Dichloromethylbenzene

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

Study period:

2016

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:

yes

Remarks:

the organic matter deviated from the recommended value of ≥ 1% (was: 0.93%; 0.94%)

Principles of method if other than guideline:

The microbial biomass of the soil was determined to represent 0.93% (Test 1) and 0.94% (Test 2) of the organic matter and thereby slightly deviated from the recommended value of ≥ 1%. The nitrification, however, confirmed a metabolic active microbial community in soil. Hence, it is concluded that the deviation had no impact on the integrity of the study.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

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. Fertilisation within the last four years was performed four times: 3500 kg/ha CaO were applied in June, in September and in December 2014 and 1463 kg/ha MgO were applied in December 2014 only.

SAMPLING CONDITIONS:
- Depth: approx. 20 cm; Date: July 15, 2015; weather conditions: cloudy

Vehicle:

yes

Details on preparation and application of test substrate:

PREPARATION
- Air drying (only until sievable) and final sieving to 2 mm

APPLICATION OF TEST SUBSTANCE TO SOIL:
- The test item was insoluble in water. Therefore, a stock solution was prepared by dissolving the test item in acetone
- The test item was mixed via spiked quartz sand into sieved field soll (Lufa standard soll type F2.3) that was amended with powdered lucerne meal at a concentration of 5.0 g/kg soll dry weight, and incubated for a test period of 28 d at 20±2 °C in the dark
- A stock solution was prepared by dissolving the test item in acetone and test solutions were prepared by a dilution series of the stock solution
- The test solutions were used to soak defined amounts of quartz sand which were then mixed into portions of soil
- Five test concentrations (31.3 - 500 mg test item/kg soil dry weight), a solvent control (SC), an untreated control (C) and a treatment control (TC) were tested with four replicates, each, and ranged from 31.3 mg test item/kg soil dry weight to 500 mg test item/kg soil dry weight
- Soil moisture of test soils was kept within a range of ± 5 % of 45 % of the maximum water-holding capacity (WHCmax)

Test organisms (inoculum):

soil

Total exposure duration:

28 d

Test temperature:

20 ± 2°C

Moisture:

Max. water holding capacity (WHCmax) [g/100 g]: 35.4 ± 1.5
Initial soil moisture [% dry mass]: 5.5
Soil moisture adjusted for test [% dry mass]: 15.9, equivalent to 45 % of WHCmax

Details on test conditions:

CHARACTERIZATION OF THE TEST SYSTEM:
- Test system: autochthonous microbial community of a freshly sampled field soil (Lufa standard soil type F2.3)
- Preparation: Air drying (only until sievable) on September 07, 2015; final sieving to 2 mm on September 10/11, 2015
- Microbial biomass: the microbial biomass carbon (Cmic) of the bulk soil (without lucerne meal added) was determined at the beginning of 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; C(mic) was estimated by the difference between the amounts of calciumdichlorideextractable carbon from fumigated and non-fumigated soil sample aliquots
- Batch number: F2.33715
- pH (0.01 M CaCI2): 5.7 ± 0.6
- Cation exchange capacity [meq/1 00 g]: 7.5 ± 0.9
- Particle size according to USDA [mm]: < 0.002 (clay) (6.5 ± 1.6 %); 0.002 0.05 (silt) (33.8 ± 0.1 %); 0.05 2.0 (sand) (59.7 ± 1.6 %)
- Soil type (USDA): sandy loam
- Microbial Biomass Carbon (Cmic) [μg Cmic g-1 soil]: 62.12
- Cmic [% of Corg]: 0.93

LUCERNE-GRASS-GREEN MEAL:
- Date of purchase: May 19, 2009
- Identification: Luzerne Gruenmehl Pellets
- C/N Analysis: LUFA Speyer, Germany
- Nitrogen content(%): 2.79
- Carbon content (% ): 41.03
- C/N ratio: 14.7

TEST SET-UP:
- Five test item concentrations (31.3, 62.5, 125, 250 and 500 mg/kg soil dw), a solvent control (SC) and an untreated control (C) were tested with 4 replicates
- 2122.3 mg of the test item were weighed into a 10 mL volumetric flask and made up with acetone resulting in a nominal test item concentration of 212 mg/mL in the stock solution; the stock solution was also used as test solution for treatment with 500 mg test item/kg soil dry mass
- The treatment control (TC) (500 mg/kg soil dry mass) was prepared from soil without lucerne meal
- For each treatment a volume of 2.0 mL of the appropriate test solution was used to spike 8.0 g of fine quartz sand
- After complete evaporation of the solvent under mild conditions the sand was mixed into 800 g (dry weight) of soil which had been amended with finely ground lucerne meal at 5.0 g/kg soil dm
- Immediately before the mixing procedure the soil had been amended with finely ground lucerne meal at 5.0 g/kg dw soil except the soil portion for treatment TC
- The soil was moistened with deionised water to adjust the soil moisture to 45% of the maximum water holding capacity (WHCmax) and distributed equally to the four test vessels per treatment

TEST VESSELS:
- Glass jars with lids (volume 370 mL, diameter 100 mm, Weck, Germany)
- The lids allowed for some gas exchange
- Each test vessel was loaded with 233 +/-2 g soil fresh weight

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
- NOEC was determined by applying either the Student's t-test or the Welch t-test for inhomogeneous variances (p~0.05, two-sided)
- Effective concentrations (ECx) could not be calculated due to the lack of a monotonous concentration-effect relationship in the nitrate concentration in treated soil compared to the solvent control on day 28
- All statistical tests were performed using the ToxRat® statistical software Version 2.1 0

Nominal and measured concentrations:

DT1: 32.7, 65.3, 131, 261 and 523 mg/kg soil dry mass
DT2: 31.3, 62.5, 125, 250 and 500 mg/kg soil dry mass
a solvent control and a untreated control

Reference substance (positive control):

no

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

261 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

nitrate formation rate

Since an abnormally low nitrate concentration in the control soil was observed at the end of the test (DT1) which could not be explained scientifically, it was decided to repeat the test (DT2).

In both tests, DT1 and DT2, the test item had no adverse effect on the N-transformation in soil at concentrations up to and including 261 mg/kg soil dry mass, up to which the nitrate concentration was increased. At a test item concentration of 500 mg/kg soil dry mass, nitrate concentration was strongly reduced. Hence, the Lowest Observed Adverse Effect Concentration (LOAEC) was 500 mg/kg soil dry mass, which was the highest tested concentration. The No Observed Adverse Effect Concentration (NOAEC) was 261 mg/kg soil dry mass. Effective concentrations (ECx) could not be calculated due to the lack of a monotonous concentration-effect relationship.

Summarising the results of both tests, it is concluded that the test item has no long term adverse effect on nitrogen transformation in soil at concentrations up to and including 261 mg/kg soil dry mass.

The microbial biomass of the soil was determined to represent 0.93% (DT1) and 0.94% (DT2) of the organic matter and thereby slightly deviated from the recommended value of ≥ 1%. The nitrification, however, confirmed a metabolic active microbial community in soil. Hence, it is concluded that the deviation had no impact on the integrity of the study.

Calculation of the test solutions in test DT1 were based on soil fresh weight instead of dry weight. Therefore the nominal test item concentrations were about 5% higher than given in the study plan (32.7, 65.3, 131, 261 and 523 mg/kg soil dry weight).

Validity criteria fulfilled:

yes

Remarks:

(variability of less than 15% within the control) fulfilled for both tests. The max. coeff. of variation (CV) for nitrate of control and solvent control samples was 1.6 % on day 0 and 2.6 % on day 28)

Conclusions:

Dichlorotoluene Mixture had no long term adverse effect on nitrogen transformation in soil at concentrations up to and including 261 mg/kg soil dry mass.

Executive summary:

According to the OECD Guideline for the Testing of Chemicals No. 216 "Soil Microorganisms: Nitrogen Transformation Test" the study was conducted in order to determine possible effects on soil microorganisms through measuring microbial nitrate formation in treated versus untreated soils after 28 days of incubation.

In both tests Dichlorotoluene Mixture had no adverse effect on the N-transformation in soil at concentrations up to and including 261 mg/kg soil dry mass and 250 mg/kg soil dry mass, respectively, as the nitrate concentration was increased at these test item concentrations. At a test item concentration of 500 mg/kg soil dry mass, nitrate concentration was strongly reduced. Hence, the Lowest Observed Adverse Effect Concentration (LOAEC) was 500 mg/kg soil dry mass, which was the highest tested concentration. The No Observed Adverse Effect Concentration (NOAEC) was 261 mg/kg soil dry mass. Effective concentrations (ECx) could not be calculated due to the lack of a monotonous concentration-effect relationship. Summarising the results of both tests, it is concluded that the test item has no long term adverse effect on nitrogen transformation in soil at concentrations up to and including 261 mg/kg soil dry mass.

This toxicity study is classified as acceptable and satisfies the guideline requirements for the soil microorganisms, nitrogen transformation test.

Description of key information

Dichlorotoluene Mixture had no long term adverse effect on nitrogen transformation in soil at concentrations up to and including 261 mg/kg soil dry mass.

Key value for chemical safety assessment

Long-term EC10 or NOEC for soil microorganisms:

261 mg/kg soil dw

Additional information

At a test item concentration of 500 mg/kg soil dry mass, nitrate concentration was strongly reduced.

Since there was no monotonous dose response relationship in nitrate concentration in the test item treated soil compared to the control, effective concentrations (ECx) could not be determined.