Due to trifluralin chemical structure, this herbicide tends to receive two electrons, which significantly increases its toxicity, since the group NH2 hydrogen of trifluralin tends to bind the polar group of cellular membranes and cause disorganization to its structure, eventually bringing function disorders [63]. This disorganization in the membrane structure seems to interfere mainly in the permeability of plasma and mitochondrial membranes. Trifluralin changes the permeability of membranes because it promotes a collapse in their electric potential, making Ca+2 efflux of the mitochondrial inner membranes and Ca+2 go from the outer to the inner surface of the cell membrane via uniporters, thus increasing the concentrations of

Since low levels of calcium are needed for polymerization, Hepler [64] ascertains that mitotic spindles may undergo disorders due to the high levels of this ion. Low concentrations of free calcium in the cytoplasm (0.1-0.2 μM) are essential to prevent phosphorus precipitation,

According to Alberts et al. [46], Ca+2 is important for regulating mitochondrial enzyme activity, and it is imported from the cytosol through an H+ electrostatic gradient. It is also believed that this process is important to remove Ca+2 from the cytosol when cytosolic Ca+2 levels get

**Figure 7.** Comparisons among sequences of α-tubulin amino acids of species *Zea mays* (vegetable)*, Hordeumvulgare* (vegetable)*, Arabidopsis thaliana* (vegetable) *Prunus amygdalus* (vegetable)*, Pisum sativum* (vegetable),Leishmania d*onovani* (parasite)*, Trypanosoma cruzi* (vegetable),*Mus musculus* (vertebrate)*, Sus scrofa* (vertebrate) and Homo sapi‐ *ens*. The sequences were obtained from the data base at NCBI (National Center of Biotechnology Information) in ac‐ cordance with the codes P14641, Y08490, P29511, P33629, U12589, U09612, M97956, P05213, P02550 and P04687, respectively [55]. The sequences were aligned by means of the ClustalW program [56], using default parameters. The

compete with Mg2+ for binding sites and act as a secondary messenger [65].

Ca2+ ions in cytoplasm, influencing onion root mitosis.

500 Herbicides - Current Research and Case Studies in Use

such ions in the inner cytoplasmic membrane.

alignment was then analyzed using the MPALign program [57].

dangerously high.

Anilines are compounds that cause a variety of toxic effects depending on the structural changes they undergo. Several studies demonstrate that anilines and halogens can induce metahemoglobin formation and also be toxic to the kidneys and the liver, either treated *in vitro* or *in vivo* [69] and [70]. Aminophenols, the primary products of aniline metabolism, are compounds related to neurotoxicity induction [69].

### **5. Trifluralin toxic effect**

Although many researchers and international governmental agencies have investigated and published trifluralin toxic effects on different fields, whether they are related to either acute or chronic toxicity, cytotoxicity, genotoxicity, mutagenicity and carcinogenicity, the results shown are confusing and often contradictory.

According to the W.H.O (World Health Organization) [70], trifluralin causes hemoglobin oxidation (by forming metahemoglobin), red blood cell destruction, besides being toxic to the kidneys and the liver, and stimulating depression in the central nervous system. It may cause vomiting, diarrhea, weakness, profuse sweating, loss of sight, memory and concentration, and dermatitis as well. This herbicide is considered to be neurotoxic and gastrointestinal irritant. It can lead to death because of ventricular fibrillation [71], although several authors [10], [72], [73], [74], [75] and [76], ascertain that trifluralin is a low toxicity substance.

Trifluralin lethal concentrations and doses for vertebrates and invertebrates are shown in Table 2.

According to the Occupational Health Service [79], prolonged skin contact with trifluralin may cause allergic dermatitis. The WSSA [80] states that administering trifluralin to dogs while washing them for two years does not cause toxic effects. However, in trifluralin chronic assays conducted with 60 animals (F344 mice), which received 0.813, 3250 and 6500 ppm dietary does

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Worthing [71] states that trifluralin is highly toxic and neurotoxic. The author ascertains that the herbicide is capable of accumulating in the adipose tissue and inhibiting the immunologic function of the thymus. Trifluralin is regarded as possibly teratogenic and fetal toxicity.It has the property of altering the endocrine and reproductive system, and it reduces the quantity of

In studies conducted by Ovidi et al. [82], they tested trifluralin concentration of 1.53 mg/ml and observed that the herbicide exerts a specific effect on the reproductive system in plants, by direct action on the formation of the pollinic tubes, since it causes complete microtubule depolymerization. The authors even suggest that pollinic microtubule cytoskeleton may be used as bioindicators for studies on toxicity induced by aneugenic agents such as trifluralin.

As a general rule, the effects of pesticides may be diversified, such as the direct reaction with nuclear DNA; incorporation of DNA during cellular replication; interference in mitosis or

Genotoxic effects may lead to DNA breaks, causing loss of genetic material and mutations which lead to cell death or result in carcinogenesis. Genotoxicity is assessed by different tests, carry out with several organisms and provide safe, precise information regarding their potential to damage the DNA.There are a number of reports evaluating trifluralin for geno‐ toxicity, immunotoxicity, and reproductive toxicity, although the results are not entirely

Chromosome aberration tests have shown evidences of trifluralin mutagenicity for different plant species [85], [86], [87], [88], [89] and [90]. Könen and Çavas [91], Peña [92] and Canevari [93] ascertained that the herbicide is capable of inducing significant microtubule rates in *Oreochromis niloticus*. Kaya et al. [94] also ascertained that the herbicide may be considered genotoxic to *Drosophila melanogaster*, since it exhibited positive outcomes for the Somatic Mutation and Recombination Test (SMART). Tests conducted in the bone marrow of mice exposed to trifluralin showed that it is potentially genotoxic [95] and it is also capable of influencing serum concentration of reproductive and metabolic hormones, especially thyroxin [96]. Nonetheless, tests performed on bacteria [14], on *Drosophyla melanogaster* conducted by Bryant and Murnik [97] and Foureman [98], on cells taken from the bone marrow of mice conducted by Nehéz et al. [99], Pilinkaya [100], Gebel et al., [95], and on cell culture conducted by IARC [101] and Ribas et al. [35 and 102] demonstrated contradictory results. According to Chan and Fong [103], Bhattacharya et al. [104] and Esteves et al. [105], due to its characteristics, mechanisms of action and, especially its reduced effects on human cells, trifluralin can be regarded as a promising substance for fighting Leishmaniasis. There is also research that confirms the use of trifluralin as a powerful antiparasitic to treat *Trypanosoma* [106] and [107],

for two years, damage to their liver and kidneys were observed [81].

semen, besides increasing the number of abnormal sperm.

meiosis, resulting from incorrect cell division [83].

*Toxoplasma* [48] and *Plasmodium* [108].

consistent, trifluralin does not appear to be strongly genotoxic [84].

**Table 2.** TrifluralinCL50 and DL50 for different organisms

Meister [78] conducted tests with animals and verified that trifluralin does not have any toxic effect on them when they are exposed to the product either through ingestion, inhalation or when in contact with the skin. Nauseas and severe gastrointestinal discomfort may occur after trifluralin ingestion. When placed in the rabbit eyes, it produced a mild irritation, which was reverted within seven days. In humans, it may induce skin allergies and, when inhaled, it may irritate the throat and the lungs.

Table 3 shows some information regarding trifluralin chronic, sub-acute and sub- chronic toxicity to different organisms.

\*Liver weight of the animals submitted to the 50 and 100mg Kg-1 diet somehow showed to be higher, when compared to the control animals. Data extracted from Gangolli [77].

**Table 3.** Data on trifluralin sub-acute, chronic and sub-chronic toxicity.

According to the Occupational Health Service [79], prolonged skin contact with trifluralin may cause allergic dermatitis. The WSSA [80] states that administering trifluralin to dogs while washing them for two years does not cause toxic effects. However, in trifluralin chronic assays conducted with 60 animals (F344 mice), which received 0.813, 3250 and 6500 ppm dietary does for two years, damage to their liver and kidneys were observed [81].

**Treatment Species Group Popular Name Toxicity** Cl50 (48h) *Lepomis macrochirus* Fish Bluegill 19 µg L-1 CL50 (48h) *Mola mola* Fish Ocean sunfish 19µg L-1 CL50 (48h) *Cyprinus carpio* Fish Common carp 1.0mg L-1 CL50 (96h) *Oncorhynchus mykiss* Fish Rainbow trout 0,21mgL-1

(Young)fish

Meister [78] conducted tests with animals and verified that trifluralin does not have any toxic effect on them when they are exposed to the product either through ingestion, inhalation or when in contact with the skin. Nauseas and severe gastrointestinal discomfort may occur after trifluralin ingestion. When placed in the rabbit eyes, it produced a mild irritation, which was reverted within seven days. In humans, it may induce skin allergies and, when inhaled, it may

Table 3 shows some information regarding trifluralin chronic, sub-acute and sub- chronic

*Ratus norvegicus* Mammal Mouse 25, 50 e 100

\*Liver weight of the animals submitted to the 50 and 100mg Kg-1 diet somehow showed to be higher, when compared

CL50 (48h) *Daphnia magma* Micro-crustacean - 0,56 mgL-1 CL50 (96h) *Procambarus clarkia* Crustacean Lobster 12mgL-1 DL50 (oral) *Apis mellifera* Insect Honey bee 0,011mg bee-1 DL50 (oral) *Mus musculus* Mammal Laboratory mice >500 mg kg -1 DL50 (oral) *Ratus norvegicus* Mammal Laboratory mice > 10.000 mg kg-1 DL50 (oral) - Mammal Dog > 200 mg kg-1 DL50 (oral) - Mammal Rabbit > 200 mg kg-1 DL50 (oral) - Bird Hen > 200 mg kg-1

Rainbow trout, Bluegill, Ocean sunfish

**Name Toxicity Symptoms**

mg kg-1 dia-1

erythema

no effects produced on either survival or appearance \*

10-90µg L-1

CL50 (96h)

*Oncorhynchus mykiss*, *Lepomis macrochirus, Mola*

**Table 2.** TrifluralinCL50 and DL50 for different organisms

**Treatment Species Group Popular**

to the control animals. Data extracted from Gangolli [77].

**Table 3.** Data on trifluralin sub-acute, chronic and sub-chronic toxicity.

LOEC *Amphiprion percula* Fish clownfish 5µg L-1 - NOEL *Amphiprion percula* Fish clownfish 2µ L-1 - CE50 (10 days) *Chlorococcum* sp Protozoa - 2,5 mg L-1 -

*mola*

502 Herbicides - Current Research and Case Studies in Use

Data extracted from Gangolli [77].

irritate the throat and the lungs.

toxicity to different organisms.

Sub-acute(dermis

chronic(ingestion - 3 months)

Sub-

Worthing [71] states that trifluralin is highly toxic and neurotoxic. The author ascertains that the herbicide is capable of accumulating in the adipose tissue and inhibiting the immunologic function of the thymus. Trifluralin is regarded as possibly teratogenic and fetal toxicity.It has the property of altering the endocrine and reproductive system, and it reduces the quantity of semen, besides increasing the number of abnormal sperm.

In studies conducted by Ovidi et al. [82], they tested trifluralin concentration of 1.53 mg/ml and observed that the herbicide exerts a specific effect on the reproductive system in plants, by direct action on the formation of the pollinic tubes, since it causes complete microtubule depolymerization. The authors even suggest that pollinic microtubule cytoskeleton may be used as bioindicators for studies on toxicity induced by aneugenic agents such as trifluralin.

As a general rule, the effects of pesticides may be diversified, such as the direct reaction with nuclear DNA; incorporation of DNA during cellular replication; interference in mitosis or meiosis, resulting from incorrect cell division [83].

Genotoxic effects may lead to DNA breaks, causing loss of genetic material and mutations which lead to cell death or result in carcinogenesis. Genotoxicity is assessed by different tests, carry out with several organisms and provide safe, precise information regarding their potential to damage the DNA.There are a number of reports evaluating trifluralin for geno‐ toxicity, immunotoxicity, and reproductive toxicity, although the results are not entirely consistent, trifluralin does not appear to be strongly genotoxic [84].

Chromosome aberration tests have shown evidences of trifluralin mutagenicity for different plant species [85], [86], [87], [88], [89] and [90]. Könen and Çavas [91], Peña [92] and Canevari [93] ascertained that the herbicide is capable of inducing significant microtubule rates in *Oreochromis niloticus*. Kaya et al. [94] also ascertained that the herbicide may be considered genotoxic to *Drosophila melanogaster*, since it exhibited positive outcomes for the Somatic Mutation and Recombination Test (SMART). Tests conducted in the bone marrow of mice exposed to trifluralin showed that it is potentially genotoxic [95] and it is also capable of influencing serum concentration of reproductive and metabolic hormones, especially thyroxin [96]. Nonetheless, tests performed on bacteria [14], on *Drosophyla melanogaster* conducted by Bryant and Murnik [97] and Foureman [98], on cells taken from the bone marrow of mice conducted by Nehéz et al. [99], Pilinkaya [100], Gebel et al., [95], and on cell culture conducted by IARC [101] and Ribas et al. [35 and 102] demonstrated contradictory results. According to Chan and Fong [103], Bhattacharya et al. [104] and Esteves et al. [105], due to its characteristics, mechanisms of action and, especially its reduced effects on human cells, trifluralin can be regarded as a promising substance for fighting Leishmaniasis. There is also research that confirms the use of trifluralin as a powerful antiparasitic to treat *Trypanosoma* [106] and [107], *Toxoplasma* [48] and *Plasmodium* [108].

Studies carried out by Peña [92] and Canevari [93] indicate that low trifluralin concentrations may induce mutagenic effects. These authors observed significant presence of micronuclei in erythrocytes of fish submitted to acute treatments with this herbicide. When the micronuclei diameters were measured by Canevari [93], data indicated that they could be derived from losses of whole chromosomes, thus proving the aneugenic effect of the herbicide due to the pesticide interference in the mitotic spindle.

Fernandes et al. [89] ascertained that, among the root meristems of *Allium cepa* under division, trifluralin promotes a significant increase in the irregular metaphase rate. These data corrob‐ orate the statement of Lignowski and Scott [85], Lee et al. [109], Dow et al. [110], Werbovetz et al. [111] and Ovidi et al. [82], who characterized trifluralin as a powerful microtubule inhibitor, which is therefore capable of accumulating a large amount of meristematic cells in metaphase.

Characterization, Modes of Action and Effects of Trifluralin: A Review

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Genotoxicity tests using the comet assay in human lymphocyte cultures showed that trifluralin produced a significant increase in the length of the comet's tail. This increase is due to DNA breaks, since there is an induction of nucleotide excision repair, resulting from damage caused by the herbicide action [103]. As for the frequency of comet-bearing cells, the author observed that, after 48 hours of exposure to the herbicide, few tailed nucleoides were found. These results

According to Ribas et al. [35], trifluralin has a genotoxic effect on human cell cultures because it causes a decrease in cell proliferation. The same author ascertains that this herbicide has not revealed carcinogenic effects, since it caused little induction exchange between sister chroma‐ tids.The micronucleus test conducted by Ribas et al. [35], used for detecting aneugenic activity, has also produced a negative response, which contradicts studies carried out by several other authors [88], [89], [91], [92], [97], [112], among others) who ascertain that trifluralin brings about chromosome aberrations and nuclear alterations resulting from problems in the mitotic spindle

According to Kang et al. [113], trifluralin is not associated with bladder, kidney, liver, leukemia, colorectal or hematopoietic-lymphatic cancers. The authors only suggest a possible connection between trifluralin exposure and the risk of colon cancer in human beings, but the inconsis‐ tency per exposure level and a small number of colon cancers indicate that this could be an

Data from the National Cancer Institute (NCI) [114] report that mice subjected to trifluralin chronic exposure, at low concentrations, had an increase in hepatocellular carcinoma and higher incidence of alveolar bronchial adenomas. An increase in bladder cancer was also verified in mice exposed to low trifluralin concentrations. It was observed that, when male mice were submitted to high doses of trifluralin, they presented higher incidence of follicular cell and thyroid gland tumors [115]. Trifluralin has been reported to cause a significant increase

**Figure 10.** Meristematic cells of *Allium* cepa treated with trifluralin. **A.** cell with micronucleus; **B.** cell with micronu‐

proved to be statistically significant, though.

cleus and an adjacent mini cell; **C.**polynucleated cell.

incidental finding.

*Allium cepa*meristematic cells treated with trifluralin also presented problems during mitosis, such as polyploidies, C-metaphases, multipolar anaphases, anaphase-telophase chromatin bridges, chromosome delay and loss of genetic material [89]. (Figure 9).

**Figure 9.** Meristematic cells of *Allium cepa* treated with trifluralin. **A.** C-metaphase; **B.**polyploid cell; **C.** multipolar cell; **D**. loss of genetic material; **E.**chromosome bridge; **F.**telophase with chromosome delay.

According to Fernandes et al. [88], in the bioassays with root meristems of *Allium cepa* treated with trifluralin, a large amount of interphase cells with more than one nucleus and cells with micronuclei and a mini cell were observed (Figure 10).

Lignowski and Scott [85] observed C-metaphases, micronuclei, amoeboid nuclei and poly‐ ploidies in root meristems of wheat and onion submitted to trifluralin action. Due to the occurrence of irregular metaphases, they concluded that the mitotic spindle might have been broken owing to the herbicide action on it.

Bioassays performed with trifluralin, using *Pisum sativum* as test material revealed a positive action of the herbicide with the increase in chromosome alterations, C-mitosis and anti-mitosis effects [87].

Fernandes et al. [89] ascertained that, among the root meristems of *Allium cepa* under division, trifluralin promotes a significant increase in the irregular metaphase rate. These data corrob‐ orate the statement of Lignowski and Scott [85], Lee et al. [109], Dow et al. [110], Werbovetz et al. [111] and Ovidi et al. [82], who characterized trifluralin as a powerful microtubule inhibitor, which is therefore capable of accumulating a large amount of meristematic cells in metaphase.

Studies carried out by Peña [92] and Canevari [93] indicate that low trifluralin concentrations may induce mutagenic effects. These authors observed significant presence of micronuclei in erythrocytes of fish submitted to acute treatments with this herbicide. When the micronuclei diameters were measured by Canevari [93], data indicated that they could be derived from losses of whole chromosomes, thus proving the aneugenic effect of the herbicide due to the

*Allium cepa*meristematic cells treated with trifluralin also presented problems during mitosis, such as polyploidies, C-metaphases, multipolar anaphases, anaphase-telophase chromatin

**Figure 9.** Meristematic cells of *Allium cepa* treated with trifluralin. **A.** C-metaphase; **B.**polyploid cell; **C.** multipolar cell;

According to Fernandes et al. [88], in the bioassays with root meristems of *Allium cepa* treated with trifluralin, a large amount of interphase cells with more than one nucleus and cells with

Lignowski and Scott [85] observed C-metaphases, micronuclei, amoeboid nuclei and poly‐ ploidies in root meristems of wheat and onion submitted to trifluralin action. Due to the occurrence of irregular metaphases, they concluded that the mitotic spindle might have been

Bioassays performed with trifluralin, using *Pisum sativum* as test material revealed a positive action of the herbicide with the increase in chromosome alterations, C-mitosis and anti-mitosis

**D**. loss of genetic material; **E.**chromosome bridge; **F.**telophase with chromosome delay.

micronuclei and a mini cell were observed (Figure 10).

broken owing to the herbicide action on it.

effects [87].

bridges, chromosome delay and loss of genetic material [89]. (Figure 9).

pesticide interference in the mitotic spindle.

504 Herbicides - Current Research and Case Studies in Use

Genotoxicity tests using the comet assay in human lymphocyte cultures showed that trifluralin produced a significant increase in the length of the comet's tail. This increase is due to DNA breaks, since there is an induction of nucleotide excision repair, resulting from damage caused by the herbicide action [103]. As for the frequency of comet-bearing cells, the author observed that, after 48 hours of exposure to the herbicide, few tailed nucleoides were found. These results proved to be statistically significant, though.

According to Ribas et al. [35], trifluralin has a genotoxic effect on human cell cultures because it causes a decrease in cell proliferation. The same author ascertains that this herbicide has not revealed carcinogenic effects, since it caused little induction exchange between sister chroma‐ tids.The micronucleus test conducted by Ribas et al. [35], used for detecting aneugenic activity, has also produced a negative response, which contradicts studies carried out by several other authors [88], [89], [91], [92], [97], [112], among others) who ascertain that trifluralin brings about chromosome aberrations and nuclear alterations resulting from problems in the mitotic spindle

According to Kang et al. [113], trifluralin is not associated with bladder, kidney, liver, leukemia, colorectal or hematopoietic-lymphatic cancers. The authors only suggest a possible connection between trifluralin exposure and the risk of colon cancer in human beings, but the inconsis‐ tency per exposure level and a small number of colon cancers indicate that this could be an incidental finding.

Data from the National Cancer Institute (NCI) [114] report that mice subjected to trifluralin chronic exposure, at low concentrations, had an increase in hepatocellular carcinoma and higher incidence of alveolar bronchial adenomas. An increase in bladder cancer was also verified in mice exposed to low trifluralin concentrations. It was observed that, when male mice were submitted to high doses of trifluralin, they presented higher incidence of follicular cell and thyroid gland tumors [115]. Trifluralin has been reported to cause a significant increase

**Figure 10.** Meristematic cells of *Allium* cepa treated with trifluralin. **A.** cell with micronucleus; **B.** cell with micronu‐ cleus and an adjacent mini cell; **C.**polynucleated cell.

in thyroid follicular cell tumors in male Fischer 344 rats only at the highest dietary dose of 6500ppm in a 2-year chronic study [115].

[3] Baird, C. QuímicaAmbiental. Porto Alegre: Bookman, 2002.

search Triangle Park v. 105, p. 308-314, 1997.

Neurology, Madras v.50, p.1346-1350, 1998.

Rev. Environ. Contam. Toxicol. v. 153, p. 1-64, 1997

nhas. Jaboticabal: FUNEP, 1992.

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2005.

EditoraPlantarum. 1990.

[4] Lorenzi, H. Manual de identificação e controle de plantas daninhas. Nova Odessa:

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[5] Oliveira Jr., R.S. Mecanismo de açãoherbicidas. In: Biologia e Manejo de PlantasDani‐

[6] Munger, R., Isacson, P., Hu, S., Burns, T., Hanson, J., Lynch, C.F., Cherryholmes, K., Vandorpe, P., Hausler, Jr. W. J. Intrauterine growth redardation in Iowa communities with herbicides-contaminated drinking watersupplies. Environ. Health Perspect., Re‐

[7] Gorell, J.M., Jhonson, C.C., Rybicki, B.A., Peterson, E.L., Ricchardson, R.J. The risk of Parkinson's disease with exposure to pesticides, farmin, well water, and rural living.

[8] Deuber, R. Botânica das plantasdaninhas. In: DEUBER, R. Ciência das plantasdani‐

[9] Grover, R., Wolt, J.D., Cessna, A. J., Schiefer, H.B. Environmental fate of trifluralin.

[10] Rodrigues, B. N., Almeida, F. S.Guia de herbicidas, 5ª ed., Grafmarke: Londrina,

[11] Sanders Pf, Seiber Jn. A chamber for measuring volatilization of pesticides for model soil and water disposal system. Chemosphere, Oxford v.12, p. 999-1012, 1983.

[12] Calderon, M.J., Hermosín, M.C., Cornejo, J. Y Moreno, F. Movilidad de trifluralina en laboreo tradicional y de conservación. Estudios de la Zona No Saturada del Suelo.

[13] Bellinaso M De. L., Henrique L.A., Gaylarde C.C., Greer C.W. Genes similar tonaph‐ thalenedioxygenase genes in trifluralin-degrading bacteria. Pest Manag. Sci., Sus‐

[14] U.S. Environmental Protection Agency. 1987. Trifluralin health advisory. Office of

[15] Cooper, M.T., Porter, T.D. Mutagenicity of nitrosamines in methyltransferase-defi‐ cient strains of *Salmonella typhimurium*coexpressing human cytochrome P450 2E1 and

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nhas. Oliveira Jr., R.S., Constantin, J., Inoue, M.H (Eds) Omnipax, 2011.

### **6. Final considerations**

The increase in agricultural productivity has occurred thanks to several factors, among which are improvements in genetics, agricultural machinery and the use of substances that allow control of weeds in agriculture.

The use of pesticides has generated discussions and controversy among the scientific com‐ munity and its users, registering advantageous and disadvantageous recommendations in different ways. Among contrary recommendations to the use of pesticides, we can point out lack of detailed studies on the action of such chemicals on the exposed organisms, making it impossible to associate their action with the emergence of eventual problems. In the soil, trifluralin is moderately persistent, which might jeopardize organisms that are eventually exposed to it. Trifluralin is a substance that has a microtubule-depolymerizing activity, which prevents cell division, a fact that might compromise organism development.

Existing reports characterize trifluralin as a highly acute toxic substance to fish, but there are not enough descriptions of its chronic toxicity and cytotoxic effect. Studies mainly related to its genotoxic, mutagenic and carcinogenic potential are mostly inconclusive or even contra‐ dictory. There is little information about the toxicity of products derived from trifluralin degradation and its effects on the organisms.

### **Author details**

Thaís C. C. Fernandes, Marcos A. Pizano and Maria A. Marin-Morales\*

\*Address all correspondence to<EMAIL_ADDRESS>
Universidade Estadual Paulista, IB-Campus de Rio Claro, Rio Claro/SP, Brasil

### **References**

[3] Baird, C. QuímicaAmbiental. Porto Alegre: Bookman, 2002.

in thyroid follicular cell tumors in male Fischer 344 rats only at the highest dietary dose of

The increase in agricultural productivity has occurred thanks to several factors, among which are improvements in genetics, agricultural machinery and the use of substances that allow

The use of pesticides has generated discussions and controversy among the scientific com‐ munity and its users, registering advantageous and disadvantageous recommendations in different ways. Among contrary recommendations to the use of pesticides, we can point out lack of detailed studies on the action of such chemicals on the exposed organisms, making it impossible to associate their action with the emergence of eventual problems. In the soil, trifluralin is moderately persistent, which might jeopardize organisms that are eventually exposed to it. Trifluralin is a substance that has a microtubule-depolymerizing activity, which

Existing reports characterize trifluralin as a highly acute toxic substance to fish, but there are not enough descriptions of its chronic toxicity and cytotoxic effect. Studies mainly related to its genotoxic, mutagenic and carcinogenic potential are mostly inconclusive or even contra‐ dictory. There is little information about the toxicity of products derived from trifluralin

prevents cell division, a fact that might compromise organism development.

Thaís C. C. Fernandes, Marcos A. Pizano and Maria A. Marin-Morales\*

Universidade Estadual Paulista, IB-Campus de Rio Claro, Rio Claro/SP, Brasil

[1] Londres, F. Agrotóxicos no Brasil: um guiaparaaçãoemdefesa da vida. – Rio de Ja‐ neiro: AS-PTA – Assessoria e Serviços a ProjetosemAgriculturaAlternativa, 2011. 190

[2] Kotaka, E. T., Zambrone, F.A.D. Contribuições para a construção de diretrizes de avaliação do risco toxicológico de agrotóxicos. Campinas, SP: ILSI Brasil, 2001.

6500ppm in a 2-year chronic study [115].

506 Herbicides - Current Research and Case Studies in Use

**6. Final considerations**

control of weeds in agriculture.

degradation and its effects on the organisms.

\*Address all correspondence to<EMAIL_ADDRESS>
**Author details**

**References**

p.

[17] Laabs, V., Amelung, W., Pinto, A., Altstaedt, A., Zech, W. Leaching and degradation of corn and soybean pesticides in an Oxisol of the Brazilian Cerrados Chemosphere. v. 41, p. 1441-1449, 2000.

[31] Grover, R., Cessna, A.J., Waite, D.T. Volatilization losses na transport in air of tria‐ zine herbicides. In: Le Baron, H.M., Gianessi, L.P., Mcfarland, J., Burnside, O.C., edi‐ tors. The triazine herbicides. Amsterdam, the Netherlands: Elservier Science B.V.,

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[32] Waite, A.D.T., Bailey, A. P. Sproull, B. J.F., Quiring, A. D.V., Chau, B.D..F J., Bailey, C. J. Cessna, C. Atmospheric concentrations and dry and wet deposits of some herbi‐ cides currently used on the Canadian Prairies. Chemosphere, Oxford. v. 58, p.693–

[33] U.S. Environmental Protection Agency. 1993. Health advisories for drinking waters

[34] Mongar, K., Miller, G.C. Vapor phase photolysis of trifluralin in an outdoor chamber:

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**Chapter 20**

**Allelochemicals as Bioherbicides — Present and**

Since the first implementation of synthetic herbicides in crop protection systems, weeds have continuously developed resistance. As a main reason of such evolution, long-lasting exploi‐ tation of herbicides with one target site in plants is considered. This has been the case with the first widely-used triazine herbicides, photosynthesis inhibitors, which have effectively eliminated a wide range of weeds. Unfortunately, inappropriate adjustment of herbicides to weed species occupying fields, application of herbicides at the incorrect developmental stage and in unsuitable weather conditions have contributed to the accumulation of active com‐ pounds in the soil, accumulation of weed species and acceleration evolution of resistant biotypes [1]. To date, there have been 211 species and 393 biotypes of herbicide resistant weeds identified [2]. Most of them are resistant to B, C1 and A groups of herbicides, inhibitors of: acetolactate synthase (ALS), photosystem II and acetyl CoA carboxylase, respectively. Ten species pose the biggest threat for crops due to causing yield losses, including the most important herbicide-resistant species which are characterized by multiple resistances: rigid ryegrass (*Lolium rigidum* Gaud.), wild oat (*Avena fatua* L.) and redroot pigweed (*Amaranthus*

Evolution of weeds resistant to herbicides demands new solutions to cope with the problem since economic losses generated by weeds can be higher than those caused by other pests. Due to the fact that abandoning chemical weed control is, with current agricultural practices, rather impossible, it is necessary to create new classes of herbicides with new mechanisms of action and target sites not previously exploited. Presently used synthetic herbicides are not approved for use in organic agriculture. Moreover, using crop protection chemicals also need public acceptance. [3]. The number of synthetic chemicals with new target sites are decreasing

> © 2013 Soltys et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 Soltys et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

distribution, and reproduction in any medium, provided the original work is properly cited.

**Perspectives**

Dorota Soltys, Urszula Krasuska,

http://dx.doi.org/10.5772/56185

**1. Introduction**

*retroflexus* L.).

Renata Bogatek and Agnieszka Gniazdowska

Additional information is available at the end of the chapter

## **Allelochemicals as Bioherbicides — Present and Perspectives**

Dorota Soltys, Urszula Krasuska, Renata Bogatek and Agnieszka Gniazdowska

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/56185

### **1. Introduction**

Since the first implementation of synthetic herbicides in crop protection systems, weeds have continuously developed resistance. As a main reason of such evolution, long-lasting exploi‐ tation of herbicides with one target site in plants is considered. This has been the case with the first widely-used triazine herbicides, photosynthesis inhibitors, which have effectively eliminated a wide range of weeds. Unfortunately, inappropriate adjustment of herbicides to weed species occupying fields, application of herbicides at the incorrect developmental stage and in unsuitable weather conditions have contributed to the accumulation of active com‐ pounds in the soil, accumulation of weed species and acceleration evolution of resistant biotypes [1]. To date, there have been 211 species and 393 biotypes of herbicide resistant weeds identified [2]. Most of them are resistant to B, C1 and A groups of herbicides, inhibitors of: acetolactate synthase (ALS), photosystem II and acetyl CoA carboxylase, respectively. Ten species pose the biggest threat for crops due to causing yield losses, including the most important herbicide-resistant species which are characterized by multiple resistances: rigid ryegrass (*Lolium rigidum* Gaud.), wild oat (*Avena fatua* L.) and redroot pigweed (*Amaranthus retroflexus* L.).

Evolution of weeds resistant to herbicides demands new solutions to cope with the problem since economic losses generated by weeds can be higher than those caused by other pests. Due to the fact that abandoning chemical weed control is, with current agricultural practices, rather impossible, it is necessary to create new classes of herbicides with new mechanisms of action and target sites not previously exploited. Presently used synthetic herbicides are not approved for use in organic agriculture. Moreover, using crop protection chemicals also need public acceptance. [3]. The number of synthetic chemicals with new target sites are decreasing

© 2013 Soltys et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Soltys et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

dramatically. Eco-friendly trends in weed management force scientists to reach for innovative sources and tools. Natural compounds pose a great field for the discovery of new environ‐ mentally safe herbicides, so called "bioherbicides", which are based on compounds produced by living organisms. According to the CAS (Chemical Abstracts Service) registry, among the 24 million organic compounds, a large group of secondary plant metabolites is represented. Some of these compounds take part in allelopathic interactions.

**9.** tannins;

**10.** steroids and terpenoids (sesquiterpene lactones, diterpenes, and triterpenoids).

juglone, benzoxazolin-2-one (BOA), 2-amino-3-H-phenoxazin-3-one (APO).

these compounds on target plants are also classified as "phytotoxic".

can significantly decrease bioactivity of the whole compound.

**3. Advantages and disadvantages of allelopathins as bioherbicides**

Mode of action of some allelochemicals is similar to synthetic herbicides. These features have allowed them to be considered for possible use in weed management as bioherbicides. However, the field of knowledge is poorly studied but it is a very attractive area to explore. Allelochemicals are highly attractive as new classes of herbicides due to a variety of advan‐ tages. However, in the perspective of bioherbicides based on allelopathins, effects caused by

Most of allelopathins are totally or partially water-soluble which makes them easier to apply without additional surfactants [3, 10]. Their chemical structure is more environmentally friendly than synthetic ones. They possess higher oxygen- and nitrogen-rich molecules with relatively few so called 'heavy atoms', a halogen substitute, and are characterized by the absence of 'unnatural' rings. These properties decrease a chemical's environmental half-life, prevent accumulation of the compound in soil and eventual influence on non-target organ‐ isms. On the other hand, these properties are an allelochemical's Achille's heel due to less than satisfactory duration of activity. Structure complexity generates more stereocenters making them more reactive and unstable. Therefore, rapid degradation of one of the chemical groups

The diversity of allelopathins makes them promising tools possessing specific properties in discovering novel, specific target sites in acceptor plants. Even if they inhibit photosynthesis

Allelochemicals are released into the environment by plant organs such as roots, rhizomes, leaves, stems, bark, flowers, fruits and seeds (Figure 1a). The huge number of allelopathic interactions is typically negative in character, with positive relations being rare. Allelopathic compounds affect germination and growth of neighboring plants by disruption of various physiological processes including photosynthesis, respiration, water and hormonal balance. The underlying cause of their action is mainly inhibition of enzyme activity. Ability of an allelochemical to inhibit or delay plant growth and/or seed germination is usually defined as its "allelopathic (or phytotoxic) potential". An excellent example of allelopathic interaction is seen in soil exhaustion due to the accumulation of allelopathins that can be prevented by using fertilizers and rotating crops. Plants producing allelopathins are considered as "donor" organisms while the plants which allelopathins are directed to are referred to as "target" plants or "acceptors". The after-effects and strength of allelopathic interactions are diverse due to modifications of the allelopathins taking place in soil (Fig 1b). Most of the allelochemicals penetrate the soil as already plant-active compounds, e.g. phenolic acids, cyanamide, momi‐ lactones, heliannuols etc. Some have to be modified into the active form by microorganisms or by specific environmental conditions (pH, moisture, temperature, light, oxygen etc.), e.g.

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### **2. Allelopathic interactions and allelopathic compounds**

Allelopathy is considered a multi-dimensional phenomenon occurring constantly in natural and anthropogenic ecosystems [4]. It is defined as the interaction between plants and micro‐ organisms by a variety of compounds usually referred to as allelopathins, allelochemicals, or allelopathic compounds. This review is focused mainly on compounds taking part in complex allelopathic interactions between higher plants. However, determination of quality, quantity, direct or indirect effects of allelopathins on plant or microorganism communities in the natural environment is very difficult owing to the multi-dimensional character of those interactions. The development of analytical techniques allowing better specification of direct effects of allelopathins, have moved the exploration (or the research on) of this phenomenon from fields into laboratories. The term "allelopathy" refers rather to interactions occurring in the natural environment [5]. For studies with plant extracts, allelopathins isolated from plant tissue, collected from exudates or even synthetic compounds identical to natural ones, it was established the term ''phytotoxicity'' to distinguish allelopathy (as a phenomenon occurring in natural environment) from studies conducted in laboratory.

Allelopathins are products of the secondary metabolism and are non-nutritional primary metabolites [6,7]. These compounds belong to numerous chemical groups including: trike‐ tones, terpenes, benzoquinones, coumarins, flavonoids, terpenoids, strigolactones, phenolic acids, tannins lignin, fatty acids and nonprotein aminoacids. A wide range of these biochem‐ icals are synthesized during the shikimate pathway [8] or, in the case of essential oils, from the soprenoid pathway. Allelochemicals can be classified into 10 categories [9] according to their different structures and properties:

#### **9.** tannins;

dramatically. Eco-friendly trends in weed management force scientists to reach for innovative sources and tools. Natural compounds pose a great field for the discovery of new environ‐ mentally safe herbicides, so called "bioherbicides", which are based on compounds produced by living organisms. According to the CAS (Chemical Abstracts Service) registry, among the 24 million organic compounds, a large group of secondary plant metabolites is represented.

Allelopathy is considered a multi-dimensional phenomenon occurring constantly in natural and anthropogenic ecosystems [4]. It is defined as the interaction between plants and micro‐ organisms by a variety of compounds usually referred to as allelopathins, allelochemicals, or allelopathic compounds. This review is focused mainly on compounds taking part in complex allelopathic interactions between higher plants. However, determination of quality, quantity, direct or indirect effects of allelopathins on plant or microorganism communities in the natural environment is very difficult owing to the multi-dimensional character of those interactions. The development of analytical techniques allowing better specification of direct effects of allelopathins, have moved the exploration (or the research on) of this phenomenon from fields into laboratories. The term "allelopathy" refers rather to interactions occurring in the natural environment [5]. For studies with plant extracts, allelopathins isolated from plant tissue, collected from exudates or even synthetic compounds identical to natural ones, it was established the term ''phytotoxicity'' to distinguish allelopathy (as a phenomenon occurring

Allelopathins are products of the secondary metabolism and are non-nutritional primary metabolites [6,7]. These compounds belong to numerous chemical groups including: trike‐ tones, terpenes, benzoquinones, coumarins, flavonoids, terpenoids, strigolactones, phenolic acids, tannins lignin, fatty acids and nonprotein aminoacids. A wide range of these biochem‐ icals are synthesized during the shikimate pathway [8] or, in the case of essential oils, from the soprenoid pathway. Allelochemicals can be classified into 10 categories [9] according to their

**1.** water-soluble organic acids, straight-chain alcohols, aliphatic aldehydes, and ketones;

Some of these compounds take part in allelopathic interactions.

518 Herbicides - Current Research and Case Studies in Use

in natural environment) from studies conducted in laboratory.

**4.** quinines (benzoquinone, anthraquinone and complex quinines);

different structures and properties:

**6.** cinnamic acid and its derivatives;

**3.** long-chain fatty acids and polyacetylenes;

**2.** simple lactones;

**5.** phenolics;

**7.** coumarins; **8.** flavonoids;

**2. Allelopathic interactions and allelopathic compounds**

**10.** steroids and terpenoids (sesquiterpene lactones, diterpenes, and triterpenoids).

Allelochemicals are released into the environment by plant organs such as roots, rhizomes, leaves, stems, bark, flowers, fruits and seeds (Figure 1a). The huge number of allelopathic interactions is typically negative in character, with positive relations being rare. Allelopathic compounds affect germination and growth of neighboring plants by disruption of various physiological processes including photosynthesis, respiration, water and hormonal balance. The underlying cause of their action is mainly inhibition of enzyme activity. Ability of an allelochemical to inhibit or delay plant growth and/or seed germination is usually defined as its "allelopathic (or phytotoxic) potential". An excellent example of allelopathic interaction is seen in soil exhaustion due to the accumulation of allelopathins that can be prevented by using fertilizers and rotating crops. Plants producing allelopathins are considered as "donor" organisms while the plants which allelopathins are directed to are referred to as "target" plants or "acceptors". The after-effects and strength of allelopathic interactions are diverse due to modifications of the allelopathins taking place in soil (Fig 1b). Most of the allelochemicals penetrate the soil as already plant-active compounds, e.g. phenolic acids, cyanamide, momi‐ lactones, heliannuols etc. Some have to be modified into the active form by microorganisms or by specific environmental conditions (pH, moisture, temperature, light, oxygen etc.), e.g. juglone, benzoxazolin-2-one (BOA), 2-amino-3-H-phenoxazin-3-one (APO).

### **3. Advantages and disadvantages of allelopathins as bioherbicides**

Mode of action of some allelochemicals is similar to synthetic herbicides. These features have allowed them to be considered for possible use in weed management as bioherbicides. However, the field of knowledge is poorly studied but it is a very attractive area to explore.

Allelochemicals are highly attractive as new classes of herbicides due to a variety of advan‐ tages. However, in the perspective of bioherbicides based on allelopathins, effects caused by these compounds on target plants are also classified as "phytotoxic".

Most of allelopathins are totally or partially water-soluble which makes them easier to apply without additional surfactants [3, 10]. Their chemical structure is more environmentally friendly than synthetic ones. They possess higher oxygen- and nitrogen-rich molecules with relatively few so called 'heavy atoms', a halogen substitute, and are characterized by the absence of 'unnatural' rings. These properties decrease a chemical's environmental half-life, prevent accumulation of the compound in soil and eventual influence on non-target organ‐ isms. On the other hand, these properties are an allelochemical's Achille's heel due to less than satisfactory duration of activity. Structure complexity generates more stereocenters making them more reactive and unstable. Therefore, rapid degradation of one of the chemical groups can significantly decrease bioactivity of the whole compound.

The diversity of allelopathins makes them promising tools possessing specific properties in discovering novel, specific target sites in acceptor plants. Even if they inhibit photosynthesis

on microbial ecology and non-target plants, possible toxic properties on human health and

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A high number of limitations does not exclude allelochemicals as possible herbicides. In particular, they can be alternatives in weed management strategy. Widely developed bioin‐ formatics and cheminformatics support development of new herbicides [3, 15, 16]. Identified chemical structure of a particular allelochemical is a starting point to design a product with the compound-like properties using computer programs. Thanks to cheminformatics we are able to predict the potential structure of analogues and make several modifications, which make it more or less active, with higher environmental stability, as it was done for leptosper‐ mone. We may also predict the target site of compound action in plants due to comparison studies. Similar structure of a compound to a commercialized herbicide or other natural compound whose mode of action is well-known may allow us to predict the target site.

Plant protection is effective but rather costly and problematic due to environmental pollution. Exploration of the allelopathic potential of some species allows the introduction of alternative techniques for weed management, e.g. extracts from allelopathic plants can be applied as foliar sprays. Apart from decreasing the costs of herbicide application, this method also improves

The best known examples of natural bioherbicides are phytotoxic water extracts from herbage of sorghum (*Sorghum bicolor* (L.) Moench.) (sorgaab) and sunflower (*Helianthus annuus* L.)

Effects of sorgaab on weeds is time- and dose-depend but is typically used at 5% or 10% (w/v) concentration as double spray 20/30 and 40/60 days after sowing (DAS) or after seedling transplantation (AT) [17-19]. The best results to account for net profits have been elicited with a double spray of 10% extract in cotton (*Gossypim hirsutum* L.), soybean (*Glycine max* L.), wheat (*Triticum aestivum* L.) or rice (*Oryza sativa* L.). The highest efficacy of such extract applications has been verified in rice on reduction of barnyard grass (*Echinochloa cruss-galli* L.) biomass by 40%, without significant changes in weed density and accompanied yield increase by 18%.

Sunfaag has been widely used in wheat. The extract has been usually applied three times at 7 day intervals starting between 3-4 weeks post-emergence. This system of application has reduced biomass of the two most commonly occurring weeds, lambsquarters (*Chenopodium album* L.) and toothed dock (*Rumex dentatus* L.), by 70% and 97% respectively, although it has not eliminated all weed species in field. It has improved wheat biomass by 7-8% in comparison to weed free control without significant changes in number of tillers and total seed biomass. The herbicidal efficiency calculated as the effectiveness of sunfaag in comparison to synthetic herbicides showed a quite high value, 60% efficiency index. Weed management systems require high concentrations of sunfaag ranging up to 80% and can generate economic losses due to the necessity of cultivating higher amounts of sorghum or sunflower that also required

(sunfaag) which can be effectively used in plant protection without yield losses.

profitability of production on a commercial scale [16].

**4. Allelopathic plant extracts as bioherbicides**

crop production.

**Figure 1.** Multi-dimensional nature of allelopathic interacions. (1a) Plant A releases allelochemicals X and F which di‐ rectly affect growth of plant B. (1b) left side; Plant A releases allelochemical X which is modified or activated by micro‐ organisms to allelochemical Y that affects growth of plant B. (1b) right side; Plant A releases allelochemical X which stimulates microorganisms to produce allelochemical Z that affects growth of plant B.

or respiration, they may also bind to proteins at different sites than synthetic herbicides [11, 12]. This provides the opportunity to eliminate weeds that are already resistant to commer‐ cialized herbicides with the same mode of action. Allelochemicals are also characterized by multi-site action in plants without high specificity which is achieved in the case of synthetic herbicides. Therefore, this feature excludes the application of an allelopathic compound as a selective herbicide or totally prohibits its usage in weed management. On the other hand, effects of allelopathins in acceptor plants are highly dose-dependent [13]. This allows the opportunity to search out compounds exhibiting selectivity. Generally, monocotyledonous plants are more resistant to allelochemicals than dicotyledonous ones. Therefore, usage of a compound as a potential herbicide is possible but rather restricted to cultivation of exact crops with a defined weed composition.

The route of discovery is much more complicated with allelopathins. In contrast to synthetic herbicides where synthesis, bioassay, evaluation and quantitative structure-active relationship follow Quantitative Structure-Activity Relationship (QSAR), allelochemicals have to be first isolated from plant extracts [14]. The amount of recovered compounds is usually low in comparison to chemical synthesis. After extraction, purification and selection of the most attractive compound and determination of its mode of action in plants is done. At the end of the process, similar to synthetic herbicides, allelpathins are subjected to QSAR. The long discovery process is usually offset by a shorter, less expensive track of registration [15]. It is worth noting that before an allelochemical can become an herbicide, the following conditions have to be performed: phytotoxic activity at the range between 10-5 and 10-7 M, identified chemical structure, known mode of action in plants, time of residence in soil, possible influence on microbial ecology and non-target plants, possible toxic properties on human health and profitability of production on a commercial scale [16].

A high number of limitations does not exclude allelochemicals as possible herbicides. In particular, they can be alternatives in weed management strategy. Widely developed bioin‐ formatics and cheminformatics support development of new herbicides [3, 15, 16]. Identified chemical structure of a particular allelochemical is a starting point to design a product with the compound-like properties using computer programs. Thanks to cheminformatics we are able to predict the potential structure of analogues and make several modifications, which make it more or less active, with higher environmental stability, as it was done for leptosper‐ mone. We may also predict the target site of compound action in plants due to comparison studies. Similar structure of a compound to a commercialized herbicide or other natural compound whose mode of action is well-known may allow us to predict the target site.

### **4. Allelopathic plant extracts as bioherbicides**

or respiration, they may also bind to proteins at different sites than synthetic herbicides [11, 12]. This provides the opportunity to eliminate weeds that are already resistant to commer‐ cialized herbicides with the same mode of action. Allelochemicals are also characterized by multi-site action in plants without high specificity which is achieved in the case of synthetic herbicides. Therefore, this feature excludes the application of an allelopathic compound as a selective herbicide or totally prohibits its usage in weed management. On the other hand, effects of allelopathins in acceptor plants are highly dose-dependent [13]. This allows the opportunity to search out compounds exhibiting selectivity. Generally, monocotyledonous plants are more resistant to allelochemicals than dicotyledonous ones. Therefore, usage of a compound as a potential herbicide is possible but rather restricted to cultivation of exact crops

stimulates microorganisms to produce allelochemical Z that affects growth of plant B.

**Figure 1.** Multi-dimensional nature of allelopathic interacions. (1a) Plant A releases allelochemicals X and F which di‐ rectly affect growth of plant B. (1b) left side; Plant A releases allelochemical X which is modified or activated by micro‐ organisms to allelochemical Y that affects growth of plant B. (1b) right side; Plant A releases allelochemical X which

The route of discovery is much more complicated with allelopathins. In contrast to synthetic herbicides where synthesis, bioassay, evaluation and quantitative structure-active relationship follow Quantitative Structure-Activity Relationship (QSAR), allelochemicals have to be first isolated from plant extracts [14]. The amount of recovered compounds is usually low in comparison to chemical synthesis. After extraction, purification and selection of the most attractive compound and determination of its mode of action in plants is done. At the end of the process, similar to synthetic herbicides, allelpathins are subjected to QSAR. The long discovery process is usually offset by a shorter, less expensive track of registration [15]. It is worth noting that before an allelochemical can become an herbicide, the following conditions have to be performed: phytotoxic activity at the range between 10-5 and 10-7 M, identified chemical structure, known mode of action in plants, time of residence in soil, possible influence

with a defined weed composition.

520 Herbicides - Current Research and Case Studies in Use

Plant protection is effective but rather costly and problematic due to environmental pollution. Exploration of the allelopathic potential of some species allows the introduction of alternative techniques for weed management, e.g. extracts from allelopathic plants can be applied as foliar sprays. Apart from decreasing the costs of herbicide application, this method also improves crop production.

The best known examples of natural bioherbicides are phytotoxic water extracts from herbage of sorghum (*Sorghum bicolor* (L.) Moench.) (sorgaab) and sunflower (*Helianthus annuus* L.) (sunfaag) which can be effectively used in plant protection without yield losses.

Effects of sorgaab on weeds is time- and dose-depend but is typically used at 5% or 10% (w/v) concentration as double spray 20/30 and 40/60 days after sowing (DAS) or after seedling transplantation (AT) [17-19]. The best results to account for net profits have been elicited with a double spray of 10% extract in cotton (*Gossypim hirsutum* L.), soybean (*Glycine max* L.), wheat (*Triticum aestivum* L.) or rice (*Oryza sativa* L.). The highest efficacy of such extract applications has been verified in rice on reduction of barnyard grass (*Echinochloa cruss-galli* L.) biomass by 40%, without significant changes in weed density and accompanied yield increase by 18%.

Sunfaag has been widely used in wheat. The extract has been usually applied three times at 7 day intervals starting between 3-4 weeks post-emergence. This system of application has reduced biomass of the two most commonly occurring weeds, lambsquarters (*Chenopodium album* L.) and toothed dock (*Rumex dentatus* L.), by 70% and 97% respectively, although it has not eliminated all weed species in field. It has improved wheat biomass by 7-8% in comparison to weed free control without significant changes in number of tillers and total seed biomass. The herbicidal efficiency calculated as the effectiveness of sunfaag in comparison to synthetic herbicides showed a quite high value, 60% efficiency index. Weed management systems require high concentrations of sunfaag ranging up to 80% and can generate economic losses due to the necessity of cultivating higher amounts of sorghum or sunflower that also required an appropriate cultivation system [20, 21]. Therefore, sunfaag can be applied as a preemergence herbicide with much lower doses. The most promising application system has considered usage of 10% (w/v) extract at pre-emergence + 25 DAS + 35 DAS. Following the application, there has been noted a remarkably reduced population of wild oat, lesser swi‐ necress (*Coronopus didymus* L.) and littleseed canarygrass *(Phalaris minor* Retz.) without affecting germination of wheat and increased wheat yield in 7% [22]. However, the inhibitory effect on weed growth and crop yield is selective and highly dependent on duration or term of sorgaab and sunfaag application.

tition. Sunfaag has been applied when wheat seedlings were 3-4 weeks old while lambsquar‐

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High allelopathic potential conditioned by glucosinolates and isothiocyanates is present in Brassica sp. [27, 28]. Isothiocyanates have been strong suppressants of germination of spiny sowthistle (*Sonchus asper* L. Hill), scentless mayweed (*Matricaria inodora* L.), smooth pigweed (*Amaranthus hybridus* L.), barnyardgrass, blackgrass (*Alopecurus myosuroides* Huds.) and wheat [28]. Black mustard (*Brassica nigra* L.) extract of different plant parts like leaf, stem, flower and root have inhibited germination and radicle length of wild oat. Inhibitory effects on germina‐ tion increased with increasing concentration of extract solution of the fresh plant parts [29]. Some experiments were conducted also using garden radish (*Raphanus sativus* L.) extract on germination of 25 weed and 32 crop species [30]. Garden radish extracts totally inhibited germination of 11 weeds such as Johnsongrass (*Sorghum halelense* L. Pers.), *Alhagi* spp., blackgrass (*Alopecurus myosuroides* Huds.), shepherd's-purse (*Capsella bursa-pastoris* L. Medik.), field bindweed (*Convolvulus arvensis* L.), dodder (*Cuscuta* sp.), carrot (*Daucus carota* L.), shortpod mustard (*Hirschfeldia incana* L.), *Ochtodium aegyptiacum* (L.), and shortfruit hedgemustard (*Sisymbrium polyceratium* L.), and 4 crop species namely lettuce (*Lactuca sativa* L.), tobacco (*Nicotiana tabacum* L.), bean (*Phaseolus vulgaris* L.), and clover (*Trifolium* sp.). Garden radish extracts at different rates (100, 66, 50 and 33% of pure extract) did not affect germination of wheat, cotton, and maize (*Zea mays* L.), but affected soybean germination at the 100% extract rate in vitro. Rhizome regeneration of Johnsongrass was inhibited by 54-99% depending on extract concentration. Regeneration of bermudagrass (*Cynodon dactylon* L. Pers.) rhizomes was inhibited to a lower extent at all concentrations; for instance, 54% inhibition occurred at the highest extract concentration. Lower extract rates stimulated redroot pigweed germination, while 66 and 100% extracts inhibited germination by 21 and 42%, respectively. Inhibition reached only 56 and 49% at the highest extract concentration for common purslane (*Portulaca oleracea* L.) and cocklebur (*Xanthium strumarium* L.), respectively. Garden radish residues which were cut into pieces and incorporated into the growing medium decreased weed

Legumes crops may also be applied as a source of allelochemicals useful in weed suppression. Mulch of dead pea plants could be used to control growth of weeds. Pea cover crop has regulated germination and growth of lady's thumb (*Polygonum persicaria* L.), smooth pigweed, smallflower galinsoga, and common lambsquarters. Similarly, the aqueous leachates (1%) of all four legumes, velvetbean (*Mucuna deeringiana* (Bort.) Merr.), jackbean (*Canavalia ensifor‐ mis* (L.) DC.), jumbiebean (*Leucaena leucocephala* (Lam.) de Wit), and wild tamarind (*Lysiloma latisiliquum* (L.) Benth.), have been shown to suppress weeds [32]. These plants exhibited strong phytotoxic effects on the radicle growth of barnyardgrass, alegría (*Amaranthus* ssp.) and amaranth (*Amaranthus hypochondriacus* L.) [33]. Russian knapweed (*Acroptilon repens*) control is difficult in many crops. Allelopathic effects of extracts and plant parts of alfalfa (*Medicago sativa* L.) on Russian knapweed were reported both in Petri dishes and pot experiments [34]. Alfalfa has been recommended in fields with high mugwort (*Artemisia vulgaris* L.) infestation, as it decreased mugwort to 89% under field conditions, while extracts of alfalfa vegetative

parts inhibited mugwort germination up to 83% in Petri dish assays.

ters and toothed dock 1-week old at the stage of three to four leaf [20, 21].

intensity and increased maize yield [31].

Aqueous extracts of sorghum and sunflower are effective on weed growth but unfortunately might not be profitable enough in crop production; however, crop allelopathy can be manip‐ ulated for achieving sustainable weed management. Combination of phytotoxic crop water extracts with lower rates of herbicides may provide reduced weed control levels with reduced herbicide usage. The interesting review of allelopathic crop plants in weed management strategy is presented in reference [23]. Two field studies were conducted utilizing water extracts of sorghum, sunflower and rapeseed (*Brassica napus* L.) with reduced glyphosate dosage for controlling purple nutsedge (*Cyperus rotundus* L) in cotton [24]. Sorghum and rapeseed water extracts were tank mixed (at 15 or 18 L ha-1) in different combinations with reduced rates of glyphosate by 767 and 575 g active substance (a.s.) ha-1 and sprayed as directed post emergence at 21 DAS. Purple nutsedge density and dry weight were suppressed by 78% to 95% and 83% to 95%, respectively, when different crop water extracts were used in combi‐ nation with a reduced rate of glyphosate. Seed cotton yield was improved from 15-21% in sorgaab and rape water extract combinations with reduced rates of glyphosate (67-75%). Similar research has been conducted on water extracts of sorghum with sunflower in combi‐ nation with herbicides in wheat, soybean, rice, and canola (*Brassica* sp.) [25, 26]. Both extracts, in combination with herbicides, have the same or even better effect on inhibition of growth of the following weeds: littleseed canarygrass and lesser swinecress, compared to single synthetic herbicide applications [25, 26]. Spraying of wheat seedlings 30 DAS with sorgaab+sunfaag (18 L each ha-1) with mesosulfuron+idosulfuron (4.32 g a.s. ha-1) has the same effect on total weed density (reduction up to 90% in relation to control) as application of mesosulfuron+idosulfuron used alone, but with higher doses (120 g a.s. ha-1). Herbicidal solution has also improved yield parameters, both in relation to control and in relation to single herbicide application: fertile tillers (10%), spikelets per spike (11%) and grains per spike (10%) [26]. In cotton, application of both extracts at 18 L ha-1 each with glyphosate (767 g a.s. ha-1) 21 DAS has been the most effective in density reduction of the highly competitive weed purple nutsedge up to 93% [24]. However, the greatest benefit in wheat is the usage of a sorgaab/sunfaag combination which lowered by 70% doses of metribuzin and phenaxaprop (at 57 g a.s. ha-1), applied at 18 L each ha-1. In turn, in cotton, application of the same rates of extracts per ha with glyphosate (767 g a.s. ha-1) seems to be the most economically reasonable costs of following weed management method [24, 25].

Selectivity of plant extracts on weeds without any negative implications on crop productivity is probably due to differences in the physiological stage of plants and following plant compe‐ tition. Sunfaag has been applied when wheat seedlings were 3-4 weeks old while lambsquar‐ ters and toothed dock 1-week old at the stage of three to four leaf [20, 21].

an appropriate cultivation system [20, 21]. Therefore, sunfaag can be applied as a preemergence herbicide with much lower doses. The most promising application system has considered usage of 10% (w/v) extract at pre-emergence + 25 DAS + 35 DAS. Following the application, there has been noted a remarkably reduced population of wild oat, lesser swi‐ necress (*Coronopus didymus* L.) and littleseed canarygrass *(Phalaris minor* Retz.) without affecting germination of wheat and increased wheat yield in 7% [22]. However, the inhibitory effect on weed growth and crop yield is selective and highly dependent on duration or term

Aqueous extracts of sorghum and sunflower are effective on weed growth but unfortunately might not be profitable enough in crop production; however, crop allelopathy can be manip‐ ulated for achieving sustainable weed management. Combination of phytotoxic crop water extracts with lower rates of herbicides may provide reduced weed control levels with reduced herbicide usage. The interesting review of allelopathic crop plants in weed management strategy is presented in reference [23]. Two field studies were conducted utilizing water extracts of sorghum, sunflower and rapeseed (*Brassica napus* L.) with reduced glyphosate dosage for controlling purple nutsedge (*Cyperus rotundus* L) in cotton [24]. Sorghum and rapeseed water extracts were tank mixed (at 15 or 18 L ha-1) in different combinations with reduced rates of glyphosate by 767 and 575 g active substance (a.s.) ha-1 and sprayed as directed post emergence at 21 DAS. Purple nutsedge density and dry weight were suppressed by 78% to 95% and 83% to 95%, respectively, when different crop water extracts were used in combi‐ nation with a reduced rate of glyphosate. Seed cotton yield was improved from 15-21% in sorgaab and rape water extract combinations with reduced rates of glyphosate (67-75%). Similar research has been conducted on water extracts of sorghum with sunflower in combi‐ nation with herbicides in wheat, soybean, rice, and canola (*Brassica* sp.) [25, 26]. Both extracts, in combination with herbicides, have the same or even better effect on inhibition of growth of the following weeds: littleseed canarygrass and lesser swinecress, compared to single synthetic herbicide applications [25, 26]. Spraying of wheat seedlings 30 DAS with sorgaab+sunfaag (18 L each ha-1) with mesosulfuron+idosulfuron (4.32 g a.s. ha-1) has the same effect on total weed density (reduction up to 90% in relation to control) as application of mesosulfuron+idosulfuron used alone, but with higher doses (120 g a.s. ha-1). Herbicidal solution has also improved yield parameters, both in relation to control and in relation to single herbicide application: fertile tillers (10%), spikelets per spike (11%) and grains per spike (10%) [26]. In cotton, application of both extracts at 18 L ha-1 each with glyphosate (767 g a.s. ha-1) 21 DAS has been the most effective in density reduction of the highly competitive weed purple nutsedge up to 93% [24]. However, the greatest benefit in wheat is the usage of a sorgaab/sunfaag combination which lowered by 70% doses of metribuzin and phenaxaprop (at 57 g a.s. ha-1), applied at 18 L each ha-1. In turn, in cotton, application of the same rates of extracts per ha with glyphosate (767 g a.s. ha-1) seems to be the most economically reasonable costs of following weed management

Selectivity of plant extracts on weeds without any negative implications on crop productivity is probably due to differences in the physiological stage of plants and following plant compe‐

of sorgaab and sunfaag application.

522 Herbicides - Current Research and Case Studies in Use

method [24, 25].

High allelopathic potential conditioned by glucosinolates and isothiocyanates is present in Brassica sp. [27, 28]. Isothiocyanates have been strong suppressants of germination of spiny sowthistle (*Sonchus asper* L. Hill), scentless mayweed (*Matricaria inodora* L.), smooth pigweed (*Amaranthus hybridus* L.), barnyardgrass, blackgrass (*Alopecurus myosuroides* Huds.) and wheat [28]. Black mustard (*Brassica nigra* L.) extract of different plant parts like leaf, stem, flower and root have inhibited germination and radicle length of wild oat. Inhibitory effects on germina‐ tion increased with increasing concentration of extract solution of the fresh plant parts [29]. Some experiments were conducted also using garden radish (*Raphanus sativus* L.) extract on germination of 25 weed and 32 crop species [30]. Garden radish extracts totally inhibited germination of 11 weeds such as Johnsongrass (*Sorghum halelense* L. Pers.), *Alhagi* spp., blackgrass (*Alopecurus myosuroides* Huds.), shepherd's-purse (*Capsella bursa-pastoris* L. Medik.), field bindweed (*Convolvulus arvensis* L.), dodder (*Cuscuta* sp.), carrot (*Daucus carota* L.), shortpod mustard (*Hirschfeldia incana* L.), *Ochtodium aegyptiacum* (L.), and shortfruit hedgemustard (*Sisymbrium polyceratium* L.), and 4 crop species namely lettuce (*Lactuca sativa* L.), tobacco (*Nicotiana tabacum* L.), bean (*Phaseolus vulgaris* L.), and clover (*Trifolium* sp.). Garden radish extracts at different rates (100, 66, 50 and 33% of pure extract) did not affect germination of wheat, cotton, and maize (*Zea mays* L.), but affected soybean germination at the 100% extract rate in vitro. Rhizome regeneration of Johnsongrass was inhibited by 54-99% depending on extract concentration. Regeneration of bermudagrass (*Cynodon dactylon* L. Pers.) rhizomes was inhibited to a lower extent at all concentrations; for instance, 54% inhibition occurred at the highest extract concentration. Lower extract rates stimulated redroot pigweed germination, while 66 and 100% extracts inhibited germination by 21 and 42%, respectively. Inhibition reached only 56 and 49% at the highest extract concentration for common purslane (*Portulaca oleracea* L.) and cocklebur (*Xanthium strumarium* L.), respectively. Garden radish residues which were cut into pieces and incorporated into the growing medium decreased weed intensity and increased maize yield [31].

Legumes crops may also be applied as a source of allelochemicals useful in weed suppression. Mulch of dead pea plants could be used to control growth of weeds. Pea cover crop has regulated germination and growth of lady's thumb (*Polygonum persicaria* L.), smooth pigweed, smallflower galinsoga, and common lambsquarters. Similarly, the aqueous leachates (1%) of all four legumes, velvetbean (*Mucuna deeringiana* (Bort.) Merr.), jackbean (*Canavalia ensifor‐ mis* (L.) DC.), jumbiebean (*Leucaena leucocephala* (Lam.) de Wit), and wild tamarind (*Lysiloma latisiliquum* (L.) Benth.), have been shown to suppress weeds [32]. These plants exhibited strong phytotoxic effects on the radicle growth of barnyardgrass, alegría (*Amaranthus* ssp.) and amaranth (*Amaranthus hypochondriacus* L.) [33]. Russian knapweed (*Acroptilon repens*) control is difficult in many crops. Allelopathic effects of extracts and plant parts of alfalfa (*Medicago sativa* L.) on Russian knapweed were reported both in Petri dishes and pot experiments [34]. Alfalfa has been recommended in fields with high mugwort (*Artemisia vulgaris* L.) infestation, as it decreased mugwort to 89% under field conditions, while extracts of alfalfa vegetative parts inhibited mugwort germination up to 83% in Petri dish assays.

Application of plant extracts as pre-emergence or as early post emergence herbicides resulted in reduction of doses of synthetic herbicide due to their synergistic or additive action. How‐ ever, not all phytotoxic extracts are effective enough to inhibit weed growth or germination when applied as spray even when plants show high allelopathic potential as mulch, inter‐ cropping system or in rotation. This may be the result of masking the activity of one compound by another in water solution or other factors such as impossibility of extract penetration through the cuticle [12]. A new opportunity to enhance effectiveness of usage of bioherbicides based on natural extracts is associated with extraction of individual allelochemicals and/or its comparison with synthetic herbicides. The extraction of sesquiterpene lactone, dehydrozalu‐ zanin C (DHZ) produced among Compositae family serves as an example [34]. Comparison studies of isolated DHZ (1 mM) and the commercial herbicide Logran® showed high inhibitory activity of DHZ on dicotyledonous plants while the synthetic herbicide showed no activity [34]. Also pure 2-benzoxazolinone (BOA) isolated from several graminaceous crops such as rye (*Secale cereale* L.), maize and wheat was active similarly as herbicide but its stability in the environment was much shorter than the synthetic herbicide [35].

 Allelopathic compounds isolated from plants that exhibit inhibitory potential on seed germination and

Sarmentine pepper *(Piper* sp.) barnyard grass, redroot pigweed, crabgrass,

mustard (*Brassica* sp.)

Sorgoleone sorghum (*Sorghum bicolor* L. Moench)

Momilactone rice (*Oryza sativa L.*), moss (*Hypnum plumaeform*)

Artemisinin annual wormwood (*Artemisia annua* L.)

Leptospermone bottle brush (*Callistemon citrinus*),

growth of weeds

J.R., G. Forst)

manuka (*Leptospermum scoparium*

Glucosinolates, Isothiocyanates

### **5. Plant allelopathins as sources of bioherbicides**

Plant phytotoxic extracts, after evaluation, can be successfully used in integrated weed management. However, as was aforementioned, not all systems of its application under field conditions are suitable and profitable enough. To circumvent masking effects of one allelopa‐ thin by another in plant extract, research is now focused on isolation and application of a single, specific compound for the purpose of weed elimination. The list of allelochemicals isolated from various plants that may act as inhibitors of weed seed germination and/or weed growth are summarized in Table 1. A purified allelopathic compound may act on target plants with much higher or much lower strength. Even in situations when an allelopathin is active at unprofitably high doses but has a favorable environmental profile, it still may be a source to explore due to several reasons such as biodegradability. Modifications of chemical structure can make a compound more active on target plants while preserving desire properties.

Herein, examples of purified allelopathins with possible roles as herbicides are described. Some herbicides based on modified allelopathins already launched on the market are also included.

#### **5.1. Sorgoleone**

The inhibitory effect of sorghum on various plant species has been known for many years. Accumulation of sorghum phytotoxins in soil affects crop growth and imposes the need for a crop rotation system. Besides crops, weeds are also vulnerable to its allelopathic influence [16, 36]. Sorghum toxicity is mainly determined by both hydrophilic phenols in herbage, as well as hydrophobic sorgoleone and its analogs exuded by the root hairs [37, 38]. Therefore, sorghum herbage reach can be successfully used against weeds as a foliar spray as it is discussed in detail in the previous chapter.

Application of plant extracts as pre-emergence or as early post emergence herbicides resulted in reduction of doses of synthetic herbicide due to their synergistic or additive action. How‐ ever, not all phytotoxic extracts are effective enough to inhibit weed growth or germination when applied as spray even when plants show high allelopathic potential as mulch, inter‐ cropping system or in rotation. This may be the result of masking the activity of one compound by another in water solution or other factors such as impossibility of extract penetration through the cuticle [12]. A new opportunity to enhance effectiveness of usage of bioherbicides based on natural extracts is associated with extraction of individual allelochemicals and/or its comparison with synthetic herbicides. The extraction of sesquiterpene lactone, dehydrozalu‐ zanin C (DHZ) produced among Compositae family serves as an example [34]. Comparison studies of isolated DHZ (1 mM) and the commercial herbicide Logran® showed high inhibitory activity of DHZ on dicotyledonous plants while the synthetic herbicide showed no activity [34]. Also pure 2-benzoxazolinone (BOA) isolated from several graminaceous crops such as rye (*Secale cereale* L.), maize and wheat was active similarly as herbicide but its stability in the

Plant phytotoxic extracts, after evaluation, can be successfully used in integrated weed management. However, as was aforementioned, not all systems of its application under field conditions are suitable and profitable enough. To circumvent masking effects of one allelopa‐ thin by another in plant extract, research is now focused on isolation and application of a single, specific compound for the purpose of weed elimination. The list of allelochemicals isolated from various plants that may act as inhibitors of weed seed germination and/or weed growth are summarized in Table 1. A purified allelopathic compound may act on target plants with much higher or much lower strength. Even in situations when an allelopathin is active at unprofitably high doses but has a favorable environmental profile, it still may be a source to explore due to several reasons such as biodegradability. Modifications of chemical structure can make a compound more active on target plants while preserving desire properties.

Herein, examples of purified allelopathins with possible roles as herbicides are described. Some herbicides based on modified allelopathins already launched on the market are also

The inhibitory effect of sorghum on various plant species has been known for many years. Accumulation of sorghum phytotoxins in soil affects crop growth and imposes the need for a crop rotation system. Besides crops, weeds are also vulnerable to its allelopathic influence [16, 36]. Sorghum toxicity is mainly determined by both hydrophilic phenols in herbage, as well as hydrophobic sorgoleone and its analogs exuded by the root hairs [37, 38]. Therefore, sorghum herbage reach can be successfully used against weeds as a foliar spray as it is

environment was much shorter than the synthetic herbicide [35].

524 Herbicides - Current Research and Case Studies in Use

**5. Plant allelopathins as sources of bioherbicides**

included.

**5.1. Sorgoleone**

discussed in detail in the previous chapter.

**Table 1.** Allelopathic compounds isolated from plants that exhibit inhibitory potential on seed germination and growth of weeds

However, allelochemical sorgoleone has enormous potential as an herbicide due to its high activity against various weed species. Studies conducted under laboratory conditions have shown that low doses of sorgoleone (100 μM) inhibit growth of the following weeds by 80%, black nightshade (*Solanum nigrum* L.), redroot pigweed, common ragweed (*Am‐ brosia atrtemisiflora* L.), and by 40% of sicklepod (*Cassia obtusifolia* L.), hairy crabgrass (*Dig‐ itaria sanguinalis* L.), velvetleaf (*Abutilon theophrasti* Medik.), barnyardgrass and tef (*Eragrostis tef* Zucc., Trotter) [11, 16].

physiological effects as applications of atrazine in redroot pigweed-susceptible biotypes [11]. These properties make sorgoleone a potential early post-emergence herbicide when applied as a spray with much less environmental implications than atrazine. Therefore, inhibition of photosynthesis is the main target site of sorgoleone action in young seed‐ lings but its mode of action in older plants may be different [12]. Sorgoleone can be a useful inhibitor of *p*-hydroxyphenylpyruvate dioxygenase (HPPD), which takes part in αtocopherol and plastoquinone synthesis. Inhibition of that enzyme leads to a decreased pool of available plastoquinone and indirectly affects activity of phytoene desaturase, a key enzyme in carotenoid synthesis. Such sequence of events causes declining carotenoid levels and affects photosynthesis [45]. Currently used triketone herbicides (e.g. sulco‐ trione, isoxaflutole) have the same mechanism of action on HPPD as sorgoleone, irreversi‐ ble competitive inhibition, with I50 = 0.4 μM. Triketone herbicides are considered by the U.S. Environmental Protection Agency (EPA) to be a low environmental risk. They are usually utilized as selective herbicides to eliminate broadleaf weeds in corn [10]. It fol‐ lows, due to similar action and chemical structure and environmental friendly profile, sorgoleone might also be useful as a selective herbicide; however, such comparison stud‐ ies have yet to be conducted. Then, its mode of action also cannot explain whether it is

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527

Extracts and residues of rice, the well-known cereal plant, also have allelopathic potential. Among isolated secondary metabolites, phenolic acids, hydroxamic acids, fatty acids, terpenes and indoles were identified [46]. The key role in rice allelopathy plays momilactone A and B isolated from root exudates. High allelopathic rice varieties release up to 2-3 μg of momilactone B per day [3]. These compounds inhibited the growth of typical weeds in rice, e.g. barnyard grass and awnless barnyard grass (*Echinochloa colona* (L.) Link.) at concentrations higher than 1 μM and 10 μM, respectively. Furthermore, phytotoxic abilities of momilacton A and B were also demonstrated on livid pigweed (*Amaranthus lividus* L.), hairy crabgrass and annual bluegrass (*Poa annua* L.) at concentrations higher than 60 μM and 12 μM, respectively [47]. The experiment has shown that momilactone B is secreted by rice roots into the rhizosphere over the entire life cycle [48]. Momilactone A and B belong to the diterpenoid phytoalexins which are known as antimicrobial secondary metabolites generated in response to signal molecules called elicitors (especially biotic elicitors) [49]. Both compounds thought to be unique to rice, recently have been found in the moss (*Hypnum plumaeforme* Wils.), a taxonomically distinct plant [49]. Despite the ability of momilactone A and B to inhibit plant growth, its mode of

Artemisinin is a sesquiterpenoid lactone of annual wormwood (*Artemisia annua* L.). It is synthesized and sequestered in glandular trichomes located on the leaves and flowers [51]. It can also be excreted by the roots or root hairs, but only at the beginning of the growing season; therefore, dead leaves are the major source of artemisinin in soils [52]. Artemisinin is also lost

more or less active on broadleaf or grass weeds species [44].

**5.2. Momilactones**

action in plants is still unknown.

**5.3. Artemisinin**

Sorgoleone released into the soil may act as a pre-emergence herbicide. Its persistence in the soil during or after sorghum cultivation inhibits germination and growth of small-seeded weeds, e.g. hairy crabgrass and green bristlegrass (*Setaria viridis* (L.) Beauv.), due to its better absorption and translocation within the small seeds than in large seeds [39]. However, strength and final effect on seeds or seedling physiology is multifactor-dependent. Sorgoleone sorbs strongly to the organic matter. This allows an extended persistence in the soil but unfortu‐ nately, significantly reduces its bioavailability. Moreover, the dynamics of decomposition significantly influences sorgoleone bioactivity, e.g. the methoxy- group of the aromatic ring is decomposed by 26% 48 h after exudation; however, some amounts of sorgoleone are also extractable after 6 weeks [40, 41]. Nevertheless, constitutive production of the compound allows a continuous supply and accumulation in the soil around 1.5 cm of root zone [42].

Inhibition of H+ -ATPase in plant roots makes sorgoleone an effective growth inhibitor and potential post-emergence herbicide [43]. Decreased activity of that enzyme affects ion uptake and water balance by decreasing water uptake and affecting plant growth. Re‐ droot pigweed, Jimson weed (*Datura stramonium* L.) and tef grown in hydroponic culture with 10 μM sorgoleone were characterized by lower H+ -ATPase activity in roots. Presence of sorgoleone in nutrient solution significantly suppressed growth and evoked brown col‐ oration and necrosis [43, 44].

Sorgoleone may be taken up by roots but cannot be translocated acropetally by xylem due to high lipophilic properties. Therefore, its application as a post-emergence herbicide may be limited. However, as a spray (0.6 kg ha-1), it has inhibited growth by 12% of green foxtail (*Setaria faberi* Herrm.), by 40-50% purslane, hairy crabgrass and velvetleaf, and up to 80-90% of common ragweed, redroot pigweed, and black nightshade [40].

Due to the structural similarity of sorgoleone to plastoquinon, it acts as a photosystem II (PSII) inhibitor [11, 43]. It binds to the niche of the D1 protein in PSII, gathers electrons and does not allow reoxidation of plastoquinon A by the secondary electron acceptor, plastoquinone B. Competition studies under sorgoleone *versus* synthetic herbicides such as atrazine, diuron, metribuzin and bentazon have shown that sorgoleone is an atrazine competitive inhibitor [11, 12]. Moreover, the I50 of sorgoleone is 0.1 μM and similar to other PSII inhibitors. It is worth mentioning that sorgoleone belongs to the His215 family of PSII inhibitors, while atrazine belongs to Ser264. Mutation in Ser264 of the D1 protein is responsible for resistance to triazines as well as other non-triazine herbicides, leading to cross-resistance. However, plants resistant to atrazine, with a QB binding site on PSII mutation (Ser264), are not resistant to sorgoleone. Application of sorgoleone is particular‐ ly justified in the case of triazine-resistant biotypes of redroot pigweed, due to the same physiological effects as applications of atrazine in redroot pigweed-susceptible biotypes [11]. These properties make sorgoleone a potential early post-emergence herbicide when applied as a spray with much less environmental implications than atrazine. Therefore, inhibition of photosynthesis is the main target site of sorgoleone action in young seed‐ lings but its mode of action in older plants may be different [12]. Sorgoleone can be a useful inhibitor of *p*-hydroxyphenylpyruvate dioxygenase (HPPD), which takes part in αtocopherol and plastoquinone synthesis. Inhibition of that enzyme leads to a decreased pool of available plastoquinone and indirectly affects activity of phytoene desaturase, a key enzyme in carotenoid synthesis. Such sequence of events causes declining carotenoid levels and affects photosynthesis [45]. Currently used triketone herbicides (e.g. sulco‐ trione, isoxaflutole) have the same mechanism of action on HPPD as sorgoleone, irreversi‐ ble competitive inhibition, with I50 = 0.4 μM. Triketone herbicides are considered by the U.S. Environmental Protection Agency (EPA) to be a low environmental risk. They are usually utilized as selective herbicides to eliminate broadleaf weeds in corn [10]. It fol‐ lows, due to similar action and chemical structure and environmental friendly profile, sorgoleone might also be useful as a selective herbicide; however, such comparison stud‐ ies have yet to be conducted. Then, its mode of action also cannot explain whether it is more or less active on broadleaf or grass weeds species [44].

#### **5.2. Momilactones**

However, allelochemical sorgoleone has enormous potential as an herbicide due to its high activity against various weed species. Studies conducted under laboratory conditions have shown that low doses of sorgoleone (100 μM) inhibit growth of the following weeds by 80%, black nightshade (*Solanum nigrum* L.), redroot pigweed, common ragweed (*Am‐ brosia atrtemisiflora* L.), and by 40% of sicklepod (*Cassia obtusifolia* L.), hairy crabgrass (*Dig‐ itaria sanguinalis* L.), velvetleaf (*Abutilon theophrasti* Medik.), barnyardgrass and tef

Sorgoleone released into the soil may act as a pre-emergence herbicide. Its persistence in the soil during or after sorghum cultivation inhibits germination and growth of small-seeded weeds, e.g. hairy crabgrass and green bristlegrass (*Setaria viridis* (L.) Beauv.), due to its better absorption and translocation within the small seeds than in large seeds [39]. However, strength and final effect on seeds or seedling physiology is multifactor-dependent. Sorgoleone sorbs strongly to the organic matter. This allows an extended persistence in the soil but unfortu‐ nately, significantly reduces its bioavailability. Moreover, the dynamics of decomposition significantly influences sorgoleone bioactivity, e.g. the methoxy- group of the aromatic ring is decomposed by 26% 48 h after exudation; however, some amounts of sorgoleone are also extractable after 6 weeks [40, 41]. Nevertheless, constitutive production of the compound allows a continuous supply and accumulation in the soil around 1.5 cm of root zone [42].

and potential post-emergence herbicide [43]. Decreased activity of that enzyme affects ion uptake and water balance by decreasing water uptake and affecting plant growth. Re‐ droot pigweed, Jimson weed (*Datura stramonium* L.) and tef grown in hydroponic culture

of sorgoleone in nutrient solution significantly suppressed growth and evoked brown col‐

Sorgoleone may be taken up by roots but cannot be translocated acropetally by xylem due to high lipophilic properties. Therefore, its application as a post-emergence herbicide may be limited. However, as a spray (0.6 kg ha-1), it has inhibited growth by 12% of green foxtail (*Setaria faberi* Herrm.), by 40-50% purslane, hairy crabgrass and velvetleaf, and up to 80-90% of

Due to the structural similarity of sorgoleone to plastoquinon, it acts as a photosystem II (PSII) inhibitor [11, 43]. It binds to the niche of the D1 protein in PSII, gathers electrons and does not allow reoxidation of plastoquinon A by the secondary electron acceptor, plastoquinone B. Competition studies under sorgoleone *versus* synthetic herbicides such as atrazine, diuron, metribuzin and bentazon have shown that sorgoleone is an atrazine competitive inhibitor [11, 12]. Moreover, the I50 of sorgoleone is 0.1 μM and similar to other PSII inhibitors. It is worth mentioning that sorgoleone belongs to the His215 family of PSII inhibitors, while atrazine belongs to Ser264. Mutation in Ser264 of the D1 protein is responsible for resistance to triazines as well as other non-triazine herbicides, leading to cross-resistance. However, plants resistant to atrazine, with a QB binding site on PSII mutation (Ser264), are not resistant to sorgoleone. Application of sorgoleone is particular‐ ly justified in the case of triazine-resistant biotypes of redroot pigweed, due to the same

with 10 μM sorgoleone were characterized by lower H+

common ragweed, redroot pigweed, and black nightshade [40].

(*Eragrostis tef* Zucc., Trotter) [11, 16].

526 Herbicides - Current Research and Case Studies in Use

Inhibition of H+

oration and necrosis [43, 44].

Extracts and residues of rice, the well-known cereal plant, also have allelopathic potential. Among isolated secondary metabolites, phenolic acids, hydroxamic acids, fatty acids, terpenes and indoles were identified [46]. The key role in rice allelopathy plays momilactone A and B isolated from root exudates. High allelopathic rice varieties release up to 2-3 μg of momilactone B per day [3]. These compounds inhibited the growth of typical weeds in rice, e.g. barnyard grass and awnless barnyard grass (*Echinochloa colona* (L.) Link.) at concentrations higher than 1 μM and 10 μM, respectively. Furthermore, phytotoxic abilities of momilacton A and B were also demonstrated on livid pigweed (*Amaranthus lividus* L.), hairy crabgrass and annual bluegrass (*Poa annua* L.) at concentrations higher than 60 μM and 12 μM, respectively [47]. The experiment has shown that momilactone B is secreted by rice roots into the rhizosphere over the entire life cycle [48]. Momilactone A and B belong to the diterpenoid phytoalexins which are known as antimicrobial secondary metabolites generated in response to signal molecules called elicitors (especially biotic elicitors) [49]. Both compounds thought to be unique to rice, recently have been found in the moss (*Hypnum plumaeforme* Wils.), a taxonomically distinct plant [49]. Despite the ability of momilactone A and B to inhibit plant growth, its mode of action in plants is still unknown.

#### **5.3. Artemisinin**

Artemisinin is a sesquiterpenoid lactone of annual wormwood (*Artemisia annua* L.). It is synthesized and sequestered in glandular trichomes located on the leaves and flowers [51]. It can also be excreted by the roots or root hairs, but only at the beginning of the growing season; therefore, dead leaves are the major source of artemisinin in soils [52]. Artemisinin is also lost from annual wormwood by rain runoff but to a minor degree (<0.5%),. This allelopathin is well known as a promising anti-malaric agent but also as a phytotoxin selective mainly to broadleaf weeds. Artemisinin (at 33 μM) significantly reduced shoot and root growth of lettuce, redroot pigweed, pitted morning-glory (*Ipomoea lacunose* L.) common purslane and annual wormwood [53]. However, the same treatment had no effect on sorghum or velvetleaf. Several studies have been aimed at identifying the molecular target site of this compound as well as the structural requirements for herbicidal activity [53-55].The effect of artemisinin is most evident on root growth and chlorophyll content. In onion root tips, artemisinin (10 - 100 μM) decreased the mitotic index, provoked abnormal mitotic figures and caused structural modifications of chromosomes [55]. However, no definite target site has yet been identified. The most recent studies on rice sprayed with 1.86 μM artemisinin indicated its inhibiting abilities on photo‐ synthetic electron transport [56]. Artemisinin site of action is probably plastoquinone B in photosystem II. Interestingly, as authors suggest, this effect is caused not directly by artemi‐ sinin itself, but rather by an unidentified artemisinin-metabolite occured in the plant after artemisinin application [56].

to give acceptable weed control. Such high doses excluded leptospermone from commercial development. The structure of this allelochemical was used as a basis for development of synthetic analogues including mesotrione (trade name Callisto), an herbicide produced by Syngenta AG. Mesotrione is applied for control of broadleaved weeds in maize. The rates of mesotrione are in the range from 75 to 225 g a.s. ha-1 (around 100 times more potent than

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529

However, leptospermone has lately been found as the main herbicidal component of manuka oil (*Leptospermum scoparium* J.R., G. Forst) [61]. Manuka oil (1%) applied as post-emergence spray, significantly decreased growth and dry weight of redroot pigweed, barnyardgrass, velvetleaf and hairy crabgrass. Though, hairy crabgrass seedlings that emerged after manuka oil application were totally blanched. Pre-emergence application of 0.17% manuka oil which corresponds to 0.2 L ha-1 of leptospermone inhibited hairy crabgrass growth by 50%. The preemergence effects are mainly dependent on its persistence in soil. Average time of leptosper‐ mone half-life in soil was calculated at 15 days while applied as a compound of manuka oil time extended by 3 days. This clearly shows that half-life of active compounds may be longer in mixture than applied alone due to additive or synergistic action. This type of leptospermone application poses another possibility of usage for this compound in its natural form without

Lately, there has been a growing interest for using essential oils as allelopathins with bioher‐ bicide potential. Some of them have already been commercialized and successfully launched in organic agriculture. They disrupt the cuticle and contribute to desiccation or burn down young tissues. Examples of this are the commercially available bioherbicide with the trade name of GreenMatch EX which consists of lemongrass (*Cymbopogon* sp.) oils or InterceptorTM with 10% pine (*Pinus sylvestris* L.) oil [3]. Essential oils are complex mixtures of monoterpenes, sesquiterpenes, and aromatic phenols, oxides, ethers, alcohols, esters, aldehydes and ketones [62]. The main terpenoids of volatile essential oils are monoterpenes (C10) and sesquiterpenes (C15). It has been well documented that essential oils found in foliage of eucalyptus (*Eucalyp‐ tus* sp.) show phytotoxic potential. During field experiments it has been reported that common weeds such as coffee senna (*Cassia occidentalis* L.) and barnyardgrass sprayed with different concentrations of eucalyptus oil (from 5 % to 10 % v/v with 0.05 % v/v Tween-80) exhibited dose-dependent and species-dependent levels of injury. Coffee senna plants were more sensitive to the eucalyptus oil than barnyardgrass [62]. Phytotoxicity of eucalyptus oil is due to the components such as 1,8-cineole, citronellal, citronellol, citronellyl acetate, p-cymene, eucamalol, limonene, linalool, α-pinene, γ-terpinene, α-terpineol, alloocimene, and aroma‐ dendrene [62]. Pre-emergence herbicidal activity of 1,8-cineole 3, and 1,4-cineole 4 were tested against rigid ryegrass and garden radish var. Long Scarlet in laboratory-based bioassays. 1,8 cineole and its derivatives showed a dose-dependent herbicidal activity against both weed species [64]. Laboratory studies [64, 65] also have shown that soil-applied 1,8-cineole sup‐ pressed the growth of several weeds. However, field reports demonstrated that 1,8-cineole alone has poor herbicidal activity [67, 68]. The commercial herbicide cinmethylin is a 2-benzyl

leptospermone) [60].

**5.5. Essential oils**

chemical modification of the structure [61].

Other controversies around the phytotoxic potential of artemisinin arose when the dichloro‐ methane extracts of annual wormwood leaves containing artemisinin showed a stronger phytotoxic effect on redroot pigweed seed germination and seedling growth than pure artemisinin [57]. Moreover, aqueous extract with disposed artemisinin had equal inhibitory effects on both physiological processes as allelopathin alone. This experiment suggests a marginal role of artemisinin in plant extract and joint action of other allelochemicals. Although, most studies analyzing allelopathic weed–crop interferences using annual wormwood were conducted under laboratory and greenhouse conditions [58].

Toxic studies on duckweed (*Lemna minor* L.) and the fresh water algae *Pseudokirchneriella subcapitata* (Korshikov) had EC50 values 0.24 and 0.19 mg L−1 respectively, with growth rate as endpoint corresponding to those of the herbicide atrazine [59]. These profiles questioned environmental safety of artemisinin for the purpose as a bioherbicide. It may be a result of its complex chemical structure, but this compound may be used as the ba‐ sis for a new herbicide, based on artemisinin chemical structure. Such attempts have al‐ ready been made using artemisinin's analogues [55]. Four of the tested 12 analogues inhibited germination and root growth of lettuce, *Arabidopsis thaliana* (L.) and duckweed at extremely low concentrations (3 μM).

#### **5.4. Leptospermone**

Leptospermone (1-hydroxy-2-isovaloryl-4,4,6,6-tetramethyl cyclohexen-3,5-dione) is a natural triketone produced by the roots of the bottlebrush (*Callistemon citrinus* Curtis) [60]. In its pure form, it was tested both pre- and post-emergence on a range of plant species including: hairy crabgrass, yellow foxtail *(Setaria glauca* (L.) P. Beauv.), barnyard grass, California red oat (*Avena sativa* L.), redroot pigweed, Indian mustard (*Brassica juncea* L.) and curly dock (*Rumex crispus* L.). Leptospermone is a strong p- hydroxyphenylpyruvate dioxygenase (HPPD) inhibitor with *I50* values 3 μg mL-1[61]. Inhibition of this enzyme leads to disruption in carotenoid biosynthesis and loss of chlorophyll. Unfortunately, a pure compound rate of 9000 g a.s. ha-1 was required to give acceptable weed control. Such high doses excluded leptospermone from commercial development. The structure of this allelochemical was used as a basis for development of synthetic analogues including mesotrione (trade name Callisto), an herbicide produced by Syngenta AG. Mesotrione is applied for control of broadleaved weeds in maize. The rates of mesotrione are in the range from 75 to 225 g a.s. ha-1 (around 100 times more potent than leptospermone) [60].

However, leptospermone has lately been found as the main herbicidal component of manuka oil (*Leptospermum scoparium* J.R., G. Forst) [61]. Manuka oil (1%) applied as post-emergence spray, significantly decreased growth and dry weight of redroot pigweed, barnyardgrass, velvetleaf and hairy crabgrass. Though, hairy crabgrass seedlings that emerged after manuka oil application were totally blanched. Pre-emergence application of 0.17% manuka oil which corresponds to 0.2 L ha-1 of leptospermone inhibited hairy crabgrass growth by 50%. The preemergence effects are mainly dependent on its persistence in soil. Average time of leptosper‐ mone half-life in soil was calculated at 15 days while applied as a compound of manuka oil time extended by 3 days. This clearly shows that half-life of active compounds may be longer in mixture than applied alone due to additive or synergistic action. This type of leptospermone application poses another possibility of usage for this compound in its natural form without chemical modification of the structure [61].

#### **5.5. Essential oils**