Qualifying and quantifying linuron degradation capacity with colorimetric reaction? Test case on a linuron treated agricultural soil

1 
 
 
1. INTRODUCTION 
 
1.1. Crop protection products   
  
Agriculture and industry increasingly need to use a wide variety of synthetically produced chemicals such as 
insecticides, fungicides, herbicides, and other crop protection products (Don et al., 1981).The use of crop 
protection products is necessary to meet the world‟s demand on foodstuff and so far there are no alternatives 
that can compete with them (Gonçalves et al.,  2005). The Food and Agriculture Organization of the United 
Nations (FAO) defines a crop protection product as any substance or mixture of substances intended for 
preventing, destroying or controlling any pest, including vectors of human or animal disease, unwanted 
species of plants or animals causing harm during or otherwise interfering with the production, processing, 
storage or marketing of food, agricultural commodities, wood and wood products or animal feedstuff or 
which may be administered to animals for the control of insects, arachnids or other pests in or on their 
bodies. The term includes substances intended for use as a plant-growth regulator, defoliant, desiccant or 
fruit-thinning agent or agent for preventing the premature fall of fruit and substances applied to crops either 
before or after harvest to protect the commodity from deterioration during storage and transport. It is evident 
that crop protection products, so defined, are used for the variety of benefits it provides to mankind, but 
unfortunately it also has some side effects (Jeyaratnam 1990). Slow degradation of crop protection products 
in the environment and extensive or inappropriate usage by farmers can lead to environmental contamination 
of the water, soil, air, several types of crops and indirectly to humans (Gonçalves et al., 2005). Crop 
protection products may enter the soil either by direct applications (e.g. agricultural practices) or indirect 
applications (e.g. accidental spillage, leaks at pesticide dump sites, discharge of wastes from production 
facilities, or urban pollution) (Sannino et al., 2001). 
   Herbicides are one of the main categories of crop protection products and they kill weeds and other plants 
that grow where they are not wanted (U.S.EPA ,1995). Among these substances there are the phenylurea 
herbicides that are a group of herbicides used mainly in either pre- or post emergence treatment of cotton, 
fruit, cereal, or other agricultural crops ( Sørensen et al., 2008). Some of the phenylurea herbicides such as 
diuron are also applied as total herbicides on non-crop areas such as roads and railway lines (Giacomazzi et 
al., 2004). This group of herbicides was introduced shortly after Second World War and became one of the 
most important classes of crop protection products (Sørensen et al., 2003). In recent years researchers are 
paying greater attention to this family of herbicides because of their high biotoxicity and possible 
carcinogenic properties and they  require several weeks to months for their removal from the environment 
(Benitez et al., 2009). Due to their extensive use, the phenylurea herbicides are detected in surface water and
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groundwater (Nitschke et al., 1998, Caux et al., 1998 ) and this problem is evident in countries where clean 
drinking water is a limited resource and there is an intensive agricultural production. For example linuron 
concentrations up to 1100 and 2800 μg l
-1
 have been detected in Canadian surface waters and ground waters, 
respectively (Caux et al., 1998). The public concern about the impact on environmental and human health is 
increasing (Rasmussen et al., 2005) and this has led to restrictions in their use ( Turnbull et al., 2001). Some 
of these phenylurea herbicides (isoproturon and diuron) are considered  as priority hazardous substances by 
the Water Framework Directive of the European Commission, and are included in Decision No. 
2455/2001/EC of the European Parliament and of the Council of November 20
th
 2001, in which a list of 
priority substances in the field of water policy is established ( Benitez et al., 2009). 
1.2. Phenylurea herbicides 
 
The phenylurea herbicides include widely used compounds such as diuron, isoproturon (IPU) and linuron 
(Rasmussen et al.,2005). Phenylurea herbicides are systemic herbicides. Following field application, plants 
take up substituted urea herbicides primarily by their root system after which they are transported passively 
via the xylem to the leaves where they inhibit photosynthesis by blocking electron transfer at the level of 
photosystem II. Ultimately, this results in chlorosis and necrosis of the plant (Caux et al., 1998). Physical and 
chemical properties of linuron, diuron and isoproturon are represented in table 1. 
 
 Linuron Diuron Isoproturon 
Substance name  
3-(3,4-dichlorophenyl)-1-
methoxy-1-methylurea 
N‟-(3,4-dichlorophenyl)-N,N-
dimethylurea 
3-(4-isopropylphenyl)-1,1-
dimethylurea 
Molecular formula C
9
H
10
Cl
2
N
2
O
2
 C
9
H
10
Cl
2
N
2
O C
12
H
18
N
2
O 
Molecular structure 
 
 
 
 
Molecular weight 249.1 233.1 206.28 
Colour/form 
White crystalline solid, fine 
flakes, coarse powder, or 
colourless crystals 
White crystalline solid, fine 
flakes, coarse powder, or 
colourless crystals 
slightly yellowish powder 
Odour Odourless Odourless Odourless 
Melting point (°C) 93–94°C 158 - 159°C 155–156 °C 
Vapour preassure 0.002 Pa at 24°C 0.009 mPa at 25 °C 2.8 - 8.1x10-6 at 20°C 
Henry’s Law costant 2.0 × 10
-4
 Pa m
3
 mol
-1
 0.000051 Pa m
3
 mol 
-1
 1.46x10
-5
 at 22°C 
Solubility in water 63.8 mg l
-1
 at pH 7, 20°C 42 mg l
-1
at 20°C 70 mg l
-1
 at 20°C 
Octanol-water 
coefficient(log Kow) 
3 2.6 2.5 
Table 1. Physical and chemical properties of the phenyl urea herbicides linuron, diuron and isoproturon
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Phenylureas are considered to be moderately persistent in the environment. Under field conditions soil half 
life (DT
50
)(degradation time for 50% of the substance) varies widely for different soil types. Diuron is 
persistent in soil from 30 to 365 days (Field et al., 2003; Okamura et al., 2003) although extrapolated values 
of greater than 3000 d have been reported (Madhun et al., 1987). IPU half-life in soil was found to vary 
between 6 and 30 days, with degradation rates linked to soil pH ( Bending et al., 2003). Linuron has a 
variable DT
50
 value in the field in a range from 30 to 150 days ( Cullington et al., 1999). Experiments in 
laboratory for different agricultural soils incubated at 10–20 °C have shown values ranging from a few days 
up to several years (Rasmussen et al., 2005). Field studies in the United Kingdom and Germany have shown 
that normal agricultural application of phenylurea herbicides such as diuron, and IPU can result in surface 
runoff and leaching to underlying groundwater bodies after heavy precipitation (Schuelein et al., 1996; 
Johnson et al., 2001; Gooddy et al., 2002). They can be detected in lakes, rivers and groundwater, in marine 
waters and sediments and in rain collected at urban and rural sites (Eriksson et al., 2007, Green et al., 2006, 
Lapworth et al., 2006, Sørensen et al., 2005, Thomas et al., 2002, Scheyer et al., 2007). Some of this 
phenylureas ,such as IPU, are frequently detected in ground and surface waters in concentrations exceeding 
the drinking water limit of 0.1 μg L
−1
 for pesticides set by the European Community (European Union, 
1998). Degradation products of phenylureas such as 3,4-dichloroaniline (3,4-DCA) have been detected in 
groundwater and surface water (Claver et al., 2006). These metabolites may be even more persistent and bind 
strongly to soil constituents (Johannesen et al., 2003). The EU has included linuron as well as diuron in a list 
comprising endocrine-disrupting chemicals which are suspected of interfering with hormone systems of 
humans and wildlife, causing birth defects, sexual abnormalities and reproductive failure in offspring (EC 
COM(2001)262). Environmental effects of linuron mainly concern aquatic ecosystems since linuron has 
been shown to be highly toxic to aquatic plants (Kegley et al., 2007) and invertebrates (U.S. EPA, 1995). 
Ecotoxicological data suggest that IPU and some of its metabolites are harmful to aquatic invertebrates 
(Mansour et al., 1999), freshwater algae (Pérés et al., 1996) and microbial activity (Remde et al., 1994).  
 
1.3. Degradation of phenylurea herbicides 
 
The phenylurea herbicides can be photodegraded, hydrolysed and biodegraded and persist for periods 
ranging from days to weeks. They are mobile in soil (WHO, 2003). The environmental fate of these 
herbicides in soil, is influenced by several factors such as agricultural practices, environmental 
characteristics, texture and hydrology condition of the soil. Depending on these factors, herbicides can 
evaporate into the atmosphere, adsorb onto the soil, run off into rivers, be leached to ground water, 
accumulate in biota and/or degrade via abiotic and biotic processes (Guzzella et al., 2006). The degradation 
processes (photochemical, chemical and biological) affect the amount of pesticide available for leaching ( 
Cox et al., 1999). The chemical procedures are based on the application of oxidizing reagents such as UV 
radiation, ozone, hydrogen peroxide, etc., or combinations of oxidants in the advanced oxidation processes
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(AOPs) (Benitez et al., 2009). Linuron, isoproturon and diuron are stable to chemical degradation in aqueous 
solution under moderate temperatures and within a pH range of 4-10, so the chemical degradation is of minor 
importance in most agricultural soils (Gerecke et al., 2001, Hill et al., 1955, Salvestrini et. al., 2002 ). As has 
been said above phenylureas have a high hydrolytic stability at neutral pH (e.g. linuron: 1460 days) but may 
become unstable when conditions become more acidic or alkaline. The photochemical degradation may 
occur when the herbicides are exposed to sunlight ( Gerecke et.al., 2001). These photochemical processes 
lead to only partial degradation, creating products that may accumulate in the environment (Sørensen et al., 
2003). Degradation products of phenylureas such as 3,4-dichloroaniline (3,4-DCA) have been detected in 
groundwater and surface water (Claver et.al., 2006). These metabolites may be even more persistent and bind 
strongly to soil constituents (Johannesen et.al., 2003). The 3,4-DCA (derived from linuron or diuron) 
displayed a much higher toxicity than their mother compounds (Tixier et.al., 2001). 
 
   Although (photo)chemical and physical processes could be involved in the degradation of phenylurea 
herbicides on soil or plant surfaces, evidence suggests that the degradation in agricultural soils occurs 
predominantly through the activities of soil micro-organisms, biodegradation is reported to be the most 
significant mechanism for their dissipation from soil (Dejonghe et al.,2003). Biodegradation is, in fact, 
recognized as the primary force in transformation and mineralization of phenylurea herbicides (Turnbull et 
al., 2001). 
  
   The rate of pesticide biodegradation does not remain constant with time, and is dependent on the physico- 
chemical properties of the soil and of the pesticide as well as on the biology of the soil. Prolonged or 
repeated contact between soil microbes and pesticides has been shown to result in an increase in the rate and 
extent of biodegradation (Fenlon et al., 2010). Biodegradation may develop differently due to various 
biological, chemical and/or physical limitations, including the level of microbial activity and the degree of 
pesticide bioavailability (Doick et al., 2003), which may change with time (Ahmad et al., 2004). 
Considerable spatial variability in the degradation potential has been observed in different agricultural soils 
for different phenylurea herbicides. Enrichment-culture techniques have been used with varied success in 
attempts to enrich and isolate phenylurea- degrading micro-organisms (Sørensen et al., 2002, Roberts et al., 
1993, Cullington et al., 1999 ). Several enrichment cultures were set up by inoculating a mineral salt 
medium, containing a phenylurea herbicide as sole source of carbon and nitrogen, with an agricultural soil 
sample (Cullington et al., 1999, El-Fantroussi 2000, Roberts et al., 1993). Attempts to isolate 
microorganisms able to degrade phenylurea herbicides in pure culture have often failed, possibly due to the 
involvement of bacterial consortia rather than single strains (El-Fantroussi 2000, Sørensen et al., 2001) or 
because most soils used in several studies were previously not treated with these herbicides. It was 
hypothesized that prior exposure is necessary for potent bacteria to proliferate or to adapt and evolve the 
necessary catabolic enzymes (Sørensen et al., 2003).
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   Wallnöfer (1969) isolated the strain Bacillus sphaericus ATCC12123, which could metabolize methoxy-
methyl-substituted phenylureas such as monolinuron, linuron and metobromuron but not diuron. 
Streptomycetes sp. PS1/5 was isolated by Shelton et al. in 1996. This strain was able to partially metabolize 
diuron and 11 other phenylureas including linuron. Esposito et al. (1998) isolated three diuron-transforming 
actinomycete strains from a 2,4-dichlorophenoxyacetate (2,4-D)-treated soil, which has been able to degrade 
diuron in the soil after seven days and stated that there is no production of metabolites. Cullington et al. 
(1999) isolated  Arthrobacter globiformis D47 from the Deep Slade agricultural field, that is able to 
transform the phenylurea herbicides diuron, linuron, monolinuron, metoxuron, and IPU ( in the order, by 
rate, linuron>diuron>monolinuron>metoxuron>isoproturon ) to their respective aniline derivatives, that 
accumulated in the medium. El-Deeb et .al. (2000) obtained the first organism capable of complete diuron 
degradation from a phenylurea-treated soil and proved it to be a Pseudomonas sp Bk8. Another Arthrobacter 
sp., the N2 strain, has been found to degrade diuron (Widehem et al., 2001), chlorotoluron and isoproturon 
(Tixier et al., 2002). Sørensen et al.(2001) isolated Sphingomonas sp strain SRS2 that was capable to 
mineralize [phenyl-U-
14
C]isoproturon to [
14
C] to carbon dioxide and biomass from a previously isoproturon-
treated British agricultural field. A stable linuron-degrading enrichment culture, which could not degrade 
monuron, diuron and metoxuron has been obtained by Roberts et al. (1993).Other studies showed that 
Sphingomonas sp strain F35 was responsible for IPU metabolism (Bending et al., 2003). In the last years 
other strains capable of complete diuron-degradation were isolated from diuron-contaminated surface water. 
These strains, are Pseudomonas sp. IB78 and Stenotrophomonas sp. IB93 (Batisson et al., 2007). Dejonghe 
et al. (2003) isolated Variovorax sp. WDL1 as part of a consortium which was responsible for linuron 
mineralization. This strain performed a crucial role by converting linuron into 3,4-DCA and also being able 
of the further mineralization of this metabolite. El Sebai (2004) obtained a Methylophila strain from a French 
agricultural soil which could mineralize IPU but not diuron or linuron. Variovorax sp. SRS16 was isolated as 
a linuron-mineralizing strain from a Danish linuron-treated agricultural field, by Sørensen et al. (2005). 
Recently, Sørensen et al.(2008) showed that this strain is also able to mineralize diuron in significant 
amounts (up to 65%) when growth substrates such as yeast extract were added to the medium. 
Mycobacterium brisbanense strain JK1, a bacterium capable of degrading the herbicide diuron, was isolated 
from herbicide exposed soil (Khurana et al., 2009). 
 
   The lack of success in isolating individual phenylurea-degrading bacteria from undefined cultures has been 
attributed to the involvement of metabolically co-operating microbial communities. In fact it was observed 
that the combined action of several species enhances or is even required for complete mineralization of 
phenylurea herbicides. One reason for incomplete microbial degradation is the presence of a variety of 
substituted groups on the aromatic backbone, each of which requires slightly different catabolic enzymes for 
total breakdown. The collection of these enzymes would more likely be present in consortia than in single 
bacteria (Dejonghe et al., 2003). Sorensen et al. (2002) described a co-culture performing rapid and extensive
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growth-linked mineralization of IPU when provided as the sole carbon and nitrogen source. The co-culture 
consisted of a Sphingomonas sp. (designated strain SRS2) and an unknown soil bacterium (designated strain 
SRS1) both enriched and isolated from a British agricultural soil. Other studies showed that the strain SRS1 
might supply SRS2 with amino acids (L-methionine) resulting in an efficient IPU degrading consortium. 
Dejonghe et.al., (2003) observed  that  there is a complex synergistic interaction between different bacteria in 
the complete linuron degradation (Fig.1). This authors isolated five bacteria from the consortium, identified 
as Variovorax paradoxus WDL1, Delftia acidovorans  WDL34, Pseudomonas sp WDL5, Comamonas 
testosteroni WDL7 and Hyphomicrobium sulfonivorans WDL6. Theirs study  showed that the corresponding 
bacterial strain Variovorax sp WDL1 may be the key player in the initial transformation of linuron. This 
strain used  linuron as the sole source of C, N and energy and first converted linuron to 3,4- dichloroaniline 
(3,4-DCA), which transiently accumulated in the medium but was subsequently degraded. This metabolite is 
degraded by Comamonas testosteroni WDL7 and Delftia acidovorans WDL34. The strain WDL1 also 
released an amount of N,O-DMHA, which is very efficiently taken up by H. sulfonivorans WDL6, the only 
member of the linuron-degrading culture that can grow on this compound. The strain  WDL5 was not 
actively involved in linuron metabolism but the linuron degradation rate increased when WDL1 was co-
cultured with WDL5. It was suggested that  Pseudomonas sp WDL5 provided WDL1 with nutrients or 
growth factors.