Mecoprop

Toxicity to soil microorganisms

Link to relevant study record(s)

Reference

Endpoint:

toxicity to soil microorganisms

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

The effect of the test material on soil microorganisms was assessed in field trials using wheat monoculture.

GLP compliance:

not specified

Remarks:

A Dissertation for the attainment of the degree of Doctor in Agricultural Sciences.

Analytical monitoring:

yes

Details on sampling:

After the application of the test material, samples of soil were removed at regular intervals from a depth of 0 – 5 cm and 5 – 10 cm using a Pürkhauer boring rod. For each test plot at each sampling time, four samples were taken which were distributed evenly over the whole plot and taken from between the rows of wheat. The soil samples from the four repetitions of the field trial were combined for each arm of the trial into one mixed sample. Before the first pesticide application in the spring (of 1979 and 1981 respectively) samples were taken in all plots so as to determine any possible test material residues remaining from cultivation prior to the start of the current field trial in 1976.
The soil samples were stored in polythene bags at -20 °C until required for analysis. The preparation of the deep-frozen soil samples and the gas chromatographic detection of the test martial residues in the soil were performed as below.

Vehicle:

no

Test organisms (inoculum):

soil

Moisture:

Estimation of the water content was done gravimetrically by drying in a drying oven for 24 hours at 105 °C.

Organic carbon content (% dry weight):

1.27

Details on test conditions:

TEST SYSTEM
- Testing facility: The field trials were carried out on the test sites of the Institute for Phytomedicine. Two test plots were available for the investigations into the degradation kinetics of the test material under field conditions. These lay immediately next to each other and could be considered as one plot as far as soil characteristics were concerned.

SOIL INCUBATION
- Treatment of the soil: About 14 days before each series of trials, an appropriate amount of soil was sieved to ≤ 2 mm and stored again at 18 °C in closed plastic buckets, which were regularly ventilated. Thereby possible variations in the microbial activity - caused by temperature changes and mechanical handling - should be evened out. The amount of test material corresponding to the formulation in question was pipetted together with 50 mL water over the sample of soil - as a rule amounting to 4 to 5 kg - which had been spread out flat. Next this was well mixed, sieved again to ≤ 2 mm, and brought to the desired water content. Estimation of the water content was done gravimetrically by drying in a drying oven for 24 h at 105 °C. The soil which had been treated in this way was stored for the whole of the duration of the test in closed polythene bags under constant conditions (40 % maximum WC; 20 ± 1°C). On taking each sample the soil was aerated and re-mixed. The sampling intervals were chosen in accordance with the requirements of the trial and the samples which were withdrawn were kept in polythene bags at -20 °C until analysed by gas chromatography for residues of the test material.

SOURCE AND PROPERTIES OF SUBSTRATE
- Geographical reference of sampling site: The field trials were carried out on the test sites of the Institute for Phytomedicine.
- History of site: Two test plots were available for the investigations into the degradation kinetics of the test material under field conditions. These lay immediately next to each other and could be considered as one plot as far as soil characteristics were concerned.
On test plot I, in the winter of 1976, a five-year wheat monoculture trial was initiated, in which a plant protection programme was to be carried out with graded degrees of intensity. In the third and fifth growing years (1979 and 1981 respectively), soil samples were taken to evaluate the rate of degradation of the test material in relation to repeated applications of pesticides over several years.
On test plot II, a one-year trial with winter wheat was performed during the growing season of 1980/1981 using the same experimental arms as were used in Trial 1, so that the degradation of the test material could also be studied after going through the trail programme for the first time.
The plant protection programme used in these field trials embraced a total of nine arms repeated four times and included, as well as an untreated control which was only cut by hand, 3 herbicides, 2 fungicides and 1 growth regulator. The pesticides were applied in part separately but mostly in combination as part of a varied spraying programme. The application was performed with a van der Weijt plot spraying apparatus at the intervals, times and dosages which are usual in practice.
- Vegetation cover: On test plot I, in the winter of 1976, a five-year wheat monoculture trial was initiated, in which a plant protection programme was to be carried out with graded degrees of intensity.
- Treatments with pesticides or fertilisers: Yes
- Depth of sampling: 0 - 5 cm and 5 - 10 cm
- % clay: 27.9 %;
- Soil taxonomic classification: Sandy loam
- pH: The pH value was 6.8
- Maximum water holding capacity (in % dry weight): Maximum water capacity of 45 g/100 g for a particle size fraction of ≤ 2 mm.
- Pretreatment of soil: About 14 days before each series of trials, an appropriate amount of soil was sieved to ≤ 2 mm.
- Storage: A quantity of soil sufficient for the whole of the period of the trial was taken in the winter of 1979 from the control plots which had not been treated with pesticides. After the soil had been lightly dried and mechanically ground down, it was stored loosely covered with a plastic sheet in an unheated room until the start of the trial.
- Initial microbial biomass as % of total organic C: The physicochemical method for the quantitative estimation of the microbial biomass in soils which was used in these trials consists of measuring the amount of CO2 (as mL CO2/h/100 g soil) which is generated by the metabolically active microorganisms in the soil, and converting this to microbial biomass (mg microbial C/100 g soil) using a regression equation.
The influence of one or several applications of pesticides on the developing biomass in the field trial plots, was determined during the growing season of 1981 at three different periods during the investigation. The soil samples which were needed for investigation were taken at a soil depth of 0 – 10 cm from the individual plots in each case, and for each arm of the trial were combined to make an average sample (of ca. 8 – 10 kg of soil). These were lightly dried until they were capable of being sieved to ≤ 2 mm. In order to provide comparable conditions, all the soil samples were moistened to 40 % of their max. water capacity, and subsequently stored for at least 14 days in plastic buckets at 22 ± 1 °C and regularly aerated.
From these were taken 100 g samples of soil (dried weight) and 2 000 µg glucose per g of soil was added. The optimum concentration of glucose for the test soils in question was determined in a preliminary test. The requisite amount of glucose was ground together with talc (0.5 g /100 g soil) in a mortar, to produce a homogeneous fine powdery mixture. This was then sprinkled over the soil, which had been spread out flat, and blended together thoroughly. The incubated soil samples were packed into glass tubes (23 cm long; 3 cm d), both ends were stoppered with rubber bungs, and the tubes connected to an "Ultragas 3" CO2 analyser made by the Vosthoff company. The temperature was maintained at 22 ± 1 °C during the incubation period. Each sample was measured in quadruplicate.
To calculate the microbial biomass in the soil (mg microbial C/ 100 g soil) the amounts of CO2 (mL CO2/h/100 g soil) determined after incubation for 3 h were substituted into the following regression equation derived by Anderson and Domsch (1978):

X = 40.04 Y + 0.37

Where:
Y = mL CO2/h/100 g soil
X = mg microbial C/100 g soil


EFFECT PARAMETERS MEASURED:
- Trials with differing concentrations of the test material and varying experimental conditions: For experiments on the kinetics of degradation of the test material, the agent was applied in the form of the commercial product [test material: 560 g/L] as an aqueous solution. The influence of differing concentrations was studied, by applying the corresponding solutions with graded concentrations of the agent and working up the test soils in the same way, so that the soil samples contained concentrations of the test material at 1, 5, 10, 100 and 250 µg/g of soil. The soil samples which had been treated in this way were stored under constant conditions (40 % max. WC; 20 ± 1 °C). In a further series of trials the influence of variations in the experimental conditions on the kinetics of degradation of the test material were verified. After equal weights of the test soil had been treated with 10 µg test material/g of soil, aliquots were brought to differing water contents of 25 %, 40 % and 75 % of the max. water capacity and stored at 20 ± 1 °C. Further samples were brought to a constant water content (40 % of max. WC) and then stored at different temperatures of 12°C, 20 °C and 35 °C.
- Repeated treatments of the soil: The first application to the soil was carried out with either 1, 10 or 100 µg/g of test material. Until the second application, the treated samples and an untreated control soil sample were stored under constant conditions in closed polythene bags, which were regularly aerated. After 2, 6 or 12 months as the case may be, a sufficiently large sample was removed and treated with 10 μg/g test material. The soil samples which had been twice treated with the test material, were compared for the degradation behaviour of this agent against the originally untreated control soil sample.

VEHICLE CONTROL PERFORMED: Not applicable

Nominal and measured concentrations:

1, 5, 10, 100 and 250 µg/g of soil

Remarks on result:

not measured/tested

Details on results:

- The degradation of the test material in soil in relation to the initial concentration in the soil (under laboratory conditions) was assessed. The course of degradation can be considered as three phases. In the first phase no degradation occurs, or only to a very slight extent. This is usually described as the lag (or induction) phase. The second phase is characterised by a gradually increasing fall in concentration which then levels off to a constant rate of decrease.
When about 10 – 15 % of the initial concentration in the soil remains, the third phase begins, in which the rate of decrease in concentration slows down again. The duration of the lag-phase (tlag) was dependent on the initial concentration of the test material.
Degradation of 1 μg/g began immediately after application, without any measurable lag phase, so that after 4 h only 95 % of the applied test material could be extracted from the soil. After 5 days under the chosen conditions of degradation, the agent could no longer be detected when applied at an initial concentration of 1 μg/g. At higher concentrations, a lag-phase was always observed, whose duration increased with rising initial concentration. Within the concentration range of 5 – 100 μg/g test material in the soil there was a linear relationship between the concentration applied and the observed duration of the lag-phase, which no longer held good at still higher concentrations. Because of the practically equal slopes of the degradation curves after the end of the lag phase the percentage rate of degradation per unit time (in relation to the initial concentration within the range 5 - 100 μg/g was constant. At a concentration of 250 μg/g the rate of degradation was distinctly less.
For the further course of this work, the period during which the rate of degradation remained constant is designated the degradation phase. The duration of the degradation phase is calculated as the difference t90 % - tlag, in which t90 % signifies the time (days) up to which 90 % degradation of the amount of agent recovered on day 1 occurred, and tlag denotes the duration of the lag-phase (in days).
An interval of two months between the first and the second applications to the repeatedly treated soil had no effect on degradation of the test material if the concentration of the first application was 1 μg/g. In repeatedly treated soils there was no lag-phase and in comparison to corresponding controls significantly accelerated degradation was established if on the first application 10 or 100 μg/g respectively of the test material was used, In the case of the last mentioned concentrations, a re-treatment affect could be established after 6 and 12 months respectively, with the difference that for the higher concentration the lag-phase it was 1 – 2 days longer. After repeated treatment there was no essential difference between the two pre-treatment procedures.
A significant effect due to storage conditions could be established with the control soils which had remained untreated up to the time of the first application even though during the whole duration of the test, as with other samples of the trial they were stored damp (at 40 % of their maximum water capacity).
During the storage period of two months for degradation of the test material there was no difference in comparison with the values for normal conditions, while after storage of the damp soil for 5 months and 12 months respectively the lag-phase was slightly prolonged whilst the degradation phase (t90% - tlag) was significantly extended.

- Influence of the degradation kinetics of the test material in soil: The course of the decrease in concentration of the test material under field conditions after the first application in 1981 (Trail Plot II) showed on the first day after application comparable amounts of residues (1.14 – 1.56 μg/g) could be detected in the soil layer from 0 – 5 cm from the various limbs of the test. The degradation of the test material started immediately after the application, without a lag-phase, so that 4 days later 50 – 60 % of the concentration found on the first day had disappeared, and 15 days after application not more than 0.1 μg/g test material could be detected in any of the test limbs. The elevated residue found on the fifth day after application are probably attributed to heavy rainfall which had washed residues of the agents from the surface of the plants. The highest values were 6 – 8 days after application and were all in the limbs of the trial lower than 0.1 μg/g test material.
The residue values for the test material for the third (1979) and fifth (1981) consecutive year of the test programme carried out on trial plot I show that in the third year the test material residues extractable one day after application amounted to 1.29 – 1.55 μg/g, while in the fifth year they turned out to be somewhat lower at 0.97 – 1.23 μg/g. In both test years, altered kinetics of the test material degradation could be established. The lag phases measured on plots 6, 8 and 9 in the third year of the test and on plots 8 and 9 and in the fifth years, lasted 3 and 2 days respectively. In the rest of the limbs of the trial no lag-phase was observed in either of those years.
Fifteen days after application, regardless of the limb of test or the year, ≤ 0.1 μg/g test material was undetectable. The increased (compared with the day before) residues values five days after application, as were observed in the fifth year of the trial (1981) must again be attributable to the heavy rainfall which occurred and the consequent leaching of the agents from the surface of the plants. The test material residues which were found at a depth of 5 – 10 cm in the soil in no case, either in the third or fifth year, amounted to > 0.1 μg/g.
Repeated treatment under field conditions showed that in the case of the soil samples taken from the various field trial plots five months after the last application of the test material and then re-treated in the laboratory with 10 μg/g test material, a shortening of the lag-and degradation phases of about one day occurred in those limbs of the trial which had already been treated with the test material under field conditions. The same procedure with the soil samples which in the spring of 1981 (11 months after the last treatment with the test material) yielded no differences regarding the duration of the lag-phase between the formerly treated and untreated soils. Only the degradation phase was 1 – 2 days shorter in the treated plots. The different crop protection programmes carried out under field conditions had no influence on the effects of repeated treatment either at the first or the second stage of observation.

- Microbial biomass in the soil: The microbial biomass content which had been determined during the growing season of 1981 in two field trial plots showed a distinct relationship to both the duration of the trial (year) to the crop protection measures carried out on the individual plots and the time of sampling.
On trial plot II with winter wheat in the first year, the biomass concentration in the untreated plot at the beginning of April 1981 amounted to ca. 105 mg microbial C/100 g soil, however it had diminished by about 14 % at the second time of sampling in the middle of May and had risen again by the time for harvesting the wheat (mid-August) to almost 100 mg C/100 g soil. On trial plots 2 – 9 the crop protection measures taken up to the sampling time in min-May (inc. the application of the test material) had no influence on the biomass content in the soil.
In the individual trial plots of field trial I, on which winter wheat was grown in five consecutive years on average 61 mg microbial C/100 g of soil was established at the first occasion of sampling. At this time no measurable differences were found between the limbs of the trial.
In the soil of the untreated plot (test limb 1) the biomass increased continuously during the growing season and at the time of harvesting amounted to ca. 74 mg microbial C/100 g soil. At the fifth year of the trial the individual and combined applications of herbicides, including the test material in trial limbs 2 to 9 had no effect on the biomass content in the soil.

- Percentage share of bacteria and fungi in the microbial activity in the soil: A comparison of the untreated plots (trial limb 1) shows that both when growing wheat for one year and also in five years of wheat monoculture, the percentage share of bacteria and fungi in the microbial activity of the soil is the same and in the ratio of 1:3 in favour of the fungi. In the course of the growing season from spring of 1981 up to the time of harvesting the wheat, on these untreated plots no displacement in the ratio of bacteria to fungi was observed. On the trial plots 2 – 9, which in the fifth year was planted with wheat and in the preceding four years had been treated with pesticides at varying intensities, at the first sampling no change in the percentage ratio or bacteria to observed when compared with the untreated control. This ratio was also constant during the growing season 1981 since on the industrial trial plots the different crop protection measures were carried out both in the one year and the five year trial programme.

Function: 1st Order

t (d)	R (mg/kg)	Rmod
1.0	1.56	1.551
2.0	1.29	1.309
3.0	0.89	1.105
4.0	0.70	0.933
5.0	0.96	0.788
6.0	0.93	0.665
8.0	0.52	0.474
12.0	0.26	0.241
15.0	0.12	0.145

Sum sq. residuals: 0.2041

Slope b: -0/0735

Intercept a: 0.2650

Modified likelihood: 0.8807

CI from -0.0904 to – 0.0567

CI from 0.1354 to 0.3927

Significance: 0.2720 at 95 %

T/1: 4.09 d CI: 3.16 – 5.03

T/10: 13.60 d CI: 10.48 – 16.72

Ratio 1st order / sqrt 1st order 85.99 %

Validity criteria fulfilled:

not applicable

Conclusions:

Under the conditions of the study, no displacement in the ratio of bacteria to fungi was observed following application of the test material. Individual and combined applications of herbicides, including the test material, had no effect on the biomass content in the soil.

Executive summary:

The effect of the test material on soil microorganisms was assessed in field trials using wheat monoculture. The microbial biomass content which had been determined during the growing season in two field trial plots showed a distinct relationship to both the duration of the trial (year) to the crop protection measures carried out on the individual plots and the time of sampling. On a plot with winter wheat in the first year, the biomass concentration in the untreated plot amounted to ca. 105 mg microbial C/100 g soil, however it had diminished by about 14 % at the second time of sampling in the middle of May and had risen again by the time for harvesting the wheat (mid-August) to almost 100 mg C/100 g soil. On other trial plots the crop protection measures taken up to the sampling time in mid-May had no influence on the biomass content in the soil.

In the individual trial plots on which winter wheat was grown in five consecutive years, on average 61 mg microbial C/100 g of soil was established at the first occasion of sampling. At this time no measurable differences were found between the limbs of the trial.

In the soil of the untreated plot the biomass increased continuously during the growing season and at the time of harvesting amounted to ca. 74 mg microbial C/100 g soil. At the fifth year of the trial the individual and combined applications of herbicides, including the test material had no effect on the biomass content in the soil.

A comparison of the untreated plots shows that both when growing wheat for one year and also in five years of wheat monoculture, the percentage share of bacteria and fungi in the microbial activity of the soil is the same and in the ratio of 1:3 in favour of the fungi. In the course of the growing season from spring of 1981 up to the time of harvesting the wheat, on these untreated plots no displacement in the ratio of bacteria to fungi was observed. On the trial plots which in the fifth year were planted with wheat and in the preceding four years had been treated with pesticides at varying intensities, at the first sampling no change in the percentage ratio of bacteria observed when compared with the untreated control. This ratio was also constant during the growing season 1981 since on the industrial trial plots the different crop protection measures were carried out both in the one year and the five year trial programme.

Under the conditions of the study, no displacement in the ratio of bacteria to fungi was observed following application of the test material. Individual and combined applications of herbicides, including the test material, had no effect on the biomass content in the soil.

Description of key information

Amrein (1982)

Under the conditions of the study, no displacement in the ratio of bacteria to fungi was observed following application of the test material. Individual and combined applications of herbicides, including the test material had no effect on the biomass content in the soil.

Additional information

Amrein (1982)

The effect of the test material on soil microorganisms was assessed in field trials using wheat monoculture. The study was awarded a reliability score of 2 in accordance with the criteria set forth by Klimisch et al. (1997).

The microbial biomass content which had been determined during the growing season in two field trial plots showed a distinct relationship to both the duration of the trial (year) to the crop protection measures carried out on the individual plots and the time of sampling. On a plot with winter wheat in the first year, the biomass concentration in the untreated plot amounted to ca. 105 mg microbial C/100 g soil, however it had diminished by about 14 % at the second time of sampling in the middle of May and had risen again by the time for harvesting the wheat (mid-August) to almost 100 mg C/100 g soil. On other trial plots the crop protection measures taken up to the sampling time in mid-May had no influence on the biomass content in the soil.

In the individual trial plots on which winter wheat was grown in five consecutive years, on average 61 mg microbial C/100 g of soil was established at the first occasion of sampling. At this time no measurable differences were found between the limbs of the trial.

In the soil of the untreated plot the biomass increased continuously during the growing season and at the time of harvesting amounted to ca. 74 mg microbial C/100 g soil. At the fifth year of the trial the individual and combined applications of herbicides, including the test material had no effect on the biomass content in the soil.

A comparison of the untreated plots shows that both when growing wheat for one year and also in five years of wheat monoculture, the percentage share of bacteria and fungi in the microbial activity of the soil is the same and in the ratio of 1:3 in favour of the fungi. In the course of the growing season from spring of 1981 up to the time of harvesting the wheat, on these untreated plots no displacement in the ratio of bacteria to fungi was observed. On the trial plots which in the fifth year were planted with wheat and in the preceding four years had been treated with pesticides at varying intensities, at the first sampling no change in the percentage ratio of bacteria observed when compared with the untreated control. This ratio was also constant during the growing season 1981 since on the industrial trial plots the different crop protection measures were carried out both in the one year and the five year trial programme.

Under the conditions of the study, no displacement in the ratio of bacteria to fungi was observed following application of the test material. Individual and combined applications of herbicides, including the test material had no effect on the biomass content in the soil.