Registration Dossier

Data platform availability banner - registered substances factsheets

Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Ecotoxicological information

Toxicity to soil microorganisms

Currently viewing:

Administrative data

Link to relevant study record(s)

Reference
Endpoint:
toxicity to soil microorganisms
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 February 2017 to 04 May 2017
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:
OECD Guidelines for Testing Chemicals: Soil Microorganisms: Nitrogen transformation test No. 216 adopted 21 January 2000.
Deviations:
yes
Remarks:
see "Any other information" for details.
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
No further details specified in the study report.
Analytical monitoring:
not specified
Details on sampling:
As soon as possible after dosing, approximately 50 g (dry weight equivalent), a sub-sample was taken from each replicate for the determination of nitrate, ammonium and nitrite concentration. Further sub-samples were taken after 28 days.
Vehicle:
yes
Remarks:
acetone
Details on preparation and application of test substrate:
TEST SYSTEM
The study was carried out with a single, common agricultural soil as described in OECD Guideline 216 (2000). The soil used was LUFA standard soil type 2.3 and was supplied by LUFA-Speyer, Obere Langgasse 40, 67346 Speyer, Germany.
The soil sampling was carried out according to ISO 10381-6 (1993) as recommended in OECD 216 (2000). Details of the soil characterisation data and soil collection, site history and soil treatment (Appendix 3 for the range finder test and Appendix 4 for the definitive test) were provided by LUFA-Speyer. The soils were stored in the dark in a refrigerator (nominally at 4oC) until required.
The test soils had a sand content of 59.6 %, pH of 6.8 for the range finder (RF) and 7.1 (measured in water) for the definitive test, a microbial biomass (238 mg Cmic/kg dry soil for the range finder and 310 mg Cmic/kg dry soil for the definitive test) which was not less than 1 % of the total organic carbon content (actual value 3.05 % for the RF and 4.62 for the definitive test) and both soils had an organic carbon content of 0.67 %. In most cases, soils with these characteristics represents an worst-case situation, since adsorption of the test chemical to soil is minimal and consequently its availability to the microflora is maximal.

Soil Acclimation
Acclimation of the soil used for the definitive phase of the study was started on 30 March 2017, when the soil was brought to a moisture content of 35 % (+/- 5 %) of the maximum water holding capacity (MWHC) of the soil, and left to acclimate in a test cabinet in the dark at 20 ± 2 deg C for six days before treatment.
Test organisms (inoculum):
soil
Total exposure duration:
28 d
Test temperature:
20 °C ± 2 °C.
Moisture:
The moisture content of the soil was maintained at 42 % ± 5% MWHC
Details on test conditions:
Preparation of Test Treatments, Amendment and Treatment Application
For the range finding test the treatment rates of 0.1, 1.0, 10, 100 and 1000 mg/kg dry soil, LOWINOX® 44B25 solutions were prepared in acetone, applied onto fine sand, and mixed thoroughly and the acetone allowed to evaporate. The control treatment soils were treated with the equivalent amount sand (acetone treated and untreated) and deionised water. Treated sand for each treatment group was incorporated into the soil at a rate of 10 g sand/kg dry soil.
For the definitive test, the soil treatment was prepared by direct addition of LOWINOX® 44B25 to dry fine quartz sand at a rate of 10 g sand/kg dry soil and the weights used are presented in the table below. Direct addition was used as the preferred method of soil incorporation of the test item. Although the test item was soluble in acetone at the test concentration of 1000 mg/kg dry soil used in the range finder the test item did not coat the fine sand effectively once evaporated, drying to a cake that had to be ground further to produce a homogenous mix. A more even distribution was achieved by direct addition and mixing. LOWINOX® 44B25 was added carefully to the sand and mixed thoroughly before incorporation in to the soil.
Treated sand was added to the pre-acclimated soil along with a volume of deionised water required to achieve the test moisture of 40 % (± 5 %) of the MWHC.
The deionised water control soil was treated with the equivalent amount of deionised water and undosed quartz sand.
The test soil was amended with powdered dried ground lucerne. The lucerne was added at a rate of 2.5 g of lucerne/500 g of soil (dry weight equivalent) to the control and treatment groups on Day 0. This was mixed into the soil by hand prior to addition of the sand and deionised water. The lucerne had a carbon content of 45.21% w/w and a nitrogen content of 3.14% w/w giving a C:N ratio of 14.4:1.
The soil was bulk dosed, mixed in a Crypto Peerless mixer and then split equally into replicate test vessels (6 x ca. 500g). Treatments were mixed in the following order: deionised water control, then test item treatment at 1000 mg Lowinox/kg dry soil.

Study Conditions
The vessels were maintained in the dark at 20 °C ± 2 °C. Temperature was monitored throughout the study using IceSpy (Skye Instruments, Powys, UK) electronic environmental data loggers and data was recorded on an hourly basis.

Moisture Content Maintenance
A satellite moisture control vessel was included in the test for the sole purpose of monitoring moisture content. The moisture content of the soil was maintained at 42 % ± 5% MWHC and on a weekly basis, the weight of the moisture control vessel was determined as a guide to test vessel water loss. Weight loss since the previous sampling occasion was restored by the addition of deionised water to all replicates in all treatments, based on the assumption that all vessels had experienced similar moisture change.

Sampling and Analysis
Nitrogen transformation test
As soon as possible after dosing, approximately 50 g (dry weight equivalent), a sub-sample was taken from each replicate for the determination of nitrate, ammonium and nitrite concentration. Further sub-samples were taken after 28 days. Inorganic ammonium, nitrate and nitrite were extracted in 250 mL of 2 M potassium chloride (KCl) solution, shaken for 2 hours and then centrifuged. Approximately 15 mL of the supernatant was collected and these extracts were stored in the dark, under refrigeration (nominally at 4 °C), prior to analysis. The extracted solution was analysed for nitrate, ammonium and nitrite using the Bran + Luebbe Autoanalyzer AA3 system (method described in SOP CEM-3394: Determination of Nitrate-N, Ammonium-N and Nitrite-N in soil solutions and water).

Analysis of nitrate and nitrite
Total oxidised nitrogen (TON, sum of nitrate and nitrite) was determined, where nitrate was reduced to nitrite after being passed through a copperised cadmium reduction column. The resulting nitrite (and any nitrite already present) was then diazotized with sulphanilamide and N-(1-napthyl)-ethylenediamine dihydrochloride to form a highly coloured azo dye, the absorbance of which was measured colorimetrically using a photometer fitted with a 550 nm wavelength filter. Nitrite in the extracts was determined, and in addition, to ensure that the total oxidised nitrogen present was derived only from nitrate and can be reported as such.

Analysis of ammonium
Ammonium in the solutions was reacted with salicylate and dichloro-isocyanuric acid (nitroprusside was used as the catalyst for the reaction), to produce a blue compound, the absorbance of which was measured colorimetrically using a photometer fitted with a 660nm filter.

Determination of Soil pH and Moisture Content
Soil was taken from the bulk sample from each treatment for pH (in water) determination at Day 0 and from one replicate on Day 28. Soil pH was determined using SOP CEM-3373: Determination of the pH of water, soil and sediment samples in water and/or salt solutions (0.01M CaCl2, 0.1M KCl, 1.0M KCl).
Soil was taken from one replicate from each treatment for moisture and dry matter content determination on Day 28, using SOP CEM-3088: Determination of the Dry Matter and Moisture Content of Soils and Plant Materials.
Nominal and measured concentrations:
A range finder test was conducted at 0.1, 1.0, 10, 100 and 1000 mg LOWINOX® 44B25 /kg dry soil. The soil was amended with powdered lucerne (Medicago sativa) over a 28 day incubation period. As a result of this test, the study definitive phase of the study was run as limit test and soil was treated with the test item at a nominal concentration of 1000 mg LOWINOX® 44B25 /kg dry soil. The number of replicates in the definitive test was increased to six to provide a more powerful test.
Reference substance (positive control):
not specified
Key result
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
nitrate formation rate
Details on results:
Summary of Range Finder Test
There was no dose response at the rates of 0.1, 1.0, 10, 100 and 1000 mg Lowinox/kg dry soil tested in the range finder test and no statistically significant difference between the controls and any of the test treatments.

Nitrate, Nitrite and Ammonium Determinations
Changes in nitrate transformation rates (mg nitrate/kg/day) over the duration of the study are given in Table 4, as the changes in nitrate production from Day 0-28.
During the 0-28 day period the soil nitrate transformation rate (mg/kg dry soil/day) in the soil treated at 1000 mg Lowinox/kg dry soil varied by -2.17% from the mean of the untreated water control treatments. The final concentrations of the products of mineralisation, nitrate, in the soils at Day 28 in the untreated control and 1000 mg Lowinox/kg dry soil were 182.6 and 186.3 mg/kg dry soil, respectively.
There was no evidence of ammonium accumulation in the control or soil treated with LOWINOX® 44B25 at 1000 mg/kg dry soil and the nitrite concentrations were all below the LOD for the method.

Soil pH and Moisture Content
Soil pH values at Day 0 and Day 28 are given in Table 5. On Day 28, LOWINOX® 44B25 did not affect soil pH in the 1000 mg/kg treatment compared to the control.
The MWHC of the soil was determined as 31.9 %. Soil moisture levels for the study were maintained at 42 %  5% of the MWHC of the soil.
Results with reference substance (positive control):
Not specified
Reported statistics and error estimates:
The nitrate rate of production over the 28 day incubation period and nitrate concentration by Day 28 data were subject to statistical analysis using ToxCalc™ (1999) to determine any statistical significance between the treatment groups and control and to establish the no observed effect concentration (NOEC) and lowest concentration effect (LOEC) of LOWINOX® 44B25 in soil.
The data were analysed for ANOVA and Dunnett’s Hypothesis test was used for both data sets (both two sided, p=0.05).

Summary Results of the Range Finder Test

Treatment

Mean nitrate on day 28 mg/kg day 0-28

Mean nitrate production mg/kg/day day 0-28

% variation from solvent control

Control (water)

Control (solvent) 0.1 mg/kg

1 mg/kg

10 mg/kg

100 mg/kg

1000 mg/kg

197

200

201

200

204

203

198

4.88

5.04

5.04

4.98

5.12

5.09

4.93

-3.06

NA

0.16

-1.05

1.69

1.13

-2.10

All results on dry soil basis

No statistical differences found for the rate of nitrate production and either control (Dunnett’s Hypothesis Test, two-sided, p≥ 0.05).

NA = Not applicable

 

Soil Nitrogen Concentrations: Day 0

Treatment

Replicate

Soil Nitrite

(mg/kg dry soil)

Soil Ammonium

(mg/kg dry soil)

Soil Nitrate

(mg/kg dry soil)

Water Control

1

-

3.81

56.16

2

-

3.81

56.16

3

-

3.81

56.39

4

-

3.87

57.07

5

-

3.87

56.84

6

-

3.87

56.84

Mean value:

Within treatment variation (%):

-

-

3.84

-0.9 to 0.9

56.6

-0.7 to 0.9

1000 mg/kg

1

-

3.74

57.30

2

-

3.94

57.53

3

-

4.01

57.76

4

-

3.94

57.76

5

-

4.01

57.30

6

-

3.87

57.76

Mean value:

Within treatment variation (%):

-

-

3.92

-4.5 to 2.3

57.6

-0.5 to 0.3

All rates as mg Lowinox/kg dry soil basis.

Some calculations made using un-rounded values.

- = Nitrite value was not calculated as below LOD.

 

Soil Nitrogen Concentrations: Day 28

Treatment

Replicate

Soil Nitrite

(mg/kg dry soil)

Soil Ammonium

(mg/kg dry soil)

Soil Nitrate

(mg/kg dry soil)

Water Control

1

-

0.69

186.74

2

-

0.69

191.76

3

-

0.56

186.28

4

-

0.56

180.35

5

-

0.56

173.50

6

-

0.56

177.15

Mean value:

Within treatment variation (%):

-

-

0.6

-7.4 to 14.7

182.6

-5.0 to 5.0

1000 mg/kg

1

-

1.09

178.98

2

-

1.22

188.57

3

-

1.22

193.13

4

-

1.49

189.94

5

-

1.22

181.26

6

-

0.96

186.28

Mean value:

Within treatment variation (%):

-

-

1.2

-20.3 to 24.0

186.4

-4.0 to 3.6

All rates as mg Lowinox/kg dry soil basis.

Some calculations made using un-rounded values.

- = Nitrite value was not calculated as below LOD.

 

Soil Nitrate Transformation Rates Day 0 – 28

Treatment

Replicate

Day 0-28

(mg/kg/day)

Water Control

1

4.66

2

4.84

3

4.64

4

4.40

5

4.17

6

4.30

Mean:

4.50

% difference from water control:

NA

1000 mg/kg

1

4.35

2

4.68

3

4.83

4

4.72

5

4.43

6

4.59

Mean:

4.60

% difference from water control:

-2.17

N/A = Not applicable

Notes: Some values are calculated using un-rounded values.

All rates as mg/kg/day dry soil basis.

 

Soil pH (measured in water)

Treatment

Day 0*

Day 28*

Water Control

6.9

6.9

1000 mg Lowinox/kg dry soil

7.0

6.9

*Taken from bulk sample on Day 0 and replicate 1 on Day 28.

 

Soil Moisture Levels at Day 28

Targeted Moisture Level

Mean Dry Matter Day 28*

(% w/w)

Mean Moisture Day 28*

(% w/w)

Achieved

% MWHCb

42% MWHC

86.6

13.4

41.9

*Taken from the mean of replicate 1 vessel from each treatment.

bMean moisture content divided by the MWHC of the soil (31.9%), multiplied by 100.

Validity criteria fulfilled:
yes
Conclusions:
There was no statistically significant difference in the rate of nitrate production from 0 to 28 days compared to the control treatment at 1000 mg product/kg dry soil. There was no statistically significant difference in the nitrate concentration at 28 days compared to the control treatment at 1000 mg product/kg dry soil (Dunnett’s two-tail, p≤ 0.05, ToxCalc version 5.0.23, 1999).
As the difference in the rates of nitrate production between the control treatment and 1000 mg Lowinox/kg dry soil treatment was <25 % it is therefore considered that LOWINOX® 44B25 will have no long-term influence on the nitrogen transformation pathways in soils at concentrations ≤1000 mg Lowinox/kg dry soil.
Executive summary:

This study determined the effects of 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol, hereafter referred to as Lowinox, on nitrogen transformation in a LUFA standard soil type 2.3 according to procedures described in OECD Guideline No.216 (2000).

 

A range finder test was conducted at 0.1, 1.0, 10, 100 and 1000 mg LOWINOX® 44B25 /kg dry soil. The soil was amended with powdered lucerne (Medicago sativa) over a 28 day incubation period. As a result of this test, the study definitive phase of the study was run as limit test and soil was treated with the test item at a nominal concentration of 1000 mg LOWINOX® 44B25 /kg dry soil. The number of replicates in the definitive test was increased to six to provide a more powerful test. The products of the process of mineralisation and nitrification (nitrate, nitrite and ammonium) were extracted from the soil on day 0 and 28 days after treatment. The average nitrate content (mg/kg soil dry weight) was calculated for each treatment rate and nitrate production (mg/kg soil dry weight per day) was calculated over the 28 day period.

 

Effect on Nitrogen Transformation in Soil after Treatment

Treatment

Day 28 Concentration and Day 0 to 28 rate data

Mean Nitrate

(mg/kg dry soil)1

% Variation (Nitrate) from water control

Mean Nitrate Production

(mg/kg/day)1

% Variation (from water control)

Water Control

182.6

-

4.50

-

1000 mg Lowinox/kg

186.312

-2.003

4.602

-2.173

1All concentrations on a dry soil basis

2Not statistically different to water control (Dunnett’s Test, two-tail, p≤ 0.05).

3Nitrate concentration and rate data variation from the control - = stimulation and + = inhibition

Some values are calculated using un-rounded values.

 

The percent variation from the water control treatment was calculated for the ammonium and nitrate concentrations and the rate of nitrate production.

 

During the 0-28 day period the soil nitrate transformation rates (mg/kg dry soil/day) in the soil treated at the concentration of 1000 mg LOWINOX® 44B25 /kg dry soil varied by -2.17% from the mean control treatment.

 

Summary of Statistical Analysis Nitrogen Transformation Soil treated with Lowinox

 

NOEC

LOEC

Day 28 Nitrate concentration

1000 mg/kg

>1000 mg/kg

Day 0-28 Nitrate Production

1000 mg/kg

>100 mg/kg

NOEC and LOEC = No Observed Effect Level and Low Observed Effect Level (Dunnett’s Test, two-tail, p≤ 0.05).

 

The study met the validity criteria as the variation in the nitrate concentrations between replicate solvent control samples was less than ±15% (actual range was -5.0% to +5.0%) on Day 28.

 

CONCLUSION

There was no statistically significant difference in the rate of nitrate production from 0 to 28 days compared to the control treatment at 1000 mg product/kg dry soil. There was no statistically significant difference in the nitrate concentration at 28 days compared to the control treatment at 1000 mg product/kg dry soil (Dunnett’s two-tail, p≤ 0.05, ToxCalc version 5.0.23, 1999).

 

As the difference in the rates of nitrate production between the control treatment and 1000 mg Lowinox/kg dry soil treatment was <25 % it is therefore considered that LOWINOX® 44B25 will have no long-term influence on the nitrogen transformation pathways in soils at concentrations ≤1000 mg Lowinox/kg dry soil.

Description of key information

LOWINOX® 44B25 will have no long-term influence on the nitrogen transformation pathways in soils at concentrations ≤1000 mg Lowinox/kg dry soil.

Key value for chemical safety assessment

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

Additional information

This study determined the effects of 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol, hereafter referred to as Lowinox, on nitrogen transformation in a LUFA standard soil type 2.3.

 

A range finder test was conducted at 0.1, 1.0, 10, 100 and 1000 mg LOWINOX® 44B25 /kg dry soil. The soil was amended with powdered lucerne (Medicago sativa) over a 28 day incubation period. As a result of this test, the study definitive phase of the study was run as limit test and soil was treated with the test item at a nominal concentration of 1000 mg LOWINOX® 44B25 /kg dry soil. The number of replicates in the definitive test was increased to six to provide a more powerful test. The products of the process of mineralisation and nitrification (nitrate, nitrite and ammonium) were extracted from the soil on day 0 and 28 days after treatment. The average nitrate content (mg/kg soil dry weight) was calculated for each treatment rate and nitrate production (mg/kg soil dry weight per day) was calculated over the 28 day period.

 

The percent variation from the water control treatment was calculated for the ammonium and nitrate concentrations and the rate of nitrate production.

 

During the 0-28 day period the soil nitrate transformation rates (mg/kg dry soil/day) in the soil treated at the concentration of 1000 mg LOWINOX® 44B25 /kg dry soil varied by -2.17% from the mean control treatment.

 

CONCLUSION

There was no statistically significant difference in the rate of nitrate production from 0 to 28 days compared to the control treatment at 1000 mg product/kg dry soil. There was no statistically significant difference in the nitrate concentration at 28 days compared to the control treatment at 1000 mg product/kg dry soil (Dunnett’s two-tail, p≤ 0.05, ToxCalc version 5.0.23, 1999).

 

As the difference in the rates of nitrate production between the control treatment and 1000 mg Lowinox/kg dry soil treatment was <25 % it is therefore considered that LOWINOX® 44B25 will have no long-term influence on the nitrogen transformation pathways in soils at concentrations ≤1000 mg Lowinox/kg dry soil.