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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:
10 June 2013 to 08 July 2013
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)
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
no
Details on sampling:
- Sampling method: Soil samples were taken from all replicates at intervals of 3 hours and 7, 14 and 28 days after application.
Vehicle:
no
Details on preparation and application of test substrate:
APPLICATION OF TEST SUBSTANCE TO SOIL
- Method: The test item was first homogenised in a mortar and then thoroughly mixed with quartz sand. The quartz sand premix was then added and mixed with the soil by means of a hand stirrer. The test item quartz sand mixture was applied to the soil at a ratio of about 10 g/kg soil d.w. Water was added to the soil to achieve a water content of approximately 45 % of the water-holding capacity (WHC)

VEHICLE:
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): No vehicle was reported.
Test organisms (inoculum):
soil
Total exposure duration:
28 d
Test temperature:
19.8 - 21.5 °C in a climatic room
Moisture:
At test start: 17.02 - 17.46 g/100 g soil d.w. (equivalent to 48.40 - 49.65 % of WHC)).
The water content of the soil in each test vessel was determined at the test start (after application) and adjusted once a week to the required range of 40 - 50 % of WHC.
Details on test conditions:
TEST SYSTEM
- Test container (type, material, size): 4 L steel test vessels with lids which permitted air exchange.
- Amount of soil: 1250 g d.w.
- No. of replicates per concentration: 3
- No. of replicates per control: 3

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Soil removal: An agricultural soil (loamy sand) was selected, supplied and analysed by the test laboratory (batch no. 2/2013). The soil was removed to a depth of 20 cm as mixed samples on 21.05.2013
- History of site: Fallow ground at soil removal 2013 and pre-culitivation 2012
- Treatments with pesticides or fertilizers: No organic or inorganic fertilisers applied since 2003. No plant protection products applied since 1990.
- Soil texture (based on % particle size distribution)
- % sand: 54.9 (0.050 - 2.0 mm) (USDA) / 53.3 (0.063 - 2.0 mm) (ISO 11277)
- % silt: 35.5 (0.02 - 0.050 mm) (USDA) / 35.5 (0.002 - 0.063 mm) (ISO 11277)
- % clay: 9.6 (< 0.002mm) (USDA and ISO 11277)
- Soil taxonomic classification: Sandy loam (USDA) / loamy sand (DIN 4220)
- pH (in water): 6.5 (ISO 10390). pH: 6.2-6.5
- N min (mg nitrate/100 g soil d.w.): 0.60 (ISO 14256-2)
- Total N (%): 0.14 (ISO 11261)
- Maximum water holding capacity (g/100 g soil d.w.): 35.17 (ISO 14240-2)
- Water content [g/100 soil dw]: 10.96 (ISO 11465)
- Cation exchange capacity (cmol+/kg soil): 9.2 (ISO 11260)
- Pretreatment of soil: The soil was dried and passed through a 2 mm sieve (21.05.2013 - 22.05.2013)
- Storage (condition, duration): The soil was stored at a temperature of approximately 4 °C in containers (22.05.2013 to 05.06.2013). Before application, the soil was adapted to test conditions.
- Initial microbial biomass as % of total organic C: 2.58 % (38.40 mg C/100 g soil d.w.) (ISO14240-1)

DETAILS OF PREINCUBATION OF SOIL (if any): Before application the soil was adapted to test conditions under aerobic conditions in the dark (05.06.2013 to 10.06.2013)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Carbon transformation was determined over a measurement period of 12 hours on sampling days 0 (3 hours after application), 7, 14 and 28 days after application. On each sampling occassion, 100 g d.w. samples of soil were taken, mixed with glucose by means of a hand-stirrer and placed into glass reaction flasks (500 mL). Small glass vessels containing 18 mL of 1 M NaOH solution were placed in the reaction flasks which were tightly closed and then connected with a respirometer system that determined the O2 - consumption over a period 12 hours at different sampling interval.

VEHICLE CONTROL PERFORMED: no

TEST CONDITIONS
- Illumination: darkness
Nominal and measured concentrations:
Test concentrations: Nominal concentration: 0 (control), 62.5, 125, 250, 500 and 1000 mg test item/kg soil d.w. Test concentrations relate to a soil depth of 5 cm and a soil density of 1.5 g/cm2.
Control was left untreated (i.e. prepared with quartz sand only).
Reference substance (positive control):
yes
Remarks:
Dinoterb, CAS: 1420-07-1, IUPAC name: 2-tert-Butyl-4,6-dinitrophenol. Application rates: 5.10, 12.00 and 20.25 kg/ha (6.80, 16.00 and 27.00 mg/kg soil d.w. respectively) with 3 replicates per treatment group.
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
respiration rate
Remarks:
Carbon transformation
Remarks on result:
other: No adverse effects on carbon transformation in soil (measured as O2 consumption in a substrate-induced respiration, (SIR) test ) were observed at all test concentrations (62.5, 125, 250, 500 and 1000 mg test item/kg soil d.w).
Details on results:
No differences greater than 25% in the carbon transformation (measured as O2 consumption in a substrate-induced respiration, (SIR) test ) were found for any test concentrations of the test item at the end of the 28 day incubation period in comparison to the control. Statistically significant differences (Student-t-test for homogeneous variance, 2-sided, p≤ 0.05) were identified between the control and treatment groups 125, 250, 500 and 1000 mg test item/kg soil d.w. In all cases the difference was ≤ 25% (see table below).
Results with reference substance (positive control):
The toxic standard dinoterb caused effects of -39.3 and -45.2 % (required >= 25 %) on carbon transformation in a field soil at the test concentrations of 16.00 and 27.00 mg dinoterb/kg soil d.w. respectively, 28 days after the application and thus demonstrated the sensitivity of the test system.
Reported statistics and error estimates:
Calculation of the cumulative O2 consumption after 12 hours using regression analysis was conducted. The goodness of fit (R2) was > 0.98 in all replicates and on all days of measurement. The rate of O2 consumption (mg/kg soil d.w./h) in reach treatment group was compared to that of the control. The standard deviation and coefficient of variation as well as the percent deviation from the control were calculated for each sampling date. Statistical evaluation of the test results (2-sided Student t-test for homogeneous variances at 5% significant level) was performed.

A NOEC value was not reported in the study. The value reported in the summary was calculated from the available data.

Calculation of the deviation (D) from control for each sampling date by the following equation:

 

D [%] = ((Ct/ Cc) – 1) * 100%

 

Ct = O2 released in the treated group in mg O2/kg soil d.w./h

Cc = O2 released in the control group in mg O2/kg soil d.w./h

Effects on Carbon Transformation in Soil after Treatment with the Test Item

Treatment group

(mg test item/kg soil dry weight)

Days after application

0

7

14

28

Control

O2Consumption

 [mg/kg soil d.w./h]

14.0

13.86

11.78

10.76

62.5 mg/kg

O2Consumption

 [mg/kg soil d.w./h]

14.47*

14.26*

12.07

11.08

Deviation from control [%]1

+3.3

+2.9

+2.5

+3.0

125 mg/kg

O2Consumption

 [mg/kg soil d.w./h]

14.27

13.63

12.56*

10.61*

Deviation from control [%]1

+1.9

-1.7

+6.7

-1.4

250 mg/kg

O2Consumption

 [mg/kg soil d.w./h]

15.20*

14.37*

13.08*

10.94*

Deviation from control [%]1

+8.6

+3.7

+11.1

+1.6

500 mg/kg

O2Consumption

 [mg/kg soil d.w./h]

15.92*

15.85*

13.25*

11.11*

Deviation from control [%]1

+13.7

+14.3

+12.5

+3.2

1000 mg/kg

O2Consumption

 [mg/kg soil d.w./h]

16.29*

17.76*

14.15*

11.42*

Deviation from control [%]1

+16.3

+28.1

+20.1

+6.1

The calculations were performed with non-rounded values

1) based on O2consumption; - = inhibition; + = stimulation

* statistically significantly different to control (Student-t-test for homogeneous variances, 2-sided, p0.05)

Validity criteria fulfilled:
yes
Remarks:
The coefficient of variation in the control group of the carbon transformation tests were maximum 1.1 % (demanded range ≤ 15 %). The toxic standard dinoterb caused a reduction in carbon transformation that demonstrated the sensitivity of the test system.
Conclusions:
28-day NOEC = 1000 mg/kg soil dry weight (no adverse effects, i.e. deviation from control <25 %, OECD 216/217)
Executive summary:

The sensitivity of soil micro-organisms to the test substance was measured in a GLP-compliant test following OECD guideline 217. Five concentrations were tested in the range from 62.5 to 1000 mg test item/kg soil d.w. over a 28 day incubation period. No adverse effect on the soil carbon transformation (measured as oxygen consumption) occurred at any test concentration up to the highest tested concentration of 1000 mg/kg soil dry weight.

 No adverse effects of the test item on carbon transformation (deviation from the control < 25 %) in soil were observed at any tested concentration ( 62.5, 125, 250, 500 and 1000 mg test item/kg soil dry weight) after 28 days.

Endpoint:
toxicity to soil microorganisms
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 June 2013 to 08 July 2013
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)
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
no
Details on sampling:
- Sampling method: For NH4-N, NO3-N and NO2-N analysis, soil samples (10 g soil d.w. per replicate) were taken at intervals of 3 hours and 7, 14 and 28 days after application. Soil was extracted by adding 50 mL 1 M KCl solution to the equivalent of 10 g soil d.w. and mixing on a rotator at 150 rpm for 60 minutes.
- Sample storage conditions before analysis: Mixtures were centrifuged and stored deep frozen prior to analysis at -20 ± 5 °C. Samples were analysed within one week after day 28.
Vehicle:
no
Details on preparation and application of test substrate:
AMENDMENT OF SOIL
- Type of organic substrate: Lucerne meal (5 g/kg soil d.w.) was added to each 200 g soil sample to provide 1.250 g lucerne meal per 250 g soil d.w. and mixed by means of a hand-stirrer.
APPLICATION OF TEST SUBSTANCE TO SOIL
- Method: The test item was first homogenised in a mortar and then thoroughly mixed with quartz sand. The quartz sand premix was then added and mixed with the soil by means of a hand stirrer. The test item quartz sand mixture was applied to the soil at a ratio of about 10 g/kg soil d.w. Water was added to the soil to achieve a water content of approximately 45 % of the water-holding capacity (WHC)

VEHICLE:
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): No vehicle was reported.
Test organisms (inoculum):
soil
Total exposure duration:
28 d
Test temperature:
19.8 - 21.5 °C in a climatic room
Moisture:
At test start: 16.31 - 17.09 g/100 g soil d.w. (equivalent to 46.39 - 45.58 % WHC)).
The water content of the soil in each test vessel was determined at the test start (after application) and adjusted once a week to the required range of 40 - 50 % of WHC.
Details on test conditions:
TEST SYSTEM
- Test container (type, material, size): 500 mL wide mouth glass flask. The screw caps of the flasks permitted an air exchange.
- Amount of soil: 250 g
- No. of replicates per concentration: 3
- No. of replicates per control: 3

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Soil removal: An agricultural soil (loamy sand) was selected, supplied and analysed by the test laboratory (batch no. 2/2013). The soil was removed to a depth of 20 cm as mixed samples on 21.05.2013
- History of site: Fallow ground at soil removal 2013 and pre-culitivation 2012
- Treatments with pesticides or fertilizers: No organic or inorganic fertilisers applied since 2003. No plant protection products applied since 1990.
- Soil texture (based on % particle size distribution)
- % sand: 54.9 (0.050 - 2.0 mm) (USDA) / 53.3 (0.063 - 2.0 mm) (ISO 11277)
- % silt: 35.5 (0.02 - 0.050 mm) (USDA) / 35.5 (0.002 - 0.063 mm) (ISO 11277)
- % clay: 9.6 (< 0.002mm) (USDA and ISO 11277)
- Soil taxonomic classification: Sandy loam (USDA) / loamy sand (DIN 4220)
- pH (in water): 6.5 (ISO 10390). pH: 6.2-6.5
- N min (mg nitrate/100 g soil d.w.): 0.60 (ISO 14256-2)
- Total N (%): 0.14 (ISO 11261)
- Maximum water holding capacity (g/100 g soil d.w.): 35.17 (ISO 14240-2)
- Water content [g/100 soil dw]: 10.96 (ISO 11465)
- Cation exchange capacity (cmol+/kg soil): 9.2 (ISO 11260)
- Pretreatment of soil: The soil was dried and passed through a 2 mm sieve (21.05.2013 - 22.05.2013). Before application, the soil was adapted to test conditions.
- Storage (condition, duration): The soil was stored at a temperature of approximately 4 °C in containers under aerobic conditions in the dark (22.05.2013 to 05.06.2013).
- Initial microbial biomass as % of total organic C: 2.58 % (38.40 mg C/100 g soil d.w.) (ISO14240-1)

DETAILS OF PREINCUBATION OF SOIL (if any): Before application the soil was adapted to test conditions (05.06.2013 to 10.06.2013)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : NH4-N, NO3-N and NO2-N were determined, with soil analysed at 0, 7, 14 and 28 days after treatment.

VEHICLE CONTROL PERFORMED: no

TEST CONDITIONS
- Illumination: darkness
Nominal and measured concentrations:
Test concentrations: Nominal concentration: 0 (control), 62.5, 125, 250, 500 and 1000 mg test item/kg soil d.w. Test concentrations relate to a soil depth of 5 cm and a soil density of 1.5 g/cm2.
Control was left untreated (i.e. prepared with quartz sand only).
Reference substance (positive control):
yes
Remarks:
Dinoterb, CAS: 1420-07-1, IUPAC name: 2-tert-Butyl-4,6-dinitrophenol. Application rates: 5.10, 12.00 and 20.25 kg/ha (6.80, 16.00 and 27.00 mg/kg soil d.w. respectively) with 3 replicates per treatment group.
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
other: Nitrogen transformation
Remarks on result:
other: No adverse effects on nitrogen transformation in soil (measured as NO3-N production) were observed at all test concentrations (62.5, 125, 250, 500 and 1000 mg test item/kg soil d.w).
Details on results:
No differences greater than 25% in the nitrogen transformation (measured as NO3-N production) were found for any concentration of the test item at the end of the 28 day incubation period in comparison to the control. Statistically significant differences (Student-t-test for homogeneous variance, 2-sided, p≤ 0.05) were identified between the control and treatment groups 250, 500 and 1000 mg test item/kg soil d.w after 28 days. In all cases a stimulation in nitrogen transformation was observed and in all cases the difference was ≤ 25% (see table below).
Results with reference substance (positive control):
At day 28, dinoterb had deviations from the control of +33.7 and +42.6 % based on nitrogen transformation (required ≥ 25 %) at concentrations of 16.00 and 27.00 mg/kg soil d.w. respectively. The reference item thus demonstrates the sensitivity of the test system.
Reported statistics and error estimates:
Calculation of the average N-content in mg/kg soil d.w. per day (separately for NO3-N) and if appropriate for NO2-N and NH4-N, were conducted for each treatment rate and assessment date. The formed NO3-N in each treatment was compared with the NO3-N in the control group. The standard deviation and coefficient of variation as well as the percent deviation from the control were calculated for each sampling date. Statistical evaluation of the test results (2-sided Student t-test for homogeneous variances at 5% significant level) was performed.

A NOEC value was not reported in the study. The value reported in the summary was calculated from the available data.

The percent deviation (D) from control was calculated for each sampling date by the following equation:

 

D [%] = ((Nt / Nc) – 1) x 100

 

Nt = nitrate formation rate in the treated group in mg NO3-N/kg soil d.w.

Nc = nitrate formation rate in the control group in mg NO3-N/kg soil d.w.

Effects on Nitrogen Transformation in Soil after Treatment with the Test Item

Treatment group

(mg test item/kg soil dry weight)

Days after application

0

7

14

28

Control

NO3-N [mg/kg soil d.w.]

11.4

38.1

47.5

60.6

62.5 mg/kg

NO3-N [mg/kg soil d.w.]

11.1

39.0

45.1*

62.5

Deviation from control [%]1

-2.6

+2.4

-5.1

+3.1

125 mg/kg

NO3-N [mg/kg soil d.w.]

10.7

37.7

46.5

60.6

Deviation from control [%]1

-5.6

-1.1

-2.2

-0.1

250 mg/kg

NO3-N [mg/kg soil d.w.]

11.0

38.2

47.3

65.2*

Deviation from control [%]1

-3.2

+0.2

-0.4

+7.6

500 mg/kg

NO3-N [mg/kg soil d.w.]

10.8

38.5

48.2

66.2*

Deviation from control [%]1

-5.0

+0.9

+1.3

+9.2

1000 mg/kg

NO3-N [mg/kg soil d.w.]

10.9

39.1

53.8

70.9*

Deviation from control [%]1

-4.4

+2.5

+13.2

+16.9

The calculations were performed with non-rounded values

1) based on NO3-nitrogen production; - = inhibition; + = stimulation

* statistically significantly different to control (Student-t-test for homogeneous variances, 2-sided, p0.05)

Validity criteria fulfilled:
yes
Remarks:
The coefficient of variation in the control group of the nitrogen transformation tests were maximum 3.6 % (demanded range ≤ 15 %). The toxic standard dinoterb caused enhanced nitrogen transformation that demonstrated the sensitivity of the test system.
Conclusions:
28-day NOEC = 1000 mg/kg soil dry weight (no adverse effects, i.e. deviation from control <25 %, OECD 216/217)
Executive summary:

The sensitivity of soil micro-organisms to the test substance was measured in a GLP-compliant test following OECD guideline 216. Five concentrations were tested in the range from 62.5 to 1000 mg test item/kg soil d.w. over a 28 day incubation period. No adverse effects (i.e. deviation from the control <25%) of the test item on nitrogen transformation (measured as NO3-N production) in soil were observed at any tested concentration after 28 days. Therefore, the 28-day NOEC in this study was 1000 mg/kg soil dry weight.

Description of key information

28-day EC50 carbon transformation: >1000 kg/kg soil dw (OECD 217, Schulz 2013)
28-day NOEC carbon transformation: 1000 kg/kg soil dw (OECD 217, Schulz 2013)
28-day EC50 nitrogen transformation: >1000 kg/kg soil dw (OECD 216, Schulz 2013)
28-day NOEC nitrogen transformation: 1000 kg/kg soil dw (OECD 216, Schulz 2013)

Key value for chemical safety assessment

Long-term EC10 or NOEC for soil microorganisms:
1 000 mg/kg soil dw

Additional information

Fully reliable GLP and guideline compliant studies on nitrogen transformation (OECD TG 216) and on carbon transformation (OECD TG 217) have been conducted. The 28-day NOEC was found to be 1000 mg/kg soil dry weight in both studies, and this can be considered as a long-term result for microbial populations.

The influence of cloquintocet-mexyl on soil microorganisms was also investigated in a 28-day study performed according to the BBA Part VI, 1-1 guideline (Morgenroth, 1992). The study was GLP compliant and is considered to be reliable without restrictions. This study showed that the substance had no inhibitory effects on soil microorganisms at typical or exaggerated application rates of the substance (based on Good Agricultural Practice) of up 0.27 mg/kg soil dw.