Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides

Toxicity to soil microorganisms

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to soil microorganisms

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From January 26, 2004 to March 09, 2004

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:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Sampling: Soil samples were analysed for nitrate at the beginning and at the end of the exposure period

Vehicle:

yes

Details on preparation and application of test substrate:

AMENDMENT OF SOIL
- Type of organic substrate: Soil was amended with Glucose at various concentrations i.e. 1, 10, 50, 100 and 200 micor mol per g soil


APPLICATION OF TEST SUBSTANCE TO SOIL
- Method: Glucose: Talcum powder (1 :5) mixtures were added to the soils. CO2 evolution from the soil was measured every hour for 6 h

Test organisms (inoculum):

soil

Total exposure duration:

28 d

Remarks:

-

Test temperature:

20 °C

Moisture:

43 %

Details on test conditions:

TEST SYSTEM
- Testing facility: Landwirtschaftliche Untersuchungs- und Forschungsanstalt Speyer, Speyer, Germany and Blgg Oosterbeek, The Netherlands
- Test container (type, material, size): Flasks made of glass (0.25 L)
- Amount of soil: 50 g
- No. of replicates per concentration: 3

SOIL INCUBATION
- Method: bulk / series of individual subsamples: Aerobic conditions

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Geographical reference of sampling site (latitude, longitude): Sandy loam; Fallow soil of garden in Offenbach, Rheinland Pfalz, Germany
- Soil taxonomic classification: Sandy loam
- Soil classification system: US Comprehensive Soil Classification System of the Soil Conservation Service
- pH (in water): 6.3±0.4
- Maximum water holding capacity (in % dry weight): 35.2 ± 3.4
- Cation exchange capacity (mmol/kg): 10± 2
- Storage (condition, duration): Stored in the refrigerator in polyetheylene bags until use in the experiment for 10 wk
- Initial microbial biomass as % of total organic C: 0.23

DETAILS OF PREINCUBATION OF SOIL (if any): Preconditioned by incubating the soil for 7 d at 20 at 10 % of its water holding capacity

Nominal and measured concentrations:

0, 50, 100, 200, 400, 800, 1600, 3200 and 6400 mg a.i./kg

Reference substance (positive control):

yes

Remarks:

Sodium nitrate

Key result

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

ca. 120 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

nitrate formation rate

Remarks on result:

other: 0.07-0.16 mg/g

Key result

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

ca. 70 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

nitrate formation rate

Details on results:

The activity of the microorganisms transforming nitrogen in soil was slightly inhibited at 50 mg a.i. /kg DDAC. The EC50 calculated was 120 mg a.i./kg with 95% confidence limits of 70 mg a.i./kg and 160 mg a.i./kg. The EC10, EC20 and EC80 of DDAC were 70, 80 and 160 mg a.i./kg respectively. (175, 200 and 400 mg of test substance)

Results with reference substance (positive control):

No data

Reported statistics and error estimates:

No data

The dose response relationship shows a normal pattern. According to the OECD guidelines the variation between the three control replicates should be less than 15 %. This criteria was met because the variation was less than 10 %. Therefore the test is considered valid. In soil not only formation of nitrate occurs but also reduction of nitrate to nitrogen gas by denitrifying
microorganisms. Decrease of the nitrate concentrations in the soil at DDAC concentration of 400 mg a.i. /kg and higher after 28 d is probably the result of the activity of these denitrifying microorganisms.  Denitrifying microorganisms are not affected by DDAC at concentrations ranging from 400 to 3200 mg a.i. /kg whereas the microorganisms responsible of the formation of nitrate are inhibited at these concentrations. The denitrifying microorganisms are inhibited at 6400 mg a.i./kg of DDAC because  after  28 d only a limited amount of the nitrate was removed.

Validity criteria fulfilled:

yes

Conclusions:

Under the study conditions, the EC50 and EC10 values for nitrogen transformation in soil was determined to be 120 and 70 mg a.i./kg, respectively.

Executive summary:

A study was conducted to evaluate the effects of test substance, DDAC (40% active) in soil microorganisms nitrogen transformation test, according to OECD Guideline 216, in compliance with GLP. The activities of the microorganisms mineralizing nitrogen-containing organic substances and nitrifying bacteria present in the soil exposed to various concentrations were compared to the control to calculate the inhibition in percentages. The microorganisms were exposed to the following test concentrations which was added to sandy loam: 50, 100, 200, 400, 800, 1600, 3200 and 6400 mg a.i./kg wet weight (ww). The soil samples were analysed for nitrate at the beginning (Day 0) and at the end of the exposure period (Day 28). Form the relation between the concentrations and the respective inhibition percentages, EC values have been determined. Nominal concentrations were used to calculate the EC values because nominal and actual concentration corresponded well. The activity of the microorganisms transforming nitrogen in soil was slightly inhibited at 50 mg a.i. /kg test substance. The EC50 calculated was 120 mg a.i. /kg with 95% confidence limits of 70 mg a.i. /kg and 160 mg a.i. /kg. The EC10, EC20 and EC80 of DDAC were 70, 80 and 160 mg a.i. /kg respectively (equivalent to 175, 200 and 400 mg of test substance). The test met the validity criteria, as the variation between the control replicates were less than 10%. Under the study conditions, the EC50 and EC10 values were determined to be 120 and 70 mg a.i./kg (nominal), respectively (Van Ginkel & Van der Togt, 2004).

Reference 2

Endpoint:

toxicity to soil microorganisms

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

From January 26, 2004 to March 09, 2004

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

KL2 due to RA

Justification for type of information:

Refer to section 13 of IUCLID for details on the read-across justification. The study with the read across substance is considered sufficient to fulfil the information requirements.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 216 (Soil Microorganisms: Nitrogen Transformation Test)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Sampling: Soil samples were analysed for nitrate at the beginning and at the end of the exposure period

Vehicle:

yes

Details on preparation and application of test substrate:

AMENDMENT OF SOIL
- Type of organic substrate: Soil was amended with Glucose at various concentrations i.e. 1, 10, 50, 100 and 200 micor mol per g soil


APPLICATION OF TEST SUBSTANCE TO SOIL
- Method: Glucose: Talcum powder (1 :5) mixtures were added to the soils. CO2 evolution from the soil was measured every hour for 6 h

Test organisms (inoculum):

soil

Total exposure duration:

28 d

Remarks:

-

Test temperature:

20 °C

Moisture:

43 %

Details on test conditions:

TEST SYSTEM
- Testing facility: Landwirtschaftliche Untersuchungs- und Forschungsanstalt Speyer, Speyer, Germany and Blgg Oosterbeek, The Netherlands
- Test container (type, material, size): Flasks made of glass (0.25 L)
- Amount of soil: 50 g
- No. of replicates per concentration: 3

SOIL INCUBATION
- Method: bulk / series of individual subsamples: Aerobic conditions

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Geographical reference of sampling site (latitude, longitude): Sandy loam; Fallow soil of garden in Offenbach, Rheinland Pfalz, Germany
- Soil taxonomic classification: Sandy loam
- Soil classification system: US Comprehensive Soil Classification System of the Soil Conservation Service
- pH (in water): 6.3±0.4
- Maximum water holding capacity (in % dry weight): 35.2 ± 3.4
- Cation exchange capacity (mmol/kg): 10± 2
- Storage (condition, duration): Stored in the refrigerator in polyetheylene bags until use in the experiment for 10 wk
- Initial microbial biomass as % of total organic C: 0.23

DETAILS OF PREINCUBATION OF SOIL (if any): Preconditioned by incubating the soil for 7 d at 20 at 10 % of its water holding capacity

Nominal and measured concentrations:

0, 50, 100, 200, 400, 800, 1600, 3200 and 6400 mg a.i./kg

Reference substance (positive control):

yes

Remarks:

Sodium nitrate

Key result

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

ca. 120 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

nitrate formation rate

Remarks on result:

other: 0.07-0.16 mg/g

Key result

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

ca. 70 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

nitrate formation rate

Details on results:

The activity of the microorganisms transforming nitrogen in soil was slightly inhibited at 50 mg a.i. /kg DDAC. The EC50 calculated was 120 mg a.i./kg with 95% confidence limits of 70 mg a.i./kg and 160 mg a.i./kg. The EC10, EC20 and EC80 of DDAC were 70, 80 and 160 mg a.i./kg respectively. (175, 200 and 400 mg of test substance)

Results with reference substance (positive control):

No data

Reported statistics and error estimates:

No data

The dose response relationship shows a normal pattern. According to the OECD guidelines the variation between the three control replicates should be less than 15 %. This criteria was met because the variation was less than 10 %. Therefore the test is considered valid. In soil not only formation of nitrate occurs but also reduction of nitrate to nitrogen gas by denitrifying
microorganisms. Decrease of the nitrate concentrations in the soil at DDAC concentration of 400 mg a.i. /kg and higher after 28 d is probably the result of the activity of these denitrifying microorganisms.  Denitrifying microorganisms are not affected by DDAC at concentrations ranging from 400 to 3200 mg a.i. /kg whereas the microorganisms responsible of the formation of nitrate are inhibited at these concentrations. The denitrifying microorganisms are inhibited at 6400 mg a.i./kg of DDAC because  after  28 d only a limited amount of the nitrate was removed.

Validity criteria fulfilled:

yes

Conclusions:

Based on the results of the read across study, the EC50 and EC10 values for nitrogen transformation in soil determined for test substance are considered to be 120 and 70 mg a.i./kg, respectively.

Executive summary:

A study was conducted to evaluate the effects of read across substance, DDAC (40% active) in soil microorganisms nitrogen transformation test, according to OECD Guideline 216, in compliance with GLP. The activities of the microorganisms mineralizing nitrogen-containing organic substances and nitrifying bacteria present in the soil exposed to various concentrations were compared to the control to calculate the inhibition in percentages. Inhibition of microbial nitrogen transformation through nitrate measurement at beginning and end of test on Day 28. Form the relation between the concentrations and the respective inhibition percentages, EC values have been determined. The dose response relationship showed a normal pattern. According to the OECD guidelines the variation between the three control replicates should be less than 15 %. This criteria was met because the variation was less than 10 %. Therefore, the test is considered valid. In soil not only formation of nitrate occurred but also reduction of nitrate to nitrogen gas by denitrifying microorganisms. Decrease of the nitrate concentrations in the soil at DDAC concentration of 400 mg a.i. /kg and higher after 28 d was probably the result of the activity of these denitrifying microorganisms.  Denitrifying microorganisms were not affected by read across substance at concentrations ranging from 400 to 3200 mg a.i. /kg, whereas the microorganisms responsible of the formation of nitrate were inhibited at these concentrations. The denitrifying microorganisms were inhibited at 6400 mg a.i./kg of read across substance because after 28 d only a limited amount of the nitrate was removed. The activity of the microorganisms transforming nitrogen in soil was slightly inhibited at 50 mg a.i. /kg read across substance. The EC50 calculated was 120 mg a.i. /kg with 95% confidence limits of 70 mg a.i. /kg and 160 mg a.i. /kg. The EC10, EC20 and EC80 of DDAC were 70, 80 and 160 mg a.i. /kg respectively (equivalent to 175, 200 and 400 mg of read across substance) (Van Ginkel & Van der Togt, 2004). Based on the results of the read across study, the EC50 and EC10 values for nitrogen transformation in soil determined for test substance are considered to be 120 and 70 mg a.i./kg, respectively.

Description of key information

Based on the results of the read across studies, the 28-day EC50 and EC10 values for effects on nitrogen transformation in soil was considered to be 120 and 70 mg/kg soil dw (nominal).

Key value for chemical safety assessment

Short-term EC50 for soil microorganisms:

120 mg/kg soil dw

Long-term EC10 or NOEC for soil microorganisms:

70 mg/kg soil dw

Additional information

No toxicity to soil microorganism study could be located on C12 -18 DAQ. Therefore, read across studies available with the structurally similar substance DDAC and C16-18 DAQ are presented. Both the test and read across substances are di-alkyl dimethyl ammonium chloride compounds. While DDAC is structurally the same but only differs in having a slightly lower average alkyl chain length, the C16-18 DAQ contains higher average alkyl chains. Data from both substances together is expected to adequately cover both the variables of environment toxicity i.e., bioavailability and intrinsic toxicity which are inversely related to alkyl chain length. The impact of the bioavailability on the terrestrial toxicity may be different, as there is additional exposure via ingestion of sorbed test substance from soil.

A study was conducted to evaluate the effects of read across substance, DDAC (40% active) insoil microorganisms nitrogen transformation test,according to OECD Guideline 216, in compliance with GLP.The activities of the microorganisms mineralizing nitrogen-containing organic substances and nitrifying bacteria present in the soil exposed to various concentrations were compared to the control to calculate the inhibition in percentages. Inhibition of microbial nitrogen transformation through nitrate measurement at beginning and end of the test on Day 28. Form the relation between the concentrations and the respective inhibition percentages, EC values have been determined.The dose response relationship showed a normal pattern. According to the OECD guidelines the variation between the three control replicates should be less than 15 %. This criteria was met because the variation was less than 10 %. Therefore, the test is considered valid. In soil not only formation of nitrate occurred but also reduction of nitrate to nitrogen gas by denitrifying microorganisms. Decrease of the nitrate concentrations in the soil at DDAC concentration of 400 mg a.i. /kg and higher after 28 d was probably the result of the activity of these denitrifying microorganisms.  Denitrifying microorganisms were not affected by read across substance at concentrations ranging from 400 to 3200 mg a.i. /kg, whereas the microorganisms responsible of the formation of nitrate were inhibited at these concentrations. The denitrifying microorganisms were inhibited at 6400 mg a.i./kg of read across substance because after 28 d only a limited amount of the nitrate was removed. The activity of the microorganisms transforming nitrogen in soil was slightly inhibited at 50 mg a.i. /kg read across substance. The EC50 calculated was 120 mg a.i. /kg with 95% confidence limits of 70 mg a.i. /kg and 160 mg a.i. /kg. The EC10, EC20 and EC80 of DDAC were 70, 80 and 160 mg a.i. /kg respectively (equivalent to 175, 200 and 400 mg of read across substance) (Van Ginkel & Van der Togt, 2004). Based on the results of the read across study, the EC50 and EC10 values for nitrogen transformation in soil determined for test substance are considered to be 120 and 70 mg a.i./kg, respectively.

Further, the DDAC biocides assessment report for Product Type 8 conducted under Directive 98/8/EC (evaluating Competent Authority: Italy, June 2015, attached in Section 13 of the IUCLID dataset), reported an additional study on DDAC, apart from the above EQC study (Van Ginkel & Van der Togt, 2004). This study resulted in a 28d EC10 >1000 mg/kg dw and 28d EC50 >1000 mg/kg dw for both carbon and nitrogen mineralization tested in two soils (sandy loam and low humic content sand). The RMS further considered the lower value from the EQC study as the endpoint for the product authorisation.

These studies can be further supported by the higher effect concentrations derived in two studies with C16-18 DAQ ,which has been reported in the EU RAR on DODMAC (or C18 DAQ) (EU, 2002) and has been summarised below:

Two studies of C16-18 DAQ toxicity to soil microorganisms are cited in the ECETOC report (1993) for which no test protocols are available. However, they provide a rough indication on possible effects. Soil respiration was measured with soil samples amended with 12.3 g activated sludge and 365 mg / per kg standard soil (Täuber et al. (1986). After 28 days no depression of oxygen uptake could be measured. In a study of Procter & Gamble (Procter & Gamble (1992). unpublished study, cited in ECETOC 1993) two different soils containing 400 mg/kg produced 96 and 119% carbon dioxide compared to the controls over 14 weeks. Therefore, the 28 day and 14 week NOECs were established at >365 and >400 mg/kg respectively.

Therefore, as a conservative approach and following the same approach as the biocides assessment report, the EC50 of 120 and EC10 of 70 mg/kg soil dw based on the effects on nitrogen transformation of the read across substance, has been considered further for hazard/risk assessment.