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Content archived on 2024-05-15

Fate and toxicity of allelochemicals (natural plant toxins) in relation to environment and consumer

Deliverables

A risk assessment was performed on the fate of wheat allelochemicals in soil, applying empirical degradation and sorption studies as well as QSAR modelling studies. The risk assessment is very relevant when allelopathic properties of cereals are exploited. Exploitation of allelopathic properties is expected to increase as a means of reducing the use of synthetic pesticides. However, it is of importance to assure that no harmful allelochemicals or transformation products hereof will leach to the groundwater. The risk assessment was performed in relation to the Uniform Principles (Directive 91/414/EEC) that regulates the use of synthetic pesticides.
We developed a data set that provides a measure of toxicity for a group of compounds by selecting synthetic pesticides with structures similar to that of allelochemicals. In particular, all compounds contain an aromatic and/or heterocyclic ring with N and O. We collected for them different species (rat, daphnia, quail, duck. trout) toxicity values comparing three different databases. We evaluated the minimum energy conformation for each compound and two different approaches to generate data of molecular descriptors were applied. We calculated atom based (some hundred) and field based (some thousands) descriptors.
The analysis, bioactivity evaluation and determination of fate and toxicity of wheat allelochemicals and their main degradation products needs useful protocols for their synthetic obtaining up to the multigram scale. Thus, several methodologies including optimisations from previously reported works, and new procedures have been developed in order to get amounts of the compounds enough for all of these purposes. The optimised synthetic methodologies have yielded compounds belonging to three different structural families: benzoxazinones, hydroxyphenylmalonamic acids and aminophenoxazinones that derive from natural allelochemicals DIMBOA and DIBOA. Benzoxazinones have been obtained from synthetic methods optimised from literature. These procedures have been modified with the intention of increasing reaction yields and obtaining starting materials of diverse structure, in order to get new leads to the development of new herbicide models based on their structures. Several of these compounds have been used as chemical standards in the development of analytical procedures towards the determination of soil degradation dynamics of allelochemicals. Hydroxyphenylmalonamic acids are fungal detoxification products from allelochemicals DIMBOA and DIBOA. Their synthesis has been developed in order to have enough amounts for their bioactivity evaluation, and to discover their ecological role. Aminophenoxazinones have been synthesized in order to determine their presence and concentration in soil after the release of allelochemicals DIMBOA and DIBOA by plants. Their bioactivity evaluation is an interesting research topic since these compounds could important to understand the allelopathic behaviour of plants that produce high amounts of benzohydroxamic acids. New synthetic procedures have been developed to get access to some derivatives in large scale.
Three different techniques were used to develop the models: Multiple Linear Regression (MLR), Partial Least Square (PLS) and Group Method of Data Handling (GMDH) Neural Networks. These studies have been done in collaboration with F. Lemke (KnowledgeMiner, Berlin) and G. Gini (Politecnico di Milano). Thus it was possible to develop several models and compare them. We also used Biochemical analysis (Docking and GRID methodology) to compare and increase the information obtained from field-based descriptors. The role of biochemical analysis is to understand biological processes and try to analyse and model the interaction of the ligand with the binding site and the formation of the ligand-macromolecule complex. 3D models, using CoMFA, GRID and Docking approaches, gave good results, which also indicate important likely molecular toxic metabolism processes. The use of atom based descriptors and GMDH vice versa is more related on automatic extraction of knowledge, independent on mechanism. Even if it gives lower performances, it can be more general and applicable to more heterogeneous structures.
The activity of the benzoxazinone allelochemicals DIMBOA and DIBOA, their main degradation products and several synthetic analogues were studied on a range of plant species including crops as well as common weed species. Studies were done using standardised laboratory tests measuring the effect of the benzoxazinones, their degradation products and synthetic analogues on germination, shoot and root growth. In general root growth was more susceptible to the allelochemicals than shoot growth and germination was the least affected parameter. Some of the metabolites were more phytotoxic than their parent compounds and particularly APO, a metabolite of DIBOA, was highly active. In the field plants will often be exposed simultaneously to several allelochemicals hence besides studying the effects of single allelochemicals we also studied the joint action of mixtures of two or three allelochemicals including the benzoxazinones, their metabolites and phenolic acids. It was found that the mixtures were either additive or slightly antagonistic and synergism, although commonly claimed, was not observed.
The wide variety of methods employed in isolation of benzoxazinoid allelochemicals has been optimised. The phytochemical constituents of wheat varieties in study afforded 250 fractions belonging to DCM and EtOAc extracts. We have studied them providing 40 major compounds, belonging to the flavonoids, simple phenolic and steroid families and one very abundant monoterpene. Needs addressed The methodology has led us to get access to the target compounds (DIMBOA, DIBOA and their glycosides). A complete phytochemical study of selected wheat varieties has been achieved. These compounds have to be obtained in amounts and purities enough to complete their bioactivity profiles and to get analytical standards to complete their determination in plant, soil and bioassay experiments.
The effects of the cereal allelochemicals and naturally occurring degradation products which had been identified in the course of the project FATEALLCHEM on the two soil organisms Folsomia candida (collembola) and Poecilus cupreus (carabid beetle) and on the aquatic organisms Daphnia magna (micro-crustaceae), Pseudokirchneriella subcapitata (former: Selenastrum capricornutum, freshwater algae) Chlorella pyrenoidosa (soil algae) and Vibrio fischeri (marine bacterium) were assessed to detect potential adverse effects on the non target organisms representing four different trophic levels and two ecotoxicological compartments. The results can be implemented into a risk assessment procedure, such as for the authorization of plant protection products (Directive 91/414/EEC) to evaluate, whether plants with increased allelochemical properties could pose a risk to soil non targets organisms, which are known as beneficials for the soil ecosystem especially in arable crop production.
A framework has been developed which describes the test needs for risk assessment of plant allelopathic properties. The framework is of importance for national legislators as well as legislators at European level (DG Agriculture, DG Environment, DG Consumer Health). Already in the project preparation phase, a framework was described for the evaluation of the allelopathic properties of wheat and used throughout the project. The project results proved that a broad range of tests is needed when risk assessment of allelopathic properties is performed.
The activity of benzoxazinoid allelochemicals have been measured against wheat pathogenic fungi: Gaeumannomyces graminis var tritici, Fusarium culmorum and Cephalosporium gramineum. The data relate to antifungal activity of unique group of naturally occurring compounds as well as their degradation products.
Wheat varieties have been tested for their qualities for reproduction of cereal aphids. No significant difference was found between small plants. A significant difference was found between mature plants. However, this significance cannot be correlated to the content of hydroxamic acids or concentrations. Different compounds and concentrations were tested in artificial food for their effect to aphids, leaf beetles and beneficials. A concentration of MBOA at 0.25mM will reduce the intrinsic rate of increase to 50%. BOA seems to have a stronger effect. This concentration is more than 10 times higher than found in the tested wheat varieties. All other allelochemicals compounds are found in lower concentrations. There is a tendency to reduce occurrence of parasitoids if content of MBOA is high inside cereal aphids. A whole range of hydroxamic acids were tested to cereal aphids for their insecticidal contact effect. The tested hydroxamic acids used in concentrations as conventional insecticides show only little insecticidal effect.
Since it has been found that benzoxazinoid allelochemical compounds and their decomposition products play an important role in the resistance of plant to insect pests and plant pathogenic fungi it has increased scientific interest for allelopathy meaning a potential for selective biological weed management. Analytical methodology have been developed and validated to quantify the content of hydroxamic acids, lactams, benzoxazinones in wheat cultivars in different (organic and conventional) farming systems and benzoxazinone derivatives as aminophenoxazinones and corresponding malonamic acids in surrounding soils. Accurate methodology was achieved by an inter-laboratory study, which involved the laboratories, which carried out benzoxazinone analyses. The results obtained from the project FATEALLCHEM will be very useful to implement the use of such chemicals in agriculture instead of pesticides. In this respect Directive 91/414/EEC concerns the placing on the market of plant protection products and the revision of this directive, specially concerning the zonal authorisation of pesticides will complicate the necessary risk assessment. By using alternative and more friendly compounds, like allelochemicals, it is expected that many of the problems encountered with the use of traditional pesticides will be solved and it will be easier to comply with Directive 91/414/EEC.
The database is constructed in the programme Access and will be available to the public in the project homepage http://www.fateallchem.dk by the end of the project period. The information given in the database has been collected from all known international papers dealing with microbial transformation of benzoxazolinone allelochemicals and published in a review in the journal Chemosphere. Additionally the database will summarize the results obtained in the FATEALLCHEM project concerning transformation products.
The content of benzoxazinoid allelochemicals has been quantified in foliage and/ or roots of 25 wheat varieties. For six varieties of wheat cultivated under conventional as well as organic farming conditions in North and South Europe the content of benzoxazinoids in foliage as well as roots has been quantified at 5 growth stages and for nineteen varieties cultivated in central Europe the content in roots has been quantified at 3 growth stages. The result provides a unique collection of data concerning variations in natural content of benzoxazinoid allelochemicals in a wide range of different varieties of wheat at different stages of development.

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