1,3-bis(3-methyl-2,5-dioxo-1H-pyrrolinylmethyl)benzene

Toxicity to soil microorganisms

Link to relevant study record(s)

Reference

Endpoint:

toxicity to soil microorganisms

Type of information:

experimental study

Adequacy of study:

key study

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:

January 21, 2000

Deviations:

yes

Remarks:

see 'Principles of method'

Principles of method if other than guideline:

According to the OECD Guideline 216 carbon content of the microbial biomass in the test soil should be at least 1% of the total soil organic carbon. Microbial biomass was determined according to DIN/ISO 1420-2 “Soil quality - Determination of soil microbial biomass - Part 2: Fumigation-extraction method”. The guideline states that the soil moisture should be >30% of the WHCmax to ensure good distribution of chloroform during the fumigation process. During determination of soil microbial biomass the soil moisture was 22.3%. Evacuation of the desiccator after fumigation was done three times and at least 7 minutes each instead of six times and 2 minutes each since after the third ventilation chloroform had already been totally evaporated as confirmed olfactorily. Results do not indicate an impact on the fumigation process. Hence, it is concluded that this deviation had no impact on the integrity of the study.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

yes

Remarks:

acetone

Details on preparation and application of test substrate:

1- Pre-TREATMENT OF THE TEST ITEM:
The test item was dissolved in acetone and applied to fine quartz sand. After complete evaporation of the solvent the spiked sand was mixed into sieved field soil (Lufa standard soil type 2.3) that was amended with powdered lucerne meal at a concentration of 5.0 g/kg soil dry weight, and incubated for a test period of 28 d at 18.4 to 21.0 °C in the dark.

2. DOSE LEVELS AND TEST SET-UP:
Five test item concentrations, a solvent control and an untreated control were tested with four replicates, each, and ranged from 12.8 mg a.s./kg soil dry weight to 500 mg a.s./kg soil dry weight with a spacing factor of 2.5. Due to the fact that the test item contains a considerable amount of nitrogen, N-transformation from lucerne could have beeen masked by nitrate originating from nitrogen of the test item. Therefore three additional treatments with two replicates each, were prepared from soil which was not amended with lucerne meal.

The test-item concentration had been defined by the study director according to the results of a previously performed non-GLP range-finding test.

Following test item application all test vessels were incubated for 28 days in the dark at 18.4 to 21.0 °C. Test vessels were weighed once per week and weight loss was compensated by adding deionised water. By doing so, the soil moisture was kept within a range of ± 5 % of the water content at test start.

3. TEST VESSELS:
Glass jars with lids (volume 370 mL, Weck, Germany) were used as test vessels. The lids allowed for some gas exchange. Each test vessel was loaded with 175±5 g soil fresh weight.

Test organisms (inoculum):

soil

Total exposure duration:

28 d

Test temperature:

18.4 to 21.0 °C

Moisture:

15.7 [% dry mass], equivalent to 45% of WHCmax

Details on test conditions:

1. TEST SYSTEM:
- Supplier: Landwirtschaftliche Untersuchungs- und Forschungsanstalt Speyer, Obere Langgasse 40, 67346 Speyer, Germany.
- Sampling location: Germany, Rhineland-Palatinate, Offenbach, field name „rechts der Landauer Str.“ field number 826/7; The sampling location of the soil was uncultivated during the last four years and has not received pesticides within the last 4 years; in 2014, the sampling location was fertilised three times (June, September and December) with 3500 kg/ha CaO, each, and once (December) with 1463 kg/ha MgO.
- Sampling conditions: Depth: ca. 20 cm; Date: 18 June 2018; weather conditions: cloudy
- Preparation: Air drying (only until sievable) from 18 June to 20 June 2018; final sieving to 2 mm on 20 June 2018.
- Microbial biomass: The microbial biomass carbon (Cmic) of the bulk soil (without lucerne meal added) was determined during the test by applying the Fumigation-Extraction (FE) method according to ISO (1997); Carbon was extracted from three soil aliquots, each of non-fumigated soil and of fumigated soil. Cmic was estimated by the difference between the amounts of CaCl2-extractable carbon from fumigated and non-fumigated soil sample aliquots.

2. LUCERNE-GRASS-GREEN MEAL:
- Identification: Semhof’s Bio Luzernepellets Turbo; Batch number: 151030; C/N Analysis: LUFA Speyer, Germany (Report BM 04/15); Nitrogen content (%): 2.81; Carbon content (%): 35.5 C/N ratio: 12.6
- Lucerne-green-meal pellets had been purchased from Semhof, 86753 Möttingen, Germany and were ground to a fine powder with an electric mill.
- The finely ground lucerne meal was incorporated in to the bulk test soil on day 0 of the test (test start) at a dosing of 5.0 g/kg dry weight soil immediately before test item application.

3. MEASUREMENT OF NITRATE:
The concentration of nitrate was determined in aqueous extracts from soil aliquots on day 0, and day 28 after test item application. Before taking a sub-sample out of a test vessel, the soil in the test vessel was mixed thoroughly using a spatula.
Aqueous extracts were prepared by shaking 20 g dry weight equivalent of soil with 100 mL of a 0.01 M CaCl2 solution in a polyethylene flask at 150 rpm for 60 min followed by filtration through folded cellulose filters of type Whatman 595½ (VWR International GmbH, Darmstadt, Germany).
Nitrate in the filtrate was measured photometrically by the use of Dr. Lange Kuvette test LCK 339 (Hach Lange GmbH, Berlin, Germany) with two separate measurements (two separate cuvettes) per specimen.
The principle of the cuvette test is based on the transformation of nitrate with 2,6-dimethylphenol to 4-Nitro-2,6-dimethylphenol in sulphur-phosphorous acid solution.
Volumes of 1.0 mL of the extract followed by 0.2 mL of a specific reagent (LCK 339) is pipetted into a LCK 339 cuvette. The cuvette is closed and inverted a few times until no more streaks can be seen.
After incubation for 15 minutes at ambient temperature the cuvette is placed into the photometer for evaluation.
At both measurement time points of the test, i.e on day 0 and on day 28, the measurement of a NO3 standard solution (LCA addista 703, Dr. Lange GmbH, Berlin, Germany) confirmed the correct measurements of NO3 by the cuvette test.

4. MEASUREMENT OF NITRATE:
Nitrate concentration in soil was determined in soil extracts on day 0 and day 28.

5. STATISTICAL EVALUATION:
Normal distribution and homogeneity of variances was tested by the Kolmogorov Smirnov test on normal distribution and the Cochran’s test on variance homogeneity, respectively. The NOEC was determined by applying the Williams multiple t-test (p≤0.05, one-sided smaller).
All statistical tests were performed using the ToxRat® statistical software Version 3.3.

Nominal and measured concentrations:

Five nominal test item concentrations of 12.8, 32.0, 80, 200 and 500 mg a.s./kg soil dry weight with a spacing factor of 2.5 were tested.

Reference substance (positive control):

no

Key result

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

200 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

nitrate formation rate

Details on results:

Nitrate concentration in the soil was statistically significantly reduced at 500 mg a.s./kg soil dry weight which was the highest tested test item concentration. Due to the structure of the data no valid ECX values could be determined. Therefore, the test substance is considered to have no long term adverse effect on nitrogen transformation in soil at concentrations up to and including 200 mg a.s./kg soil dry weight.

Validity criteria fulfilled:

yes

Remarks:

Coefficient of variation (CV) of control samples (nitrate concentration): 1.2% (day 0), 1.8% (day 28)

Conclusions:

The NOAEC was 200 mg a.s./kg soil dry weight after 28 days of exposure. Due to the structure of the data no valid ECX values could be determined. Therefore, Perkalink 900 is considered to have no long term adverse effect on nitrogen transformation in soil at concentrations up to and including 200 mg a.s./kg soil dry weight.

Executive summary:

A study was conducted to assess the effects on Soil Microorganisms according to OECD Guideline for the Testing of Chemicals No. 216 “Soil Microorganisms: Nitrogen Transformation Test". The test item was dissolved in acetone and applied to fine quartz sand. After complete evaporation of the solvent the spiked sand was mixed into sieved field soil (Lufa standard soil type 2.3) that was amended with powdered lucerne meal at a concentration of 5.0 g/kg soil dry weight, and incubated for a test period of 28 d at 18.4 to 21.0 °C in the dark. Five test item concentrations, a solvent control and an untreated control were tested with four replicates, each, and ranged from 12.8 mg a.s./kg soil dry weight to 500 mg a.s./kg soil dry weight with a spacing factor of 2.5. Due to the fact that the test item contains a considerable amount of nitrogen, N-transformation from lucerne could have been masked by nitrate originating from nitrogen of the test item. Therefore three additional treatments with two replicates each, were prepared from soil which was not amended with lucerne meal. Soil moisture of test soils was kept within a range of ± 5% of 45% of the maximum water-holding capacity (WHCmax). Nitrate concentration in soil was determined in soil extracts on day 0 and day 28. The study was valid (Coefficient of variation (CV) of control samples (nitrate concentration): 1.2% (day 0), 1.8% (day 28)). On day 0, nitrate concentration of test-item treated soils did not differ significantly from nitrate concentration of the solvent control. On day 28, nitrate concentration was significantly reduced at 500 mg a.s/kg soil dw. The NOAEC was 200 mg a.s./kg soil dry weight after 28 days of exposure. Due to the structure of the data no valid ECX values could be determined. Therefore, Perkalink 900 is considered to have no long term adverse effect on nitrogen transformation in soil at concentrations up to and including 200 mg a.s./kg soil dry weight.

Description of key information

After 28 days of exposure a No-Observed-Adverse-Effect-Concentration (NOAEC) of 200 mg a.s./kg soil dry weight was measured. Perkalink 900 is considered to have no long term adverse effect on nitrogen transformation in soil at concentrations up to and including 200 mg a.s./kg soil dry weight.

Key value for chemical safety assessment

Long-term EC10 or NOEC for soil microorganisms:

200 mg/kg soil dw

Additional information

Due to the structure of the data no valid ECX values could be determined. Therefore, Perkalink 900 is considered to have no long term adverse effect on nitrogen transformation in soil at concentrations up to and including 200 mg a.s./kg soil dry weight.