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Pesticides Research, 57

Pesticides in air and in precipitation and effects on plant communities

Effects of herbicides in precipitation on plants and plant communities

Indhold

Summary

In several European studies pesticides have been found in precipitation. The potential risk of adverse effects of herbicides in precipitation on plants and plant communities in the agro-ecosystem and adjacent ecosystem can be assessed if the No Observable Effect Level (NOEL) is known. In this project NOEL was defined as the dose required to reduce fresh or dry weight by 10% (ED₁₀). Mecoprop-P was selected as model pesticide and the ED₁₀ dose was determined on a number of susceptible plant species at different growth stages. In addition, the competition indeces between selected species grown in binary mixtures were compared without herbicide treatment and following application of mecoprop-P doses close to ED_10. The ED₁₀ dose of the most susceptible plant species included in this study was more than 3 times higher than the maximum yearly deposition of mecoprop in Danish rain water. In the study, different methods were applied to assess effects of the low doses of mecoprop-P. The results showed that biomass production was as susceptible a parameter as seed production. The substitution rate between plant species with similar competitive ability but different susceptibility to mecoprop was not significantly influenced of doses close to ED₁₀.

Sammendrag

I såvel internationale som danske undersøgelser er der påvist forekomst af pesticider i nedbør. For at kunne vurdere hvorvidt disse forekomster udgør en risiko i økotoksikologisk sammenhæng, er der behov for viden om hvor lave koncentrationer af pesticider, der kan forventes at resultere i målbare effekter i naturen. Af hensyn til den statistiske bearbejdning af resultaterne har vi valgt at definere NOEL (No Observable Effect Level), som den dosering, der resulterer i 10% reduktion af frisk- eller tørvægten på de behandlede planter (=ED₁₀). Mechlorprop-P er anvendt som modelstof ,og ED₁₀ doseringen af dette herbicid er bestemt overfor en række følsomme plantearter (Agersennep, Pengeurt, Hyrdetaske, Agertidsel, Haremad, Hvidmelet gåsefod og Fuglegræs). Bestemmelsen er foretaget ved behandling på forskellige udviklingstrin, ved forskellige høsttidspunkter og ved måling af biomasse og frøproduktion. Desuden er det undersøgt, om konkurrenceevnen af en følsom art overfor en mindre følsom art påvirkes ved behandling med doseringer af mechlorprop-P omkring ED₁₀. Resultaterne viste, at der ikke er målbare effekter af de mængder af mechlorprop-P, som er fundet i opsamlede regnvandsprøver , idet ED₁₀ doseringen på den mest følsomme planteart var ca. 10 gange højere end det maksimale fund i en enkelt udtagning og 3 gange højere end den gennemsnitlige årlige deponering pr. arealenhed. Undersøgelsen har desuden vist, at hverken frøproduktion eller konkurrenceevnen er mere følsomme parametre end biomasseproduktion.

1. Introduction

Several European studies have revealed that pesticides can occur in precipitation (Sibers et al., 1995; Jaeschke et al., 1995, Lode et al., 1995; Kreuger, 1995). In the years 1992-94 rain water collected at two locations in Denmark was analysed for the content of 10 pesticides but only phenoxyalkanoic acid herbicides were found and this group of herbicides occured at concentrations up to 0.4 mg/l (Kirknel & Felding, 1995). For this study we therefore selected mecoprop as model herbicide. Mecoprop was extensively used in Denmark in 1996 when the project was initiated, however in the following year the usage of phenoxyalkanoic acid herbicides was severely restricted resulting in a 80-85% reduction in the consumption from 1996 to 1997.

It seems probable that herbicides in precipation can affect the growth and reproductivity of terrestrial plants. However no attempts have been made so far to assess the possible effects of herbicides in precipitation on plants and plant communities in the agro-ecosystems and adjacent ecosystems.

The objective of this part of the project was to assess the potential risk of adverse effects of mecoprop-P present in precipitation on plants and plant communities in the agro-ecosystem.

The No Observable Effect Level (NOEL) is a recognized ecotoxicological term designating the highest dose having no effects on the growth of the test organism. As the experimental design and number of replications can have a marked influence on the estimation of NOEL it is more convenient to define NOEL as the dose required to reduce growth by 5 or 10%. In this project the dose resulting in a 10% reduction in growth of the test plants (ED10) has been used as a measure for NOEL.

2. Materials and methods

The effects of low doses of mecoprop were examined in two different types of experiments:

1. The ED₁₀ dose of mecoprop-P was determined on a number of susceptible plant species
2. The competition indeces between selected species grown in binary mixtures were compared following no application of herbicide and application of mecoprop-P doses close to the ED_{10 .}

2.1 Experiments for determining ED₁₀ doses

Test plants

The ED₁₀ doses of mecoprop-P were determined on Capsella bursa-pastoris, Thlaspi arvense, Sinapis alba, Circium arvense, Lapsana communis Chenopodium album and Stellaria media. All species are known to be very susceptible to phenoxyalkanoic acid herbicides.

The experiments were carried out in a glasshouse in the period from April 15 to October 15. Seeds were sown in 2 L pots in a soil/ sand/ peat mixture (2:1:1 w/w) containing all necessary macro and micro nutrients. After emergence the number of plants per pot was reduced to a similar number.

Application and doses

The treatments with mecoprop were carried out on different growth stages of the plants varying from two to eight leaves. In one experiment Thlaspi arvense was also exposed to mecoprop-P at the flowering stage. Mecoprop-P was applied using a laboratory pot sprayer equipped with two Hardi 4110-14 nozzles in a spray volume varying between 130 and 165 L ha^-1. Each plant species was treated with 7 to 10 doses of mecoprop-P (Duplosan MP, 600 g L^-1 mecoprop-P). In order to obtain responses from 0 to 100% effect the doses were varied between plant species and growth stages. Each treatment was replicated three times using a completely randomised layout

Plants were harvested 3-4 weeks after treatment and fresh and dry weights were collected. In one experiments the influence of time of harvest was examined by harvesting at two different time intervals (2 respectively 4 weeks) after treatment. This experiment included also a comparison of the effect of mecoprop-P on seed versus biomass production.

Model used for estimating dose response curves

For each plant species and growth stage dose response curves were estimated using non-linear regressions. A four parameter logistic model was used to describe the relationship between plant weight and dose :

In this model U is the fresh- or dry weight of the plants, D denotes the upper limit of plant biomass at zero dose, C the lower limit at high doses, b is the slope, z the dose and ED₁₀ is the dose resulting in 10% reduction of growth.

The regression model was evaluated by a test for lack of fit comparing the sum of residuals from the regression and variance analyses.

2.2 Competition experiments

Test plants

In the competition experiments different densities of C. bursa-pastoris and G. dissectum were grown in monoculture and in binary mixtures varying the ratio and density of the two species.

At the 1-2 leaf stage plants of the two species were transplanted to 40 by 40 cm polystyren boxes (growth area 35 by 35 cm) filled with a soil/sand/peat mixture (2:1:1 w/w) containing all necessary micro- and macro nutrients.

The experiments were based on a complete additive design as proposed by Cousens (1991). Plants were transplanted in a geometric series of plant densities using regular patterns (Figure 1). The density of each plant species varied between 1 and 32 plants per box covering scenarios from no competition to high competition intensity. For each plant species, 5 different densities were used giving 25 different combinations. In addition a high density (64 plants per box) of each species alone was included in order to assess the intraspecific competition at a high density.

The plants were placed in a glasshouse and watered daily. The boxes were placed in blocks according to the total number of plants in the boxes. One block included boxes with less than 20 plants, another included boxes with 20 to 40 plants and in the last block boxes with more than 40 plants were placed.

Figure 1
Composition of two component mixtures of G. dissectum and C. bursa-pastoris in the experiments. The axes show the number of plants per box (growth area 35 by 35 cm).

Sammensætning af to-komponent blandinger af G. dissectum og C. bursa-pastoris i forsøgene. Akserne viser antallet af hver planteart pr. kasse (vækstareal 35 x 35 cm).

Application and doses

Mecoprop-P was applied to the plants at the 6-8 leaves stage. Each combination of plant densities and ratios were represented by an untreated and one or two doses of mecoprop-P close to the expected ED₁₀ dose of C. bursa-pastoris. Each treatment was replicated twice. Mecoprop-P was applied using a laboratory pot sprayer equipped with two Hardi 4110-14 flat fan nozzles producing a spray volume of 150 L ha^-1.

Concurrently with the competition experiments the ED₁₀ doses of each of the two plant species were estimated in pot experiments.

The plants were harvested 3 weeks after treatment. The outer 5 cm of the boxes was not harvested. The number of plants of each species was counted and harvested separately. Fresh- and dry weights were collected.

The biomass per plant y₁ and y₂ of species 1 (C. bursa-pastoris) and species 2 (G. dissectum) can be calculated when the number of plants n₁ and n₂ and the total plant biomass x₁ and x₂ are known.

Competition model

If however we wish to describe the biomass y₁ as a function of n₁ and n₂ the invers linear model suggested by Spitters (1983) can be used:

In this model B₀, B₁ and B₂ are unknown parameters. As y₁ and y₂ are the most relevant factors in this context the equation was rewritten as:

Madsen et al. (1995) reparameterized this equation in order to give all parameters a biological meaning:

y₁ is now the biomass per plant of species 1 when grown in a binary mixture containing n₁ plants of species 1 and n₂ plants of species 2 per box. A is the biomass per plant at an arbitrar zero density (n₀), which in the analyses was set to 8. B denotes the maximum yield per box at infinite plant densities and C is the substitution rate. If plants compete for the same resources C can be used to decribe the competition rate of species 2 against species 1. If C >1 species 2 is more competitive than species 1 and vise versa. The arbitrary value for n_o is used to avoid extrapolation to non-observed densities as well as devoting a welldefined biological explanation for B (Fredshavn, 1994).

When the substitution rate C is known the effective density n_e can be calculated:

ne = n1 + Cn2

3. Results and discussion

3.1 Determining ED₁₀ doses

Susceptibility of different plant species

Table 1 shows the estimated ED₁₀ doses. The most susceptible species were S. alba and C. bursa-pastoris with ED₁₀ doses lower than 1 g a.i. ha^-1 while the ED₁₀ doses of T. arvense, C. album and C. arvense were 3 to 4 g a.i. ha^-1. L. communis and S. media were the most tolerant species with ED₁₀ doses higher than 5 g a.i. ha^-1. One would expect the ED₁₀ dose to increase with growth stage, however as the experiments were carried out at different times of the year factors other than growth stage could have an influence on the susceptibility of the plants.

Table 1
Estimated ED₁₀ doses of mecoprop on different species and growth stages. The figures in parentheses are minimum and maximum of the estimated mean values.

Estimerede ED10 doseringer af mechlorprop på forskellige arter og udviklingstrin. Tallene i parentes er minimum og maximum af de estimerede værdier.

Deposition of mecoprop by rain

The maximum yearly deposition of mecoprop calculated on basis of the analysis of the rain samples was 0.2 g mecoprop ha^-1 (Table 3.15). Consequently, the ED₁₀ dose of the most susceptible species was more than 3 times higher than the maximum yearly deposition by rain and 10 times the maximum concentration found in one sample.

Influence of time of harvest

In the experiments shown in Table 1 the plants were harvested approximately 3 weeks after treatment. In practise plants will often grow for a longer period after exposure to herbicides which raises the question whether they will gradually recover. Here the influence of the time of harvest on the final effect was determined in one experiment where plants were harvested at different time intervals after treatment. In addition the susceptibility to mecoprop-P at the flowering stage as well as the effect on seed production was examined. The results are shown in Table 2.

Table 2
The influence of growth stage at treatment and time of harvest on ED₁₀ doses of Thlaspi arvense estimated on biomass and seed production, respectively. Figures in parentheses are standard errors.

Betydning af udviklingstrin på behandlingstidspunktet og høsttidspunkt for e ED₁₀ doseringer på Thlaspi arvense beregnet udfra henholdsvis biomasse og frøproduktion. Tallene i parentes er standardafvigelser.

When treated at the 8 to 10 leaves stage no significant differences were found between harvest 2 and 4 weeks after treatment. Surprisingly biomass production was inhibited more when plants were exposed to mecoprop-P at the flowering stage compared to the 8 to 10 leaves stage. At both growth stages the ED₁₀ doses estimated on basis of seed production were significantly higher than those estimated on basis of biomass production.

3.2 Competition experiments

Characteristics of test plants

Three competition experiments were carried out. The purpose of the first
experiment was to determine plant species and densities to be used in the subsequent experiment (results not shown). C. bursa-pastoris and G. dissectum were selected as two plant species possessing the same competitive ability but differing in susceptibility to mecoprop. The growth habit of these two plant species differ widely. C. bursa-pastoris is a relative small plant which elongate at an early stage where as G. dissectum forms a vigorous roset at an early growth stage. C. bursa-pastoris is known to be much more susceptible to mecoprop than G.dissectum.

Susceptibility to mecoprop

In subsequent experiments, the competition between the two species without herbicide and after treatment with 1 or 2 doses of mecoprop-P was determined. In one trial a dose of 3 g a.i. ha^-1 of mecoprop-P was applied while in the second trial 0.5 and 2.0 g a.i. ha^-1 were applied. The ED₁₀ doses estimated on the pot-grown plants grown in monoculture revealed that the ED₁₀ doses of mecoprop-P on C. bursa-pastoris and G. dissectum were respectively 0.4 g a.i. ha^-1 and 8.2 g a.i. ha^-1.

Susceptibility of A and B parameters

Table 3 shows the estimated parameters in the two competition experiments. In both experiments the A parameter (weight per plant at n₀=8 plants per box) of C. bursa-pastoris was significantly higher than the A parameter of G. dissectum. In experiment 1, the applied dose of mecoprop-P was much higher than the ED₁₀ dose of C. bursa-pastoris and the A parameter of C. bursa-pastoris was significantly reduced while no effect was found on G. dissectum. A tendency to a reduction of the B parameter of C. bursa-pastoris (the maximum production per box) was also seen.

In experiment 2 the A and B parameters were unaffected of the lowest dose of mecoprop-P while on C. bursa pastoris both parameters tended to decrease with increasing dose. The B parameter of G. dissectum also tended to decrease with increased dose.

Susceptibility of C parameter

The influence of herbicide treatments on the interspecific competition can be assessed by the C parameter. No significant differences in C parameters were found between the species and doses indicating that the interspecific competition was equal. In experiment 1, the estimated C parameters on G. dissectum was 1.7 for untreated and 0.9 after treatment with 3 g a.i. ha^-1. Consequently, 1 plant of C. bursa-pastoris can be replaced by 1.7 and 0.9 plants of G. dissectum respectively. Based on the C. bursa-pastoris data the results showed that when untreated 1 plant of G.dissectum could be replaced by 1.4 plants of C. bursa-pastoris while the substitution rate after treatment with 3 g a.i. ha^-1 of mecoprop was 1. Herbicide treatment halves the substitution rate of G. dissectum while the change of the substitution rate of C.bursa-pastoris was lower indicating that the applied mecoprop dose had more influence on the growth of C. bursa-pastoris than of G. dissectum. However, as none of the estimated C parameters differed significantly from 1 and as they were not significantly affected by the applied doses it can be concluded that the competitiveness of the species was not significantly influenced by the applied mecoprop-P dose.

In experiment 2 the C parameter of G. dissectum tended to decrease when 0.5 g a.i. mecoprop was applied confirming that C. bursa-pastoris was more susceptible than G. dissectum. Similarly the number of G. dissectum plants necessary to substitute one C. bursa-pastoris plant was lower after herbicide treatment. However when the dose was increased to 5 times the ED₁₀ dose the C parameter tended to increase, which we can not explain. None of the differences were significant.

Figure 2 shows the observed and predicted A values (biomass per plant) as a function of n_e (effective plant density). The curves of C. bursa-pastoris are more steep than the corresponding curves of G. dissectum, indicating that C. bursa pastoris was more susceptible to increasing plant density than G. dissectum. This was not reflected as a difference in the competition ability because intraspecific competition played a major role with C. bursa-pastoris.

In the competition experiments, the two selected plant species, although differing widely in growth habits, were expected to possess the same competition ability whereas the susceptibility to mecoprop differed. Following application of a mecoprop-P dose that would influence the growth of the most susceptible species, one would expect a change in the competition index favourizing the most tolerant species. However, no significant differences in the competitive ability of C. bursa-pastoris and G. dissectum were observed when applying a mecoprop-P dose close to the ED₁₀ dose of C. bursa-pastoris or a dose 5 times higher. Therefore it can be concluded that when using the experimental design adopted in this study competition was not a more susceptible factor to study than biomass production.

Table 3
Estimated parameters in the competition experiments (fresh weight). The figures in parentheses are 95% confidence intervals.

Estimerede parametre i konkurrenceforsøgene (friskvægt). Tallene i parentes er 95% konfidens intervaller.

Figure 2
The influence of effective plant number on production of biomass per plant (A) of C. bursa-pastoris (a-c) and G. dissectum (d-f) without herbicide and after treatment with 0.5 or 2 g a.i. ha^-1 of mecoprop. Experiment 2.

Indflydelse af effektivt plantetal på biomasseproduktionen pr. plante af C. bursa-pastoris (a-c) og G. dissectum (d-f) uden herbicid og efter behandling med 0.5 og 2 g ha-1 mechlorprop

Figure 2 (continued)
The influence of effective plant number on production of biomass per plant (A) of C. bursa-pastoris (a-c) and G. dissectum (d-f) without herbicide and after treatment with 0.5 or 2 g a.i. ha^-1 of mecoprop. Experiment 2.

Indflydelse af effektivt plantetal på biomasseproduktionen pr. plante af C. bursa-pastoris (a-c) og G. dissectum (d-f) uden herbicid og efter behandling med 0.5 og 2 g ha^-1 mechlorprop.

4. Conclusions

The potential risk of adverse effects of mecoprop in precipitation has been assessed by applying different methods. The results have revealed that biomass is as susceptible a parameter as seed production. The substitution rate between species with similar competitive ability but different susceptibility to the herbicide was not significantly influenced by doses close to ED₁₀ .

The results have shown that the No Observable Effect Level (in this study defined as the ED₁₀ dose) of mecoprop-P on the most susceptible plant species included in this study was more than 3 times higher than the maximum yearly deposition of mecoprop in Danish rain water and 10 times higher than the maximum deposition within a two weeks period. Consequently, effects of mecoprop in precipitation is not very likely.

In this project we only studied the influence of a single herbicide, however the analyses of rain water have revealed the presence of several herbicides. A question to address in a future project is the possible effects of such ‘pesticide cocktails’ on terrestrial plants.

5. Acknowledgement

We wish to thank Dr Jens Erik Jensen at the Royal Veterinary and Agricultural University for statistical assistance in analyzing the competition experiments

6. References

Cousens R. (1991). Aspects of the design and interpretation of competition (interference) experiments. Weed Technology, 5 (3), 664-673.

Fredshavn J.R. (1994). Use of substitution rates to describe competition in mixed plant populations. Acta Agricultura Scandinavica, Section B, Soil and Plant Science, 44 (1), 47-54.

Jaeschke W., Gath B. & Pfäfflin D. (1995). Deposition of pesticides in Southern Germany. In: Pesticides in precipitation and surface water, Tema Nord, 1995:558. Ed. A. Helweg, 65-74.

Kirknel E. & Felding G. (1995). Analysis of selected pesticides in rain in Denmark. In: Pesticides in precipitation and surface water, Tema Nord, 1995:558. Ed. A. Helweg, 45-54.

Kreuger J. (1995). Pesticider i regnvatten i Sverige. In: Pesticides in precipitation and surface water, Tema Nord, 1995:558. Ed. A. Helweg, 33-44.

Lode O., Eklo O.M., Holen B., Svensen A & Johnson Å.M. (1995). Pesticides in precipitation in Norway. In: Pesticides in precipitation and surface water, Tema Nord, 1995:558. Ed. A. Helweg, 19-32

Madsen K.H., Poulsen G.S., Fredshavn J.R., Jensen J.E. & Steen P. (1995). A method to study competitive ability of seabeet hybrids (Beta vulgaris ssp. maritima) and glyphosate tolerant sugarbeet (Beta vulgaris ssp. vulgaris). Acta Agricultura Scandinavica, Section B, vol. 48, no. 3, 170-174.

Sibers J., Gottschild D. & Nolting H.-G. (1995). Deposition of pesticides in Nothers Germany. In: Pesticides in precipitation and surface water, Tema Nord, 1995:558. Ed. A. Helweg, 55-64.

Spitters C.J.T. (1983). An alternative approach to the analysis of mixed cropping experiments. 1. Estimation of competition effects. Netherlands Journal of Agricultural Sciences, 31, 1-11.