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Toxicity to soil microorganisms

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to soil microorganisms

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 October 2017 to 24 May 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 216 (Soil Microorganisms: Nitrogen Transformation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Exceptions to GLP compliance: test soil collection and analysis by CEMAS, Ltd.; and analysis of carbon:nitrogen content of lucerne meal and nitrogen content of the test soil by Butterworth Laboratories

Analytical monitoring:

no

Details on sampling:

Triplicate samples of 20 g soil were withdrawn from each of the non-treated control soil and treated soil group replicates within 6 hours of treatment on Day 0 and 28 days after preparation (Day 28). In addition, on Day 0, a 20 g soil sample was removed from each of the non-treated control replicates (Group 1) and used for spike recoveries. Bulk soils were thoroughly mixed prior to sub-sampling at each time point. The soil samples were extracted on the same day of sampling and sample extracts were stored frozen (at approximately -20°C) until analysis.

The soil samples were extracted with 100 mL of a 0.1M potassium chloride solution by shaking on a mechanical shaker at approximately 150 rpm for 60 minutes. The supernatant solutions were filtered through filter paper and then analysed for their concentrations of nitrate spectrophotometrically. The soil sample extract filtrates were processed by measuring 35 mL of acid mixture into a suitable glass container, adding 5 mL of soil extract filtrate, then adding 5 mL of 2,6-dimethylphenol solution . Samples were mixed thoroughly and then allowed to stand for 10 – 60 minutes.

Vehicle:

yes

Remarks:

acetone

Details on preparation and application of test substrate:

The group mixtures were prepared in triplicate replicates of 1 kg wet soil for each group (e.g., N1A, N1B, N1C). Lucerne meal was distributed evenly in several aliquots over the surface of the test soil (spread as a thin layer of approximately 2.5 cm) and then thoroughly mixed after each aliquot addition. To achieve satisfactory incorporation of the test item into the test soil, the test item was dissolved in acetone and 20 mL of an appropriate concentration of the dissolved test item was added to sand, evaporated to dryness, ground in a mortar and pestle and then the treated sand was distributed evenly in several aliquots over the surface of each soil replicate at a rate of 10 g treated sand per kg dry weight soil (spread as a thin layer of approximately 2.5 cm). The treated test soil was thoroughly mixed after each aliquot addition to give the final nominal soil concentrations. Sufficient distilled water was sprinkled onto the test soil to increase the soil moisture content to 45% of the soil water holding capacity (WHC). The control and reference item soil mixtures were similarly treated with the same volume of acetone on the same amount of sand, evaporated, mixed, distributed evenly over the surface of the soil and water added to bring moisture content to 45% of WHC.

Test organisms (inoculum):

soil

Total exposure duration:

28 d

Test temperature:

20°C ± 2°C

Moisture:

45% of the soil water holding capacity (WHC)

Nitrogen content (% dry weight):

0.3

Details on test conditions:

TEST SYSTEM
- Test container: 5 L polypropylene with perforated lids fitted to limit water loss and allow gas exchange
- Amount of soil: 1 kg
- No. of replicates per concentration: three
- No. of replicates per control: none
- No. of replicates per vehicle control: three

SOIL INCUBATION
- Method / Conditions: Incubated in the dark in a temperature controlled room set at 20°C ± 2°C. Moisture content of the soil groups checked weekly by weight and moisture adjusted by adding sufficient distilled water to bring the samples back to original moisture content following removal of samples.

SOURCE AND PROPERTIES OF SOIL
- Sampling source: Landlook (Midlands), Leamington Spa, Rugby, Warwickshire, England
- Soil sample details: Landlook (Midlands) soil series/number: Bromsgrove 317, CEMAS Ltd. Study No. CEMS-8056
- Soil reference number: Bromsgrove 317/13.08.17
- Vegetation cover: fallow agricultural site with grass cover
- Treatments with pesticides or fertilizers: no pesticides or fertilisers for 5 years prior to sampling
- Depth of sampling: 20-25 cm
- Soil texture class: Sandy loam
- % sand: 73% w/w
- % silt: 16% w/w
- % clay: 11% w/w
- pH (in water): 6.3
- Initial nitrate concentration (mg nitrate/kg dry weight): See Table 1 (Day 0)
- Maximum water holding capacity (in % dry weight): 37.2% w/w
- Cation exchange capacity: 10.3 meq/100 g
- Organic carbon (Walkley Black method): 1.34 % w/w
- Pre-treatment of soil: none specified
- Storage: ambient temperature prior to delivery to testing facility, then stored at 2-8 °C in the dark in aerobic conditions
- Initial microbial biomass as % of total organic C: 3.19% (428 mg C/kg soil)

DETAILS OF PREINCUBATION OF SOIL (if any): acclimatised at 20 ± 2°C for 3 days prior to testing
- Amendment of test soil with a nitrogen source: Lucerne meal (Batch 112)
- Lucerne meal analysis (Butterworth Laboratories): Carbon 42.53%, Hydrogen 6.23%, Nitrogen 3.28%
- Carbon:Nitrogen ratio: 13:1

EFFECT PARAMETERS MEASURED:
The means of the replicate analyses (e.g., N1A, N1B and N1C) were calculated and used to plot the nitrate concentrations per gram dry weight soil at Day 0 and at Day 28. The percentage inhibition of nitrogen transformation activity was also calculated by comparing the nitrate formation rate in the treated soil with that in the non-treated control vessels. The EC50, EC25 and EC10 values and the No Observed Effect Concentration (NOEC) were determined by statistical analysis of the Day 28 data.

Nominal and measured concentrations:

Nominal concentrations : 10, 32, 100, 320 and 1000 mg/kg dry weight soil.

Reference substance (positive control):

yes

Remarks:

Nitrapyrin, a nitrification inhibitor (10 mg/kg dry weight soil)

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

nitrate formation rate

Remarks on result:

other: Estimate outside the range of the data, cannot be relied upon

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

2.08 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

nitrate formation rate

Remarks on result:

other: 95% Confidence Interval (-0,08 - 203,47 mg/kg dw)

Duration:

28 d

Dose descriptor:

EC25

Effect conc.:

117.98 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

nitrate formation rate

Remarks on result:

other: 95% Confidence Interval (14.59 - 949.12 mg/kg dw)

Key result

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

32 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

nitrate formation rate

Details on results:

Nitrogen transformation in non-treated control soil (Group 1) and Groups 2 and 3 (10 mg and 32 mg/kg dry weight soil, respectively) was evident from the increase in nitrate concentrations over the study period. At the end of the 28 day study period, the percent inhibition in soil nitrogen transformation activity between non-treated control soil (Group 1) and Groups 2 and 3 was <25%. The 100, 320 and 1000 mg/kg dry weight soil treated groups showed statistically significantly lower nitrate concentrations (p<0.01) and showed >25% inhibition of nitrogen transformation compared to the non-treated control group at the end of the 28 day study period.

The EC50 and 95% confidence intervals were estimated, but because they fall outside the range of the data they should not be relied upon. The estimates for the EC10 and EC25 have large confidence intervals and the values should be used with caution.

The No Observed Effect Concentration (NOEC) was determined to be 32 mg/kg dry weight soil.

Results with reference substance (positive control):

Treatment of the test soil with Nitrapyrin, a nitrification inhibitor, resulted in significant inhibition of nitrogen transformation activity in the test soil as expected, confirming the sensitivity of the test system.

Reported statistics and error estimates:

The EC10, EC25 and EC50 values were estimated with a logistic sigmoid curve fitted to the log transformed data using Marquardt’s iterative compromise method. Results from treated groups were compared to the control group using two-tailed Dunnett's test. The No Observed Effect Concentration (NOEC) was determined as the maximum test concentration at which no significant inhibition of transformation activity compared to untreated control was observed on Day 28.

Table1: Inhibition of nitrogen transformation activityin soil

Treatment Group	Mean Nitrate Concentration (mg/kg dry weight soil)^a		Percent Inhibition (%)^b
	Day 0	Day 28	Day 0	Day 28
Group 1: Non-treated Control	77.53	136.66	--	--
Test item
Group 2: 10 mg/kg	85.13	117.35	[9.80]	14.13
Group 3: 32 mg/kg	86.96	121.83	[12.16]	10.85
Group4: 100 mg/kg	92.27	92.45	[19.01]	32.35**
Group5: 320 mg/kg	88.42	93.64	[14.05]	31.48**
Group 6: 1000 mg/kg	84.76	89.98	[9.33]	34.16**
Nitrification Inhibitor (Nitrapyrin)
Group 7: 10 mg/kg	75.61	8.42	2.48	93.84

 

^a  Nitrate concentrations in soil expressed asa mean figure derived from 3 replicate analyses.

^b Values in brackets reflect negative percent inhibition, indicating a stimulation of carbon transformation relative to the non-treated control.

**p< 0.01

Validity criteria fulfilled:

yes

Remarks:

The variation between triplicate control soil samples (Group 1) for nitrate analysis (with a coefficient of variance of 2.73%) was less than +/- 15% at Day 28.

Conclusions:

Based on the effects of the test item on nitrogen transformation activity of the soil microorganisms in this study, the EC50 was determined to be greater than 1000 mg/kg dry weight soil and the No Observed Effect Concentrations (NOEC) was determined to be 32 mg/kg dry weight soil.

Executive summary:

This study according to GLP was performed to assess the long-term effect of the test item, after a single exposure, on the nitrogen transformation activities of soil micro-organisms. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2000) No. 216, "Soil Microorganisms: Nitrogen Transformation Test", and Method C.21 of Commission Regulation (EC) No. 440/2008.

 

Soil microorganisms were exposed to the test item at five treatment concentrations (10, 32, 100, 320 and 1000 mg/kg dry weight soil) for 28 days at temperatures of between 18 to 22°C in the dark. The test soil was a sandy loam soil obtained from a site in Warwickshire, England that had received no pesticides or fertilisers for 5 years prior to sampling. 

 

Nitrogen transformation was assessed in non-treated control soil and soils treated with the test item, prepared as 3 replicate samples per group. Each soil group replicate was supplemented with lucerne meal as a nitrogen source. Triplicate portions of each soil group replicate were analysed for soil nitrate concentrations in samples on Day 0 and 28 days after treatment.

 

Nitrogen transformation in non-treated control soil (Group 1) and Groups 2 and 3 (10 mg and 32 mg/kg dry weight soil, respectively) was evident from the increase in nitrate concentrations over the study period. Percent inhibition in soil nitrogen transformation activity between non-treated control soil (Group 1) and Groups 2 and 3 was less than 25% at the on Day 28. At higher treatment concentrations (100, 320 and 1000 mg/kg dry weight soil), statistically significant inhibition of nitrogen transformation in soil was observed (p=0.003), with nitrate concentrations that were at least 25% lower compared to the non-treated control group at the end of the 28­day study period.

 

Treatment of the test soil with Nitrapyrin, a nitrification inhibitor, resulted in significant inhibition of nitrogen transformation activity in the test soil as expected, confirming the sensitivity of the test system. 

 

The EC50 (6403.41 mg/kg) and 95% confidence intervals were estimated for the test item, but they fell outside the range of the data. The EC50 for nitrogen transformation is therefore considered to be greater than 1000 mg/kg dry weight soil. The estimates for the EC10 (2.08 mg/kg) and EC25 (117.98 mg/kg) have large confidence intervals and the values should be used with caution. The No Observed Effect Concentration (NOEC) for nitrogen transformation was determined to be 32 mg/kg dry weight soil.

Reference 2

Endpoint:

toxicity to soil microorganisms

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 October 2017 to 24 May 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 217 (Soil Microorganisms: Carbon Transformation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Exception to GLP compliance: test soil collection and analysis by CEMAS, Ltd.

Analytical monitoring:

no

Details on sampling:

Carbon transformation was determined by the glucose induced respiration method, based on the procedure described by Anderson and Domsch(4) 1978.

Samples (100 g) of each of the untreated control replicates and all treated soil replicates were withdrawn on Day 0 and 28 days after preparation (Day 28). Glucose-induced respiration rates were determined on each sampling day by mixing sufficient glucose into the soil of each replicate to elicit an “immediate maximum respiratory response”. The amount of glucose required to elicit this response was determined in a preliminary test to be 0.2 g glucose/100 g soil. The glucose was first ground to a powder in the presence of clean quartz sand and then homogenously mixed into the soil at a rate not exceeding 10 g sand/kg dry weight soil.

The glucose amended soil samples (100 g) were distributed into respirometer flasks and incubated in a temperature controlled room set to maintain 20 +/- 2 deg C for the duration of the glucose induced respiration analysis.

Vehicle:

yes

Remarks:

acetone

Details on preparation and application of test substrate:

The group mixtures were prepared in triplicate bulk lots of 1 kg wet soil for each group (e.g., C1A, C1B, C1C). To achieve satisfactory incorporation of the test item into the test soil, the test item was dissolved in acetone and 20 mL of an appropriate concentration of the dissolved test item was added to sand, evaporated to dryness, ground in a mortar and pestle and then the treated sand was distributed evenly in several aliquots over the surface of each soil replicate at a rate of 10 g treated sand per kg dry weight soil (spread as a thin layer of approximately 2.5 cm). The treated test soil was thoroughly mixed after each aliquot addition to give the final nominal soil concentrations. Sufficient distilled water was sprinkled onto the test soil to increase the soil moisture content to 45% of the soil water holding capacity (WHC). The control and reference item soil mixtures were similarly treated with the same volume of acetone on the same amount of sand, evaporated, mixed, distributed evenly over the surface of the soil and water added to bring moisture content to 45% of WHC.

Test organisms (inoculum):

soil

Total exposure duration:

28 d

Test temperature:

20°C ± 2°C

Moisture:

45% of the soil water holding capacity (WHC)

Organic carbon content (% dry weight):

1.43

Details on test conditions:

TEST SYSTEM
- Test container: 5 L polypropylene with perforated lids fitted to limit water loss and allow gas exchange
- Amount of soil: 1 kg
- No. of replicates per concentration: three
- No. of replicates per control: none
- No. of replicates per vehicle control: three

SOIL INCUBATION
- Method / Conditions: Incubated in the dark in a temperature controlled room set at 20°C ± 2°C. Moisture content of the soil groups checked weekly by weight and moisture adjusted by adding sufficient distilled water to bring the samples back to original moisture content following removal of samples.

SOURCE AND PROPERTIES OF SOIL
- Sampling source: Landlook (Midlands), Leamington Spa, Rugby, Warwickshire, England
- Soil sample details: Landlook (Midlands) soil series/number: Bromsgrove 317 (CEMAS Ltd. Study No. CEMS-8467)
- Soil reference number: Bromsgrove 317/30.01.18
- Vegetation cover: fallow agricultural site with grass cover
- Treatments with pesticides or fertilizers: no pesticides or fertilisers for 5 years prior to sampling
- Depth of sampling: 20-25 cm
- Soil texture class: Sandy loam
- % sand: 71% w/w
- % silt: 17% w/w
- % clay: 12% w/w
- pH (in water): 6.5
- Maximum water holding capacity (in % dry weight): 41.3% w/w
- Cation exchange capacity: 10.4 meq/100 g
- Pre-treatment of soil: none specified
- Storage: ambient temperature prior to delivery to testing facility, then stored at 2-8 °C in the dark in aerobic conditions
- Initial microbial biomass as % of total organic C: 4.24% (606 mg C/kg soil)

DETAILS OF PREINCUBATION OF SOIL: acclimatised at 20 ± 2°C for 3 days prior to testing

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
the means of the replicate analyses (eg. C1A, C1B and C1C) at each hourly interval over 12 hours were calculated and used to plot graphically the mean carbon dioxide formation rates, expressed in mg CO2/kg dry weight soil/hour, at Day 0 and at Day 28. The EC50, EC25 and EC10 values and the No Observed Effect Concentration (NOEC) were determined by statistical analysis.

Nominal and measured concentrations:

Nominal concentrations in both tests were non-treated control, 10, 32, 100, 320 and 1000 mg/kg dry weight soil.

Reference substance (positive control):

no

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

respiration rate

Key result

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

respiration rate

Details on results:

On Days 0 and 28, the percent inhibition of carbon transformation in Groups 2 through 5 compared to the non-treated control soil (Group 1) was <25%. Statistically significant carbon transformation inhibition was observed during the first 2 to 3 hours following the addition of glucose for Groups 4 through 6 on Day 0, but this was not considered to be biologically significant as the observed inhibition was not clearly dose-dependent and was not sustained over the 12 hours of measurements on Days 0 and 28. Statistically significant increases (p<0.001) in rates of carbon transformation were observed in treatment Groups 5 and 6 at the end of the 28­day exposure period compared to non-treated control soil (Group 1).

The EC10, EC25 and EC50 values and associated confidence intervals could not be estimated as there was an insufficient inhibition across the treatment groups. The NOEC was determined to be 1000 mg N-octadecylstearamide/kg dry weight soil.

Reported statistics and error estimates:

For all time points on both Day 0 and Day 28, the EC10, EC25 and EC50 values and the associated confidence intervals could not be estimated as there was an insufficient dose response relationship.

Results from treated groups were compared to the control group using two-tailed Dunnett's test. The No Observed Effect Concentration (NOEC) was determined as the maximum test concentration at which no significant inhibition of transformation activity compared to untreated control was observed on Day 28.

Table1: Carbon Transformationin Soil - Day 0 and 28 Group Mean Respiration Values

 

Hourly interval after glucose addition	Carbon Transformation (mg C/kg dry weight)^a
	Group 1 (Non-treated control soil)	Group 2 (10 mg/kg dw)	Group 3 (32 mg/kg dw)	Group 4 (100 mg/kg dw)	Group 5 (320 mg/kg dw)	Group 6 (1000 mg/kg dw)
Day 0
1	8.85	8.86	8.77	7.99	7.82	7.98
2	7.92	7.87	7.83	7.35	7.26	7.43
3	7.63	7.56	7.56	7.26	7.19	7.35
4	7.56	7.48	7.51	7.60	7.57	7.69
5	7.65	7.55	7.56	7.74	7.71	7.88
6	8.10	7.97	7.98	8.06	8.04	8.22
7	8.42	8.26	8.30	8.22	8.26	8.42
8	8.95	8.76	8.79	8.85	8.89	9.12
9	9.43	9.20	9.22	9.15	9.22	9.45
10	10.15	9.87	9.93	10.07	10.10	10.42
11	11.03	10.65	10.71	11.52	11.58	11.93
12	12.02	11.52	11.60	12.88	13.00	13.42
Day 28
1	6.93	6.69	6.62	7.50	7.72	8.52
2	6.57	6.37	6.35	6.89	7.16	8.07
3	6.48	6.38	6.35	6.90	7.12	8.16
4	6.57	6.48	6.50	6.90	7.12	8.28
5	6.76	6.58	6.66	7.10	7.36	8.62
6	7.01	6.79	6.87	7.29	7.58	9.03
7	7.26	7.09	7.13	7.46	7.74	9.42
8	7.53	7.38	7.39	7.64	7.94	9.83
9	7.79	7.56	7.64	8.06	8.36	10.46
10	8.15	7.86	7.99	8.53	8.87	11.31
11	8.65	8.35	8.49	9.00	9.39	12.17
12	9.25	8.97	9.09	9.60	10.01	13.26

^a Values expressed as mg C as CO₂per kg dry weight soil/hour. Each value is a mean figure derived from three replicate analyses for Groups 1, 3-6; each value for Group 2 (10 mg/kg) on Day 28 is a mean of two replicate analyses. 

 

Table2: Percent Inhibition ofCarbon Transformationin Soil - Day 0 and 28

 

Hourly interval after glucose addition	Percent Inhibition (%)^a
	Group 2 (10 mg/kg dw)	Group 3 (32 mg/kg dw)	Group 4 (100 mg/kg dw)	Group 5 (320 mg/kg dw)	Group 6 (1000 mg/kg dw)
Day 0
1	[0.1]	0.9	9.7**	11.7***	9.8**
2	0.7	1.1	7.1**	8.4**	6.2*
3	0.8	0.9	4.8*	5.8*	3.7
4	1.1	0.7	[0.4]	[0.0]	[1.7]
5	1.4	1.3	[1.1]	[0.8]	[2.9]
6	1.6	1.4	0.5	0.7	[1.5]
7	1.9	1.4	2.4	2.0	[0.0]
8	2.1	1.8	1.2	0.8	[1.8]
9	2.5	2.2	2.9	2.2	[0.2]
10	2.7	2.1	0.7	0.5	[2.7]
11	3.5	2.9	[4.4]	[4.9]	[8.2]
12	4.1	3.5	[7.2]	[8.1]	[11.7]*
Day 28
1	3.4	4.5	[8.3]	[11.4]**	[22.9]***
2	3.0	3.4	[4.8]	[9.0]**	[22.8]***
3	1.5	2.0	[6.5]	[9.8]**	[25.9]***
4	1.4	1.2	[5.0]	[8.3]*	[26.0]***
5	2.6	1.5	[5.1]	[8.9]**	[27.6]***
6	3.2	2.0	[4.0]	[8.1]*	[28.8]***
7	2.3	1.7	[2.8]	[6.6]	[29.8]***
8	2.0	1.9	[1.4]	[5.5]	[30.6]***
9	3.0	1.9	[3.4]	[7.3]	[34.2]***
10	3.6	1.9	[4.7]	[8.9]	[38.8]***
11	3.4	1.9	[4.1]	[8.5]	[40.7]***
12	3.0	1.7	[3.8]	[8.3]	[43.4]***

^a Values in brackets reflect negative percent inhibition, indicating a stimulation of carbon transformation relative to the non-treated control.

 *p<0.05, **p<0.01, ***p<0.001

Validity criteria fulfilled:

yes

Remarks:

The variation between triplicate control soil samples (Group 1) for nitrate analysis (0.27% to 2.85%) was less than +/- 15% at Day 28.

Conclusions:

The test item showed no significant adverse effects on the carbon transformation activity of soil microorganisms at test concentrations up to and including 1000 mg/kg dry weight soil following the 28-day exposure period and therefore is considered to have no long-term effect on carbon transformation in soil. The No Observed Effect Concentration (NOEC) was determined to be 1000 mg/kg dry weight soil.

Executive summary:

This study according GLP was performed to assess the long-term effect of the test item, after a single exposure, on the carbon transformation activities of soil micro-organisms. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2000) No. 217, "Soil Microorganisms: Carbon Transformation Test", and Method C.22 of Commission Regulation (EC) No. 440/2008.

 

Soil microorganisms were exposed to the test item at five treatment concentrations (10, 32, 100, 320 and 1000 mg/kg dry weight soil) for 28 days at temperatures of between 18 to 22 deg C in the dark. The test soil was a sandy loam soil obtained from a site in Rugby, Warwickshire, England that had received no pesticides or fertilisers for 5 years prior to sampling. 

 

Carbon transformation was assessed in non-treated control soil and soils treated with the test item, prepared as three replicate samples per treatment group. Triplicate portions of each soil group replicate were sampled on Day 0 and 28 days after treatment and glucose-induced respiration rates were determined on each sampling day by mixing sufficient glucose into the soil of each replicate to elicit an “immediate maximum respiratory response”. The glucose amended soil samples (100 g) were distributed into respirometer flasks and incubated in a temperature controlled room set to maintain 20 +/- 2 deg C. Carbon dioxide formation was determined hourly for 12 hours using an Infrared Gas Analyser.

 

On Days 0 and 28, the percent inhibition of carbon transformation in Groups 2 through 5 compared to the non-treated control soil (Group 1) was less than 25%. Statistically significant carbon transformation inhibition was observed during the first 2 to 3 hours following the addition of glucose for Groups 4 through 6 on Day 0, but this was not considered to be biologically significant as the observed inhibition was not clearly dose-dependent and was not sustained through the 12 hours of measurements on Days 0 and 28. Statistically significant increases (p<0.001) in rates of carbon transformation were observed in treatment Groups 5 and 6 at the end of the 28­day exposure period compared to non-treated control soil (Group 1). 

 

The EC10, EC25 and EC50 values and associated confidence intervals could not be estimated as there was an insufficient inhibition across the treatment groups. The test item showed no significant adverse effects on the carbon transformation activity of soil microorganisms at test concentrations up to and including 1000 mg/kg following the 28-day exposure period and therefore is considered to have no long-term effect on carbon transformation in soil. The NOEC for carbon transformation in soil was determined to be 1000 mg/kg dry weight soil. 

Description of key information

In an OECD 216 study, conducted according to GLP, the long-term effects of the test item on nitrogen transformation activity of the soil microorganisms was investigated and the EC50 was determined to be greater than 1000 mg/kg dry weight soil. The No Observed Effect Concentrations (NOEC) was determined to be 32 mg/kg dry weight soil. 

 

In an OECD 217 study, conducted according to GLP, no significant adverse effects on the carbon transformation activity of soil microorganisms were observed following exposure to the test material at concentrations up to and including 1000 mg/kg dry weight soil following the 28-day exposure period and the test item therefore is considered to have no long-term effect on carbon transformation in soil. The NOEC for carbon transformation was determined to be 1000 mg/kg dry weight soil.

Key value for chemical safety assessment

Long-term EC10 or NOEC for soil microorganisms:

32 mg/kg soil dw

Additional information

For the purposes of the Chemical Safety Assessment, the more sensitive endpoint (nitrogen transformation activity of the soil microorganisms) was reported as the key value.