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Toxicity to soil microorganisms

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Reference
Endpoint:
toxicity to soil microorganisms
Type of information:
experimental study
Remarks:
Read-across approach, refer to Section 13.
Adequacy of study:
key study
Study period:
2018-01-09 to 2018-02-07
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Well documented guideline study, with GLP and no deviations.
Qualifier:
according to
Guideline:
OECD Guideline 216 (Soil Microorganisms: Nitrogen Transformation Test)
Version / remarks:
January 2000
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 217 (Soil Microorganisms: Carbon Transformation Test)
Version / remarks:
Janurary 2000
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Test item: reaction mass of ethylbenzene and mixed xylenes (called as mixed xylenes in the report)
Supplier: Sigma Aldrich
Batch number: SHBJ3208
CAS RN (a.s.): 1330-20-7
Molecular formula: C8H10
Molecular weight: 106.17 g/mol
Content (certified): 83.2 % (mixed xylenes), 16 % (ethylbenzene)
Specific gravity: 0.864 (15 °C)
Appearance: Colourless liquid
Expiry date: 2022-10-16
Analytical monitoring:
yes
Details on sampling:
Observation intervals: Measurements of inorganic nitrate and respiration were carried out after 0, 7, 14 and 28 days. The pH values and water contents were determined on day 0 and 28. The room temperature was measured and recorded continuously.
Details on preparation and application of test substrate:
A field fresh, silty sand soil (LUFA standard soil 2.3) was used in the study. Nominal soil parameters were:
- Sand content 50 - 75 %
- pH value 5.5 - 7.5
- Organic carbon content 0.5 - 1.5 %
- Microbial biomass (expressed as carbon content) at least 1 % of the total soil organic carbon.
The amount of total inorganic nitrogen was determined at test start.

Origin: LUFA SPEYER, Obere Langgasse 40, 67346 Speyer, Germany

Reason for the choice: The soil met the requirements of the test guideline.

Sampling date: 2017-10-09

Soil history Cultures:
2013 - 2017: uncultivated

Fertilisation:
2017: none
2016: none
2015: none
2014: 3500 kg/ha CaO (2014-06-05 and 2014-09-24), 3500 kg/ha CaO; 1463 kg/ha MgO (2014-12-15)
2013: none

Pesticides: No crop protection products applied during the year of sampling and 4 preceding years.

Soil handling LUFA Speyer: The soil was manually cleared of large objects and then sieved to a particle size of up to 2 mm. The maximum water holding capacity and the pH value were determined.

Test facility: Prior to final moistening at test start, the soil was adjusted to about 42 % of its maximum water holding capacity with demineralized water. Drying out of the soil was prevented by moistening with demineralized water as necessary. The amount of total inorganic nitrogen was determined. The soil was checked for a detectable microbial biomass (result expressed in terms of percentage of total organic carbon). This determination was done in parallel to the measurement of respiration rate but with soil without addition of Lucerne-green-grass-meal.

The soil amounts were amended with powdered lucerne-green-grass-meal (0.46 % of soil dry weight).
Origin:
Heucobs
Burgstraße 9
16259 Falkenberg / M.
Germany

Total Nitrogen Content: 29.3 g/kg
Total Carbon Content: 41.8 %
C / N ratio: 14.3

Soil storage: The soil was stored for 60 days (2017-10-24 to 2017-12-22) in the dark at 4 ± 2 °C in a climatic room. Subsequently, the soil was pre-incubated at room temperature (ca. 20 °C) for 18 days (2017-12-22 to 2018-01-10) to adapt the micro-organisms to test conditions before the start of the study.

Test organisms (inoculum):
soil
Total exposure duration:
28 d
Test temperature:
20.1-21.7 °C
Moisture:
Moisture content as % of maximum water holding capacity 45.3 - 48.3 %
Details on test conditions:
Control: Untreated soil was tested under the same conditions as the test replicates.

Test concentrations: 1000 - 316 - 100 - 31.6 - 10 mg /kg soil dry weight (factor √10)

Replicates: Triplicates

Test vessels: Plastic boxes (volume 6.25 L, food grade) with perforated tops to enable gas exchange. Incubation was performed in bulk and sub-samples were taken.

Application: The respective test item amount for each test item concentration were pipetted into a graduated flask, mixed with demineralized water and applied to the surface of the soil. Additional demineralized water was added to adjust the moisture of the soil to about 45 % MWHC. Afterwards, the soil was mixed with a handheld blender (max. 2 minutes) to ensure a homogeneous distribution of the test item in the soil and divided into replicates. Compacting of the soil whilst mixing was avoided.

Frequency of application: The application was carried out once at the start of the experiment (day 0).

Temperature 20 ± 2 °C (nominal)

Photoperiod: Dark

Soil moisture content: At the start of the experiment the pre-moistened soil was adjusted to approximately 45 % of its maximum water holding capacity. All replicates were weighed once per week to check for water losses by evaporation (recommended maximum 5 %, actual 0.2 - 0.4 %). Demineralized water was added as necessary.

Aeration: The soil was incubated in the above mentioned boxes. The tops of the boxes were perforated to enable gas exchange.
Reference substance (positive control):
yes
Remarks:
Nitrogen Transformation: Cyanoguanidine. Carbon Transformation: Dinoterb (2-(2-Methyl-2-propanyl)-4,6-dinitrophenol).
Duration:
7 d
Dose descriptor:
EC25
Remarks:
inhibition of the nitrate-N formation rate
Effect conc.:
669 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
nitrate formation rate
Key result
Duration:
7 d
Dose descriptor:
EC50
Remarks:
inhibition of the nitrate-N formation rate
Effect conc.:
867 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
nitrate formation rate
Duration:
28 d
Dose descriptor:
EC25
Remarks:
inhibition of the nitrate-N formation rate
Effect conc.:
> 1 000 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:
EC50
Remarks:
inhibition of nitrate-N formation
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
nitrate formation rate
Duration:
28 d
Dose descriptor:
EC25
Remarks:
glucose induced respiration rate
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
respiration rate
Key result
Duration:
28 d
Dose descriptor:
EC50
Remarks:
glucose induced respiration rate
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
respiration rate
Details on results:
After 7 days of exposure, EC25- and EC50-values for inhibition of the nitrate-N formation rate were 669 and 867 mg/kg soil dry weight, respectively.

After 14 and 28 days of exposure no EC25- and EC50-values were calculated, since no inhibition of the nitrate-N formation > 25 % was determined.

For the carbon transformation no EC25- and EC50-values were calculated, since no inhibition of the glucose induced respiration rate > 25 % was determined over the test duration.
Results with reference substance (positive control):
Nirtrogen Transformation
Cyanoguanidine is tested yearly at a concentration of 50 mg/kg soil dry weight (laboratory ID: TBN71703, duration: 2017-03-14 to 2017-04-11).

Lot (Origin) / Expiry date: WXBC1139V (Sigma Aldrich) / 2022-01-10
Purity: 99.8 %
CAS-RN: 461-58-5

Carbon Transformation
Dinoterb (2-(2-Methyl-2-propanyl)-4,6-dinitrophenol) is tested yearly as toxic reference item with a concentration of 13.75 kg/ha, corresponding to 18.3 mg/kg DW (laboratory ID TBC71703, 2017-03-06 to 2017-04-03).
Lot (Origin) / Expiry date SZBD226XV (Sigma Aldrich) / 2018-08-14
Purity 99.9 % (area)

Definitive test

Table 4. Changes of Nitrate-N Content in Soil Containing Xylenes (mixed Xylenes)

Nitrate-N Content

Test concentration

[%] compared to untreated control

[mg/kg soil dry weight]

0 d

7 d

14 d

28 d

10

5

6

9

-3

31.6

18

10

12

2

100

14

8

3

-1

316

20

12

4

0

1000

20

40

7

7

negative value = promotion of nitrate-N content              positive value = inhibition of nitrate-N content

Table 5: Changes of Nitrate-N Formation Rates in Soil containing Xylenes (Mixed Isomers)

Nitrate-N Formation Rate

Test concentration

[%] compared to untreated control

[mg/kg soil dry weight]

7 d

14 d

28 d

10

9

11

-6

31.6

0

9

-3

100

2

-4

-8

316

3

-7

-8

1000

65

-2

2

negative value = promotion of nitrate-N formation rate       positive value = inhibition of nitrate-N formation rate

Table 6: Changes in Respiration Rate in Comparison to the Untreated Control

Respiration Rate

Test concentration

[%] compared to untreated control

[mg/kg soil dry weight]

0 d

7 d

14 d

28 d

10

5

4

0

3

31.6

4

6

2

7

100

-2

6

-4

-10

316

9

16

-3

-11

1000

7

14

-12

-22

negative value = promotion of respiration rate                 positive value = inhibition of respiration rate

Table 7: Moisture and pH values of the soil after 0 and 28 days.

Test concentration

Soil Dry Weights [%]

Moisture content as [%] of MWHC

pH values
(soil solution)

[mg/kg soil dry weight]

day 0

day 28

day 0

day 28

day 0

day 28

Control

86.2

85.8

45.4

46.8

6.32

6.46

10

86.2

86.0

45.3

46.1

6.37

6.28

31.6

86.0

86.0

45.9

46.1

6.39

6.34

100

86.0

85.7

46.1

47.1

6.47

6.26

316

85.8

85.7

46.9

47.2

6.50

6.28

1000

85.8

85.4

46.7

48.3

6.48

6.28

Table 8: Room temperature throughout test duration.

Temperature [°C]

nominal

measured

20 ± 2

20.1 - 21.7

Reference test

Table 9: Changes of Inorganic-N Contents and N Formation rate following dosing with Cyanoguanidine

Test concentration

[mg/kg DW]

Nitrate-N Contents

Nitrate-N Formation Rate

[%] compared to untreated control

 [%] compared to untreated control

0 d

7 d

14 d

28 d

7 d

14 d

28 d

50

-2

21

31*

15

n.d.

n.d.

75*

Table 10: Changes in Respiration Rates following dosing with Dinoterb (2 -(2-Methyl-2 -propanyl)-4,6 -dinitrophenol)

Reference Item Concentration

[%] compared to untreated control

[kg/ha]

0 d

7 d

14 d

28 d

13.75

-8

-18

19

56*

*)Differences in respiration rate values>25 % are considered to be a reference item effect.
negative value = promotion of respiration rate
                       positive value = inhibition of respiration rate

Validity criteria fulfilled:
yes
Conclusions:
After 7 days of exposure, EC25- and EC50-values for inhibition of the nitrate-N formation rate were 669 and 867 mg/kg soil dry weight, respectively, based on nominal test item concentration. After 14 and 28 days of exposure no ECx-values were calculated, since no inhibition of the nitrate-N formation > 25 % was determined. For the carbon transformation no ECx-values were calculated, since no inhibition of the glucose induced respiration rate > 25 % was determined over the test duration.
Executive summary:

The effects of Xylenes (Mixed Xylenes) (Batch no.SHBJ3208) on the metabolic activity of soil micro-organisms (nitrogen and carbon transformation) were determined according to OECD Guideline 216 and 217. The test item was applied via demineralized water at nominal concentrations of 0, 10, 31.6, 100, 316 or 1000 mg test item/kg soil dry weight. Untreated field soil, adjusted to a moisture content corresponding to nominally 45 % of its maximum water holding capacity and mixed with a source of organic nitrogen (lucerne meal), was tested as the control under the same test conditions as the test item replicates. The effects of the test item on the microbial soil activity (nitrogen and carbon transformation) were analysed on the day of treatment (day 0) and subsequently after 7, 14 and 28 days.

After 7 days of exposure, EC25-and EC50-values for inhibition of the nitrate-N formation rate were 669 and 867 mg/kg soil dry weight, respectively, based on nominal test item concentration. After 14 and 28 days of exposure no ECx-values were calculated, since no inhibition of the nitrate-N formation > 25 % was determined. For the carbon transformation no ECx-values were calculated, since no inhibition of the glucose induced respiration rate > 25 % was determined over the test duration.

Description of key information

The 7-day EC50 (nitrate formation) of reaction mass of ethylbenzene and mixed xylenes was 867 mg/kg soil. At 28 days, EC50s for respiration and nitrate formation were reported to be >1000 mg/kg soil dry weight.

Key value for chemical safety assessment

Short-term EC50 for soil microorganisms:
867 mg/kg soil dw
Long-term EC10 or NOEC for soil microorganisms:
1 000 mg/kg soil dw

Additional information

In a 28 day study, soil microorganisms were exposed to mixed xylenes (or the reaction mass of ethylbenzene and mixed xylenes). After 7 days of exposure, EC25-and EC50-values for inhibition of the nitrate-N formation rate were 669 and 867 mg/kg soil dry weight, respectively, based on nominal test item concentration. After 14 and 28 days of exposure no ECx-values were calculated, since no inhibition of the nitrate-N formation > 25 % was determined. For the carbon transformation (respiration) no ECx-values were calculated, since no inhibition of the glucose induced respiration rate > 25 % was determined over the test duration.

A supporting study that investigates the effects of o-xylene on soil microorganisms is available. This study reported a 10-hour EC50of 0.00022 mg/kg nominal. Although this study does not follow the standard guidelines for measuring respiration inhibition of soil microorganisms it is well documented and scientifically acceptable. The test design uses a closed system and analytical monitoring. The exposure period is only 10 hours compared to the standard 28 days.