Diquat dibromide

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:

08 Jun 2007 to 10 Jul 2007

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)

Version / remarks:

2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on preparation and application of test substrate:

AMENDMENT OF SOIL
- Type of organic substrate: Agriculturally utilised soil

APPLICATION OF TEST SUBSTANCE TO SOIL
- Method: The test item was mixed with water and the test solution was then mixed with the soil by means of a hand stirrer. The application of the test item to the test soil was performed 7 days before test start (day -7). After an ageing period of 7 days, the test was started by adding lucerne-meal to the soil (day 0) as nitrogen source for the nitrogen transformation

Test organisms (inoculum):

soil

Total exposure duration:

28 d

Remarks:

7 days aging and 28 days incubation

Test temperature:

20.3 - 21.1 °C

Moisture:

16.84 - 17.52 g/100 g soil d.w. (equivalent to 42.43 - 44.14 % of WHC)

Organic carbon content (% dry weight):

1.49

Nitrogen content (% dry weight):

0.15

Details on test conditions:

TEST SYSTEM
- Testing facility: Tightly closed wide-mouth glass flasks (500 mL)
- Amount of soil: 200 g dry weight
- No. of replicates per concentration: 3
- No. of replicates per control: 3

SOIL INCUBATION
- Method: The incubation of the soil samples was performed as series of individual and equally sized sub-samples of each treatment group. After applying the test substance, water was added to the soil to achieve a water content of approximately 45 % of WHC. After an ageing period of 7 days the test was started by adding and mixing of 0.5 % lucerne-meal (i.e.1.0 g/200 g soil d.w.) to the soil by means of a hand-stirrer (the C/N ratio of the lucerne meal was 15/1). One additional soil sample (without lucerne meal) was used for determination of the initial NH4-N- and NO3-Ncontent. The NO3-N content was 2.94 mg N/100 g soil d.w.
- Aeration: To avoid anaerobic conditions the vessels were briefly opened twice to three times a week for aeration.
- Water content determination: The water content of the soil in each test vessel was determined at test start (day 0) and checked once a week. During the test, the water content of the soil of each test vessel was in the required range of 40 - 50 % of WHC.
- pH determinations: The pH-values of the soil used in the test were measured at test start (day 0 after application) and at the sampling on day 28.

SOURCE AND PROPERTIES OF SUBSTRATE
- Geographical reference of sampling site: 12.694435960 degrees East, 51.403774567 degrees North
- Treatments with fertilizers: none since 2003
- Depth of sampling: The soil was removed to a depth of 20 cm as mixed samples.
- Soil classification: Loamy sand soil
- % sand (2 - 0.063 mm): 51.9%
- % silt (0.063 - 0.002 mm): 38.0%
- % clay (< 0.002 mm): 10.0%
- pH (water): 6.6
- Humus content: 2.56%
- Water holding capacity: 39.70 g/100g soil dry weight
- Water content: 10.12 g/100 g soil dry weight
- Cation exchange capacity (cmol+/kg soil dry weight): 9.4
- Initial minimal nitrate concentration: 1.23 mg nitrate/100 g dry weight
- Microbial biomass: 37.57 mg C/100 g soil dry weight (equivalent to 2.52% compared to organic carbon)
- Pretreatment of soil: Because the soil was wet, it was carefully dried at room temperature. Afterwards, the soil was passed through a 2 mm mesh sieve.
- Storage (condition, duration): The soil stored at 4 ± 2 °C for a maximum of three months before test start was to be exposed to room temperature (20 ± 2 °C) for at least 48 hours and max. 21 days before application to allow the microflora to adjust to the corresponding test conditions.

DETAILS OF PREINCUBATION OF SOIL
Before the experiment started, the test substance treated soil was experienced a 7-day aging period in darkness. This aging process was carried out under 20.1 - 20.8 °C in a climatic room. The water content of the soil was approximately 45% WHC.

EFFECT PARAMETERS MEASURED
Soil samples (10 g soil d.w. per replicate) were taken at intervals of 3 hours, 7, 14 and 28 days after test start and the NH4-N, NO3-N and NO2-N content was determined.
For the extraction 50 mL 1 m KCI solution (10 g soil d.w. with 50 mL KCI solution) and a rotator (150 rpm) were used. The extraction duration was 60 minutes. The mixtures were centrifuged and stored deep-frozen prior to analysis at minus (20 ± 5) °C. The analysis was performed within one week after day 28.
The intensity of the formed compound is colorimetrically measured at a wavelength of 625 nm. Nitrate is reduced to nitrite by hydrazinesulphate. The nitrite reacts with sulphanilamide in an acidic solution to form a diazocompound. The diazotized product is then coupled with naphthylamine. The intensity of the formed azodye, which is proportional to the sum of the nitrate and nitrite originally present in the sample, is colorimetrically measured at a wavelength of 525 nm. The differences between the nitrate/nitrite sum and the nitrite contents are the nitrate contents. The nitrite contents are determined without nitrate reduction. The chemicals for the calibration solutions were NaNO2, (NH4)2SO4 and KNO3. The autoanalyzer was calibrated before each measurement series by establishing a calibration curve. Each 30 samples a standard was measured for recalibration and adjusting the calibration curve. The calibration curve was calculated with linear regression. The Limits of Quantification (LOQ) for NO3-N and NH4-N / NO2-N are 0.06 mg/100 g soil d.w. and 0.1 mg/100 g soil d.w., respectively.

Nominal and measured concentrations:

Nominal rates: 0 (negative control), 62.5, 125, 187.5, 250, 500 mg cation/kg soil dry weight.

Reference substance (positive control):

yes

Remarks:

Dinoterb was tested as reference item in separate studies to verify the sensitivity of the test system.

Key result

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

>= 934 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

nitrate formation rate

Remarks on result:

other: Recalculated value, expressed as pure substance, see ‘Any other information on results incl. tables’ for respective calculation

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

>= 500 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Remarks:

cation

Basis for effect:

nitrate formation rate

Remarks on result:

other: Original value presented in study

Details on results:

An overview of the results is provided in Table 1 in ‘Any other information on results incl. tables’.
The NO3-N content in the control group was 27.8 mg/kg soil d.w. on day 0. The NO3-N in the 62.5, 125, 187.5, 250 and 500 mg cation/kg soil d.w test substance treated groups were 28.8, 29.1, 28.8, 26.5, 23.8 mg/ kg soil dry weight, respectively (which were 3.7, 4.9, 3.8, -4.6 and -14.3% deviation from the control, respectively). After 28 days incubation, the control group showed 64.9 mg NO3-N mg/kg soil d.w.. The NO3-N in the 62.5, 125, 187.5, 250 and 500 mg cation/kg soil d.w test substance treated groups were 63.7, 65.4, 62.8, 61.9 and 60.0 mg/kg soil d.w., respectively (which were -1.8, 0.9, -3.2, -4.6 and -7.5% deviation from the control, respectively).
No differences greater than 15 % in the nitrogen transformation were found for the tested concentrations of the test item at any time interval in comparison to the respective control. The coefficients of variation during the experiment were within the demanded limit (control <15 %).

Results with reference substance (positive control):

In a separate study, the reference item Dinoterb caused a stimulation of nitrogen transformation of + 44.8%, + 58.2% and + 23.7% at 6.8 mg, 16.00 mg and 27.00 mg Dinoterb per kg soil dry weight, respectively, 28 days after application.

Reported statistics and error estimates:

Student-t-test for Homogeneous Variances and Welch-t-test for Inhomogeneous Variances were used in this study.

Table 1. Effects on nitrogen transformation in soil after treatment with the test item

Treatment group (mg cation/kg soil dry weight)		Days after test start
		0	7	14	28
Control	NO3-N [mg/kg soil d.w.]	27.8	49.0	56.5	64.9
	Coefficient of Variation [%]	4.0	1.5	1.6	2.9
62.5	NO3-N [mg/kg soil d.w.]	28.8	51.6	57.9	63.7
	Coefficient of Variation [%]	2.2	2.6	0.6	2.1
	Deviation from control [%]^1	+ 3.7	+ 5.2^*s	+ 2.4	-1.8
125	NO3-N [mg/kg soil d.w.]	29.1	53.2	57.5	65.4
	Coefficient of Variation [%]	2.6	3.5	3.9	1.9
	Deviation from control [%]^1	+4.9	+8.6^*s	+1.7	+0.9
187.5	N03-N [mg/kg soil d.w.]	28.8	52.0	57.5	62.8
	Coefficient of Variation [%]	4.4	1.3	6.9	1.6
	Deviation from control [%]^1	+ 3.8	+ 6.1^*s	+ 1.7	-3.2
250	NO3-N [mg/kg soil d.w.]	26.5	49.1	54.2	61.9
	Coefficient of Variation [%]	2.1	2.2	3.5	1.5
	Deviation from control [%]^1	- 4.6	+ 0.1	- 4.1	- 4.6
500	NO3-N [mg/kg soil d.w.]	23.8	43.6	51.0	60.0
	Coefficient of Variation [%]	0.7	2.8	2.7	3.1
	Deviation from control [%]^1	- 14.3^*w	- 11.0^*s	- 9.8^*s	- 7.5^*s

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

*s statistically significantly different to control (Student-t-test for homogeneous variances, 2-sided, p < 0.05)

*W statistically significantly different to control (Welch-t-test inhomogeneous variances, 2-sided, p < 0.05)

Calculation of key result

The original effect levels were expressed as cation species of the registered substance. The key effect level is re-calculated and corrected to include the counterion species by multiplying with 1.868 (344.0 g/mol molecular weight of registered substance divided by 184.2 g/mol molecular weight of cation species).:

1.868 x 500 mg cation/kg soil dw = 934 mg registered substance/kg soil dry weight

Validity criteria fulfilled:

yes

Remarks:

See Validity criteria in 'Any other information on materials and methods incl. tables'

Conclusions:

The test item caused no adverse effects (deviation from control < 25 %) on the soil nitrogen transformation (measured as NO3-N production) after 7 days aging treatment followed with 28-day incubation period. Therefore, the NOEC was determined to be ≥ 500 mg cation /kg soil dry weight (equivalent to ≥ 934 mg test substance/kg soil dry weight).

Executive summary:

The purpose of this study was to determine the effects of the test item on the activity of soil microflora with regard to nitrogen transformation in a laboratory test over a period of 28 days of exposure. The test was performed in accordance to the OECD TG 216 (2000) and in compliance with GLP criteria. Application of the test item was performed 7 days before test start (day -7). After an ageing period of 7 days, the test was started (day 0) by adding lucerne meal (concentration in soil 0.5 %) as nitrogen source. The study was carried out at 20 – 21 ˚C in darkness. The water content of the soil was 42.43 – 44.14% WHC. The nominal test rate were0 (negative control), 62.5, 125, 187.5, 250, 500 mg cation/kg soil dry weight.The effect onNO3-nitrogenproduction in soil was determined on0, 7, 14 and 28 days after test start.NH4-, NO3- and NO2-nitrogen was determined by using the Autoanalyzer II. After 7 days of ageing, the NO3-N content in the control group was 27.8 mg/kg soil d.w.. The NO3-N in the 62.5, 125, 187.5, 250 and 500 mg cation/kg soil d.w test substance treated groups were 28.8, 29.1, 28.8, 26.5, 23.8 mg/ kg soil dry weight, respectively (which were 3.7, 4.9, 3.8, -4.6 and -14.3% deviation from the control, respectively). After 28 days incubation, the control group showed 64.9 mg NO3-N mg/kg soil d.w.. The NO3-N in the 62.5, 125, 187.5, 250 and 500 mg cation/kg soil d.w test substance treated groups were 63.7, 65.4, 62.8, 61.9 and 60.0 mg/kg soil d.w., respectively (which were -1.8, 0.9, -3.2, -4.6 and -7.5% deviation from the control, respectively). No differences greater than 15 % in the nitrogen transformation were found for the tested concentrations of the test item at any time interval in comparison to the respective control. The coefficients of variation during the experiment were within the demanded limit (control <15 %). In a separate study, Dinoterb was tested as reference item to verify the sensitivity of the test system. After 28 days application, Dinoterb caused a stimulation of nitrogen transformation of + 44.8%, + 58.2% and + 23.7% at 6.8 mg, 16.00 mg and 27.00 mg Dinoterb per kg soil dry weight. Based on the findings, the NOEC of the test substance was determined to be ≥ 500 mg cation /kg soil dry weight (equivalent to ≥ 934 mg test substance/kg soil dry weight).

 

Reference 2

Endpoint:

toxicity to soil microorganisms

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 Apr 2017 to 05 May 2017

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)

Version / remarks:

2000

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

no

Details on preparation and application of test substrate:

APPLICATION OF TEST SUBSTANCE TO SOIL
- Method: The test item was mixed with deionised water and the test solution was subsequently mixed with the soil in the laboratory mixer.

Test organisms (inoculum):

soil

Total exposure duration:

28 d

Test temperature:

19.7 - 21.3 °C

Moisture:

16.57 - 17.06 g/100 g soil d.w. (equivalent to 43.60 - 44.89 % of WHC)

Organic carbon content (% dry weight):

1.48

Nitrogen content (% dry weight):

0.16

Details on test conditions:

TEST SYSTEM
- Testing facility: 4 L steel test vessels (lids on the vessels permitted air exchange)
- Amount of soil: 1000 g soil dry weight
- No. of replicates per concentration: 3
- No. of replicates per control: 3

SOURCE AND PROPERTIES OF SUBSTRATE
- Geographical reference of sampling site: 12.694435960 degrees East, 51.403774567 degrees North
- Cultivation: Fallow ground
- Treatments with fertilizers: None since 2003
- Last application of plant protection products: 1990
- Soil removal: 02 March 2017
- Drying and sieving (at room temperature): 02 March 2017 - 07 March 2017
- Storage (at approx. 4 °C): 07 March 2017 - 31 March 2017
- Soil conditioning (under test conditions): 31 March 2017 - 06 April 2017
- Application: 06 April 2017
- Depth of sampling: Depth of 20 cm as mixed sample
- pH (in water): 6.6
- Initial nitrate min: 2.96 mg nitrate/100g dry weight
- Water content: 9.74 g/100 g soil d.w.
- Cation exchange capacity: 9.9 cmol+/kg soil
- Carbon content of microbial biomass: 55.31 mg C/100g soil d.w. (equivalent to 3.74% of organic carbon)
- Pretreatment and storage of soil: The soil was carefully dried at room temperature, passed through a 2 mm mesh sieve and then stored at a temperature of approx. 4 °C in containers under aerobic conditions in the dark.

According to USDA:
- % sand (0.050 - 2.0 mm): 51.1%
- % silt (0.002 - 0.050 mm): 37.7%
- % clay (< 0.002 mm): 11.1%
- Soil taxonomic classification: USDA
- Soil classification: Loam

According to DIN 4220:
- % sand (0.063 - 2.0 mm): 55.9%
- % silt (0.002 - 0.063 mm): 33.8%
- % clay (< 0.002 mm): 10.3%
- Soil taxonomic classification: DIN 4220
- Soil classification: Loamy sand

DETAILS OF PREINCUBATION OF SOIL
Before test start, the following parameters were determined, based on the respective ISO guidelines: pH, water content, WHC and carbon content of microbial biomass. Water was added to the soil to achieve a water content of approximately 45 % of WHC. The water content of the soil in each test vessel was determined at test start (after application) and adjusted once a week to the required range of 40 - 50 % of WHC. The method is based on the initial respiratory response of microbial populations to which glucose as carbon and energy source has been added (substrate-induced respiration, SIR). Before test start, the optimal glucose concentration was determined as 0.4 %.

EFFECT PARAMETERS MEASURED
The 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 occasion, 100 g samples of soil (d.w.) were taken, mixed with glucose by means of a hand-stirrer and placed into glass reaction flasks (500 mL). Then 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. The respiration of micro-organisms leads to O2-consumption and formation of CO2 that is absorbed in NaOH solution. The absorption of CO2 decreased the pressure in the reaction flask, which is compensated with O2 delivered by the respirometer. The respirometer determines the cumulative O2-production (corresponding to the O2-consumption by micro-organisms) over a 12-hour measurement period.

Nominal and measured concentrations:

- Nominal concentrations: 0 (negative control), 235, 471, 942, 1883 mg test item/kg soil dry weight

Reference substance (positive control):

yes

Remarks:

Dinoterb was tested as reference item in separate studies to verify the sensitivity of the test system.

Key result

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

>= 605 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

respiration rate

Remarks on result:

other: Recalculated value, expressed as pure substance, see ‘Any other information on results incl. tables’ for respective calculation

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

>= 1 883 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

respiration rate

Remarks on result:

other: Original value presented in study

Remarks:

(equivalent to ≥ 320 mg cation/kg soil dw

Details on results:

An overview of the results is provided in Table 1 and Table 2 in ‘Any other information on results incl. tables’.

The O2 consumption in the control group was 21.59 mg/kg soil d.w. on day 0. The O2 consumption in the 235, 471, 942, and 1883 mg test item/kg soil d.w treated groups were 22.13, 20.90, 20.19 and 20.25 mg/ kg soil dry weight, respectively (which were +2.5, -3.2, -6.5 and -6.2% deviation from the control, respectively). After 28 days incubation, the control group showed 16.77 mg/kg soil d.w.O2 consumption. The O2 consumption in the 235, 471, 942, and 1883 mg test item/kg soil d.w treated groups were 16.58, 15.95, 5.37 and 15.46 mg/kg soil d.w. O2 consumption, respectively (which were -1.1, -4.9, -8.4 and -7.9% deviation from the control, respectively). No differences greater than 25 % in the carbon transformation were found for the tested concentrations of the test item at the end of the 28-day incubation period in comparison to the respective controls.

Results with reference substance (positive control):

In a separate study, the reference item Dinoterb caused a inhibition on the carbon transformation of -20.7%, -34.8% and -43.8% at 6.8 mg, 13.60 mg and 27.20 mg Dinoterb per kg soil dry weight, respectively, 28 days after application.

Reported statistics and error estimates:

The cumulative O2-consumption after 12 hours was calculated (using regression analysis; the goodness of fit (R2) was > 0.99 in all replicates and on all days). Furthermore, standard deviation and coefficient of variation were calculated for each treatment group and sampling date.
For evaluation of the results the relative deviations (%) of the test item treatment groups from the control were calculated for each sampling date. Statistical evaluation of the test results (2-sided Student-t-test at 5 % significance level) was performed.

Table 1. Effects on Carbon Transformation in Soil after Treatment with the test substance

Treatment group (mg test item/kg soil dry weight)		Days after application
		0	7	14	28
Control	O2 consumption [mg/kg soil d.w./h]	21.59	19.24	17.77	16.77
235 mg test item/kg	O2 consumption [mg/kg soil d.w./h]	22.13	19.97	17.75	16.58
	Deviation from control [%]^1	+2.5	+3.8	-0.1	-1.1
471 mg test item/kg	O2 consumption [mg/kg soil d.w./h]	20.90	18.87	16.84*	15.95
	Deviation from control [%]^1	-3.2	-1.9	-5.2	-4.9
942 mg test item/kg	O2 consumption [mg/kg soil d.w./h]	20.19*	18.29*	16.17*	15.37*
	Deviation from control [%]^1	-6.5	-4.9	-9.0	-8.4
1883 mg test item/kg	O2 consumption [mg/kg soil d.w./h]	20.25	18.02*	16.53*	15.46*
	Deviation from control [%]^1	-6.2	-6.4	-7.0	-7.9

The calculations were performed with non-rounded values.

CV [%] = Coefficient of Variation

1) based on O2-consumption; - = inhibition; + = stimulation

* = statistically significant differences between the control and the test item treatments were calculated.

Table 2. Results and Calculations – Carbon Transformation

Days after appli- cation	Treatment group	Repl.	Measured values O2-consumption [mg/kg] 12 h^1	O2-consumption	Mean²	SD	CV   [%]	Deviation from control [%]
				mg/kg soil d.w./h
0	Control	1	251.64	20.97	21.59	0.53	2.5	-
		2	263.01	21.92
		3	262.47	21.87
	235 mg test item/kg	1 2 3	255.82 266.56 274.20	21.32 22.21 22.85	22.13	0.77	3.5	+2.5
	471 mg test item/kg	1 2 3	249.23 248.10 255.07	20.77 20.68 21.26	20.90	0.31	1.5	-3.2
	942 mg test item/kg	1 2 3	236.64 244.50 245.58	19.72 20.38 20.47	20.19	0.41	2.0	-6.5
	1883 mg test item/kg	1 2 3	235.60 242.38 251.10	19.63 20.20 20.93	20.25	0.65	3.2	-6.2
7	Control	1	225.91	18.83	19.24	0.40	2.1	-
		2	235.36	19.61
		3	231.53	19.29
	235 mg test item/kg	1 2 3	235.42 240.15 243.22	19.62 20.01 20.27	19.97	0.33	1.6	+3.8
	471 mg test item/kg	1 2 3	222.28 224.12 233.02	18.52 18.68 19.42	18.87	0.48	2.5	-1.9
	942 mg test item/kg	1 2 3	217.76 221.10 219.67	18.15 18.43 18.31	18.29	0.14	0.8	-4.9
	1883 mg test item/kg	1 2 3	219.31 221.80 207.58	18.28 18.48 17.30	18.02	0.63	3.5	-6.4
14	Control	1	213.26	17.77	17.77	0.12	0.7	-
		2	211.84	17.65
		3	214.63	17.89
	235 mg test item/kg	1 2 3	213.39 211.74 214.00	17.78 17.65 17.83	17.75	0.10	0.5	-0.1
	471 mg test item/kg	1 2 3	199.36 202.81 204.09	16.61 16.90 17.01	16.84	0.20	1.2	-5.2
	942 mg test item/kg	1 2 3	195.16 189.80 197.17	16.26 15.82 16.43	16.17	0.32	2.0	-9.0
	1883 mg test item/kg	1 2 3	198.16 196.51 200.57	16.51 16.38 16.71	16.53	0.17	1.0	-7.0
28	Control	1	197.82	16.49	16.77	0.26	1.6	-
		2	204.05	17.00
		3	202.00	16.83
	235 mg test item/kg	1 2 3	200.19 195.61 201.21	16.68 16.30 16.77	16.58	0.25	1.5	-1.1
	471 mg test item/kg	1 2 3	192.42 183.97 197.92	16.04 15.33 16.49	15.95	0.59	3.7	-4.9
	942 mg test item/kg	1 2 3	185.54 180.66 187.03	15.46 15.06 15.59	15.37	0.28	1.8	-8.4
	1883 mg test item/kg	1 2 3	184.85 185.17 186.36	15.40 15.43 15.53	15.46	0.07	0.4	-7.9

1) Calculated with regression analysis over 12 hours

2) Mean O2-consumption per hour

SD = Standard Deviation

CV [%] = Coefficient of Variation

Repl. = Replicate

The calculations were performed with non-rounded values.

Calculation of key result

The original effect levels were expressed as aqueous solution of the cation species (17.2 % w/w) of the registered substance. The key effect level is re-calculated and corrected for the amount of water, and corrected to include the counterion species by multiplying with 1.868 (344.0 g/mol molecular weight of registered substance divided by 184.2 g/mol molecular weight of cation species).:

17.2% x 1883 mg test item/kg soil dw x 1.868 = 605 mg registered substance/kg soil dw

Validity criteria fulfilled:

yes

Conclusions:

Based on the findings, the NOEC of the test substance on carbon transformation was determined to be ≥1883 mg test item/kg soil dw.

Executive summary:

The purpose of this study was to determine the effects of the test item on soil microflora with regard to carbon transformation in a laboratory test over a period of 28 days of exposure. The test was performed in accordance to the OECD TG 217 (2000) and in compliance with GLP criteria. Loamy sand soil was used in the test with 1.48% organic carbon and 38.00 g/100g dry soil water holding capacity (WHC). The study was carried out at19.7 – 21.3˚C in darknessat pH 6.4 – 6.6. The water content of the soil was approximately 45% of its maximum WHC during the test. The nominal test rate were 0 (negative control), 235, 471, 942 and 1883 mg test item/kg soil dryweight.A respirometer system was used to determine the O2-consumption over a period of 12 hours at different sampling intervals. The soils were analysed at 0, 7, 14 and 28 days after treatment.

The O2 consumption in the control group was 21.59 mg/kg soil d.w. on day 0. The O2 consumption in the 235, 471, 942, and 1883 mg test item/kg soil d.w treated groups were 22.13, 20.90, 20.19 and 20.25 mg/ kg soil dry weight, respectively (which were +2.5, -3.2, -6.5 and -6.2% deviation from the control, respectively). After 28 days incubation, the control group showed 16.77 mg/kg soil d.w.O2 consumption. The O2 consumption in the 235, 471, 942, and 1883 mg test item/kg soil d.w treated groups were 16.58, 15.95, 5.37 and 15.46 mg/kg soil d.w. O2 consumption, respectively (which were -1.1, -4.9, -8.4 and -7.9% deviation from the control, respectively). No differences greater than 25 % in the carbon transformation were found for the tested concentrations of the test item at the end of the 28-day incubation period in comparison to the respective controls. In a separate study, the reference item Dinoterb caused a inhibition on the carbon transformation of -20.7%, -34.8% and -43.8% at 6.8 mg, 13.60 mg and 27.20 mg Dinoterb per kg soil dry weight, respectively, 28 days after application. Based on the findings, the NOEC of the test substance on carbon transformation was determined to be ≥ 1883 mg test item/kg soil dw.

Description of key information

28-d NOEC ≥ 934 mg pure test substance/kg dry soil (loamy sand soil), Nitrogen transformation, OECD TG 216, Schulz 2007

28-d NOEC ≥ 605 mg pure test substance/kg dry soil (loamy sand soil), Carbon transformation, OECD TG 217, Schulz 2017

Key value for chemical safety assessment

Long-term EC10 or NOEC for soil microorganisms:

605 mg/kg soil dw

Additional information

Nitrogen transformation

The effects of the substance on the activity of soil microflora with regard to nitrogen transformation were studied under GLP to OECD TG 216 (2000) in a laboratory test over a period of 28 days of exposure. After an ageing period of 7 days following test substance application, the test was started (day 0) by adding lucerne meal (concentration in soil 0.%) as nitrogen source. Soil treatment at nominal concentrations of 62.5, 125, 187.5, 250, 500 mg cation/kg soil dry weight had a minor effect on the NO3-N content and no differences greater than 15% in the nitrogen transformation were found at any test concentration and time interval in comparison to the respective control. The coefficients of variation during the experiment were within the demanded limit (control <15%). In a separate study, Dinoterb was tested as positive reference item to verify the sensitivity of the test system, which caused a stimulation of nitrogen transformation of +44.8%, +58.2% and +23.7% at 6.8 mg, 16.00 mg and 27.00 mg Dinoterb per kg soil dry weight after 28 days.

With regards to nitrogen transformation, a NOEC value of ≥500 mg cation/kg soil dry weight was determined for the test substance. This is equivalent to ≥934 mg registered substance/kg soil dry weight.

Carbon transformation

The effects of the substance on soil microflora with regard to carbon transformation were studied under GLP to OECD TG 217 (2000) in a laboratory test over a period of 28 days of exposure. Loamy sand soil was used in the test with 1.48% organic carbon. Soil treatment at nominal concentrations of 235, 471, 942 and 1883 mg test item/kg soil dry weight had a minor effect on oxygen consumption and CO2 production, resulting in differences of less than 25% in the carbon transformation at all tested concentrations at the end of the 28-day incubation period in comparison to the respective controls. In a separate study, the positive reference item Dinoterb caused an inhibition of the carbon transformation of -20.7%, -34.8% and -43.8% at 6.8 mg, 13.60 mg and 27.20 mg Dinoterb per kg soil dry weight, respectively, 28 days after application. With regards to carbon transformation, a NOEC value of ≥1883 mg test item/kg soil dw was determined. This is equivalent to ≥605 mg registered substance/kg soil dw.