Phytochemical responses to herbicide exposure and effects on herbivorous insects
11. General summary and discussion
11.1 General phytochemical trends in relation to biotic and abiotic factors
11.1.1 Growth conditions
Three different experimental set-ups were used, resulting in different growth conditions for plants (and herbivores), i.e. controlled-environment chamber (after treatment) (Chapters 3, 4, 5, 6, 7 and 10), greenhouse with or without supply of UV-B light (Chapters 4 and 9) and field-like conditions on tables outside the greenhouse (Chapter 4). Since phytochemicals were not measured in plants from Chapters 9 and 10, they are excluded from this part of the discussion. Here only results of growth conditions are presented, i.e. phytochemical concentrations in control plants not subjected to herbivory. The collective term’laboratory’ is used for greenhouse and controlled-environment chamber.
The concentration of compound 1 was higher in plants grown under field conditions than in plants grown in greenhouse or controlled-environment chamber (Chapters 2, 4, 5, 6). Concentrations consistently increased from bottom to top leaves, i.e. was largest in younger leaves (Chapters 2, 3, 4) both in controlled-environment-chamber and under field conditions. Supply of UV-B caused a slight increase in middle leaves of greenhouse plants (Chapter 4), but not enough to approach the content to that of field plants. In Chapter 4, time had no effect on the vertical distribution of compound 1, but when the same leaf position was followed over time, the concentration of compound 1 decreased (Chapter 2). In Chapter 7, in which the plants are followed for a longer period than in Chapter 4, the concentration of compound 1 in the middle (third) leaf first increased and then decreased. Chapter 5 was not comparable. Thus, it seems likely that compound 1 can be found in higher concentrations in rather young leaves than in older leaves, as a consequence of either dilution due to growth, transport out of the ageing leaves, or formation of the compound mainly taking place in growing (younger) leaves.
For compound 2, concentrations were also higher in field plants than in laboratory and greenhouse plants (Chapters 2, 3, 4, 6, 7, 10). UV-B had a large impact on greenhouse plants, increasing the concentration to the same level as seen in field plants (Chapter 4). This strongly indicates that light is one of the major factors affecting compound 2. Vertical distribution within the plants was similar for field and laboratory plants, with the highest concentrations in the top leaves, except for Chapter 3, where concentrations tended to be equal or higher in middle leaves. Concentrations in the middle (third) leaf tended to increase with time and then decrease (Chapters 2, 4 and 7). This was somewhat opposite to the effect in field plants (Chapter 4), in which the content in the middle leaf did not decrease with time, but then again the time-span was shorter for field plants than for laboratory plants.
Compound 3 was never found in untreated laboratory plants, whereas (only small) concentrations were found in plants grown under field conditions (Chapter 4). Supplying greenhouse plants with UV-B light (Chapter 4) could not mimic this difference.
Compound 4 occurred in comparable concentrations in field and controlled-environment-chamber plants (Chapters 2, 4, 6, 7), whereas the corresponding data for controlled-environment-chamber in Chapter 3 and greenhouse plants in Chapter 4 were about twice as high. Since effects of time (Chapters 2, 4 and 7) were small (decrease) or absent, and UV-B light (Chapter 4) had no effect on compound 4, the differences between the various experiments cannot be explained. Concentrations increased from bottom to top (or middle) leaves for both field and laboratory plants.
Compound 5 was found in much larger concentrations in greenhouse plants (Chapter 4) than in plants grown in controlled-environment chamber (Chapter 2). We have no obvious explanation for this discrepancy, since neither time nor UV light seemed to have any major impact on this compound, which was not identified in the other experiments.
Compound 6 was found in larger concentrations in field plants than in laboratory plants (Chapters 2, 3, 4, 6, 7, 10). UV-B supply increased the similarity in concentrations of compound 6 in middle leaves between greenhouse and field plants, but there were still considerable larger concentrations in field plants, indicating that other factors differing between laboratory and field conditions have a major impact on this compound. In laboratory plants, compound 6 was found almost exclusively in the top leaves (Chapters 2, 3, 4), whereas the vertical distribution in field plants was more uniform, although concentrations still were highest in top leaves (Chapter 4). Concentrations decreased slightly with time in laboratory plants (Chapters 2, 4, 7), whereas no time effect was seen in field plants (Chapter 4).
In conclusion, the occurrence and distribution of the selected phytochemical compounds in control plants were fairly similar in experiments performed under laboratory conditions. With the exception of compound 4, concentrations were higher in field plants than in laboratory plants, and the vertical distribution also was somewhat different from plants grown indoor. Light conditions proved to be an important factor in the phytochemical difference between growth conditions for compounds 1, 2 and 6, which has also been found for phenolic compounds in e.g. birch (Lavola, 1998), rice (Ambasht and Agrawal, 1997) and several other species, as reviewed by Waterman and Mole, 1989).
11.1.2 Chlorsulfuron treatment
Concentrations of compound 1 decreased in both laboratory and field plants after treatment with the herbicide chlorsulfuron, especially in top leaves, but the same trend was seen in middle leaves (Chapters 3, 4, 6 and 7). There were hardly any effects of time in laboratory plants (Chapters 4 and 7), whereas for field plants the herbicide effect increased with time (Chapter 4). Supply of UV-B did not change the general picture in greenhouse plants (Chapters 4 and 7).
Chlorsulfuron treatment generally increased the content of compound 2 in laboratory plants (Chapters 3, 4, 6, and 7), whereas herbicide effects were almost absent in field plants (Chapter 4). Effects were most pronounced in bottom and middle leaves of laboratory plants, whereas concentrations in top leaves were unaffected or reduced (Chapters 3 and 4).
As already mentioned, in laboratory plants compound 3 only occurred after herbicide treatment, whereas in field plants the compound was also found in untreated plants. In laboratory plants, the highest concentrations of compound 3 following herbicide treatment was found in bottom (Chapter 3) or middle leaves (Chapter 4), whereas in field plants the highest concentrations in herbicide treated plants were found in top leaves (Chapter 4). The herbicide effects on compound 3 in middle leaves of greenhouse plants were enhanced by UV-B light (Chapter 4).
The content of compound 4 was almost unaffected in both laboratory and field plants in Chapter 4, but there was a tendency of a decrease at chlorsulfuron dosages up to 0.5 time the recommended field rate, whereas at full dosage the concentration increased again. A similar effect of time on phytochemical response following chlorsulfuron treatment was found in sunflower seedlings by (Suttle et al., 1983). In contrast to this, both 0.5 times the field rate (Chapters 5, 6 and 7) and full field rate (Chapter 3) resulted in a decrease in compound 4 in laboratory plants. Since time does not seem to have an effects on the effect of herbicide treatment on compound 4, an explanation of the mentioned discrepancies seems difficult on basis of the presented studies.
Compound 5 was only observed after herbicide treatment in one experiment (Chapter 4), and consequently comparisons between studies are not possible.
Concentrations of compound 6 were generally reduced following chlorsulfuron treatment (Chapters 3, 5 and 6), particularly in top leaves of laboratory plants (Chapters and 4) and middle leaves of field plants (Chapter 4). As was the case for control leaves, UV-B supply caused the concentration of compound 6 in middle leaves of laboratory plants to approach that of field plants (Chapter 6).
All in all, consistency is good between the different laboratory experiment concerning the effects of chlorsulfuron treatment on the selected phytochemical compounds, except for compound 4. Herbicide effects on phytochemicals were generally less evident in field plants than in laboratory plants. Differences in herbicide effects between growth conditions were largest for compounds 2, 3 and 6. Supply of UV-B light in the laboratory reduced the difference between laboratory and field. This may be a consequence of induced changes in the plant, as discussed above, or a result of UV- mediated changes of the chemistry of the herbicide (Gold et al., 1994).
However, part of the difference between field and laboratory plants is due to differences in vertical distribution of the phytochemicals within the plants, and the effect of UV-B on this aspect remains unsolved. Other possible explanations of the observed differences in levels and distribution between laboratory and field plants may include the habitus of the plants. Field plants were very compact compared to laboratory plants, and as a consequence herbicide deposition may be more uneven in field plants, assuming that the top leaves exerted a "shadowing" effects on the lower leaves. This may have led to a lower exposure of middle and bottom leaves, which may explain the tendency of compounds 2 and 3 to concentrate more in the top leaves of field plants than in laboratory plants. Furthermore, field plants are expected to have a thicker wax layer than laboratory plants, and especially the lower (older) leaves of field plants may thus have experienced a lower internal concentration of the herbicide, which may also add to the difference in vertical distribution compared to laboratory plants (Schreiber and Schönherr, 1992). On the other hand, wind and heavy rainfall may damage the wax layer, resulting in an increased penetration of the herbicide (MacKerron, 1976) as referred in Bonnet and Bossharert, 1994).
11.2 Implications
11.2.1 Laboratory versus field
From the above it follows that laboratory experiments may provide a fairly good background for predictions of the effects of chlorsulfuron on the selected phytochemicals under field conditions concerning the general pattern. However, numerical values are generally underestimated in laboratory, and there seems to be a tendency to overestimate herbicide effects on concentrations of phytochemicals. The UV experiments revealed that laboratory conditions may approach field condition if UV-B light is supplyed in combination with ordinary lamps. There are, of course, other parameters differing between laboratory and field, which may affect the phytochemical profile both in control plants, and in herbicide treated plants. It was noted in Chapter 4 that field plants have a different growth form than laboratory plants, i.e. are more compact and therefore experience a different herbicide and light exposure. In addition, colour differences are often seen. Both characteristics are likely to stem from differences in light intensity, possibly combined with wind and precipitation. Furthermore, these parameters may also affect other plant characteristics, such as the thickness of the wax layer (as discussed above) and the persistence and translocation of herbicides within the plants (Shaner, 1994), which may in turn affect the activity of the herbicide. A solution for approaching the mentioned parameters to the situation in the field may be semi-field experiments, like the ones described in the present report, where plants are grown outside, but with controlled supply of water etc., and sprayed under controlled conditions. However, this limits the experimental season greatly compared to laboratory experiments. Furthermore, the remaining discrepancies from real field conditions (soil characteristics, water supply and exposure conditions) may also indirectly affect the phytochemical response. This may happen through their effects on physical plant characteristics and metabolism, but also because of differences in actual exposure, as indicated in Chapter 9, where spraying with dosages at the recommended field rate resulted in c. 40 % deposition right beneath the sprayer.
11.2.2 Indicator of exposure
In Chapter 3 we found that treatment with all the tested herbicides, i.e. both sulfonylurea herbicides and herbicides with other modes of action, caused a response in the concentrations of compounds 1, 4 and 6 in F. convolvulus. These compounds may thus be general herbicide stress indicators, and for compounds 1 and 6 this is supported by the rather uniform reaction in the different laboratory studies (see start of this chapter).
Concentrations of compounds 2 and 3 in F. convolvulus leaves only changed when the plants were sprayed with sulfonylurea herbicides (Chapter 3). In the presented laboratory experiments, compound 3 was only found in sprayed plants, not in the control plants. However, in field plants compound 3 was found in low concentrations in controls (Chapter 4). Consequently, the use of this compound as an indicator of plant exposure to sulfonylurea herbicides may not be as promising as indicated by the laboratory experiments. However, if the simple relationship between chlorsulfuron dosage and leaf concentrations of compound 3 found for laboratory plants in Chapter 5 also holds for the field situation, this compound may still possess an indicator potential.
In Chapter 5 we found that there was a "window" of 2-3 weeks under laboratory conditions of 20° C in which the effect of chlorsulfuron on compound 3 was detectable and the response linear with herbicide dosage. Under field conditions, this would correspond to a window of approximately 1.5 months in field. We also found that the phytochemical response took at least 4 days to be induced (Chapter 2 and 4), which would correspond to a delay of approximately one week under field conditions. The lowest chlorsulfuron dosage at which the response of compound 3 was induced was 6.25 % (1/16) of the field rate (Chapter 5). At the lowest dosage tested, i.e. 3.125 % of the field rate, no response could be detected. In relation to spray drift (Chapter 9), this means that a phytochemical response in the plant tested here can only be expected within the first meter or so from the spray boom, assuming the sensitivity under field conditions equal the one found under laboratory conditions.
11.2.3 Indicator of effects on herbivores in agricultural fields and in the spray drift zone
In Chapter 8 a model for the concentration of compounds 2 and 3 was presented. The model mediated calculations of body burden with respect to compounds 2 and 3 were related to the survival of the insect larvae. Compound 2 was highly correlated with the survival of the larvae. The phenolic compounds we have focused on may co-vary with other compounds, which are the actual elicitors of the observed effects. Such a general response would be caused by the general side effect of sulfonylurea herbicides, i.e. reduced transport out of the leaves (Bestman et al., 1990; Vanden Borne et al., 1998). Other compounds that might be relevant encompass both primary (e.g. nitrogenous compounds) and secondary plant metabolites. It is intriguing that compound 2 explains not only the mortality of insects on treated plants but also the high mortality of insect placed on control plants. Compound 3 also responded to herbicide dosage, but the control plants weakened the correlation, because compound 3 is not present in control plants.
Another explanation for the observed disappearance/mortality of G. polygoni is that a feeding attractant is reduced so that the beetle stops eating. For this to happen it is a prerequisite that the insect does not recognise the plant as a host even under the no-choice conditions of starvation.
In the project, two field experiments was carried out in which the survival of G. polygoni larvae was followed over time without any measures of phenolic compounds. In the first experiment, no herbicide treatment was involved, whereas in the other, herbicide treatment was included in three dosages. In all experiments, irrespective of herbicide dosage, only 2 to 4 individuals per replicate (out of 20) survived a full larval development. In order to confirm if this high mortality should be expected from the concentrations of phenolic compounds, we modelled the mortality from the content of compound 2 on the basis of the established regression line between survival and concentration of compound 2 in laboratory experiments (Chapter 8). The content of compound 2 was modelled with the assumption of 20 larvae from the start, all larvae present on the third leaf counted from the bottom of the plant and UV-B radiation present. The computations revealed that only minor effects of chlorsulfuron dosage are predicted (Figure 1.1), with the number of survivors approximately the same as observed in the experiments.
Figure 11.1
Model estimates of number of larvae surviving as a function of chlorsulfuron dosages
applied to host plant.
The calculations presented above may also be used in a prediction of G. polygoni- survival on plants placed in the spray drift zone. In Chapter 9 it was measured that plant in a 5 m zone downwind of the spray swath would receive between 0.01 and 0.06 times the recommended field rate. A slight reduction in the number of surviving larvae should therefore be expected (Figure 11.1).
The calculations presented above suggest that nearly all G. polygoni would die under normal conditions in the agricultural field. This is, however, not the case (Kjær et al., 1998a). The reason for the discrepancy could be that normally F. convolvulus plants, unlike our test plants, are growing in the shadow of the crop plants. Therefore, they do not receive the same amount of UV-B light. The larvae are primarily found on the lower parts of the plant, i.e. on parts receiving low UV-B radiation. Consequently, the UV parameter in the model is too high for these conditions, but not for the test condition. To make this model more precise and relevant for prediction of field effects, an experiment should be performed with a range of light intensities and qualities, incorporating the behaviour of the larvae.
The spray drift measures reported in Chapter 9 were performed under low wind conditions and with only one swath. The use of multiple swaths has been observed to increase the downwind deposition with a factor approximately 1.4 (Dobson et al., 1983; Gilbert and Bell, 1988), and the boom height and wind speed also affect the spray drift (Nordby and Skuterud, 1975). The volume of spray solution as predictor of effect can be questioned, because droplets evaporate and the resulting drop can be more concentrated with respect to the spray and smaller droplet also tend to be better withheld by diverse structures. This may explain the 10 times difference in effect of the same volume spray solution in the spray zone compared to observation under the sprayer observed by (Nordby and Skuterud, 1975). If this observation is valid a safety factor of 10 should be added to the actual deposition measures in order to estimate biological effects. The study, unfortunately, had different effect assessments for the two treatment conditions, i.e. lab and field assessment, rendering the estimate controversial. But if the measure in fact does express an ecotoxicological difference the spray drift zone is greatly expanded.
11.3 Conclusion
In general terms, the implications of the present study are:
 | The use of phytochemical exposure indicators specific of single herbicides or groups of herbicides seems promising for plants treated with low-dosage herbicides. |
| For herbivorous insects in and outside the field, the main effect of low-dosage herbicides is likely to be the possible loss of food source. The effects so far documented for one herbicide-group out of six and one insect-plant system out of three, is not a general phenomenon, how ever, it is likely to be found in other systems as well. |
| Drift effects of low dosage herbicides are likely to occur within a distance of 5 m from the sprayed field on plants in the nearby hedge/field margin and in adjacent crops. |
| The study has underlined the difficulties in making reliable predictions of the concentration of phenolic compounds in plants grown in the field on basis of laboratory data. The inclusion of UV-light in laboratory set-ups reduce the difference between lab and field significantly |