1

CHAPTER ONE

1.0 INTRODUCTION

1.1 Background of Study

Synthetic pyrethroids are analogues of the natural pyrethrin found in the Chrysanthemum

cinerariaefolium plant of the Asteraceae family. Synthetic pyrethroids can be divided into first

generation and second generation. First generation pyrethroids are mainly ester of chrysanthemic

acid derivatives and alcohols with furan ring with terminal side change moiety, these class of

pyrethroids are extremely sensitive to light and air oxidation. While second generation

pyrethroids with 3-phenoxybenzyl alcohol as one of the moiety is quite photo-stable and thus can

be used against agricultural pest. Further second generation pyrethroids can be divided into type

I and type II pyrethroids, with latter containing cyano group and thus increasing its insecticidal

properties. Type I pyrethroids are generally used as household pesticides, veterinary medicines

because of their low photo-stability while type II can be used even as agriculture pesticides (Xu

et al., 2019).

Lamda (λ)-cyhalothrin is a widely used synthetic pyrethroid insecticide and its persistence in

plant, soil and water exerts a detrimental effect on humans as well as the environment (Cycoń et

al., 2017). One major category of insecticides is synthetic pyrethroids (SPs). The natural

pyrethrin compounds showed an effective insecticidal activity but were instable in the

environment. Modifications in the molecular structure of pyrethrins led to the synthesis of SPs

which are more stable and efficient than native pyrethrins in direct sunlight and diverse

environmental conditions. These characteristics made them more convenient for use in

agriculture. The insecticidal potency of pyrethroids relies on the induction of a toxic effect in the
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cells of the central and peripheral nervous system of insects. Pyrethroids induce insect paralysis

then eventual death of insects by permitting a fux of sodium, calcium and chloride ions

(Madrigal et al., 2021).

Many types of SPs such as λ-cyhalothrin, fenpropathrin and cypermethrin have been classified as

‘‘moderately hazardous’’ (Class II) by the World Health Organization. Hence, it is crucial to

remove SPs residues away from the ecosystem (Zhao et al., 2021). Generally, SPs can be

degraded through both abiotic pathways, including photo-oxidation and chemical oxidation and

biotic pathways mainly by soil microflora. Microorganisms-induced degradation makes it easier

for SPs remediation because it is flexible, cost-effective, and better for the environment.

Microorganisms characterized by their ability to degrade one or two kinds of pesticides have

been isolated and identified (Cycon and Piotrowska-Seget 2016).

1.2 Statement of the Research Problem

Globally, approximately 3.5 million tons of pesticides are used annually (Sharma et al., 2019).

Although insecticides protect plants and increase the crop production yield, they cause dire

environmental pollution problems because of their persistence and other detrimental effects on

plants, animals, humans, and soil microorganisms (Alengebawy et al., 2021). It has been

estimated that approximately 0.1 percent of pesticides sprayed on farms reach their intended

targets; the remainder are spread to ecosystems, contaminating land, water, and air putting lives

in jeopardy. The persistence substances at non-lethal dosages impair the brain system,

hepatocytes, lymphocytes, hormone balance, reproduction, embryogenesis, and population

increase in fish (Madrigal et al., 2021).

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1.3 Justification of the Study

 Synthetic pyrethroids (SPs), including λ-cyhalothrin, have been employed extensively in

agriculture, domestic plants, and lush landscapes during the past few decades to replace

the usage of the more hazardous and environmentally persistent organochlorine and

organophosphorus pesticides (Zhang 2018).

 Pyrethroids account for almost 25% of the global pesticide market (Xu et al., 2019).

 Synthetic pyrethroids residues have frequently been discovered in soils, sediments,

natural waterways, and agricultural goods as a result of their excessive use. Inhalation

and skin contact are the two main ways that SPs are exposed to people. However, SPs

residues were also found in prepared solid food, proving that these chemicals can easily

get to people through food chains (Morgan et al., 2018).

1.4 Aim of the Study

The aim of this study was to determine the effect of different temperature on the biodegradtion of

lamda cyhalothrin.

Specific Objectives

i. Isolate and identify lamda cyhalothrin- degrading bacterial isolates from agricultural

soils.

ii. To determine the effect of different temperature on the biodegradtion of lamda

cyhalothrin.
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CHAPTER TWO

2.0 LITERATURE REVIEW

2.1 Pyrethoids

Pyrethroids are insecticides that have a high biological activity and are used all over the world to

control pest insects in agriculture, public and commercial buildings, animal facilities,

greenhouses, and veterinary facilities (Dringenberg, 2020). Pyrethroids are also the most

common active ingredients in commercially available insect sprays and are the domain pesticide

for malaria control. The insecticidal potency of pyrethroids is connected with the induction of a

toxic effect in the cells of the nervous system of insects (Pitzer et al., 2019). By permitting a flux

of sodium ions, pyrethroids alter the activity of the sodium channels that are responsible for the

signal transmissions of nerve impulses. When pyrethroids bind to target channel proteins, they
 5

disrupt the proper function of the nervous cells thus leading to paralysis and the eventual death of

insects (Herring and Nicoll, 2016). Pyrethroids differ from many other pesticides in that they

contain one to three chiral centers; their chirality may arise from the acid moiety, the alcohol

moiety or both. A pyrethroid compound, therefore, consists of two to eight isomers. The isomers

of a chiral compound often differ from each other in their biological properties. Isomer

selectivity has been widely observed for the isomers of a pyrethroid compound in insecticidal

activity (Hossain et al., 2016). Recently, studies have shown that the biodegradation of

pyrethroids also exhibits significant isomer selectivity. Based on their toxicological and physical

properties, pyrethroids are categorized into two separate classes—type I and type II (Sharma et

al., 2019).

Type I pyrethroids, which include allethrin, bifenthrin, d-phenothrin, permethrin, resmethrin, and

tetramethrin, do not have a cyano group. Conversely the insecticides that represent Type II such

as cyhalothrin, cypermethrin, cyfluthrin, deltamethrin, fenvalerate, fluvalinate, and lambda-

cyhalothrin have a cyano group in their structure (Kumar et al., 2015). Due to their complex

chemical structure, pyrethroids are composed of two, four or eight isomers and their commercial

products may contain a mixture of these various isomers. The production of individual

pyrethroids that have varying isomeric ratios may be the reason for the variations in the toxicity

of the same compound. In addition, pyrethroids represent highly hydrophobic compounds that

are characterized by their low water solubility, which ranges from insolubility to a value of 0.1

mg/L and high octanol-water partition coefficients (Richardson et al., 2015). Although,

pyrethroids are considered to be safer than other insecticides, the common and extensive use of

these compounds in a wide variety of fields has resulted in widespread contamination of the

environment that is of ecological concern (Amaraneni et al., 2017). The results of many studies
 6

have revealed that SPs may negatively affect non-target organisms such as fish and aquatic

insects, beetles, bees, parasitic wasps and microorganisms. It is thought that some pyrethroids

may be responsible for disruptions of the endocrine system, suppression of the immune system,

reproductive damage and increased chances of cancer in humans (Amaraneni et al., 2017). To

reduce the environmental and public health risks associated with pyrethroid use, it is necessary to

develop rapid and effective methods to remove or minimize the concentrations of insecticides in

the environment. Among the variety of methods that are used for the remediation of

contaminated environments, the biological approach, which is based on the catabolic activity of

pesticide-degrading bacteria, seems to be the most promising and effective strategy (Burns and

Pastoor, 2018).

2.2 Mode of Action of Pyrethoids

Pyrethroids mainly enter the body through ingestion, commonly via contaminated food or water,

but also through ingestion of soil or dust particles, especially in children. Absorption through the

skin when exposed to products during application or when touching treated surfaces is slower but

it is also possible because pyrethroids are fat soluble and cells, such as those of the skin, are

composed of a lipid bi-layer (Zarate et al., 2017).

Hence, using flea shampoo leads to limited absorption by the skin. Finally, breathing fine

droplets or airborne dust particles may also occur, especially when using pyrethroids in an

enclosed space. Once pyrethroids have entered the body, they are transformed through

degradation into products called metabolites prior to excretion in the urine. Metabolites common

to several pyrethroids include cis- or trans-DCCA (dichlorovinyl-dimethyl-cyclopropane

carboxylic acid), 20 different pyrethroids may transform into 3BPA (3-phenoxybenzoic acid),

while Cyfluthrin may also become 4F3PBA (also known as 4-fluoro-3-phenoxybenzoic acid)
 7

(Braun et al., 2016). Hence, identifying which pyrethroid exposure corresponds to which

metabolite detected in the body is difficult. Furthermore, metabolites may only be detectable in

the blood or urine for a few hours or a few days. This is why proving a direct link between

pyrethroid exposure and clinical symptoms of toxicity is so difficult (Braun et al., 2016).

2.3 Classification of Pyrethoids

2.3.1. Classification by Structure

There are two main classes, Type I and Type II. Type I pyrethroids lack a cyano moiety at the

alpha-position, whereas Type II pyrethroids contain this group (Li et al., 2019). Pyrethroids

share alcohol and acid moieties and a central ester bond. Most pyrethroids are stereoisomers

because the acid moiety contains two chiral carbons, and the alcohol group has one chiral

carbon. Hence, there can be up to eight stereoisomers for some pyrethroids and chirality affects.

In general, cis isomers are more toxic than trans-isomers. Lamda Cyalothrin lacks a trans isomer

and exists only in a cis configuration, suggesting greater toxicity per molecule (Li et al., 2019).

2.3.2. Classification by Symptoms

At high doses, Type I and II pyrethroids induce different symptoms or syndromes (Wall et al.,

2018). In insects pyrethroids induce uncoordinated movements, convulsions, paralysis, and

death. In mammals, Type I pyrethroids, such as permethrin, at high doses produce tremors or T

syndrome; consisting of fine tremors that become dose at it is increased, along with prostration,

and sensitivity to stimuli. High doses of Type II pyrethroids in mammals produce

choreoathetosis and salivation or CS syndrome that includes writhing, salivation, pawing,

burrowing, tremors, and clonic seizure. Pyrethroids with overlapping CS and T symptoms are

designated mixed or Type III pyrethroids (Vorhees and Williams, 2016).

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2.3.3. Classification by Receptor

2.3.3.1. Sodium Channels

A third classification is by receptor binding or target. The principal binding site of pyrethroids is

on the α-subunit of voltage-gated sodium channels (VGSC), which makes up the central pore of

the channel as well as contains regions for voltage sensing and ion selectivity (Field et al., 2017).

VGSCs are composed of α- (220–260 kDa) and one or more β subunits (30–40 kDa) (Zhang et

al., 2017). The α-subunit of the VGSC shares homology with other voltage dependent channels

(i.e., calcium and potassium channels), however, the β-subunits are homologous to neural cell

adhesion molecules. β-subunits are involved in the kinetics and voltage dependence of VGSC, as

well as modulating cell adhesion and migration (Zhang et al., 2017). Several studies address

VGSC structure and its interactions with pyrethroids, such as Lamda Cyalothrin (Zhang et al.,

2017).

2.3.3.2 Calcium Channels

Pyrethroids also affect calcium channels. Voltage gated calcium channels (VGCCs) belong to the

same protein superfamily as VGSCs. VGCC are also signal transducers and control calcium

influx (King et al., 2018). There are two types and they differ in voltage dependence: lowvoltage

VGCCs that are activated by small depolarizations but rapidly inactivate; and high-voltage

VGCCs that are activated by large depolarizations but are slowly inactivated (King et al., 2018).

VGSCs and VGCCs have similar structures, with analogous α subunits (α1 for the VGCCs),

however they differ in their regulatory subunits that control the voltage-gated pore, and four

repeat domains with six transmembrane segments (S1-S6) containing a loop between S5 and S6

(. VGCCs differ from VGSCs in regulatory subunits. The high-voltage VGCCs have 4–5

subunits consisting of one pore-forming α1 subunit, an extracellular α2 subunit, a transmembrane

 9

δ subunit, an intracellular β subunit, and sometimes a transmembrane γ subunit (King et al.,

2018). The low-voltage VGCCs have the α1 subunit without additional subunits.

2.3.3.3. Chloride Channels

Soderlund et al. (2012) suggested that the CS syndrome could be a product of pyrethroids acting

on voltage-gated chloride channels (VGCLCs) (Soderlund et al., 2012). However, only some

pyrethroids inhibit VGCLCs only at high doses of Type I pyrethroids and by a few Type II

pyrethroids, including Lamda Cyalothrin, but not by esfenvalerate or λ-cyhalothrin (Burr and

Ray, 2004). Therefore, even among Type II pyrethroids there are important differences in how

they affect VGCLCs. For Type II pyrethroids that do not affect VGCLCs, they should not

contribute to the CS syndrome, and it appears that Lamda Cyalothrin is an exception because it

elicits a CS syndrome and does affect VGCLCs. The effect of pyrethroids on VGCLCs was

reviewed in detail by Soderlund (2012). However, the role of developmental Lamda Cyalothrin

exposure on VGCLCs remains an open question.

2.4 Effects of Pyrethoids on Human Health

2.4.1 Effect on Brain

The EPA estimates human exposures of pyrethroids at 3.0 μg/kg/day in children and 0.6

μg/kg/day in adults (EPA, 2020). 10 μg/kg/day is the reference dose for Lamda Cyalothrin for

acute dietary exposure. These are lower than used in animal studies that find behavioral and

cognitive deficits, however, rats metabolize and clear Lamda Cyalothrin much faster than

humans, therefore, administered dose comparisons are not meaningful. Brain concentrations

would be ideal but cannot be obtained in people, therefore, direct dose comparisons are not

possible (Bennett et al., 2020).

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2.4.2 Alteration of Hormones

Recent research involving animal testing and epidemiological studies in humans shows potential

adverse effects on human fertility, such as alterations of the male reproductive system, decreased

sperm count, and mobility and DNA damage, which all led to lower fertility and pregnancy rates

(Bunbury-Blanchette and Walker, 2019). Pyrethroids have been shown to alter hormones

(endocrine disruptors), for example, by decreasing concentrations of testosterone (an important

male hormone) and interfering with luteinizing hormone (involved in the production of sperm

and ova) or altering thyroid function. In vitro studies on Cypermethrin and Fenvalerate show that

pyrethroids may alter female and male hormones (estrogenic and antiandrogenic activity)

(Buragohain et al., 2019).

2.4.3 Cancer

Long-term pesticide exposure may lead to DNA damage and oxidative stress46 and also disrupt

the endocrine system, which may lead to cancer. The World Health Organization recognizes that

tumours have been induced in rodents which were exposed to pyrethroids during their whole life,

however, in 2019 the WHO considered that were no clear indication of carcinogenicity relevant

for human health risk assessments (WHO, 2020). Animal evidence includes initiation (but not

promotion or completion) of carcinogenic activity in mice exposed to Deltamethrin on their skin,

initiation, promotion and complete carcinogenic activity in mice exposed to Permethrin on their

skin, preputial gland adenomas and carcinomas in rats exposed to D-Phenothrin in their diet,

lung adenomas in mice exposed to Permethrin, mammary adenocarcinomas in mice exposed to

Cyhalothrin, urinary bladder haemangiomas in male mice following Bifenthrin exposure, and

follicular cell adenomas in the thyroid of female rats exposed to Etofenprox (Widyawati et al.,

2019).
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However, several of these findings were dismissed on the basis of non-statistical significance, no

difference with the history of the control population, absence of joint genotoxicity (mutation of

genes) or non-toxicological significance as assessed by groups of experts (WHO, 2017). Some

pyrethroids are considered possible human carcinogens in the US, though the International

Agency for Research on Cancer (IARC) considers them not classifiable (Deltamethrin,

Fenvalerate, Permethrin) due to inconsistent evidence in animals or absence of evidence in

humans. In a recent internal report, however, the IARC reviewed its earlier statement on

Permethrin, and in the face of new carcinogenicity evidence, assigned a high priority for the

revision of the carcinogenicity of this pesticide within the window of 2015 to 2019 (WHO,

2020).

2.4.4 Enhanced Sensitivity of Children

Children may be more sensitive than adults to pyrethroids because they have a lower body

weight but breathe and eat proportionally more than adults, and they often play on the ground,

exhibiting hand-tomouth behavior (Tsimbiri et al., 2015). Their detoxification systems may not

be as mature and their rapid development may lead to windows of particular sensitivity, for

instance, during brain development. Normally, children are primarily exposed to pyrethroids in

food; however when their homes have been recently treated, dermal absorption may be more

important. Pyrethroids may be found in 5% of food regularly consumed by children, but not all

food is systematically tested, nor is it tested on an annual basis. Unstructured eating habits of

children may enhance their exposure, for example when food is dropped on treated surfaces and

then eaten (Sharma et al., 2019).

A Montreal-based study revealed that children, compared to adults, excreted more of a

pyrethroid metabolite most common in domestic or commercial extermination applications, even

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though home treatments with pyrethroids were seldom reported (Möhring et al., 2020).

Furthermore, children are more prone to head lice infestations, hence more susceptible to being

treated with pyrethroid shampoo. Online forums contain numerous questions and testimonies of

parents using dog shampoo to treat head lice infestations in children at a lower cost. Such uses

are not evaluated in the registration of the products, are beyond the instruction label guidelines

and should be prevented; however, since inappropriate use is in response to financial and health

issues, simple recommendations to read and follow the label may not suffice (Mora et al., 2018).

2.4.5 Effect of Pyrethoids on Soil Health

Soil Health Before assigning specific problems that can result from excess use of chemical

fertilizers, it is better to define soil health and understand the different components of a healthy

soil (Al-Kaisi, 2017). Soil health is defined as the capacity of a soil to function, within natural or

managed ecosystem boundaries, to sustain biological productivity, maintain environmental

quality and promote plant, animal and human health. Soil health is established through

sustainable interactions of soil’s physical, chemical and biological soil properties and soil air,

which together determines soil fertility in agro-ecosystems. Thus, soil fertility is the capacity of

any soil to adequately provide nutrients required by plants for growth and development. This

implies that a healthy soil is a fertile soil (Lekeanju et al., 2016).

2.5 Application of Pyrethoids

2.5.1 Domestic Use

In homes, pyrethroids may be used in vaporizers or as powders to eradicate several crawling and

flying insects like cockroaches, ants and wasps. Indoor uses in confined spaces may lead to

exposure by breathing during application (inhalation) (Mohammad et al., 2018). After

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application, children who play on the floor and exhibit hand-to-mouth behaviour may further be

exposed via their skin (dermal) and digestive tract (ingestion). In a treated home, floors, sofas

and carpet fabrics are important reservoirs for pyrethroids, and may lead to exposure of

occupants. In addition, some commercial formulations of pyrethroids (with each product

containing a different assemblage of pyrethroids active ingredients and co-formulants to target

different pests) may have a repulsive effect on cockroaches (they augment locomotor activity)

(Medina and Triacchini, 2018).

This may render eradication via less toxic alternatives more difficult because the exposed insects

then migrate from their usual kitchens and bathrooms habitats to unusual zones such as

bedrooms. Permethrin was detected in all (100%) urban public housing units surveyed in Boston

(Mass., USA), while Cypermethrin and Cyfluthrin had a prevalence of more than 90% and 71%,

respectively (World Bank, 2019).

Finally, flea treatment of pets often relies on pyrethroids in shampoos and collars. These have

been linked to poisoning in children (McDonald et al., 2016). Misuse of pyrethroids in domestic

settings (e.g., using Cyfluthrin wettable powder without mixing with water) has been

documented and leads to greater than necessary exposure. Exposure to pyrethroids in homes and

other treated areas varies with the age of residents, income, but can also be affected by how

pesticides are applied, such as whether or not recommended guidelines are followed (McDonald

et al., 2016).

2.5.2 Agricultural Uses

While pyrethroids are commonly used to control several insects affecting agricultural production,

their main agricultural uses are for animal rearing. pyrethroids may be applied to several
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vegetables (sweet corn, potatoes, carrots, lettuces, onions, green onions and members of the

cabbage family) and fruits (apples, strawberries and other berries) (Gregorio et al., 2020).

Until resistance is found in certain insect pests, pyrethroids are commonly the first choice in

conventional growing systems. Agricultural uses are linked to exposure of workers, especially

when good agricultural practices are not followed. Agricultural use will leave traces of residues

on food, and this leads to exposure via ingestion (Gregorio et al., 2020). As long as pesticides are

used according to the recommended practices (within approval limitations and according to good

agricultural practices), this leads to human pesticide exposure which has been deemed safe by

Health Canada (Gregorio et al., 2020).

2.6 Lamda Cyalothrin

Lambda-cyhalothrin is a pyrethroid insecticide. Pyrethroids are synthetic chemical analogues of

pyrethrins, which are naturally occurring insecticidal compounds produced in the flowers of

chrysanthemums (Chrysanthemum cinerariaefolium). Insecticidal products containing

pyrethroids have been widely used to control insect pests in agriculture, public health, and homes

and gardens (Environment Agency, 2019). In agriculture, target crops include cotton, cereals,

hops, ornamentals, potatoes, and vegetables, with applications made to control aphid,

coleopterous, and lepidopterous pests. Pyrethroids are important tools used in public health

management where applications are made to control cockroaches, mosquitoes, ticks, and flies,

which may act as disease vectors. Residential use of pyrethroid products has increased because

of the suspension of organophosphate products containing chlorpyrifos or diazinon (Environment

Agency, 2019).
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2.7 Mode of Action of Lamda Cyalothrin

Pyrethroids are axonic poisons that affect the nerve fiber by binding to a protein that regulates

the voltage-gated sodium channel. Normally, this gate opens to cause stimulation of the nerve

and closes to terminate the nerve signal. The channels are pathways through which ions are

permitted to enter the axon and cause excitation. When the channels are left open, nerve cells

produce repetitive discharges and eventually cause paralysis. Pyrethroids bind to this gate and

prevent it from closing normally, which results in continuous nerve stimulation and tremors in

poisoned insects. Poisoned organisms lose control of their nervous system and are unable to

produce coordinated movement (EU, 2019).

There are two groups of pyrethroids with distinctive poisoning symptoms, denoted as Type I and

Type II. Chemically, Type II pyrethroids are distinguished from Type I pyrethroids by the

presence of an α-cyano group in their structure. In comparison to Type I pyrethroids (e.g.,

permethrin), which exert their neurotoxicity primarily through interference with sodium channel

function in the central nervous system, Type II pyrethroids (e.g., lambda-cyhalothrin) can also

affect chloride and calcium channels that are important for proper nerve function (Fera Science,

2018). Because of the lipophilic nature of pyrethroids, biological membranes and tissues readily

absorb them. Specifically, lambda-cyhalothrin penetrates the insect cuticle, disrupting nerve

conduction within minutes; this leads to cessation of feeding, loss of muscular control, paralysis,

and eventual death. Additional protection of the crop is provided by the insecticide’s strong

repellent effect toward insects (Fera Science, 2019).

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2.8 Ecotoxicity of Lambda-Cyhalothrin

2.8.1 Fish and Shellfish

Lambda-cyhalothrin is highly toxic to a number of fish and shellfish. The reported LC 50 (96 hr) is

210 ng/L for bluegill sunfish, 240 ng/L for rainbow trout, 360 ng/L for Daphnia magna, 4.9 ng/L

for mysid shrimp, and 0.8 ng/L for sheepshead minnow. An EC 50, the concentration at which the

effect occurs in 50% of the test population, for eastern oyster is 0.59 ng/L. A bioconcentration

factor (BCF) of 2240 has been reported in fish (species unspecified), but concentration was

confined to nonedible tissues and rapid depuration was observed (EU, 2019).

2.8.2 Macrophytes

The structure of an ecosystem determines the final effect of pesticide exposure to macrophytes

(Wendt-Rasch et al. 2004). Using a pesticide mixture containing lambdacyhalothrin applied to 10

mesotrophic aquatic ecosystems dominated by submerged macrophytes (Elodea) and 10

simulated eutrophic ecosystems with a high Lemna surface coverage, significant increases in the

biomass and alterations of species composition of the periphytic algae were observed in the

Elodea-dominated microcosms, but no effect on Myriophyllum spicatum growth was observed.

The opposite was found in the Lemna-dominated microcosms, in which decreased growth of M.

spicatum was observed but no alterations were observed in the periphytic community (FSC,

2019).

2.8.3 Effects on Soil Fauna

Species sensitivity distributions (SSD) and 5% hazardous concentrations (HC5) are distribution-

based approaches for assessing environmental risks of pollutants, e.g., lambda-cyhalothrin risks

to soil invertebrate communities. From a systematic review of literature, a total of 1950

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laboratory toxicity test results were obtained, representing 250 pesticides including lambda-

cyhalothrin and 67 invertebrate taxa (UKWIR, 2018). The majority (96%) of pesticides have

toxicity data on fewer than 5 species. Based on a minimum of 5 species, the best available

endpoint data (acute mortality median lethal concentration) enabled SSD and HC5 to be

calculated for 11 pesticides including lambda-cyhalothrin. Arthropods and oligochaetes exhibit

pronounced differences in their sensitivity to most of these pesticides. The standard test

earthworm species, Eisenia fetida sensu lato, is least sensitive to insecticides based on acute

mortality, whereas the standard Collembola test species, Folsomia candida, is among the most

sensitive species for a broad range of toxic modes of action (biocide, fungicide, herbicide, and

insecticide) (Frampton et al. 2006). To assess the effects of lambda-cyhalothrin on soil

invertebrates under tropical conditions, ecotoxicological semifield studies were conducted using

intact soil-core terrestrial model ecosystems (TMEs) (Forster et al. 2006). Earthworms, isopods,

and diplopods were added to intact soil cores and the mortality of soil invertebrates was

determined. The results indicated that lambda-cyhalothrin was toxic to isopods and millipedes,

whereas no effect on arthropods was detected in the field (UKWIR, 2019).

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CHAPTER THREE

3.0 MATERIALS AND METHODS

3.1 Sample Collection

The soil samples that was used in the present study was collected from the soil rhizosphere

(10.0 cm depth) of two different sites located in 3 different farmland Malete, Kwara State,

Nigeria with pesticide application history. Samples was collected aseptically in sterilized

polyethylene bags, labelled (date, time, and quantity) and transported to the laboratory while

stored at 4 °C until inoculation. The brand of Lambda cyhalothrin which is a broad spectrum

insecticide was karate and was purchased from agrochemical vendors Malete, Kwara State

(Arora and Verma 2017).

3.2 Sterilization of Materials

All glass wares were washed, allowed to air-dry and wrapped in aluminium foil and sterilized in

the autoclave for 121 0C for 15mins.

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3.3 Study Area

The Study area was different farms in Elemere Village, Moro Local Government, Kwara State,

Nigeria.

3.4 Isolation of Pesticides-Degrading Bacteria by Enrichment Methods

The enrichment technique was carried out in accordance with the procedures outlined by

Almuhayawi et al. (2021) by suspending five grams of soil samples in sterilized 250 ml

Erlenmeyer flasks containing 100 mL mineral salt medium (MSM) (Na2HPO4 2.0 g/L, KH2PO4

0.75 g/L, MgSO4.7H2O 0.5 g/L, NH4C The cultures was incubated on a rotating shaker at 30 °C

(150 rpm). Aliquots (10%, v/v) was inoculated into a new MSM supplemented with Lamda-

cyhalothrin (25 mg/L) and incubated for a further seven days. Samples was applied to MM agar

plates containing Lamda-cyhalothrin (25 mg/L) after numerous serial transfers. Colonies with

various morphologies was inoculated onto sterile MSM agar plates with increasing amounts of -

cyhalothrin (25, 50, 75, 100, 125 mg/L) as the only source of carbon and nitrogen. Bacterial

isolates that growth at highest concentrations was chosen, purified, and kept on agar slants at 4

°C.

3.5 Characterization and Identification of the Pesticide-Degrading Bacterial Isolates

The isolates were characterized and identified morphologically, and biochemically. Catalase,

indole, methyl red, voges proskaeur, citrate utilization, urease, motility, hydrogen sulphide

production, starch hydrolysis and carbohydrate tests were carried out as described by Arora and

Bae (2014).
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3.5.1 Identification of Bacteria

For bacterial identification, 24hour-old culture of bacteria was observed under microscope by

gram stain method and further various biochemical tests were performed for the identification of

bacteria such as catalase test, oxidase test, Urease test, Indole test, Methyl Red, Coagulase Test,

Voges Prausker Test as described in “Bergey’s Manual of Systematic Bacteriology”.

3.5.1.1 Gram Staining

A thin smear from each bacterial culture was prepared on clean grease-free slides by dissolving a

minute portion of the colony obtained from a 24hours old culture of each bacterial in one drop of

distilled water on the slide. This was subsequently air dried and heat fixed by passing over gentle

flame. Each heat-fixed smear was stained by addition of 2 drops of crystal violet solution for 60

sec and rinsed with water. The smear were again flooded with Gramʼs iodine for 30 sec and

rinsed with water, decolorized with 70% alcohol for 15 sec and were rinsed with distilled water.

They were then counter stained with 2 drops of Safranin for 60 sec and finally rinsed with water,

then allowed to air dry. The smears were mounted on a Microscope and observed under oil

immersion objective lens. Gram negative cells appeared pink or red, while Gram positive

organisms appeared purple.

3.5.1.2. Motility Test

This test was done to demonstrate whether isolate was motile or not, culture used for this test

was18- 24hours old broth culture, then 10ml of prepared nutrient agar was poured into a

universal bottles and allowed to solidify. A sterile syringe was then dipped inside the broth

culture of the organisms grown and then used to stab the solidified agar in the middle; the

universal bottle was then incubated at 37 0C for 24h and examined for motile action. If the
 21

organism diffused around the agar then it is motile but if there was no diffusion, then the

organisms was said to be non-motile.

3.5.1.3 Catalase Test

The test was carried out to detect the presence of Catalase which converts hydrogen peroxide to

water and oxygen. A wire loop was used to pick up the organism to be tested from a culture plate

and placed in a drop of hydrogen peroxide on a clean glass slide. Formation of gas bubbles

indicates a positive reaction, while absence of gas bubble indicates negative reaction.

3.5.1.4. Coagulase Test

A sterile wire loop was used to pick a colony from an overnight culture and mixed with a normal

saline placed at the end of a clean glass slide. Drop of blood plasma was added and incubated at

370C for 1–6 hours. Clumping within 1 to 6 hours (1–6hrs) indicates a positive reaction.

3.5.1.5. Oxidase Test

Culture of the bacteria was made on an agar medium and allowed to grow. After growth a freshly

prepared 1% tetra-methyl-p-phenylene-diaminedihydrochloride was poured on the plate so as to

cover the surface, this is then decanted. Oxidase positive developed purple color rapidly while

Oxidase negative do not develop the purple Color.

3.5.1.6 Citrate Test

This test detects the ability of an organism to utilize citrate as a sole source of carbon and energy.

About 2.4 g of citrate agar was dissolve in 100mL of distilled water. About nine milliliter (9mL)

of citrate medium was dispensed into each tube and covered, then sterilized and allowed to cool

in a slanted position. The tubes were inoculated by streaking the organisms once across the

surface. A change from green to blue indicates utilization of the citrate.

 22

3.5.1.7. Sugar Fermentation Test

Sugar fermentation test was carried out to determine the ability of organisms to ferment sugars

with production of acid and gas. Sugar indicator broth was prepared using peptone water

medium containing 1% sugars (Glucose, Lactose, Maltose, Fructose and Sucrose) and 0.01%

phenol red About nine millimeters of sugar broth was dispensed into each of the test tubes,

durham tube which would trap the gas if produced was inverted carefully. The test tubes were

autoclaved and inoculated with a loopful of 24 h old culture of the test organisms after then

incubated for 2-7 days at 36°C and observed daily for acid and gas production. Yellow coloration

indicates acid production while gas production was indicated by displacement of the medium in

the durham tube.

3.5.1.8. Indole Test

Tryptone broth (5mL) was placed into different test tubes after which a loopful of the bacterial

isolates was inoculated into the test tubes, leaving one of the test tubes uninoculated to serve as

control. The test tubes were then incubated at 370C for 48 h. After incubation, 3drops of Kovac's

reagent was added and shaken gently; it was allowed to stand for 20 min to permit the reagent to

rise. A red colour at the top surface of the tube indicates a positive result while yellow coloration

indicates a negative result.

3.5.1.9. Methyl Red

Five millimeters of glucose phosphate broth (1g glucose, 0.5% KH2PO4, 0.5% peptone and

100mL distilled water) were dispensed in clean test tubes and sterilized. The tubes were then

inoculated with the test organisms and incubated at 37°C for 48hrs. At the end of incubation, few

drops of methyl red solution were added to each test and colour change was observed. A red

colour indicates a positive reaction.

 23

3.6 Optimization of Bio-degradation of Lamda Cyhalothrin

At varied temperature of 1ml and 3ml optimization studies for the biodegradation of lamda

cyhalothrin was carried out. Mineral Salt Medium was used to conduct these research. Using the

techniques outlined by Almuhayawi et al. (2021), the degradation potential was calculated from

the change in absorbance values at 620 nm at days 0.3, 6, 12, and 14.

3.7 Identification of the Degradation Products

This was done using a GC-MS analysis. The GC-MS analysis was executed on a Varian CP3800

gas chromatograph (GC) (Walnut Creek, CA, 124 USA) assembled with a split/splitless injector

and linked to a Saturn 2200 ion trap mass spectrometer (ITMS). The separation was carried out

with a Varian Factor Four VF-5MS capillary column (30 m × 0.25 mm i.d., 0.25µm film

thickness) (Varian Inc, Lake Forest, CA). Helium (99.99%) was used as carrier gas at a flow rate

of 1 mL min-1. The oven temperature program was as follows: initial temperature 60 ºC (hold 1

min); rate 30 ºC/min 128 to 180 ºC (hold 3 min); rate 5 ºC/min to 280 ºC (hold 3 min). The

complete analysis time was 30 min for each sample (Rodriguez et al., 2016).

3.8 Statistical Analysis

Data obtained was entered into Microsoft ExcelTM 2016 and Microsoft Word 2019 for statistical

computation.
 24

CHAPTER FOUR

4.0 RESULTS

4.1 Morphological and Biochemical Characteristics of Bacteria Isolate

Two bacterial isolates that showed the potential to degrade lambda-cyhalothrin were obtained.

Their morphological and biochemical characteristics are shown in Table 1. They were tentatively

identified as Bacillus species.

4.2 Effects of Temperature on Biodegradation of Lamda Cyalothrin by Bacillus sp

The effects of temperature on biodegradation of lamda cyalothrin by Bacillus sp is shown in

Table 2. The effect of temperature for isolate B2 was done at 20 ⁰C, 30 ⁰C, and 40 ⁰C for 14

days. At 20 ⁰C the highest total viable count was recorded at day 6 (60x10 5CFU/ml). At 30 ⁰C

the highest total viable count was recorded at day 6 (76x10 5CFU/ml). At 40 ⁰C the highest total

viable count was recorded at day 6 (85x105CFU/ml).

 25

4.3 Gas Chromatographic-Mass Spectrometry Analysis of Metabolites

A total of 40 different metabolites were produced from the biodegradation of Lamda Cyalothrin

at different temperature. The chromatograph showed metabolites at different retention time

(Table 3-6), which are different from the pure lambda-cyhalothrin (Figure 1-3).

Isolat Oxi Cat Ci In MR VP Co U Gl L Mal Su Shape Gram Probable

e t r a stain organism

1 + + + - - + - - + + + + Rod + Bacillus
sp.

2 + + + - - + - - + + + + Rod + Bacillus
sp.

Table 1. Morphological and Biochemical Characteristics of Bacteria Isolate

KEY: + is Positive - is Negative OXI = Oxidase CAT = Catalase CIT = Citrate

In = Indole MR = Methyl red VP = Voges-Proskauer CO = Coagulase UR = Urease Gl =

Glucose LA = Lactose MAL = Maltose SU = Sucrose

 26

Table 2. Total Viable Bacteria (Temperature)

Isolate B2
DAYS TOTAL VIABLE BACTERIA COUNT (× 105CFU/ml)

Temp. 20 ⁰C Temp. 30 ⁰C Temp. 40 ⁰C

0 45 49 47

3 50 56 59

6 68 76 85

12 49 46 51

14 41 42 43
27
 28

Figure 1. Mass spectra of Degradation of Lamda Cyalothrin

 29

Sample Name: B430

Figure 2. Chromatogram of Isolate B30 during Biodegradation of Lamda Cyalothrin

 30

Sample Name: B440

Figure 3. Chromatogram of Isolate B440 during Biodegradation of Lamda Cyalothrin

 31

Table 3 Retention Time of Isolate B430 after the Biodegradation of Lambda Cyalothrin

S/N Retention Time Chemical Name Molecular Weight (mg/mol)

 32

1 2.229 3-Hexene 84.16

2 11.130 Hexasiloxane, 1,1,3,3,5,5,7,7,9,9, 11,11-dodecamethyl- 430.94

3 12.229 Tetradecanoic acid 228.37

4 13.271 Oleic acid 282.43
5 13.197 Octadec-9-enoic acid 282.46
6 14.032 6-Octadecenoic acid, (Z)- 282.46
7 14.282 9-Octadecenoic acid, (E)- 282.46
8 14.567 Oleic acid 282.46
9 15.159 Oleic acid 282.46
10 15.525 Z-(13,14-Epoxy)tetradec-11-en-1-ol acetate 268.39
11 15.947 Oleic acid 282.46

Table 4. Retention Time of Isolate B440 after the Biodegradation of Lamda Cyalothrin

S/N Retention Time Chemical Name Molecular Weight (mg/mol)

 33

1 7.975 D-Fructose, 1-O-methyl- 194.18

2 9.158 Azetidine, 1-chloro-2-methyl- 91.54
3 9.806 Cyclohexasiloxane, dodecamethyl- 444.92
4 10.229 1-Cyclohexylnonene 208.38
5 11.130 Silanamine, N-[2,6-dimethyl-4-[(trimethylsilyl)oxy]phenyl]-1,1,
1-tri-methyl- 281.55
6 11.581 9-Hexadecenoic acid 254.44
7 11.975 Phenol, 2,4-bis(1,1-dimethylethyl) 206
8 12.088 Oleic acid 282.46
9 12.229 Undecylenic acid 184.27
10 12.426 Methyl 9-heptadecenoate or 9-17:1 252.48
11 12.989 Tetradecanal 212.21
12 13.130 2-Heptadecenal 252.4
13 13.468 7,11-Hexadecadienal 236.09
14 13.581 Cyclopentadecanone, 2-hydroxy- 240.38
15 14.032 Dichloroacetic acid, undec-2-enyl 226.4
16 14.257 2-Chloroethyl linoleate 146.372
17 14.708 1,4-Cyclohexanedimethanamine 142.246
18 15.046 Z-6-Pentadecen-1-ol acetate 268.4
19 15.238 E-11-Tetradecenoic acid 226.55
20 15.722 Chloroacetic acid, 1-cyclopentylethyl ester 95.49
21 15.865 2-Propenoic acid, 2-propenyl ester 112.12
 34

CHAPTER FIVE

5.0 DISCUSSION

Biodegradation experiments in mineral salt medium media were performed with the two most

efficient strains of Bacillus sp. Several isolates that demonstrated growth on minimal medium

where lambda cyalothrin was the sole carbon source were purified. Based on preliminary studies

isolates B430 and B440 were selected for further study. These isolates were subsequently

cultivated in liquid minimal medium in an attempt to quantify cell growth. Table suggests that

growth of strain B430 and B440 could be monitored by Optical density (OD) by determining the

effect of different temperature. At 20 ⁰C the highest total viable count was recorded at day 6

(60x105CFU/ml). At 30 ⁰C the highest total viable count was recorded at day 6 (76x10 5CFU/ml).

At 40 ⁰C the highest total viable count was recorded at day 6 (85x10 5CFU/ml). Low temperature

significantly reduces the activity of pesticides degradation (Lekeanju et al., 2016). The higher the

temperature, the higher the total bacterial count and pesticide degradation. The optimum

temperature for the application of most pesticides is 18-24 °C. The effect of systemic pesticides

directly depends on the intensity of sap flow in plants. Their effectiveness decreases at both low

and high temperatures (Petrova, and Chermenskaya, 2019).

Microbial degradation is an effective strategy used in bioremediation of crude oil pollution

because microorganisms, such as bacteria, can degrade hydrocarbons to carbon dioxide and

water. Saturated hydrocarbons are characterized by low chemical reactivity and poor water

solubility, which effectively limit availability and accessibility to the degrading microorganisms

(Burns and Pastoor, 2018). Although low molecular weight saturated hydrocarbons are sparingly

available to the degrading microbe, they are very toxic. The high molecular weight saturates are

insoluble in aqueous medium. It is well established that pesticide-contaminated sites can contain
 35

significant sources of indigenous “hydrocarbon-degrading” bacteria. As pesticides is rich in

diverse chemical compounds and various hydrocarbons. Bacillus have been found to degrade

various hydrocarbons including aromatic and aliphatic hydrocarbons (Bhart et al., 2019). In this

present study, a total of 40 different metabolites were produced from the biodegradation of

Lamda Cyalothrin at different temperature. It was found out that metabolites belonging to the

aliphatic carboxylic groups were found to be degraded mostly by isolate B430. It was also found

that methyl groups belonging to aliphatic compounds was observed to be degraded mostly by

isolate B440. This is similar the study by Knott et al. (2020) who reported degradation of

aromatic hydrocarbons by Bacillus sp.

 36

5.1 Conclusion

The high environmental concentrations of lamda cyalothrin found in the vicinity of agriculture

soils in Elemere Village pose a significant health risk. Despite the apparent persistence of lamda

cyalothrin in these environments, this study shows that it can serve as a source of carbon

supporting microbial growth. Two isolates, B430 and B440, identified as Bacillus species

respectively, rapidly biodegraded lamda cyalothrin.

5.2 Recommendation

It is recommended that the soil temperature and moisture conditions should be made ideal as it

would be helpful for the microbial degradation of lambda-cyhalothrin. Ideal environmental

conditions should also be provided for supplying high-efficient degrading bacteria so as to

eliminate lambda-cyhalothrin residue in farming field.

5.3 Contribution to Knowledge

Understanding pesticide metabolism in plants and microorganisms is a key component for the

development, the safe and efficient utilization of these compounds, and for bioremediation of

these chemicals in contaminated soil and water.

 37

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