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Toxicity to soil microorganisms

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Reference
Endpoint:
toxicity to soil microorganisms
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2008-07-02 - 2008-11-04
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:
OECD Guideline 217 (Soil Microorganisms: Carbon Transformation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Vehicle:
yes
Details on preparation and application of test substrate:
The test item was mixed with deionised water and the test solution was subsequently mixed with the soil by means of a hand stirrer.
Test organisms (inoculum):
soil
Total exposure duration:
28 d
Test temperature:
20.2 - 21.5 °C in a climatic room
Moisture:
Nitrogen transformation test

16.25 - 16.69 g/100 g soil d.w. (equivalent to 42.63 - 43.80 % ofWHC)

Carbon transformation test

16.60 -17.14 g/100 g soil d.w. (equivalent to 43.57 - 44.96 % of WHC)
Details on test conditions:
Experimental procedure:

Nitrogen transformation test:
The incubation of the soil samples was performed as series of individual and equally sized sub-samples of each treatment group.
200 g soil dry weight (= one sub-sample) per test vessel was weighed. The soil was mixed with 0.5 % (i.e. 1.0 g/200 g soil d.w.) lucerne meal 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 N03- N-content. The initial NH4-N and N03-N content was 0.05 mg and 1.47 mg/100 g soil d.w, respectively. The test item was mixed with deionised water and the test solution was subsequently mixed with the soil by means of a hand stirrer. Water was added to the soil to achieve a water content of approximately 45 % of WHC. The incubation of the prepared soil was carried out in wide-mouth glass flasks (500 mL) under the conditions mentioned above. The screw caps of the flasks used permit air exchange. 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.

Carbon transformation test
The incubation of the soil samples was performed as series of individual and equally sized sub-samples of each treatment group.
1200 g soil dry weight (= one sub-sample) per vessel was weighed in the mixing vessel of a laboratory mixer ("Kitchen Aid").
The test item was mixed with water and the test solution was subsequently mixed with the soil in the laboratory mixer. Water was added to the soil to achieve a water content of approximately 45 % of WHC.
The incubation of the prepared soil was carried out in steel vessels (4 L) under the conditions mentioned above. The lids on the vessels used permit air exchange. 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.

Sampling and analysis:

Nitrogen transformation test:
Soil samples (10 g soil d.w. per replicate) were taken at intervals of 3 hours, 7, 14 and 28 days after application and the NH4-N, N03-N and N02-N content were determined.
For 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. For the quantitative determination of the mineralized part of nitrogen the Autoanalyzer II (produced by BRAN+LUEBBE, Hamburg, Germany) was used.
The Autoanalyzer II is a continuous flow analysis system. Ammonium reacts with salicylate and dichloroisocyanur acid to form an indophenoleblue compound. 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 NaN02, (NH4)2S04 and KN03 from Merck (p.a. quality). The autoanalyzer was calibrated before each measurement series by establishing a calibration curve. After every 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 N03-N and NH4-N / NQ2- N are 0.06 mg/100 g soil d.w. and 0.1 mg/100 g soil d.w., respectively.

Carbon transformation test:
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 %.
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.
For this purpose soil samples (100 g soil d.w.) were taken, mixed with glucose by means of a hand-stirrer and filled into reaction flasks (500 mL). Afterwards, small vessels containing 18 mL 1 m NaOH solution were placed in the reaction flasks, which were tightly closed and then connected with a respirometer (BSB digi SELUTEC, Mossingen-Oschingen, Germany).
The respiration of micro-organisms leads to 02 consumption and formation of C02 that is absorbed in NaOH solution. The absorption of C02 caused low-pressure in the reaction flask, which is compensated with 02 delivered by the respirometer.
The respirometer determines the cumulative oxygen production (corresponding to the 02 consumption by micro-organisms) over a 12-hour measurement period.

Nominal and measured concentrations:
Nominal:
0.53 and 2.67 mg/kg soil dw

Nitrogen transformation test: 0.11 and 0.53 mg/200g soil dw (equivalent to 400 and 2000 g/ha)

Carbon transformation test: 0.65 and 3.20 mg/1200 g soil dw (equivalent to 400 and 2000 g/ha)
Reference substance (positive control):
yes
Remarks:
Dinoterb
Key result
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
> 2.67 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
nitrate formation rate
Key result
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
> 2.67 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: carbon transformation
Results with reference substance (positive control):
Nitrogen transformation test:
The toxic standard dinoterb caused effects of +27.7 %, +60.8 % and +68.1 % (required > 25 %) on the nitrogen transformation at the tested concentrations of 6.80, 16.00 and 27.00 mg/kg soil dry weight, respectively, on day 28 and thus demonstrates the sensitivity of the test system.

Carbon transformation test:
The toxic standard dinoterb caused effects of -24.8 %, -42.0 % and -49.0 % (required > 25 %) on the carbon transformation in a field soil at the tested concentrations of 6.80 mg, 16.00 mg and 27.00 mg dinoterb per kg soil dry weight, respectively, 28 days after application and thus demonstrates the sensitivity of the test system.

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

Days after application

Control

0.53 mg test item/kg soil dry weight equivalent to 400gtest item/ha

2.67 mg test item/kg soil dry weight equivalent to 2000gtest item/ha

N03-N [mg/kg soil d.w.]

CV [%]

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

CV [%]

Deviation

from control [%]1)

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

CV [%]

Deviation

from control [%]1)

0

13.5

1.1

13.2

4.6

-2.7

13.2

4.6

-2.2

7

33.0

9.2

34.9

2.7

+5,9

32.4

5.7

-1.7

14

38.8

2.9

37.8

4.5

-2.5

40.5

1.7

+4.4

28

54.1

4.4

53.0

1.3

-2.0

54.4

1.8

+0.5

The calculations were performed with non-rounded values CV [%] = Coefficient of Variation

1)based on N03-nitrogen production; - = inhibition; + = stimulation

Effects on carbon transformation in soil after treatment with the test item

Days after application

Control

0.53 mg test item/kg soil dry weight

2.67 mg test item/kg soil dry weight

O2 consumption [mg/kg soil d.w./h]

CV [%]

O2 consumption [mg/kg soil d.w./h]

CV [%l

Deviation from control

O2 consumption [mg/kg soil d.w./h]

CV [%]

Deviation from control[%ï

0

16.36

0.7

16.36

1.0

+0.0

16.36

0.4

±0.0

7

15.42

1.2

15.42

0.9

±0.0

15.19

1.4

-1.5

14

14.94

1.0

15.03

0.8

+0.6

14.66

0.5

-1.9

28

12.97

0.8

12.80

2.6

-1.3

12.70

0.3

-2.1


Validity criteria fulfilled:
yes
Conclusions:
The test item caused no adverse effects (deviation from control < 25 %, OECD 216/217) on soil nitrogen transformation (measured as NO3-N production) and on soil carbon transformation (measured as oxygen consumption) at the end of the 28-day incubation period.
The study was performed in a field soil at concentrations equivalent up to a field application rate of 2000 g test item/ha.


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 (mineralization) and carbon transformation (respiration) in a laboratory test over a period of 28 days of exposure. The test was performed in accordance with the OECD guidelines 216 and 217 (2000) by measuring the nitrogen turnover and the short-term substrate-induced respiration.

No adverse effects of the test item on nitrogen and carbon transformation in soil were observed at both tested concentrations (0.53 mg/kg dry soil and 2.67 mg/kg dry soil) after 28 days.

Description of key information

The test item caused no adverse effects (deviation from control < 25 %, OECD 216/217) on soil nitrogen transformation (measured as NO3-N production) and on soil carbon transformation (measured as oxygen consumption) at the end of the 28-day incubation period.

The study was performed in a field soil at concentrations equivalent up to a field application rate of 2000 g test item/ha.

Key value for chemical safety assessment

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

Additional information

NOEC > 2.67 mg/kg soil dry weight