BIOACCUMULATION AND RISK ASSESSMENT OF BUTACHLOR IN THE SOIL ECOSYSTEM

ABSTRACT

Bioaccumulation of butachlor in plants following its application in the farm against weeds was evaluated using Phaseolus vulgaris (bean plant). Also, the risk of the consumption of such plants with some amount of bioaccumulated butachlor by non-target humans was studied using rabbits as animal model. The field experiments were carried out by crop cultivation with the application of 4.0 liters per hectare (2.6 kg a.i/ha), 4.4 liters per hectare (2.9 kga.i/ha) and 5.0 liters per hectare (3.2 kg a.i/ha) concentrations of butachlor at pre-emergence of the bean plant and the leaves of the plant were analyzed for the presence of butachlor residues using GC-MS , the result gave 0.10, 0.13 and 0.20 ppm bioaccumulated butachlor respectively for the concentrations of the butachlor applied to the plots of land. For 28 days three replicate groups of rabbits (4 per group) were fed the leaves containing these different concentrations (0.10, 0.13 and 0.20 ppm) of butachlor while the control groups which were composed of three replicates, were fed the plants cultivated in plots not treated with the herbicide. The rabbits were allowed access to water ad libitum. At the end of this exposure period, significant increases (p < 0.05) were observed in Cytochrome P450 (CYP) protein and increases in Glutathione S-transferase (GST) activity of the post-mitochondria liver fractions which were observed for the groups of rabbits fed the leaves having butachlor at the concentrations of 0.13 and 0.20 ppm in a time- and concentration- dependent manner; The liver marker enzymes, aspartate aminotransferase and alkaline phosphatase (AST, ALP) activities increased significantly (p < 0.05) for the rabbits fed the leaves with butachlor concentrations of 0.13 and 0.20 ppm in a time and concentration dependent manner thus suggesting a possible body defiance mechanism for herbicide detoxification. There was fluctuations in the ALT activity, with a decrease observed in group 4 rabbits that consumed the highest concentrations. Histological sections of the liver tissues of the exposed rabbits thus revealed no pathological alterations on day 28. The pesticide biomarker enzyme results is an indicative of induction and animal exposure to xenobiotics. The two enzymes (CYP and GST) could however be overwhelmed when the  concentration of the  herbicide increase, or upon chronic exposure, resulting to toxicity. The study shows that the manufacturers’ recommended application rate for butachlor (2.6 kg a.i/ha) poses no health risk; however, the application rates above the recommended rate could pose some risk when butachlor bioaccumulates in edible plants that are consumed. Thus, this work underlines the importance of strict adherence to the manufacturers’ and regulatory bodies directives, in the application of this herbicide butachlor in

CHAPTER ONE

INTRODUCTION

One of the most important tasks of the economy of developing countries such as Nigeria is to develop the agricultural sector so as to increase and generate employment, promote self- sufficiency in food, improve the standard of living, increase gross domestic production and contribute to general development. To attain food sufficiency, government encourages farmers to use improved seeds, fertilizers, irrigation and pesticides; thus leading to a higher pesticide usage,  with  many  problems  often  associated,  such  as  unsafe  use,  persistence  in  the environment, toxicity to bees, fish and wild life, contamination of water sources, persistent pesticide accumulating in food chain, negative impact on earth worms and other non-target organisms being identified (Akinloye et al., 2011).

Pesticide is the term used for a broad range of chemicals and biologicals used for pest control. The Environmental Protection Agency (EPA) defines a pesticide as any substance or mixture of substances/chemicals intended to  prevent, destroy,  repel or  mitigate pests (undesirable animals and plants) (USEPA, 2006). Pesticide also includes plant growth regulators, defoliants or desiccants (Hagtrum and Subramanyam, 2006). They are manufactured and used in most countries around the world to protect agricultural, horticultural and forestry crops as well as increasing their productivity. Pesticide types include: insecticides, herbicides, fungicides, bactericides, rodenticide nematicides, etc (Ware and Whitacre, 2004). Herbicides are agrochemicals used for controlling weeds in different crops and they are the most widely used pesticides (Senseman, 2007).

The use of herbicides has increased worldwide over the years in other to secure food supply for the teaming global population. In the  tropical regions, Nigeria in  particular, an intensive practice has led to higher herbicide usage especially where agricultural labour is scarce or expensive; herbicides save the farmer’s time by replacing laborious manual weed control. Many farmers and extension agents lack the technical skills for proper and effective use of pesticides, resulting to many unfortunate consequences which include; human and livestock exposure to pesticide poisonings, crop injuries, and environmental pollution (Dugie et al.,

2008) Butachlor (N-butoxymethyl)-2-chloro-2’,6-diethylacetamide) is a member of the chloroacetamide family of herbicides. It is a selective systemic herbicide manufactured by Monsanto USA in 1970, for the pre-emergent control of annual grasses and certain broad leaf weeds in rice, barley, wheat and some other leguminous crops (Yu et al., 2003). Butachlor is the active ingredient of formulations sold under the trade name Machete (USA), Butaforce/Butastar (Nigeria). It is thought to inhibit the synthesis of lipids, alcohols, fatty acids, proteins, isoprenoid and flavonoids in non-target plants (Ecobichon, 2001; Heydens et al., 2002; Gotz and Boger, 2004).  Butachlor can degrade rapidly under optimal conditions but in some soil that lack suitable microbial degraders, it  may remain biologically active and persist for a long time (Lin et al., 2000). The increased application of butachlor on rice ,tea, wheat, beans and other crops, has been shown to exert detrimental effects on earthworms (Muthukaruppan and Gunasekaran, 2010) and other non-target animals (Kumari et al., 2009; Debnath et al., 2002). Studies show that it is a potential threat to agro-ecosystem and human health through food chains (Tilak et al., 2007; Sinha, 1995). Ecotoxicological studies also suggested that butachlor and their  metabolites may be harmful (genotoxic, neurotoxic) to aquatic invertebrates (Ateeq et al., 2002, 2006; Tilak et al., 2007; Yin et al., 2007), microbial communities (Min et al., 2002; Debnath et al., 2002), and possibly being carcinogenic in animals including apoptosis-resulting from DNA strand breaks and chromosomal aberrations in cultured mammalian cells (Sinha et al., 1995; Panneerselvam et al., 1999; Geng et al.,

2005a). Butachlor as well as other members of the chloroacetanilide including acetochlor, alachlor and metoalchlor have been shown to be mutagenic and carcinogenic in rats/mice upon acute exposure and these have been thoroughly reviewed (USEPA, 1998; Dearfield et al.,

1999; Wilson and Takei, 1999). Butachlor contamination of 0.163ppb has been recorded in groundwater collected from tube wells adjacent rice fields in Philippines (Natarajan, 1993). Butachlor has been banned in some countries like Canada and Europe; and it is not been used for rice cultivation in USA probably because of its toxicity, hydrophobicity, bioaccumulation and its negative impact on wildlife and humans via the food chain (Kannan et al., 1996; WHO, 2010a). However it is in prevalent use in continents such as Latin America, Asia and Africa.

Many of the herbicides widely used in the 1960s and 1970s have been phased out and replaced by the  newer,  safer  and  more  potent  herbicides discovered later.  The  use  of some older

herbicides has also been restricted, reduced and even eliminated in view of environmental and toxicological problems.  Developing  countries  maintain  that  they cannot  ban  certain  older pesticides,  for  reasons  of cost  and/or  efficacy.  Thus  the  dilemma  of  cost/efficacy  versus ecological impacts,  including  long  range  impacts via  atmospheric transport  and  access  to modern pesticide formulations at low cost remains a contentious global issue (Stephenson and Solomon, 1993; FAO, 1995). According to the Stockholm Convention on Persistent Organic Pollutants (POPs), nine of the twelve most dangerous POPs are pesticides. The POPs are compounds that resist degradation and thus persist in the environment for longer periods. They have the ability to bioaccumulate, biomagnify and bioconcentrate; hence they are poisonous to non-target organisms (Stochlm, 2009). Persistence is usually described in terms of the half-life (T ½) of a chemical in water, soil, sediment, or air. The T½ is the amount of time necessary for a given amount of chemical released into the environment to decrease to one-half of its initial value. Millions of tons of pesticides are applied annually, however less than 5% of these products are estimated to reach the target organism, with the remainder being deposited on the soil and non-target organism as well as moving into the atmosphere and water (Hamilton and Crossley, 2004). The fate of an herbicide applied on the soil include: chemical and photochemical degradation,  microbial degradation, volatilization,  runoff  into  water  bodies, leaching into groundwater, adsorption by soil particles and uptake by plants/animals which may result to bioaccumulation. This is dependent on abiotic environmental conditions (temperature, moisture, soil pH, etc) microbial or plant species (or both), pesticide characteristics (chemical or physical-hydrophobicity, Kow, etc (Lyman, 1995; Schnoor, 1996).

1.1      Bioaccumulation of Herbicides

When herbicides are applied, acceptable remainders of active substances (maximum residue limit, MRL) can often be detected in cultivated plants depending on the physico-chemical properties of the active substances of herbicides and ways of detoxification, some of these pollutants tend to increase concentration while passing through organisms of higher trophic levels, thus leading to a significant bioaccumulation in food chains (Allinson and Morita,

1995). Bioaccumulation is the process by which organisms accumulate chemicals such as pesticides both directly from the abiotic environment (i.e., water, air, soil) and from dietary sources (trophic transfer) (Baron, 1995). Studies have shown that herbicides or its metabolites can enter into the human body along food chain, where they may be metabolised or accumulated in fatty tissues and this creates potential health risks to human (Hodgson and Levi, 1997). Bioaccumulation occurs when an organism absorbs a toxic substance at a rate greater than that at which the substance is lost. Thus, the longer the biological half-life of the substance, the greater the risk of chronic poisoning, even if environmental levels of the toxin are not very high (Hamilton and Crossley, 2004). Bioaccumulation is a natural process that gradually concentrates non-toxic levels of pollutants into toxic levels within a biota causing unpleasant side effects (Connell, 1990). The regular intake of sub toxic levels of persistent pollutants can gradually bioaccumulate up to toxic levels and after some time produce chronic effects. From the roots of plants, the pesticides move by translocation to stems and then often a strong bioaccumulation occurs in the leaves (Bicalho and Langenbach, 2012) or fruits and such crops where pesticides are used intensively maybe consumed by cattle, humans or wild life. A strong increase in the concentrations of these molecules can occur in a process called biomagnification. In order to minimize ecotoxicity, there is the need for the restriction of inappropriate use of pesticide, thus removing them from the food chain and water reserves.

Bioaccumulation studies are used to assess the rate and extent of contaminant accumulation in a given lower trophic level and this is important for assessing the hazard it poses for a higher trophic  level  of a  food  chain  (USEPA,  2000).  The  extent  of chemical accumulation  is expressed in form of bioaccumulation factor (BAF); this is a ratio of the concentration of the chemical in the organism (plant, animal tissue) to that in the surrounding environment (soil, air, water). It is a major criterion for assessing bioaccumulation. The greater the value of BAF, the more the chemical accumulates in the plant/animal and the higher the risk of exposure to humans. Another criterion for bioaccumulation potential is the  n- octanol/water partition coefficient (Kow); this is the ratio of a chemical’s solubility in n-octanol to its solubility in water at equilibrium. It is often used to express lipophilicity or hydrophobicity. For organic chemicals, log Kow ranges from –3 to 7. Organic chemicals that have log Kow higher than 2 are usually hydrophobic and considered liable to bioaccumulate in biota (Oliver and Charlton, 1984; Elzerman and Coates, 1987; Franke et al., 1994; USEPA, 2000). Herbicide use can result to environmental, ecological and health effects. In health effects, they may cause acute or chronic effects when exposed. In animals and humans, these effects range from simple

irritation of the skin and eyes to more severe and chronic effects affecting the nervous system (Gorell et al., 1998; Tanner et al., 2011; Mostafallou and Abdollahi, 2012), disruption of hormonal functions causing reproductive problems (Bosveld et al., 1995; Bretveld et al., 2006) ,carcinogenetic (Ou et al., 2000), teratogenic (Paganelli et al., 2010) genotoxic, or result to mortality (Dieter et al., 1996).

1.2      Risk Assessment of Herbicides

The analysis of herbicides and their residues had in the past aided objective re-evaluation and reassessment of these substances on a benefit-risk analysis basis and their subsequent withdrawal from use when found to be hazardous to human health and the environment (Achudume, 2011).  Risk  assessment  of chemicals  is  described as  a  process  intended to calculate or estimate the risk to a given target organism, system or (sub) population, including identification of attendant uncertainties, following exposure to a particular chemical taking into account the inherent characteristics of the agent of concern as well as the characteristics of the specific target system (OECD, 2003). Risk Assessment is the central component of risk analysis and provides a scientific basis for risk management decisions on measures that may be needed to protect human health. It evaluates the possible danger in the consumption of organic chemicals by animals and human. Risk  is characterized by assessing the Hazard Quotient (HQ) and the Health risk index (HRI). The HQ is a simple ratio of single exposure and effect values and may be used to express hazard or relative safety (Gerba, 2010; Wang et al., 2005). Assessments may be undertaken for acute (short term) or chronic (long term) exposures, where acute exposure covers daily average and chronic if exposure is over the entire lifetime.

In most countries, herbicides must be approved for sale and use by government agencies such as Environmental Protection Agency (EPA) and the National Agency for Food and Drug Administration (NAFDAC) under  the  Food  Quality Protection in Nigeria.  Pesticides are regulated after complex studies (10 field trials or tests) to ensure that these products do not pose adverse effects to humans or the environment. Standards are then set for the level of pesticide residue that is allowed in or on crops (USEPA, 2000; Hamilton and Crossley, 2004). Internationally, risk assessment of chemical substances present in or on food forms the core work  of  Joint  Food  and  Agricultural  Organisation  and  the  World  Health  Organisation

(FAO/WHO and Expert committee on Food Additives (JECFA). These organisations base their evaluations on scientific principles and ensure consistency in their risk assessments determination, Also the Codex Alimentarius Commission (established by FAO and WHO in

1962) together with the committee establishes maximum residue limits (MRL) for pesticide residues, veterinary drug residues, contaminants and  food additives in order to  facilitate international trade and protect the health of consumers (WHO/ FAO, 2010a). Each year,

140,000 tons of pesticides are sprayed onto crops in the European (EU) alone. Fruits and vegetables are the crops mostly contaminated. According to data from the EU’s pesticides action network in 2008, some 350 different pesticides were detected in food produced in the EU. More than 5% of products contained pesticides at levels exceeding the EU maximum permitted levels (Fenik et al., 2011). Scientific researchers have shown that several health effects due to pesticides were apparent at much lower doses than the typical levels of pesticide residues found in food (Nebeker et al., 1994; Walz, 2010). Studies have also been carried out in some other countries and the detection of pesticides/residues in food and drinks were reported; some of which are above or below the maximum residue limit (MRL), as stated by the Codex Maximum Residue limits/Extraneaous Maximum Residue limit  (MRL/EMRL) (Tadeoa et al., 2000; Tseng et al, 2002,Sun et al., 2005; Chang et al., 2005; WHO/FAO, 2006; Darko and Acquaah, 2008; Qiu et al., 2010; Etonihu et al., 2011; Fenik et al., 2011).

1.3      Bean Plant (Phaseolus vulgaris)

The bean plant is a herbaceous legume grown annually for its edible fruit, either the dry seed or the unripe fruit, both of which are referred to as beans. It belongs to the family known as leguminosae. It is a warm season growing legume that does better under subtropical and temperate conditions. It requires a moderate well-distributed rainfall (300-400 mm per crop cycle) with average temperatures range between 17.5°C and 27°C. The common bean grows well on a large variety of soils with pH ranging from 4 to 9. It does better on well-drained, sandy loam, silt loam or clay loam soils, rich in organic content. The duration of the cycle for the common bean ranges from 60–90 days depending on the variety. The leaves of this leguminous plant is also occasionally used as vegetable, the leaves and the straw can be used for fodder and fed fresh to livestock. The creeping variety is cultivated around the warm season (Ferreira et al., 1997). The black bean variety popularly called Akidi, is commonly grown in the eastern part of Nigeria for its protein rich seed and edible leaves.

1.4      Historical Use of Herbicides.

Long before 2000 BC, humans have utilized pesticides to protect their crops. The first known pesticide is elemental sulfur dusting used in ancient summer about 450 years ago in ancient Mesopotia. By the 15-19th century saw the use of toxic chemicals such as arsenic mercury, lead to nicotine and other natural pesticides like pyrethrum which is derived from chrysanthemums, and rotenone, which is derived from the roots of tropical vegetables in the

1950s (Miller, 2002). Paul Muller discovered that DDT was a very effective insecticide and the organochlorines became dominant, however later replaced by the organophosphates and carbamates by 1975. Herbicides became common in the 1940s–1950s led by the triazine and other nitrogen-based compounds, carboxylic acids and glyphosate (Miller, 2002). The first widely used herbicide was 2, 4–dichlorophenxyacetic acid (2, 4-DPNA) which was commercialized  by the  paint  company Sherwin–Williams. It  is  easy and  inexpensive to manufacture, kills  many broad leaf plants, however, high doses of 2, 4-DPNA could be harmful. The low cost of 2,4–DPNA led to continued usage today and it remains one of the most commonly used herbicides in the world. Seventy-five percent of all pesticides in the world are used by developed countries, but use in developing countries is increasing (Ritter,

2009). The triazine family of herbicides which include atriazine, was being discovered to be the herbicide family of greatest concern regarding ground water contamination. Glyphosate sold with a brand name Roundup was introduced in 1974 for non-selective weed control, and it is now a major herbicide due to the development of crop plants resistant to it. In the 1970 the  use  of chemicals  such as DDT  and  some  organochlorines   were  banned Under  the Stockholm Convention on Persistent  Organic pollutant,  because of their long persistence in the environment, although DDT is still been used in some developing nations to treat malaria and tropical  diseases (WHO, 1989). Today organochlorine pesticide levels are still detected in fish from waterways (Chindah et al., 2004). World-wide, an estimated 2.3 billion kg of 1,600 different pesticides are applied yearly (Pimentel, 1995). In Europe, the total agricultural use of pesticides is estimated to be 350 000 tons of active ingredients per year (Kreuger, 1999) It has been established that pesticide application in Nigeria ranges from 125,000 to 130,000 metric tons yearly (Asogwa and Dongo, 2009). World Health Organisation maintained that an estimated three (3) million farmers in developing countries experience acute poisoning from

pesticide and eighteen thousand (18000) of them eventually died from this (FAO, 2002). Nigeria is  not  immune to this phenomenon, one hundred and twelve (112) people were hospitalized and two (2) children died after eating beans preserved with pesticides in Bekwara Local Government Area of Cross Rivers state. Again, one hundred and twenty (120) students of a secondary school in Doma, Gombe State became sick as a result of eating food items contaminated by  pesticides  (Shaibu,  2008).  Abrahame  and  Brunt,  (1984)  explained  that though data on the amount of pesticide use generally in Africa is difficult to ascertain, it has been established that import of pesticide into the continent is on the increase. Nigeria ranked first according to Bull, (1982) among West African countries importing pesticides from the United Kingdom having imported 16,462 metric tonnes of pesticide in 1980; it accounts for about 93% of United Kingdom’s pesticide exports to West African countries. According to Lee, 2006, 75% of all pesticide is used in developed countries and yet developing countries with just 25% of global pesticide use, accounts for a disproportional number of cases of pesticide  poisoning  and  deaths.  Some  of the  inherent  problems  in  pesticide  application include: toxicity, phytotoxicity, mismanagement and maintenance of equipment, poor availability  of  pesticides/equipment,  lack  of  safety  measures,  extension  services,  wrong dosage of pesticide and pesticide misuse (Asogwa and Dongo, 2009). Problems associated with  herbicide  hazards to  man and  the  environment are  not  confined to  the  developing countries. Developed nations have already suffered these problems, and still facing some problems in certain locations. For many reasons, the severity of herbicide hazards is much pronounced in Third World Countries (Mansour, 2004). Today about 4500 pesticides are in general use all over the world, out of which 25 have high toxicity potential to a wide range of flora and fauna of economic importance (Adhikary and Sahu, 2001).

1.5      Classification of Pesticides

Pesticides can be classified according to target species; insecticides, herbicides, fungicides, bactericides, rodenticides, nematicides and avicides (Ware and Whitacre, 2004); pattern of use (defoliants, repellants), chemical structure (organochlorines, organophosphates, carbamates, chloroacetamides and phenoxyacetic acids),  mechanism of action (enzyme inhibitors and photosystem inhibitors). Pesticides can also be classified as inorganic, synthetic or biological

(biopesticides). Biological biopesticides include microbial pesticides and biochemical pesticides (Ware, 2000).

1.5.1   Classification of Herbicides

Herbicides can be classified based on: (a) Method of application/use (b) Type of vegetation controlled, (c) Mode of action/activity and (d) Chemical nature/family.

1.5.1.1 Classification based on Method of Application

They can be soil applied or foliar applied.  Soil- applied herbicides are applied to the soil and are taken up by the roots and/or hypocotyls of the target plant. There are two main types of soil applied herbicides

a.         Pre-plant incorporated herbicides which are applied prior to planting and mechanical incorporation into the soil. They are mixed with the soil before seeding. This incorporation is another way to prevent dissipation through photodecomposition and/or volatility.

b.         Pre-emergent Herbicides are applied to the soil before the crop and weed emerges thus preventing germination or early growth of weed seeds

Foliar applied herbicides 5are applied to plant foliage since they have foliar activity. Post emergent herbicides are applied to the soil or foliage after the germination of crops and weeds (Senseman, 2007).

1.5.1.2 Classification based on the Type of Vegetation:

Selectivity is  the process by which a  herbicide controls certain plants but  leaves others unharmed.  Herbicides can  be  selective  or  non-selective  i.e.  the  selective  herbicides kill specific targets (weeds) while leaving the desired crop relatively unharmed. Non selective herbicide kill all type of plant material including target and non-target plants, when in contact, hence they are commonly  used for preparation of land, industrial sites, railways and waste ground, forestry, wildlife habitat and pasture systems (Senseman, 2007).

1.5.1.3 Classification based on Activity/Mode of Action

This refers to how they are translocated in plants.

a.         Contact herbicides (non-systemic) destroy only the plant tissue in contact with the chemical, they are fast acting herbicides however less effective on perennial plants which are able to grow from rhizomes, roots or tubers.

b.         Systemic herbicides (translocated): are translocated through the plant, either  from foliar application down to the roots or from soil application up to the leaves and stems.  They are capable of controlling perennial plants and may be slower acting but ultimately more effective than contact herbicides (Rao, 2000).

1.5.1.4 Classification based on chemical nature/family

Herbicide may be divided into two groups – Inorganic and organic herbicide. The inorganic herbicides are  made up of inorganic compounds which include copper sulphate, sodium arsenate, sulphur acid and sodium chlorate. They were used between 1896–1930s, however organic herbicides dominate modern agriculture. The organic herbicides include; Phenoxy carboxylic acid herbicides e.g. 2,4-dichlorophenoxyacetic acid, Triazines (atrazine) Bipyridyliums (paraquat), Sulphonyl Ureas, Dinitroanilines and Chloroacetamides (butachlor) (Rao, 2000). Chloroacetamides belong to a subgroup of the acetanilides or acetamides (substituted acidamide), having a phenyl ring attached to the amide nitrogen.  Some of the members of the acetamides are applied post-emergence while others are pre-emergence; all are effective on germinating weeds (shoot and root inhibitors).  The herbicidal mode of action is not totally understood, however, it is known that this class of herbicides inhibits biosysnthesis of lipids, fatty acids, proteins, isoprenoids and flavonoid in plants (Heydens et al.,  2002).  Studies  showed  that  they  cause  herbicidal  effect  via  conjugation  of  acetyl coenzyme A and other sulfhydryl-containing enzymes with consequent inhibition of critical function needed for the germination or survival of seedlings. The effective shelf-life of most chloroacetamide is of the order of 6-12weeks.   The members of the acetamides include; alachlor, acetochlor, butachlor, metolachlor, propachlor and allidochlor.

1.6       Butachlor

1.6.1   Physicochemical Properties of Butachlor

Butachlor (N-butoxymethyl) -2-chloro-2’,6-diethylacetamide-C17 H26  NO2Cl) is an amber coloured liquid with a faint sweet odour, with a molecular weight of 311.9g. The solubility of butachlor in water is 20ppm at 25oC, it is semi-volatile and miscible with alcohol, ether, n- hexane, acetone and benzene. The partition coefficient (1-octanol/water)Kow  is3.16 X 1004

and logKow   is 4.50 at pH 7, while the Koc is 700mL/g and the vapour pressure is 2.90×10-6 mmHg (=3.86×10–4  Pa) (25°C). The specific gravity /density is 1.076 (25oC) and it is not hydrolyzable. The melting and boiling points are   -0.55 and 156oC respectively. The half-life

in soil due to biodegradability (aerobic degradation) is 42 -70 days (6-10weeks) (Heydens et al., 2002).The structure of butachlor and its metabolite-2,6 diethylacetamide are shown below

in figures 1a and b. CH2CH3

COCH2ClN

CH2O(CH2)3CH3

CH2CH3

a)  Structure of Butachlor

b)  Metabolite of butachlor in animal

Figure 1a: Chemical Structure of Butachlor b) Metabolite of butachlor in animal.

Butachlor was developed by Monsanto and commercialized as machete USA in 1970 for the pre-emergent control of annual grasses and certain broad leaf weeds in rice, barley, wheat and some other leguminous crops. It is the active ingredient of an emulsifiable concentrate sold under the trade name Butastar and Butaforce in Nigeria. Butachlor is a selective systemic herbicide, absorbed primarily by the germinating shoots and secondarily by the roots, with translocation throughout the plant, giving higher concentrations in vegetative parts than in the reproductive parts (Rao, 2000). It is known to inhibit cell division mainly by inhibiting lipid and protein synthesis. It’s activity is dependent on water availability such as rainfall following

treatment, overhead irrigation or applications to standing water as in rice culture. In soil its degradation is principally by microbial activity (Rao, 2000).

1.6.2   Toxicity of Butachlor

World Health Organisation (WHO) has classified butachlor under Class ΙΙΙ toxicity level. Studies on acute toxicity have been investigated and it was discovered that the acute oral LD50 for rat is 2000 mg/kg bw,   mice 4747 mg/kg bw, for rabbits >5010mg/kg bw and the LC50 for fish 0.1-0.14mg/l. Other studies done on rabbits by exposure to butachlor for 21 days at dose levels of 250mg/kg/day showed signs of dermal irritation with LD50>13000mg/kg (Wilson and Takei, 1999; Kreiger, 2001). The chronic (subchronic) toxicity of butachlor has also been evaluated in dogs, mice and rats. The primary target organs have been shown to be the liver, kidney and bladder in one or more species (Wilson and Takei, 1999).

1.7      Mode of Action of Herbicides in Target Plants

Herbicides bring about various physiological and biochemical effects on the growth and development of emerging seedlings as well as established plants, either on or after coming into contact with the plant surface or reaching the site of action within the plant tissue.  The net result is death of the plant.  These physiological and biochemical effects are followed by various  types  of  visual  injury  symptoms on susceptible plants.  These  include chlorosis, defoliation stunting, necrosis, growth stimulation, cupping of leaves,  marginal leaf  burn, desiccation, delayed emergence, germination failure etc (Rao, 2000).

The rate of appearance of these signs varies with the characteristic actions of the herbicide and depends on the degree of tolerance or susceptibility of the plant  species. Environmental factors and  soil conditions affecting  plant  growth, as well as  herbicide  formulation and application method significantly influence the effect of herbicides. Herbicides differ in their site of action, there are more than one site of action and the primary site is the most sensitive which is affected first; as the herbicide concentration builds up in the tissue, the secondary and tertiary sites may be involved.  The different physiological and chemical processes that occur within the living plant include; photosynthesis, mitochondrial activities, protein/nucleic acid biosynthesis, pigment biosynthesis, fatty acid/lipid biosynthesis, amino acid biosynthesis,

hydrolytic enzyme activities, aromatic compound biosynthesis, etc. (Rao, 2000).Studies show that butachlor acts by inhibiting the elongase responsible for the elongation of very long chain fatty acids (VLCFA) and geranyl geranyl pyrophosphate cyclisation enzymes (Matthes et al.,

1998; Gotz and Boger, 2004). A majority of VLCFAs is located in the plasma membrane and as cuticular and epicuticular waxes. When absent the membrane loses stability and becomes leaky leading to death of the herbicide-treated plant (Matthes and Böger, 2002).

Fatty Acid Biosynthesis and Elongation

Figure 2.  Simplified schematic of fatty acid synthesis and elongation in plants.  Abbreviations: ACCase, acetyl-CoA carboxylase;  ACP,  acyl  carrier  protein;  ACS,  acetyl-CoA synthase;  CoA, coenzyme A; dims, cyclohexanedione inhibitors; FAS, fatty acid synthase; fops, aryloxyphenoxy propionate inhibitors; PDC, pyruvate dehydrogenase complex.

Source:  Gronwald, (1991). Lipid biosynthesis inhibitors. Weed Sci. 39:435-449.

1.8      The Soil Ecosystem

The Soil is a complex ecosystem of many species including plants, fauna and microorganisms. It is a variable mixture of mineral and organic materials with living, dead, decaying biologic components, air and water (Tomaz, 2013). The fate of any herbicide depends on its properties, environmental factors and the properties of the soils to which it is applied.

(i)  Soil properties that affect the uptake of pesticide (herbicide)

The uptake of a xenobiotic by the crop, following a pesticide treatment, depends on the degree of exposure from both the roots and aerial parts of the plant. Pesticides on the surface of the plant or in the soil will be subject to a range of environmental factors, e.g. photolysis and microbial  activity,  which  can  result  in  degradation  of  the  pesticide.  These  degradation products and the parent pesticide are therefore available for absorption. A further factor influencing the uptake of xenobiotics from the soil is the interaction of chemicals with the soil (Hellstrom, 2004).

(a) Soil Adsorption

This process takes place when pesticides sprayed on the soil surface adhere to the soil particles and organic matter. Soil properties that affect herbicide adsorption are soil pH, organic matter content, soil moisture, soil temperature and soil colloids (Rao, 2000). Soils high in organic matter or clay are the most adsorptive. A pesticide that is adsorbed by the soil is less likely to volatilize, leach or be broken down by microbes. However, it will move with the soil if the soil is eroded. They are not strongly adsorbed to sandy soils. Herbicides are adsorbed to soil colloids to varying degrees and the colloids have negatively charged sites to which herbicides can be adsorbed. Adsorption reduces herbicide activity in soils because the molecules adsorbed to the colloids may not be available for desorption. Desorbed or non- sorbed molecules are bio-available and can move into the food chain or into ground water (Kerle et al., 2007). Adsorption/Desorption are thus the key processes controlling herbicide efficacy, dissipation and behavior in soil as well as the contamination of ground and surface– waters.    Absorption  is  the  movement  of  pesticides  into  organisms  (plants,  animals)  or structures (wood). To be absorbed into the roots, the xenobiotic needs to be bioavailable or present in the soil–water compartment, a function of the interactions of the chemical with soil

organic matter or clay particles. This interaction is measured as the adsorption coefficient, Kd or Koc  ; Koc  is the tendency of herbicide sorption to organic carbon. Higher values (greater than 1000) indicate a herbicide that is very strongly attached to soil and is less likely to move unless soil erosion occurs. Lower values (less than 300-500) indicate herbicide that tends to move with water and have the potential to leach or move with surface runoff (Monaco et al.,

2002; Connell, 1990). The Koc for butachlor is 700ml/g which indicates moderate soil sorption as compared to trifluralin with Koc of 7000ml/g which strongly attaches to soil.

The uptake of compounds by the root is therefore a function of the soil adsorption coefficient of the xenobiotic, the concentration gradient between the soil solution and that inside the root, the octanol/water partition coefficient Kow (lipophilicity), degree of ionization, and on the mass  flow  of  water.  The  most  important  property  is  hydrophobicity,  which  usually  is expressed as the 1-octanol/water partition coefficient (Kow), or more often log Kow. This is the ratio of a chemical’s solubility in n-octanol to its solubility in water at equilibrium (Bacci,

1994). Log Kow spans over a wide range for different organic compounds (Hellstrom, 2004). It is an important basis for estimating bioaccumulation factor (BAF). The Kow  also provides information on how strongly organic and inorganic compounds are likely to bind to soil or sediment particles, or to partition into lipid (octanol) versus aqueous phase liquids. Strong binding indicates a high potential to persist and accumulate in soils and sediments; soil/sediment binding also tends to be correlated with the potential to bio accumulate. Since lipophilic substances with high octanol-water coefficient remain preferencially in soils and with little bioavailability thus they have low bioaccumulation potential (Connell, 1990). More hydrophobic compounds, having a higher Kow, are sorbed more strongly to organic particles in the soil. Due to the solubility of 20ppm and high hydrophobicity of log kow  4.5 butachlor presents high adsorption in soils with medium to high organic matter (Wang et al., 1999)

(b) Soil Organic Matter (SOM)

Organic matter consists of decaying plant material. The content of organic carbon in soil is one of the most important environmental factors influencing root-uptake of non-ionic organic compounds from soil into roots. The higher the soil organic matter content, the greater the soil’s ability to hold both water and adsorbed pesticides. To further describe the distribution of a  chemical  in  soil,  the  soil-water  partition  coefficient  (Kd)  is  used.  Kd   is  generally

proportionally to the hydrophobicity of the compound and to the amount of soil organic matter (Hellstrom, 2004). The smaller the Kd value the greater the concentration of herbicide in solution. In soils with high organic matter content and clay content, lipophilic and charged pesticides are retained in the soil organic matter for longer time and the uptake into plants decreases (Trapp et al., 1990). Thus a soil with higher organic matter content will have more pesticide adsorbed to the soil, and this reduces detachment and leaching, but may have a higher runoff potential because more of the chemical is retained in the surface zone of the soil. The reverse occurs in low organic matter sandy soils with low cation exchange capacity (CEC). Some studies have also shown that soil amendment with manure compost may reduce bioavailability by retaining the toxic organic chemicals in the organic matter and therefore reduce the hazardous effects by bioaccumulation (Jiang et al., 2010; Tomaz, 2013). Ecotoxicology   depends   on   soil   organic   matter   (SOM).   When   SOM   is   high   the ecotoxicological effects are low and when the SOM is low the effects are high (Tomaz, 2013).

(c) Soil Texture and Structure

Soil texture is the relative proportions of sand, silt, and clay-sized particles. Percolating water moves faster  in sandy soils, and  fewer binding sites are available  for the adsorption of dissolved chemicals when compared to clay or silt soils. Though sandy soils are more prone to pesticide movement, leaching may also occur in clay or silt soils. Soil structure which is the shape or arrangement of soil particles plays an important role in determining the size and shape of the pores through which water moves. Small amounts of pesticides may also move through soil cracks. Light textured sandy soils do not adsorb and retain high amount of hazardous products and this  will  both bioaccumulate in  living  tissues and pollute water sources. In soils of this type plants strongly adsorb pesticides resulting in enhanced contamination with subsequent phytotoxicity and toxicological effects on fauna (Covaci et al.,

2010). Soils may function as a filter or as source of pollutants, depending mainly on the kind of soil. It has been shown that the soil infiltration capacity depends on soil texture characteristics, porosity and humidity. Soils with a sandy texture are more susceptible to the process  of  leaching  (less  adsoption),  while  clay  soils  have  greater  pesticide  adsorption potential and less leaching potential (Tomaz, 2013).

(d) Soil pH

Soil pH will affect  the electrical charge of certain pesticides. The electrical charge will determine the type and degree of adsorption. It also affects the ionic or molecular character of the chemical, the ionic character and the cation exchange capacity (CEC) of the soil colloids, as well as the activity of soil microorganisms (Rao, 2000). Non-ionic herbicides such as the chloroacetamides do not react with water and do not carry any electrical charge, but they are still affected by soil pH as they are polar in nature. Differences in the pH of the soil affect its ability to adsorb and retain herbicide molecules, thereby affecting leaching of the herbicide through the soil profile. Different herbicides respond differently to changes in soil pH and Soil pH has been shown to affect the speed of degradation of chloracetamides. Liu et al. (2002) reported that the greatest degradation of acetochlor took place under strong alkaline conditions (pH 12) and was lower under acidic conditions (pH < 5). However, soil pH also affects microbial degradation of the herbicide as it influences the microbial life in the soil. Microbe numbers tend to increase in soils with a neutral pH, resulting in a faster loss of activity in these soils due to greater microbial activity (Rao, 2000). Soil pH influences the growth of microorganism, for instance bacteria and actinomyces are favored by soils having a medium to high pH and their activity is reduced below pH 4.5. Hence the persistence, uptake as well as bio-accumulation of butachlor are enhanced at  low pH. Butachlor is easily hydrolysed at alkaline pH and as such not effective in the soil with high pH since alkaline pH favours the growth of microorganisms that enhance its degradation (WSSA, 2002).

(e) Soil Moisture

Herbicide adsorption and phytotoxicity is very dependent on soil moisture, which is important for herbicide movement, particularly the herbicide is moving through mass flow (Rao, 2000). The amount of moisture in the soil affects the amount of the herbicide particles that can be adsorbed by the soil, as these molecules tend to compete with water molecules for absorption sites on mineral colloids. The space available for herbicides to go into solution also decreases as soils dry out, as such less free herbicide is present in dry soils. Under dry conditions, plants are therefore less likely to absorb toxic concentrations of herbicide (Rao, 2000). When soil moisture is replenished, herbicide will desorb from the colloids and re-enter the soil solution.

1.9      Fate of Herbicides in Soil Ecosystem

Most herbicides are applied as water based sprays using ground equipment or applied aerially using helicopters or air plants.   The ground equipment varies in design, it could be self- propelled sprayers, towed handled, or horse drawn sprayers. The metabolic fate of herbicides is dependent on abiotic environmental conditions (temperature, moisture, soil pH etc) microbial or plant species (or both), herbicides characteristics (chemical or physical- hydrophobicity, pKa,  Kow, etc (Schnoor, 1996; Lyman, 1995). In the environment, organic pollutants such as herbicides can be degraded by Abiotic: chemical (Sarmach and Sabadie,

2002), photochemical (Nelieu et al., 2001) or by Biotic/biological processes (van Eerd et al,

2003). Other ways by which herbicides can be removed from site of application via physical processes include; surface run-offs from agricultural lands into streams or rivers, adsorption by  soil  particles  (Hamilton  and  Crossley,  2004).  Degradation rates  after  release  to  the environment vary widely between substances, with half-lives from minutes to many years. Degradation rates and the half-lives of herbicides are specific for one location and season. The following are ways of degradation of herbicides.

1.9.1   Chemical/Photochemical  Degradation:  These  are  abiotic  processes  that  result  in  the chemical or physical breakdown of the chemical components of the pesticides(herbicides) leading to reactions such as oxidation, hydrolysis of the chemicals into ground water (leaching into solution), reduction, photolysis and volatilization. Chemical decomposition of herbicides in soils is affected by soil moisture, pH, herbicide adsorption, soil temperature and types of ions that are present in the soil solution (Hamilton and Crossley, 2004)

(a) Photo decomposition: The molecules of some herbicides are unstable in light (ultraviolet) thus they are readily degraded by light when left on soil surface for an extended period of time.  This process plays an important part of the degradation of pre-emergence herbicides. Ultraviolet ion is  absorbed by the  molecules of these  light  sensitive herbicides and this destabilizes the herbicide molecules, causing them to lose their herbicidal activity or become more phytotoxic. At UV wavelengths reaching the earth’s surface, sunlight has sufficient energy to cause direct photochemical reactions by rearranging or cleaving carbonyl double- bonds, carbon–halogen, carbon–nitrogen, some carbon–carbon, and peroxide O–O bonds, but not enough to cleave most carbon–oxygen or carbon–hydrogen bonds (Mill, 1993). Lyman

(1995) states that the end result of photolysis may include such reactions as dissociation or fragmentation, rearrangement or isomerization, cyclization, photo-reduction by hydrogen-ion extraction from other molecules, dimerization and related addition reactions, photoionization and electron transfer reactions.

Photolysis is relatively insensitive to temperature and pH effects compared to hydrolysis (Mill, 1993). However, as would be expected, photolysis  is strongly affected by factors influencing the spectral distribution, intensity and duration of sunlight. Such factors include latitude, time and date, cloud cover, dust, etc. and the extent of absorption of UV–βradiation by atmospheric ozone. Laboratory studies have generally found that direct or indirect photolysis occurs more slowly on soil surfaces than in water. Only a thin layer of soil is either reached directly by photons or indirectly by diffusion of reaction products such as singlet oxygen. Hence, the extent to which photolysis occurs is affected by the amount of exposure of the soil surface to sunlight and the amount of pesticide available at the soil surface. Once incorporated into the soil by cultivation or leached in by rain or irrigation, a large proportion of pesticide is  likely to  be unavailable for photolysis, unless returned to the surface by volatilization (Hamilton and Crossley, 2004). The rate and extent of photochemical degradation depends on the chemical nature of the compound, the wave length of light and the presence of other chemicals (Stamgroom et al., 2000). The acetamides are slow to undergo photolyzed reaction particularly under  soil conditions.    Photodecomposition of butachlor involves debutoxymethylation, dechlorination followed by hydroxylation, O-dealkylation and polymerization.The photodecomposed products include 2chloro-2, 6, diethyl acetanilide, 2

Hydroxy  2,  6  diethyl  N-(butoxymethyl)  acetamide  and  N-2’,   6’  –  diethylphenyl  2,  3

dihydrosazole 4-one (Lin et al., 2000)

(b)  Volatilization: Volatility is a physical process in a substance and involves a change from a solid or liquid state to gaseous state.  Soil applied herbicides compete with soil moisture for sites on soil colloids. Herbicides molecules loosely held to soil colloids may be moved to the soil surface with water by capillary action and lost to the atmosphere by volatilization. It is highly dependent on the physical properties of the molecule; vapour pressure, octanol/water

partition coefficient, water solubility, adsorption coefficient, the type and condition of the surface on which the herbicide is deposited (Hodgson and Goldstein, 2001; Mill, 1993).

(c) Hydrolysis: This refers to the cleavage of a bond and formation of a new bond with the oxygen atom of water, i.e introducing HOH or OH into the molecule gives the generalization that hydrolysis may be important in any molecule where alkyl, carbonyl or imino carbon atoms are linked to halogen, oxygen or nitrogen atoms or groups through σ-bonds. Hydrolysis may occur abiotically or biotically and this is a major means of chemical alteration in the degradation pathways of many pesticides. Abiotic hydrolysis may be the principal means of pesticide degradation where biological activity is low. These reactions may be strongly pH-

dependent, occurring in the presence of H2O, H3O+ and OH− to varying degrees (respectively,

neutral, acid and base hydrolysis), and related to the acid–base dissociation characteristics (pKa) of the molecule. The rate of hydrolysis increases with increasing temperature, and may be affected by other environmental factors, such as whether the pesticide is present in solution or adsorbed to particles. In general, hydrolysis products are more polar than the molecules from which they are derived and may be significantly more water soluble and less subject to bioaccumulation (Holland and Sinclair, 2004).

(d)       Solubility: This process shows the capacity of a pesticide or a chemical to dissolve in water, Solubility is often expressed in milligrams per liter (mg/l) or parts per million (ppm). Pesticides with high degree of solubility enjoy greater tendency to pass through the soil and reach to groundwater. Others with solubility of less than 1.0 mg/l are normally strongly adsorbed or attached to sediment and loss to surface waters via soil erosion and is of primary environmental concern. Herbicides differ in their solubility in water; the greater the solubility of an herbicide, the greater the amount of that herbicides that gets into soil water.

(e)       Adsorption-desorption: Adsorption refers to the adhesion of pesticide molecules on the surface of soils. This adhesion is the result of physical or chemical attraction between substances.  Desorption is the reverse of adsorption and it refers to the tendency of pesticide molecules to  separate or become detached from the surfaces of soils to  which they are attached. The adsorption of a herbicide into the soil (based on Kd  or Koc) can result in a reduced ability of microorganisms to break it down, as the herbicide is less readily available

(or accessible) in the soil solution. Thus the greater the adsorption the less degradation on the herbicide (Rao, 2000)

(f)       Leaching: Leaching refers to the downward movement of water and its dissolved substances in the soil.  It is a physical process by which a herbicide may be removed from the soil profile. Leaching of herbicides is affected by the chemical properties of the herbicide, the soil texture, solubility of the herbicide, adsorption of herbicide and by the amount of water reaching the soil (Holland and Sinclair, 2004)

(g)       Persistence

Persistence is the property of pesticides, which determines how long they can survive in the environment. The degree of persistence is determined by the length of time a pesticide can remain in the environment and also its effective durability in combating the target pests. Persistence can be expressed in terms of half-life, or the time required for one-half of the pesticide to discompose to products or other molecule than the original pesticide. Pesticides with long degradation half-lives will typically have greater annual pesticide losses in runoff than pesticides with smaller half-lives due to the longer key period where significant amount of pesticides and precipitation occur (Leonard, 1990). The persistence of butachlor in soils depends on many factors like soil moisture, soil temperature, organic matter content and microbial activity (Charkroaborty et al., 1990; Kerle et al., 2007).

1.9.2   Biological Degradation: Abiotic degradation processes may be significant in the dissipation of pesticides from air, soil and water. However, in many cases pesticides or their initial degradation products are relatively stable to abiotic degradation processes. Pesticide residues may also reach environments where conditions are unfavorable for abiotic degradation to occur (e.g. unsuitable pH for hydrolysis or protection from sunlight). Fortunately, biological processes, primarily microbial metabolism, are often highly effective in assisting the dissipation of pesticides once they reach the environment. It includes biotic processes such as microbial or plant metabolism which is a major route of detoxification. However, herbicides may be biologically unavailable because of compartmentalization which occurs as a result of pesticide adsorption to soil and soil colloids without altering the chemical structure of the original molecule (Holland and Sinclair, 2004).

(i)       Microbial Degradation: Much microbial metabolism affecting herbicides in the environment occurs through co-metabolism, i.e. metabolic reactions transform the herbicide molecule incidentally, without the organism deriving energy or useful metabolites for cell growth or division, or only using a portion of the molecule. Microbial activity may also lead to polymerization involving pesticide or metabolite molecules, amide and ether hydrolysis dealkylation, dehalogenation, hydroxylation of aromatic ring, oxidation and additions such as acetylation or methylation, or conjugation with endogenous substrates such as glycosides or amino acids. Changes to the original molecule through these processes may assist in detoxification and elimination of the pesticide. Microbial Metabolism/cometabolism (Aerobically or  Anaerobically), takes place  through enzymatically  mediated oxidative or reductive reactions or other degradative processes mediated by hydrolases, amidases etc. Microorganisms such as bacteria, possess or use several enzymatic pathways including a variety of xenobiotic metabolizing enzymes to protect themselves against the potentially toxic effects  of pesticides.  The  metabolic  fate  of  xenobiotics (pesticides)  involves  two  phase metabolism including cytochrome P450 mediated- Phase I reactions (in some microorganism) and   the   transferase-mediated   conjugation   reactions   (Phase   II)   (Van   Eerd   et   al,

2003).Metabolites formed by organisms initially taking up a pesticide may be amenable to assimilation by other organisms, so enabling degradation to proceed (Holland and Sinclair,

2004).  Microbial breakdown tends to  increase  when  temperatures are  warm,  soil pH  is favourable, soil moisture and oxygen are adequate and soil fertility is good (Rao, 2000). Butachlor is readily degraded by soil microbes. Under aerobic conditions the half-life of butachlor ranges from 3-5 weeks under laboratory conditions. Under anaerobic conditions degradation of butachlor in soil was accelerated (Roberts and Hutson, 1999). Studies have shown that organic amendments hastened the degradation of butachlor due to the presence of microbes (Prakash and Suseela, 2000).

(ii)       Mineralization:  This is the microbial conversion of an compound from an organic form to an inorganic form as in pesticide degradation whereby it results to carbon dioxide, ammonia or water as a terminal metabolite.  Soil micro-organisms biotautilize the herbicide as a source of carbon or other nutrients.  Chemicals such as 2, 4-D are rapidly broken down in the soil while others are less easily broken down e.g 2,4 5-T. Though, some others such as

atrazine are very persistent and are slowly broken down (Stephenson and Solomon, 1993). Mineralization of pesticide molecules by microorganisms often occurs only slowly or to a very limited extent, although the parent molecule may be significantly altered. Mineralization may be indefinitely delayed by incorporation of residues into soil organic matter. Assimilation of useful portions of the original molecule (e.g. as protein) may also delay release of carbon as carbon dioxide(CO2), nitrogen as ammonia(NH3), etc., with those components again being assimilated by higher organisms in the food chain and potentially being incorporated into human foods without being  completely mineralized. Residues of unchanged pesticide or metabolites may persist and may accumulate with repeated use. The extent to which such residues are bioavailable depends on their water solubility and the strength of adsorption or binding to soil or sediment (Holland and Sinclair, 2004) the soil ecosystem.

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