecticidal activity and quick degradation. Acephate's stability is affected by pH. It has a comparatively shorter half-life (of 16-d, pH 9) in alkaline environments (Chevron, unpublished 1972b). Acephate is particularly mobile in coarse textured soils and has the potential to leach (Yen et al., 2000), but it is quickly degraded by microbial activity. In plants, acephate's half-life is approximately 5 to 10-d. Approximately 5 to 10% of acephate is degraded to methamidophos (which has insecticidal activity), the remainder to salts (of N, P and S) (Chevron, unpublished 1973). Acephate has both translaminar and systemic activity in plants. Acephate is a broad spectrum systemic, used for control of aphids, leaf miners, Lepidopterous larvae, sawflies, and thrips. 97.4% acephate is a soluble granular offered as an implant (Ace-Cap, Creative Sales, Fremont Nebraska) or tree injection formulation (ACE-

Imidacloprid (1-[(6-chloropyridin-3-yl) methyl]-N-nitro-4, 5-dihydroimidazol-2-amine) is a chloronicotinyl (neonicotinoid) chemistry with a water solubility of 0.51 g/L (Yen and Wendt, 1993). Imidacloprid has moderate binding activity (Ko/c of 300 to 400) to clay and organic matter (Cox et al., 1997), however there is potential for the compound to move through porous, coarse textured soils (Jenkins, 1994). Imidacloprid has translaminar and systemic activity in plants (Buchholz and Nauen, 2002). Imidacloprid controls sucking insects such as adelgids, aphids, thrips, whiteflies, and some beetles, including Cerambycids. Examples of tree injection formulations of imidacloprid are Imicide (JJ Mauget, Arcadia, CA), Xytect (Rainbow Treecare Scientific Advancements, Minnetonka,

the chemistry applied, and (3) tree condition.

acephate, (2) imidacloprid and (3) emamectin benzoate.

**9. Tree injection methodology** 

**9.1 Acephate** 

jet, Arborjet, Inc.).

**9.2 Imidacloprid** 

MN) and IMA-jet (Arborjet, Inc.).

Emamectin benzoate is a semi-synthetic compound derived from the fermentation byproduct of a soil actinomycete, *Streptomyces avermitilis* (Jansson et al., 1996). Emamectin benzoate is a mixture of the benzoic acid salt of two structurally complex heterocyclic (glycoside) compounds. It occurs as a mixture of ≥90% benzoic acid salts of 4'-epimethylamino-4'-19 deoxyavermectin B1a and <10% 4'-epi-methylamino-4'-deoxyavermectin B1b (Wood, 2010). Emamectin benzoate is poorly (0.024 g/L) soluble in water (Tomlin, 2004). It has a Ko/c of >25,000 and is immobile in soils (Mushtaq et al. 1996). Emamectin benzoate has translaminar activity, but limited plant systemic activity when applied to the foliage (Copping, 2004). A novel micro-emulsion formulation (TREE-äge, Syngenta Crop Protection, LLC, Greensboro, NC) used for systemic tree injection is registered for use in the US against specific Coleoptera and Lepidoptera pests.

#### **10. Behaviors of injected chemistries**

Injected chemistries differ in their rate of movement in the vascular system, and in their residual activity. In Avocado (*Persea americana*), Acephate peaked in foliage 2 weeks following tree injection, whereas peak imidacloprid residues were not observed for 7-9 weeks following application (Morse et al., 2008). The slow upward movement of imidacloprid may be explained by its comparatively higher carbon adsorption, and may play a role in the extended activity observed in field studies (Doccola et al., 2007; Morse et al., 2008). Studies in green ash (*Fraxinus pennsylvanica* Marsh) and white ash (*F. americana* L.) have demonstrated that imidacloprid accumulates in the canopy, but tree injection could also provide a reservoir for continued systemic activity (Cregg et al., 2005; Tanis et al., 2006, 2007, 2009). Takai et al. (2003), reported 3 years of protection in pine trees against pine wilt nematode after injecting a liquid formulation of emamectin benzoate. In the US, emamectin benzoate was reported to provide 2 or more years of protection against Lepidopterous and Coleoptera pests, including Pine cone worm (*Dioryctria*)*,* Southern pine beetle (*Dendroctonus frontalis*) and Emerald ash borer (*Agrilus planipennis*) (Grosman et al., 2002, Grosman et al., 2009; Smitley et al., 2010).

Injection into plant tissues protects the chemistry from phytolysis and microbial degradation, mechanisms that breakdown the chemistry in the environment relatively quickly. Although hydrolysis occurs within the plant, some of the metabolites have insecticidal activity (for example, olefinic-, dihydroxy- and hydroxy-imidacloprid breakdown products of imidacloprid) (Sangha & Machemer, 1992; Suchail et al., 2001). Residual activity is based on the half-life of the chemistry, but carbon adsorption may also play a role in the activity observed in perennial tissues (such as in twig, branch and stem) over time. Injected formulations that provide multiple years of activity must move (spatially) from the original injection site in the xylem tissue into new vascular tissue in order to be effective against insects that perennially attack and feed in the lateral cambium. Residual activity of an injected insecticide provides protection against insect pests that have extended emergence periods, multiple generations per year, or are epidemic (i.e., increase exponentially over time).

#### **11. When to treat trees**

Apply treatments before damage (defoliation, vascular mining) occurs for optimum results. Oak trees defoliated by gypsy moth must use stored carbohydrates for recovery

Tree Injection as an Alternative Method of Insecticide Application 69

barrier. Tyloses may be formed in older wood naturally (e.g., white oak, *Quercus alba*, forms tyloses in second year wood), or are a consequence of trauma (e.g., red oak, *Q. rubra*, forms tyloses in response to wounding) (Shigo, 1999). When a tree is physically injured, both biochemical and structural changes occur. The biochemical reactions (changes of stored carbohydrates to phenolic and terpene defense chemicals) are observed in tree sections in three dimensions. These were named reaction zones (or boundary walls) 1 – 3. Reaction zone 1 occurs in the axial direction (i.e., with the stem axis) and is the least limiting boundary. Reaction zone 2 occurs in the radial direction (i.e., with the tree radius, inward toward the pith), and reaction zone 3 occurs in the tangential direction (i.e., with the tree's circumference), and is the strongest limiting boundary of the three reaction zones. The fourth wall, referred to as the barrier zone occurs after injury, and is the strongest limiting boundary. Meristematic cells (cambium) divide to form callus tissue, which later differentiates into new woundwood (new xylem, cambium and phloem). Native insect attacks to healthy trees are fended off by the biochemistry and by the subsequent physical responses. Emerald ash borer attacks to Asian species of ash (*Fraxinus chinensis*, *F. manchurica*) do not result in tree mortality: plant defense responses effectively isolate the larva in early stages of attack and limit its progression. In *F. pennsylvanica* (a native), the larvae are compartmentalized via physical boundaries (wall 4), but the biochemistry (phenols, terpene chemistries) does not effectively stop the insect's development. Injection of an insecticidal chemistry to compensate for insufficient tree response is the basis of successful tree protection. EAB research has demonstrated that this strategy is very effective

Tree wound responses are dependent upon a number of intrinsic and extrinsic variables such as tree species, tree health, method of treatment and chemistry applied. Tree wound response is under genetic control (Santamour, 1979). For example, birch (*Betula* spp.) poplar (*Populus* spp.) and willow (*Salix* spp.) are considered weak compartmentalizers, whereas oak (*Quercus* spp.), sycamore (*Platanus* spp.) and linden (*Tilia* spp.) are considered strong compartmentalizers (Dujesiefken and Liese, 2008). Santamour (1986) described fourteen cultivars of maple (*Acer*), ash (*Fraxinus*), oak (*Quercus*) and linden (*Tilia*) that were strong wall 2 compartmentalizers. As a group, trees have evolved to resist assaults and are successful, long-lived perennial plants. Tree health is another variable with numerous contributing factors. These include the age of the tree, soil conditions (texture, structure, moisture, pH, minerals and drainage), and exposure (sun, shade). Trees require light, water and minerals for essential life functions (including defense). Photosynthesis is the basis of carbohydrate synthesis. Woundwood responses utilize energy (carbohydrate, lipid) stores. When injections are made to trees in relatively good health (preventative-early therapeutic treatments) tree woundwood development readily proceeds to close wounds. However, the prognosis for recovery is comparatively lower, when making late therapeutic (rescue) applications, because energy stores are reduced. Optimal wound responses are observed when applications are made early, relative to infestation (Doccola et al., 2011). To further manage wounds in trees, make the fewest number of injection sites to apply the dose, and whenever possible, avoid drilling in the valleys between roots (Shigo and Campana, 1977). The Wedgle Direct-Inject (ArborSystems, LLC, Omaha, NE) is a method of tree injection that does not require drilling into the sapwood. The system relies on forcing the de-lamination (slippage) of the bark from the sapwood to apply a small amount of a formulation. This method directly exposes the lateral cambium to concentrated solvents. A consequence is phytotoxicity (e.g., hypersensitive reactions, necroses) to the tissues of the lateral meristem

(Smitley et al., 2010).

(Shigo, 1989; Shigo, 1991). Furthermore, native insects are opportunistic: oaks that have been defoliated by insects such as gypsy moth (*Lymantria dispar*) are predisposed to attack by the two-lined chestnut borer (Haack & Acciavatti, 1992). Minimizing defoliation in trees is a sound practice to protect tree health. Rather than resorting to "rescue" treatments to save trees at risk of wood and bark infesting insects, treat them when they still appear visibly healthy. Late insecticide treatments (e.g., >33% canopy dieback, epicormic sprouting, bark cracks, woodpecker flecks, exit holes) are contra-indicated. This approach minimizes negative outcomes, such as canopy dieback, delayed recovery or tree mortality.

As discussed earlier, the upward movement of an injected chemistry is dependent upon plant evapo-transpiration. Therefore, tree injections are most efficiently applied when trees are transpiring. Transpiration is dependent on a number of factors, such as soil moisture, soil and ambient temperature, the relative humidity and time of day. For optimal uptake, apply when the soil is moist, soil temperatures are above 7.2°C (45F), and during the 24 hour period when transpiration is greatest.

When using insecticides with short-residual activity (an example is acephate), make the application when the pest is active. Application of chemistries with greater residual activity are somewhat less dependent upon insect feeding activity (e.g., imidacloprid, emamectin), but are typically applied 30-d or more of expected pest activity. Fall applications may be applied in some instances. For example, imidacloprid applications in evergreen trees may be applied late in the season. Imidacloprid applications for HWA applications are made in the autumn to coordinate with resumption of sistens nymphal activity following summer aestivation. Imidacloprid activity is retained in hemlock (leaves of 3-6 age classes persist in trees) for extended residual activity (Doccola et al., in press). In addition, systemic insecticides with high adsorption coefficients (>5000) may be applied in the fall (at leaf senescence) for activity in the next growing season. TREE-äge (emamectin benzoate) is an example of a fall application used to protect ash trees against EAB (Smitley et al., 2010).

#### **12. Tree defense responses**

When trees are wounded, whether by an insect boring into the tree or by a mechanical drill bit, tree defense mechanisms come into play. These defense reactions and responses were systematically described by Shigo and Marx (1977). Dujesiefken and Liese have elaborated on the (CODIT) model taking into account the role of air exposure and embolism formation in the process of walling the damage in trees (2008). Individual trees may vary considerably in the strength of their response to similar types of wounds depending on genetics or tree health (Shigo, 1999). A discussion of tree wound responses must consider basic tree anatomy, in particular the secondary vascular tissues. Of most interest is the lateral meristem (cambium). This secondary cambium is only a few cells thick and occurs between the sapwood (xylem) and inner bark (phloem). This tissue is embryonic in nature. Periclinal divisions form xylem cells inward and phloem cells outward. The cambium is not transport tissue. Sapwood consists of living (symplast) and non-living (apoplast) cells. The living cells within the sapwood are non-differentiated parenchyma. The parenchyma cells store starch, oils and ergastic substances (Esau, 1977). Parenchyma occurs both as radial and axial tissues. Radial parenchyma extends into the phloem. The conductive xylem is functional when it matures and dies. The side walls of the xylem are pitted. Parenchyma cells sometimes balloon into the lumen of the xylem through the sidewall pits to form a tylose, or a physical

(Shigo, 1989; Shigo, 1991). Furthermore, native insects are opportunistic: oaks that have been defoliated by insects such as gypsy moth (*Lymantria dispar*) are predisposed to attack by the two-lined chestnut borer (Haack & Acciavatti, 1992). Minimizing defoliation in trees is a sound practice to protect tree health. Rather than resorting to "rescue" treatments to save trees at risk of wood and bark infesting insects, treat them when they still appear visibly healthy. Late insecticide treatments (e.g., >33% canopy dieback, epicormic sprouting, bark cracks, woodpecker flecks, exit holes) are contra-indicated. This approach minimizes negative outcomes, such as canopy dieback, delayed recovery or tree

As discussed earlier, the upward movement of an injected chemistry is dependent upon plant evapo-transpiration. Therefore, tree injections are most efficiently applied when trees are transpiring. Transpiration is dependent on a number of factors, such as soil moisture, soil and ambient temperature, the relative humidity and time of day. For optimal uptake, apply when the soil is moist, soil temperatures are above 7.2°C (45F), and during the 24

When using insecticides with short-residual activity (an example is acephate), make the application when the pest is active. Application of chemistries with greater residual activity are somewhat less dependent upon insect feeding activity (e.g., imidacloprid, emamectin), but are typically applied 30-d or more of expected pest activity. Fall applications may be applied in some instances. For example, imidacloprid applications in evergreen trees may be applied late in the season. Imidacloprid applications for HWA applications are made in the autumn to coordinate with resumption of sistens nymphal activity following summer aestivation. Imidacloprid activity is retained in hemlock (leaves of 3-6 age classes persist in trees) for extended residual activity (Doccola et al., in press). In addition, systemic insecticides with high adsorption coefficients (>5000) may be applied in the fall (at leaf senescence) for activity in the next growing season. TREE-äge (emamectin benzoate) is an example of a fall application used to protect ash trees against EAB (Smitley et al., 2010).

When trees are wounded, whether by an insect boring into the tree or by a mechanical drill bit, tree defense mechanisms come into play. These defense reactions and responses were systematically described by Shigo and Marx (1977). Dujesiefken and Liese have elaborated on the (CODIT) model taking into account the role of air exposure and embolism formation in the process of walling the damage in trees (2008). Individual trees may vary considerably in the strength of their response to similar types of wounds depending on genetics or tree health (Shigo, 1999). A discussion of tree wound responses must consider basic tree anatomy, in particular the secondary vascular tissues. Of most interest is the lateral meristem (cambium). This secondary cambium is only a few cells thick and occurs between the sapwood (xylem) and inner bark (phloem). This tissue is embryonic in nature. Periclinal divisions form xylem cells inward and phloem cells outward. The cambium is not transport tissue. Sapwood consists of living (symplast) and non-living (apoplast) cells. The living cells within the sapwood are non-differentiated parenchyma. The parenchyma cells store starch, oils and ergastic substances (Esau, 1977). Parenchyma occurs both as radial and axial tissues. Radial parenchyma extends into the phloem. The conductive xylem is functional when it matures and dies. The side walls of the xylem are pitted. Parenchyma cells sometimes balloon into the lumen of the xylem through the sidewall pits to form a tylose, or a physical

mortality.

hour period when transpiration is greatest.

**12. Tree defense responses** 

barrier. Tyloses may be formed in older wood naturally (e.g., white oak, *Quercus alba*, forms tyloses in second year wood), or are a consequence of trauma (e.g., red oak, *Q. rubra*, forms tyloses in response to wounding) (Shigo, 1999). When a tree is physically injured, both biochemical and structural changes occur. The biochemical reactions (changes of stored carbohydrates to phenolic and terpene defense chemicals) are observed in tree sections in three dimensions. These were named reaction zones (or boundary walls) 1 – 3. Reaction zone 1 occurs in the axial direction (i.e., with the stem axis) and is the least limiting boundary. Reaction zone 2 occurs in the radial direction (i.e., with the tree radius, inward toward the pith), and reaction zone 3 occurs in the tangential direction (i.e., with the tree's circumference), and is the strongest limiting boundary of the three reaction zones. The fourth wall, referred to as the barrier zone occurs after injury, and is the strongest limiting boundary. Meristematic cells (cambium) divide to form callus tissue, which later differentiates into new woundwood (new xylem, cambium and phloem). Native insect attacks to healthy trees are fended off by the biochemistry and by the subsequent physical responses. Emerald ash borer attacks to Asian species of ash (*Fraxinus chinensis*, *F. manchurica*) do not result in tree mortality: plant defense responses effectively isolate the larva in early stages of attack and limit its progression. In *F. pennsylvanica* (a native), the larvae are compartmentalized via physical boundaries (wall 4), but the biochemistry (phenols, terpene chemistries) does not effectively stop the insect's development. Injection of an insecticidal chemistry to compensate for insufficient tree response is the basis of successful tree protection. EAB research has demonstrated that this strategy is very effective (Smitley et al., 2010).

Tree wound responses are dependent upon a number of intrinsic and extrinsic variables such as tree species, tree health, method of treatment and chemistry applied. Tree wound response is under genetic control (Santamour, 1979). For example, birch (*Betula* spp.) poplar (*Populus* spp.) and willow (*Salix* spp.) are considered weak compartmentalizers, whereas oak (*Quercus* spp.), sycamore (*Platanus* spp.) and linden (*Tilia* spp.) are considered strong compartmentalizers (Dujesiefken and Liese, 2008). Santamour (1986) described fourteen cultivars of maple (*Acer*), ash (*Fraxinus*), oak (*Quercus*) and linden (*Tilia*) that were strong wall 2 compartmentalizers. As a group, trees have evolved to resist assaults and are successful, long-lived perennial plants. Tree health is another variable with numerous contributing factors. These include the age of the tree, soil conditions (texture, structure, moisture, pH, minerals and drainage), and exposure (sun, shade). Trees require light, water and minerals for essential life functions (including defense). Photosynthesis is the basis of carbohydrate synthesis. Woundwood responses utilize energy (carbohydrate, lipid) stores. When injections are made to trees in relatively good health (preventative-early therapeutic treatments) tree woundwood development readily proceeds to close wounds. However, the prognosis for recovery is comparatively lower, when making late therapeutic (rescue) applications, because energy stores are reduced. Optimal wound responses are observed when applications are made early, relative to infestation (Doccola et al., 2011). To further manage wounds in trees, make the fewest number of injection sites to apply the dose, and whenever possible, avoid drilling in the valleys between roots (Shigo and Campana, 1977).

The Wedgle Direct-Inject (ArborSystems, LLC, Omaha, NE) is a method of tree injection that does not require drilling into the sapwood. The system relies on forcing the de-lamination (slippage) of the bark from the sapwood to apply a small amount of a formulation. This method directly exposes the lateral cambium to concentrated solvents. A consequence is phytotoxicity (e.g., hypersensitive reactions, necroses) to the tissues of the lateral meristem

Tree Injection as an Alternative Method of Insecticide Application 71

Today, tree injection is an alternative method of chemical application with definite advantages: (1) efficient use of chemicals, (2) reduced potential environmental exposure, and (3) useful when soil and foliar applications are either ineffective or difficult to apply (Stipes, 1988; Sanchez-Zamora and Fernandez-Escobar, 2004). Tree injection is used when trees are at risk from attack from destructive or persistent pests. It may be put to good use in tall trees. They are administered in trees growing in environmentally sensitive locations (e.g., near water, in sandy soils). Tree injection does create wounds, however the benefit of the introduced chemistry to protect trees often outweigh the drilling wound. The new paradigm weighs the potential of off target consequences of application to the consequences of the drilled wound made by tree injection. Unintended off target exposures include toxicity to earthworms, fish, aquatic arthropods, pollinators and applicator. Insecticides are by design, toxic, albeit useful, substances. Tree injection is a method to deliver specific toxicants to the injurious pest and to minimize non-intended exposures. In this chapter, three specific insecticides used in tree injection were considered, each with unique attributes for specific applications in trees. Tree injection is an alternative methodology to apply

The authors thank David Cox, Ph.D., Syngenta Crop Protection, LLC for his review, edits, and comments of this chapter. The authors also thank Ms. Monica Davis for her review and

Amman, G.D., M.D. McGregor, and R. E. Dolph, Jr. Updated 2002. Mountain Pine Beetle.

Anderson, C. 1991. Photodegradation of NTN 33893 in water. Unpublished report study

ecological risk assessment – final report. SERA TR 05-43-24-03a. 283 pp. ANSI A300 Part 3. 2006. Supplemental Support Systems. American National Standards

ANSI A300 Part 4. 2008. Lightning Protection Systems. American National Standards

Anulewicz, A.C., D.G.McCullough, D.L. Cappaert and T.M. Poland. 2008. Host range of the

 http:www.ca.uky.edu/agc/pubs/for/for38/for38.htm (website accessed 4/15/11). Buchholz, A. and R. Nauen. 2002. Translocation and translaminar bioavailability of two

Institute (ANSI) A300 Standards for Tree Care Operations.

Institute (ANSI) A300 Standards for Tree Care Operations.

Barnes, T.G. 1989. Controlling woodpecker damage. FOR-38.

Management Science, 58(1): 10-16.

Forest Insect & Disease Leaflet 2. US Department of Agriculture Forest Service.

prepared by Nitokuno, ESR, Yuki Institute. 128 pp. In SERA (Syracuse Environmental Research Associates, Inc.). 2005. Imidacloprid – human health and

Emerald ash borer (*Agrilus planipennis* Fairmaire) (Coleoptera: Buprestidae) in North America: results of multiple-choice field experiments. Environ. Entomol.

neonicotinoid insecticides after foliar application to cabbage and cotton. Pest

**14. Tree injection as an alternative** 

systemic insecticides for tree protection.

(website accessed 4/14/2011).

**15. Acknowledgements** 

37(1): 230-241

edits.

**16. References** 

(the initials for woundwood development). The small doses and exposures to the lateral cambium by this method offers no clear advantage over drilling into trees for injection. Protection of the lateral cambium is of greater consequence to tree wound response compared to drilling into the sapwood. Further, wound closure rates of trees are positively correlated with trunk growth, and greater callus is produced around larger wounds than around smaller diameter wounds (Neely, 1988). Arborjet, Inc. employs a (7 or 9 mm) diameter drill hole to efficiently deliver higher volumes of insecticides into trees. The larger diameter hole is strongly limited by boundary wall 3 (this strong boundary reduces the likelihood of girdling and is an advantage to tree survival). With this system, a plastic Arborplug is inserted into the drilled hole, which creates the injection interface. The Arborplug from a tree wound defense perspective, reduces exposure of the lateral cambium to the solvent carriers in the injection formulation and minimizes wood exposure to air. Placing backflow preventers into the bark do not function in the same manner. Further, when the Arborplug is set correctly (at the sapwood-bark plane), it provides a flat surface for callus and woundwood development and wound closure. This encapsulation is the survival strategy of trees following injury (Dujesiefken and Liese, 2008).

#### **13. Multiple-year activity**

It is possible to make applications that are effective against a persistent and destructive tree pest and not require an annual treatment. The residual activity of tree injected imidacloprid may be due to protection against photolysis and microbial degradation. Foliar half-life of imidacloprid is ~9.8-d (Linn, 1992d, unpublished). Plants metabolize imidacloprid via hydrolysis, but some of the metabolites have insecticidal activity. The predominant metabolites associated with toxicity in insects are olefinic-, dihydroxy- and hydroxyimidacloprid (Sangha & Machemer, 1992; Suchail et al., 2001). In studies of large (50 cm) diameter hemlock infested with HWA, both soil and tree injections with imidacloprid were made (Doccola et al., in press). Two methods of tree injections were employed, one using low volume micro-injection (QUIK-jet, Arborjet, Inc.) and the second using high volume micro-infusion (TREE I.V., Arborjet, Inc.). The soil applications were made using the Kioritz injector (Kioritz Corporation, 7-2, Suehirocho 1 –Chome, Ohme, Tokyo, 198 Japan). Tree injection administered 0.15 g imidacloprid per 2.5 cm dbh, micro-infusion applied 0.3 g per 2.5 cm dbh whereas soil injection applied 1.45 g per 2.5 cm dbh. In that study, data was collected on HWA infestation, tree growth and imidacloprid residues in the foliage over a three year period. Tree foliage responses were greater in the tree injection treatments. Imidacloprid residues taken annually from 70 to 1165-d were above the LC50 value of 0.30 µg/g for HWA (Cowles et al., 2006) for all the imidacloprid treatments. At 1165-d, foliage residues (of 1.35 μg/g) in the lowest dose injections continued to protect trees. This residual activity of imidacloprid was attributed to both the perennial nature (of 3-6 years) of the foliage, and to the slow, upward movement of imidacloprid. Green ash trees treated with emamectin benzoate tree injections were protected from EAB for up to four years (Smitley et al., 2010). A recently completed 3 year study using low dose injections of emamectin benzoate protected trees for three years (Deb McCullough, personal communication). These studies point to efficacy and duration of tree injection methods. The TREE-äge label is approved (by US EPA) for up to two years of control against listed arthropods, including EAB. Injection is a very efficient use of insecticidal chemistry to protect trees.

#### **14. Tree injection as an alternative**

70 Insecticides – Basic and Other Applications

(the initials for woundwood development). The small doses and exposures to the lateral cambium by this method offers no clear advantage over drilling into trees for injection. Protection of the lateral cambium is of greater consequence to tree wound response compared to drilling into the sapwood. Further, wound closure rates of trees are positively correlated with trunk growth, and greater callus is produced around larger wounds than around smaller diameter wounds (Neely, 1988). Arborjet, Inc. employs a (7 or 9 mm) diameter drill hole to efficiently deliver higher volumes of insecticides into trees. The larger diameter hole is strongly limited by boundary wall 3 (this strong boundary reduces the likelihood of girdling and is an advantage to tree survival). With this system, a plastic Arborplug is inserted into the drilled hole, which creates the injection interface. The Arborplug from a tree wound defense perspective, reduces exposure of the lateral cambium to the solvent carriers in the injection formulation and minimizes wood exposure to air. Placing backflow preventers into the bark do not function in the same manner. Further, when the Arborplug is set correctly (at the sapwood-bark plane), it provides a flat surface for callus and woundwood development and wound closure. This encapsulation is the

It is possible to make applications that are effective against a persistent and destructive tree pest and not require an annual treatment. The residual activity of tree injected imidacloprid may be due to protection against photolysis and microbial degradation. Foliar half-life of imidacloprid is ~9.8-d (Linn, 1992d, unpublished). Plants metabolize imidacloprid via hydrolysis, but some of the metabolites have insecticidal activity. The predominant metabolites associated with toxicity in insects are olefinic-, dihydroxy- and hydroxyimidacloprid (Sangha & Machemer, 1992; Suchail et al., 2001). In studies of large (50 cm) diameter hemlock infested with HWA, both soil and tree injections with imidacloprid were made (Doccola et al., in press). Two methods of tree injections were employed, one using low volume micro-injection (QUIK-jet, Arborjet, Inc.) and the second using high volume micro-infusion (TREE I.V., Arborjet, Inc.). The soil applications were made using the Kioritz injector (Kioritz Corporation, 7-2, Suehirocho 1 –Chome, Ohme, Tokyo, 198 Japan). Tree injection administered 0.15 g imidacloprid per 2.5 cm dbh, micro-infusion applied 0.3 g per 2.5 cm dbh whereas soil injection applied 1.45 g per 2.5 cm dbh. In that study, data was collected on HWA infestation, tree growth and imidacloprid residues in the foliage over a three year period. Tree foliage responses were greater in the tree injection treatments. Imidacloprid residues taken annually from 70 to 1165-d were above the LC50 value of 0.30 µg/g for HWA (Cowles et al., 2006) for all the imidacloprid treatments. At 1165-d, foliage residues (of 1.35 μg/g) in the lowest dose injections continued to protect trees. This residual activity of imidacloprid was attributed to both the perennial nature (of 3-6 years) of the foliage, and to the slow, upward movement of imidacloprid. Green ash trees treated with emamectin benzoate tree injections were protected from EAB for up to four years (Smitley et al., 2010). A recently completed 3 year study using low dose injections of emamectin benzoate protected trees for three years (Deb McCullough, personal communication). These studies point to efficacy and duration of tree injection methods. The TREE-äge label is approved (by US EPA) for up to two years of control against listed arthropods, including

survival strategy of trees following injury (Dujesiefken and Liese, 2008).

EAB. Injection is a very efficient use of insecticidal chemistry to protect trees.

**13. Multiple-year activity** 

Today, tree injection is an alternative method of chemical application with definite advantages: (1) efficient use of chemicals, (2) reduced potential environmental exposure, and (3) useful when soil and foliar applications are either ineffective or difficult to apply (Stipes, 1988; Sanchez-Zamora and Fernandez-Escobar, 2004). Tree injection is used when trees are at risk from attack from destructive or persistent pests. It may be put to good use in tall trees. They are administered in trees growing in environmentally sensitive locations (e.g., near water, in sandy soils). Tree injection does create wounds, however the benefit of the introduced chemistry to protect trees often outweigh the drilling wound. The new paradigm weighs the potential of off target consequences of application to the consequences of the drilled wound made by tree injection. Unintended off target exposures include toxicity to earthworms, fish, aquatic arthropods, pollinators and applicator. Insecticides are by design, toxic, albeit useful, substances. Tree injection is a method to deliver specific toxicants to the injurious pest and to minimize non-intended exposures. In this chapter, three specific insecticides used in tree injection were considered, each with unique attributes for specific applications in trees. Tree injection is an alternative methodology to apply systemic insecticides for tree protection.

#### **15. Acknowledgements**

The authors thank David Cox, Ph.D., Syngenta Crop Protection, LLC for his review, edits, and comments of this chapter. The authors also thank Ms. Monica Davis for her review and edits.

#### **16. References**


Tree Injection as an Alternative Method of Insecticide Application 73

Chukwudebe AC; Feely WF; Burnett TJ; Crouch LS; Wislocki PG. 1996b. Uptake of

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Tree Injection as an Alternative Method of Insecticide Application 75

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**5** 

*PlantVax Inc.* 

*USA* 

**Development of a Prophylactic** 

**Homologous Macaque Model** 

Yvonne Rosenberg, Xiaoming Jiang, Lingjun Mao, Segundo Hernandez Abanto, Keunmyoung Lee

**Against Insecticide Toxicity Using a** 

**Butyrylcholinesterase Bioscavenger to Protect** 

Organophosphorus (OP) and carbamate pesticides are extensively used to control agricultural, household and structural pests. Each year approximately 5.6 billion pounds of pesticides are used worldwide potentially exposing ~1.8 billion people who use pesticides to protect the food and commercial products that they produce (Alavanja, 2009). Although unintentional occupational poisonings represent only a small number, estimated to be ~10% (Litchfield, 2005) or 25 million agricultural workers globally (Jeyaratnam, 1990), large scale exposure of both civilian and military personnel has become an ever increasing threat, as a result of deliberate insecticide contamination of the environment and critical water supplies by terrorists. In this context, pesticide use is one of only two exposures consistently identified by Gulf War epidemiologic studies to be significantly associated with the multisymptom illness profiles described as Gulf War illness (Cao et al., 2011). Pesticide use has also been associated with neurocognitive deficits and neuroendocrine alterations in Gulf

While OP nerve agents and WHO Class I and Class II OP pesticides constitute a diverse group of chemical structures, all potentially exhibit a common mechanism of toxicity, that is, active site phosphorylation of acetylcholine (AChE) resulting in AChE inhibition and accumulation of acetylcholine, overstimulation of cholinergic receptors, and consequent clinical signs of cholinergic toxicity such as seizures, brain damage and cognitive and behavioural defects (Millard et al., 1999; Rosenberry et al., 1999; Colosio et al., 2009). The relationship between AChE inhibition and symptoms showed that prevalence ratios were significantly >1 for respiratory, eye and central nervous system symptoms for workers with >30% inhibition (Ohayo-Mitoko et al., 2000). More recent studies indicate that insecticide exposure to DFP (diisopropyl fluorophosphate) causes a prolonged increased in hippocampal neuronal Ca++ plateau which may underlie morbidity and mortality (Deshpande et al., 2010). These findings are consistent with those indicating persistent changes in locus coeruleus noradrenergic neuronal activity and lasting changes in this brain area after removal of the insecticide chlorpyrifos oxon; reminiscent of the lasting cognitive

War veterans in clinical studies conducted following the end of the war.

**1. Introduction** 


### **Development of a Prophylactic Butyrylcholinesterase Bioscavenger to Protect Against Insecticide Toxicity Using a Homologous Macaque Model**

Yvonne Rosenberg, Xiaoming Jiang, Lingjun Mao, Segundo Hernandez Abanto, Keunmyoung Lee *PlantVax Inc. USA* 

#### **1. Introduction**

78 Insecticides – Basic and Other Applications

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benzoate at efficacious concentrations in pine tissues after injection of a liquid

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in three tree species when trunk- and soil-injected. Journal of Arboriculture. 24(1):

01-99GEN: Asian Longhorned Beetle (*Anoplophora glabripennis*): A New

Organophosphorus (OP) and carbamate pesticides are extensively used to control agricultural, household and structural pests. Each year approximately 5.6 billion pounds of pesticides are used worldwide potentially exposing ~1.8 billion people who use pesticides to protect the food and commercial products that they produce (Alavanja, 2009). Although unintentional occupational poisonings represent only a small number, estimated to be ~10% (Litchfield, 2005) or 25 million agricultural workers globally (Jeyaratnam, 1990), large scale exposure of both civilian and military personnel has become an ever increasing threat, as a result of deliberate insecticide contamination of the environment and critical water supplies by terrorists. In this context, pesticide use is one of only two exposures consistently identified by Gulf War epidemiologic studies to be significantly associated with the multisymptom illness profiles described as Gulf War illness (Cao et al., 2011). Pesticide use has also been associated with neurocognitive deficits and neuroendocrine alterations in Gulf War veterans in clinical studies conducted following the end of the war.

While OP nerve agents and WHO Class I and Class II OP pesticides constitute a diverse group of chemical structures, all potentially exhibit a common mechanism of toxicity, that is, active site phosphorylation of acetylcholine (AChE) resulting in AChE inhibition and accumulation of acetylcholine, overstimulation of cholinergic receptors, and consequent clinical signs of cholinergic toxicity such as seizures, brain damage and cognitive and behavioural defects (Millard et al., 1999; Rosenberry et al., 1999; Colosio et al., 2009). The relationship between AChE inhibition and symptoms showed that prevalence ratios were significantly >1 for respiratory, eye and central nervous system symptoms for workers with >30% inhibition (Ohayo-Mitoko et al., 2000). More recent studies indicate that insecticide exposure to DFP (diisopropyl fluorophosphate) causes a prolonged increased in hippocampal neuronal Ca++ plateau which may underlie morbidity and mortality (Deshpande et al., 2010). These findings are consistent with those indicating persistent changes in locus coeruleus noradrenergic neuronal activity and lasting changes in this brain area after removal of the insecticide chlorpyrifos oxon; reminiscent of the lasting cognitive

Development of a Prophylactic Butyrylcholinesterase

proteins in weeks (Goodin et al., 2008).

25U/ml (28mg/L)

Agrobacterium-mediated infiltration

8U/ml (9mg/L)

expression systems.

Bioscavenger to Protect Against Insecticide Toxicity Using a Homologous Macaque Model 81

1997). In human serum, the association of lamellipodin proline rich peptides with the monomeric chains results in the formation of BChE tetramers (Li et al., 2008). Recombinant BChE produced in mammalian cells, in contrast, has only 10-20% tetrameric forms and therefore optimal tetramerization requires the addition of either poly(L-proline) to the culture medium or co-expression of the full length BChE monomers with the proline-rich

To date, rHuBChE and rMaBChE molecules have been produced in transgenic mammalian cells (Chilukuri et al., 2008; Rosenberg et al, 2010), goat milk (Huang et al., 2007) and in plants (Geyer et al., 2010; Jiang, unpub. data). Our approach has been to utilize two expression systems for the production of rMaBChE and rHuBChE. Initially, Chinese hamster ovary cells (CHO) were used because of their human-like glycosylation. More recently, a transient plant expression platform was adopted to increase the yield and reduce the time and cost of producing rBChE. Although CHO cells and plants are able to produce significant levels of tetrameric BChE molecules (Li et al., 2008; Geyer et al., 2010), in the present studies, co-transfection of the BChE and PRAD genes has been shown to increase both levels of tetramerization and yields in each expression system. While the CHO cell expression of recombinant proteins is very well established, recent innovations in transient plant expression systems e.g. Bayer's Magnifection system (Gleba et al., 2005) and the Cow Pea Mosaic Virus Hyper-translatable Protein Expression System (PBL Technology) (Sainsbury et al., 2008) have been shown to be some of the most rapid, cost effective and productive expression systems in existence; capable of producing grams of recombinant

CHO-derived (Stable Transfection)**\*** Plant-derived (Transient Transfection)**\***

*N. tobacum N. benthamiana*

60 U/gm (66.6 mg/kg)

140 U/gm (155.5 mg/kg)

400 U/gm (444 mg/kg)

Monomeric Tetrameric Monomeric Tetrameric Monomeric Tetrameric Tetrameric

+ BChE activity is determined spectrophotometrically (Grunwald at al., 1997), using butyrylthiocholine (BTC) (0.5 mM each) as substrate. One unit of enzyme activity is the amount required to hydrolyze 1 umol substrate/min. One mg MaBChE has 900 units of activity and one mg HuBChE has 700units. Table 1. Expression levels of different forms of rBChE using CHO-and plant-based

In addition to the tetrameric forms, a truncated monomeric form of rBChE (MW=~81KDa) that is incapable of oligomerization has also been produced by the insertion of a stop codon at G534 resulting in a monomeric form lacking 41 C-terminal residues (Blong et al., 1997). The smaller monomeric molecules may more rapidly gain access to the blood from muscle or lungs (depending on the route of delivery) with transiently higher bioavailablity in the plasma, which would be advantageous in emergency situations that require real time

45 U/ml (64.3 mg/L)

\*All tobacco plants and leaves from *Nicotiana tobacum* and *N. benthamiana* were transfected using

rMaBChE#+ rHuBChE rMaBChE#

16 U/ml (22.9mg/L)
