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Toxicity to soil microorganisms

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Endpoint:
toxicity to soil microorganisms
Type of information:
experimental study
Adequacy of study:
key study
Study period:
September 2006 to 28 November 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 216 (Soil Microorganisms: Nitrogen Transformation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.21 (Soil Microorganisms: Nitrogen Transformation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Details on sampling:
On days 7, 14, 21 and 28, triplicate control, solvent control and test vessels were sacrificed for nitrate analysis.
Vehicle:
yes
Details on preparation and application of test substrate:
APPLICATION OF TEST SUBSTANCE TO SOIL
- Method: 54 mg of test material was dissolved in acetone and the volume adjusted to 10 mL. 2 mL of this stock solution was added dropwise onto 15 g dry quartz sand and the solvent allowed to dry. The test material/sand and 7.5 g powdered Lucerne-green-grass-mean was then added to 1.5 kg (dry weight) soil and mixed prior to the addition of 56.1 mL water to give the test material concentration of 7.2 mg/kg. The test concentration was mixed again prior to splitting into 27 replicates of 50 g dry weight soil for incubation.

The above was repeated using a 2 mL aliquot of a 270 mg/10 mL stock solution to prepare the test concentration of 36 mg/kg.

VEHICLE:
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): acetone
Test organisms (inoculum):
soil
Total exposure duration:
28 d
Test temperature:
21 ± 1 ºC
Moisture:
WHC 40 ± 5 %
Details on test conditions:
TEST SYSTEM
- Testing facility: glass beakers
- Test container (type, material, size): covered with plastic film
- Amount of soil: 50 g dry weight of soil

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Geographical reference of sampling site (latitude, longitude): LUFA Speyer, Obere Langgasse 40, 67346 Speyer, Germany
- History of site: no treatment with plant protection products or organic fertilisers for at least 4 years prior to sampling
- Maximum water holding capacity (in % dry weight): 40 % of WHC
- Storage (condition, duration): 21 ºC prior to use
- Initial microbial biomass as % of total organic C:

VEHICLE CONTROL PERFORMED: yes
Nominal and measured concentrations:
7.2 and 36 mg/kg (nominal)
Reference substance (positive control):
no
Key result
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
36 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
nitrate formation rate
Details on results:
The soil characteristics satisfied the recommendations of the Test Guidelines. The microbial biomass of the soil was shown to be 205 µg C/g; this was equivalent to 2.0 % of the total soil organic carbon content.

Absorbance values determined on each sampling day are given in Table 1.

The mean nitrate concentrations in the control, solvent control and test vessel on each sampling day are given in Table 2 and percentage inhibition of nitrogen transformation activity on each sampling day is given in Table 3.

The difference in the nitrogen transformation activity of soil micro-organisms exposed to the test substance was less than 25 % after 28 days. The test material can therefore be considered to have no long-term effect on nitrogen transformation in soil.

Variation in nitrogen transformation activity between the solvent control and test material groups on sampling days was shown to be relatively high. In the majority of cases the nitrogen transformation activity in the test substance groups was shown to be higher than that in the solvent control groups and hence it was considered that the test substance exhibited no inhibitory effect on nitrogen transformation.

There were no statistically significant differences between the solvent control and 7.2 mg/kg test concentration, however the 36 mg/kg test concentration was shown to have significantly higher nitrate concentrations than the solvent control group. Given that the increase in nitrate concentrations in this concentration could have only been due to an increase in the nitrogen transformation rate it was considered that the test material exhibited no adverse effect on the nitrogen transformation rate.

Table 1: Absorbance readings on each sampling occasion

      Absorbance (324 nm)*
  Nominal concentration (mg/kg)  Day 0  Day 7  Day 14  Day 21  Day 28
 Control  R1  0.149  0.048  0.180  0.284  0.269
   R2  0.138  0.106  0.176  0.336  0.249
   R3  0.147  0.096  0.305  0.326  0.255
 Solvent control  R1  0.142  0.095  0.239  0.222  0.219
   R2  0.146  0.175  0.157  0.225  0.236
   R3  0.144  0.108  0.220  0.261  0.244
 7.2  R1  0.150  0.053  0.229  0.240  0.262
   R2  0.156  0.058  0.328  0.233  0.248
   R3  0.136  0.074  0.274  0.279  0.254
 36  R1  0.169  0.150  0.397  0.403  0.285
   R2  0.184  0.138  0.250  0.431  0.274
   R3  0.202  0.139  0.387  0.300  0.269

*corrected for blank absorbance value

Table 2: Mean nitrate concentrations on sampling occasions

      Nitrate concentration (mg NO3/kg)
  Nominal concentration (mg/kg)     Day 0  Day 7  Day 14  Day 21  Day 28
 Control  Mean  50.88 28.44 78.56 113.32 92.22
   C.V.  4.21  39.89  34.16  8.91  4.07
 Solvent control  Mean  50.64  44.05  73.08  84.30  83.20
   C.V.  1.45  35.66  21.49  9.42  5.61
 7.2  Mean  51.86  20.51  99.30  89.66  91.13
   C.V.  7.24  19.57  18.26  10.11  2.82
 36  Mean  65.64  55.51  124.05  136.25  98.93
   C.V.  9.21  15.09  24.22  18.52  3.03

Table 3: Inhibition of nitrogen transformation activity

   % inhibition
 Nominal concentration (mg/kg)  Day 7  Day 14  Day 21  Day 28
 7.2  53  [36]  [6]  [10]
 36  [26]  [70]  [62]  [19]

[increase in nitrogen transformation activity as compared to solvent control]

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the test, the test material showed no significant inhibitory effect on the nitrogen transformation activity of soil micro-organisms at 7.2 and 36 mg/kg and therefore can be considered to have no long-term effect on nitrogen transformation in soil. The NOEC was determined to be 36 mg/kg.
Executive summary:

In a GLP compliant nitrogen transformation study conducted in line with standardised guideline OECD 216, the effect of the test material on nitrogen transformation activity of soil micro-organisms over 28 days was determined. The inhibitory effect of the test material on nitrogen transformation at test concentrations of 7.2 and 36 mg/kg was assessed by the determination of nitrate concentration. Under the conditions of the test, the test material showed no significant inhibitory effect on the nitrogen transformation activity of soil micro-organisms at 7.2 and 36 mg/kg and therefore can be considered to have no long-term effect on nitrogen transformation in soil. The NOEC was determined to be 36 mg/kg.

Endpoint:
toxicity to soil microorganisms
Type of information:
experimental study
Adequacy of study:
key study
Study period:
September 2006 to 28 November 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 217 (Soil Microorganisms: Carbon Transformation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.22 (Soil Microorganisms: Carbon Transformation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Details on sampling:
On days 7, 14 and 28, triplicate control, solvent control and test vessels were sacrificed for glucose-induced respiration rate.
Vehicle:
yes
Details on preparation and application of test substrate:
APPLICATION OF TEST SUBSTANCE TO SOIL
- Method: 54 mg of test material was dissolved in acetone and the volume adjusted to 10 mL. 2 mL of this stock solution was added dropwise onto 15 g dry quartz sand and the solvent allowed to dry. The test material/sand and 7.5 g powdered Lucerne-green-grass-mean was then added to 1.5 kg (dry weight) soil and mixed prior to the addition of 56.1 mL water to give the test material concentration of 7.2 mg/kg. The test concentration was mixed again prior to splitting into 27 replicates of 50 g dry weight soil for incubation.

The above was repeated using a 2 mL aliquot of a 270 mg/10 mL stock solution to prepare the test concentration of 36 mg/kg.

VEHICLE:
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): acetone
Test organisms (inoculum):
soil
Total exposure duration:
28 d
Test temperature:
21 ± 1 ºC
Moisture:
40 % of WHV
Details on test conditions:
TEST SYSTEM
- Testing facility: glass beakers
- Amount of soil: 50 g dry weight of soil

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Geographical reference of sampling site (latitude, longitude): LUFA Speyer, Obere Langgasse 40, 67346 Speyer, Germany
- History of site: no treatment with plant protection products or organic fertilisers for at least 4 years prior to sampling
- Maximum water holding capacity (in % dry weight): 40 % of WHC
- Storage (condition, duration): 21 ºC prior to use

VEHICLE CONTROL PERFORMED: yes
Nominal and measured concentrations:
7.2 and 36 mg/kg (nominal)
Reference substance (positive control):
no
Key result
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
36 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
respiration rate
Details on results:
The soil characteristics satisfied the recommendations of the Test Guidelines. The microbial biomass of the soil was shown to be 205 µg C/g. This was equivalent to 2.0 % of the total soil organic carbon content.

The mean glucose induced respiration rates determined on each sampling day are given in Table 1 and percentage inhibition on each sampling day is given in Table 2.

Percentage inhibition of glucose induced respiration rates in soil micro-organisms exposed to the test material was less than 25 % after 28 days. The test substance can therefore be considered to have no long-term effect on carbon transformation in soil.

The respiration rates obtained for the 36 mg/kg Replicate R1 vessel on Day 0 and the 7.2 mg/kg Replicate R1 vessel on Day 28 were considered to be erroneous and hence were not used in the calculation of mean respiration rates or percentage inhibition values. The reason for the erroneous values was considered likely to be due to a leak in the respirometer system when the vessels were being incubated. Due to an instrument malfunction no respiration rates were measured for the 36 mg/kg vessels on Day 7.

The above were considered to have no adverse effect on the overall outcome of the study as it was clear that based on the definition given in the Test Guidelines the test material exhibited no long term effect on carbon transformation activity.

Both the 7.2 and 36 mg/kg test concentrations were shown to be statistically different from the solvent control. Whilst statistically the test concentrations were shown to be different from the solvent control, exanimation of the percentage inhibition of carbon transformation values showed that in biological terms there was no significant effect. The test material can therefore be considered as having shown no long-term effect on carbon transformation in soil.

Table 1: Mean glucose induced respiration rates

      Glucose induced respiration rate (mg O2/kg/h)
 Nominal concentration (mg/kg)     Day 0  Day 7  Day 14  Day 28
 Control  Mean  11.83  8.77  8.42  7.48
   C.V.  0.55  2.80  1.01  0.97
 Solvent control  Mean  11.70  8.34  8.42  7.75
   C.V.  15.85  3.57  0.81  0.99
 7.2  Mean  11.48  8.27  7.86  7.28
   C.V.  2.97  1.39  10.14  3.01
 36  Mean  11.75  *  7.67  7.26
   C.V.  1.44  *  1.11  1.40

*no value determined due to instrument malfunction

Table 2: Inhibition of carbon transformation activity

   % inhibition
 Nominal concentration (mg/kg)  Day 7  Day 14  Day 28
 7.2  1  7  6
 36  *  9  6

*no value determined due to instrument malfunction

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the test, the test material showed no significant inhibitory effect on the carbon transformation activity of soil micro-organisms at 7.2 and 36 mg/kg and therefore can be considered to have no long-term effect on carbon transformation in soil.
Executive summary:

In a GLP compliant carbon transformation study conducted in line with standardised guidelines OECD 217 and EU Method C.22, the effect of the test material on carbon transformation activity of soil micro-organisms over 28 days was determined. The inhibitory effect of the test material on carbon transformation at test concentrations of 7.2 and 36 mg/kg was assessed by the determination of glucose induced respiration rates. Under the conditions of the test, the test material showed no significant inhibitory effect on the carbon transformation activity of soil micro-organisms at 7.2 and 36 mg/kg and therefore can be considered to have no long-term effect on carbon transformation in soil.

Description of key information

Nitrogen transformation test conducted in line with GLP and standardised guideline OECD 216; the NOEC was determined to be 36 mg/kg.

Carbon transformation test conducted in line with GLP and standardised guidelines OECD 217 and EU Method C.22; no long-term effect on carbon transformation in the soil was observed.

Key value for chemical safety assessment

Long-term EC10 or NOEC for soil microorganisms:
36 mg/kg soil dw

Additional information

The available data showed that concentrations of test material equivalent to its Predicted Environmental Concentration of 7.2 mg/kg and five times that concentration did not elicit adverse effects.