Benzododecinium chloride

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to microorganisms, other

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1994

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

DIN 38412-8 (Pseudomonas Zellvermehrungshemmtest)

Deviations:

not specified

GLP compliance:

not specified

Analytical monitoring:

not specified

Vehicle:

yes

Remarks:

Water

Test organisms (species):

other: Tetrahymena pyriformis and Pseudomonas aeruginosa

Details on inoculum:

Ciliates
Stock cultures of Tetrahymena pyriformis were grown at 29°C in a liquid medium increases. The cell-rich surface layer 2- to 4-day ciliate cultures were mixed with the medium on a Cell number of about 5 x 10E4 / mL set.

Pseudomonas aeruginosa
Stock cultures of Pseudomonas aeruginosa were incubated at 37°C for 20 h Standard nutrient broth multiplied. The precultures were 1: 10,000 with a deficient medium (without amino acids and proteins) diluted.

Test type:

static

Water media type:

other: freshwater and demineralised water

Limit test:

no

Total exposure duration:

21 h

Remarks on exposure duration:

The plates were first 18 h at 37°C incubated, then after addition of a 0.5% solution of 3- (4,5-dimethylthiazol-2-yl) 2,5-diphenyl-tetrazolium bromide (MTT) incubated for a further 3 h.

Test temperature:

37 deg C

Reference substance (positive control):

not specified

Key result

Duration:

21 h

Dose descriptor:

EC50

Remarks:

Pseudomonas aeruginosa

Effect conc.:

ca. 7.103 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: cytotoxicity

Remarks on result:

other: converted from original EC50 value of 10^-4.68 mol/L using molecular mass of test substance 340 g/mol

Key result

Duration:

21 h

Dose descriptor:

EC50

Remarks:

Tetrahymena pyriformis

Effect conc.:

ca. 53.882 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: cytotoxicity

Remarks on result:

other: converted from original EC50 value of 10^-3.8 mol/L using molecular mass of test substance 340 g/mol

Validity criteria fulfilled:

yes

Conclusions:

Under the study conditions, the 21 h EC50 of test substance to Pseudomonas aeruginosa and Tetrahymena pyriformis were determined to be 7.103 and 53.88 mg/L, respectively.

Executive summary:

A study was conducted to determine growth inhibition potential of the test substance C12 ADBAC (purity not specified) to Tetrahymena pyriformis and Pseudomonas aeruginosa according to DIN 38412. For Tetrahymena pyriformis, the ciliate suspensions (5 × 10E4 cells /mL) were in the ratio 9:1 with dilution levels of the chemical (dissolved in demineralized water) and filled into microtiter plates (100 μL / well, 8 Wells per dilution step of the test substance). The plates were initially incubated for 18 h at 25°C, then after adding a 0.5% solution of 3- (4,5-dimethylthiazol-2-yl) 2.5 Diphenyl-tetrazolium bromide (MTT) incubated for a further 3 h. For Pseudomonas aeruginosa, as per DIN 38412 part 8 modified method the bacterial suspensions (10E8 cfu/mL) were in the ratio 9:1 with dilution levels of the chemical (dissolved in deficiency medium) and in microtitration platesfilled (100 μL / well, 8 wells per dilution stage of test substance). The plates were first 18 h at 37°C incubated, then after addition of a 0.5% solution of 3 -(4,5-dimethylthiazol-2 -yl) 2,5 -diphenyl-tetrazolium bromide (MTT) incubated for a further 3 h. Cytotoxicity was determined photometrically at 550 nm wavelength, by measuring the conversion of MTT to formazan. Under the study conditions, the 21 h EC50 of test substance to Pseudomonas aeruginosa and Tetrahymena pyriformis were determined to be 7.103 and 53.88 mg/L respectively (Huber, 1994).

Reference 2

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1986

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

An inoculum of sewage micro-organism is grown in a nutrient broth and rate of growth is measured by the increase in turbidity of the solution with time. Addition of a toxic chemical to the culture medium inhibits the cell growth, and the turbidity of the test solution is thus lower than that of the control.

GLP compliance:

not specified

Analytical monitoring:

not specified

Vehicle:

not specified

Remarks:

Water

Test organisms (species):

sewage, domestic

Details on inoculum:

No details given

Test type:

not specified

Water media type:

not specified

Remarks on exposure duration:

No details available on exposure duration

Details on test conditions:

An inoculum of sewage micro organism was incubated in nutrient broth. The growth rate of this culture was determined by measurement of turbidity increase at a wavelength of 530 nm. The toxicity of test substances was plotted as a function of concentration and the EC50 value obtained (concentration causing a 50% reduction in growth rate).

Reference substance (positive control):

not specified

Key result

Dose descriptor:

EC50

Effect conc.:

ca. 3.2 - ca. 7.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth inhibition

Validity criteria fulfilled:

not specified

Conclusions:

Under the study conditions, the EC50 of test substance was determined to be 3.2 to 7.5 mg/L.

Executive summary:

A study was conducted to determine the effect of test substance, C12 ADBAC (purity not specified), on the growth of sewage micro-organism. An inoculum of sewage micro organism was incubated in nutrient broth and test substance was added in different concentration (no more specific details about the test procedure available). The growth rate of this culture was determined by measurement of turbidity increase of the solution with time at a wavelength of 530 nm. The toxicity of test substances was plotted as a function of concentration and the EC50 value obtained (concentration causing a 50% reduction in growth rate). This test was performed at two different laboratories and the EC50 values determined were 3.2 and 7.5 mg/L. There was an acceptbale correlation between the results obtained by these two laboratoies. Under the study conditions, the EC50 of test substance was determined to be 3.2 to 7.5 mg/L (ECETOC, 1986).

Reference 3

Endpoint:

activated sludge respiration inhibition testing

Type of information:

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

Adequacy of study:

supporting study

Study period:

From May 12, 2000 to June 20, 2000

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 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Details on test solutions:

A stock solution of the test was prepared by dissolving 2.0008 g of the test substance in 1,000 mL of BOD dilution water.
A stock solution of the reference substance was prepared according to OECD guideline no 209.

The pH of the sludge suspension was found to be 7.1 before use. Control mixtures were prepared by combining 16 mL of synthetic sewage feed with the appropriate volumes of the test substance stock solution and dilution water to a volume of 300 mL in one litre glass beaker.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Laboratory culture: Municipal oxidation ditch, which is used to treat the domestic sewage (Hazerwoude Dorp, the Netherlands)
- Preparation of inoculum for exposure: Approximately 20 litres of activated sludge was taken from the municipal oxidation ditch, which is used to treat the domestic sewage. The sample was centrifuged and the supernatant discarded. The activated sludge was washed thrice by centrifugation and re-suspension in tap water twice and once in dilution water. The dry weight of the sludge suspension was determined. The mixed liquor suspended solids content was found to be 7.1 g/L.
- Initial biomass concentration: The dry weight was checked prior to use in the test and was found at a mixed liquor suspended solids level of 3.7 g/L.

Test type:

semi-static

Water media type:

freshwater

Total exposure duration:

3 h

Test temperature:

21.9 - 23°C

pH:

7.6 - 7.8

Nominal and measured concentrations:

Nominal concentrations: 0, 3.2, 10, 32, 100, 320 and 1,000 mg/L

Details on test conditions:

A stock solution of the test was prepared by dissolving 2.0008 g of the test substance in 1,000 mL of BOD dilution water.
A stock solution of the reference substance was prepared according to OECD guideline no 209.

The pH of the sludge suspension was found to be 7.1 before use. Control mixtures were prepared by combining 16 mL of synthetic sewage feed with the appropriate volumes of the test substance stock solution and dilution water to a volume of 300 mL in one litre glass beaker.

The test was started by adding 200 mL of sludge suspension to the mixtures with and without test substance. The incubation of mixtures was started at the intervals of 12 min and the first and last sample were controls without the test substance. The mixtures were aerated vigorously and incubated at the temperature between 21.9 and 23°C for 3h. After 3h, a sample of each mixture was poured into a BOD bottle of and stirred vigorously. An oxygen electrode (connected to a WTW OXI 2000 meter) was inserted into the sample and oxygen concentration was recorded every minute during a period of about 10 min.

Simultaneously, three concentrations (5, 12 and 30 mg/L) of the reference substance 3,5-dichlorophenol were tested. Each concentration of reference was diluted with dilution water to 284 mL, mixed with 16 mL of synthetic sewage feed and 200 mL sludge suspension.

The BOD dilution water was prepared according to NEN Standard No. 6634.
The synthetic sewage feed was prepared according to OECD guideline no. 209.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

7.75 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: 95% confidence limits of 6.35 and 9.5 mg a.i./L

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

1.6 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: calculated

Details on results:

The EC50 of the test substance for activated sludge was 7.75 mg a.i./L with 95% confidence limits of 6.35 and 9.5 mg a.i./L. The EC20 and EC80 were 3.4 and 17.8 mg a.i./L, respectively. The NOEC of the test substance was found to be 1.6 mg a.i./L.

Results with reference substance (positive control):

- Results with reference substance valid: yes
- Relevant effect levels: The 3-h EC50 of reference substance was found to be 5 mg/L, which is in the range prescribed by OECD guideline (range of 5-30 mg/L).

The respiration rates of two controls of the test were found to be 13.69 and 14.75 mg O2/g/h, respectively. The difference between the two values amounted to 3.8% being within the validity criterion (15%) of OECD guideline. Further, the 3-h EC50 of reference substance was found to be 5 mg/L, which is in the range prescribed by OECD guideline (range of 5-30 mg/L).

Validity criteria fulfilled:

yes

Conclusions:

Based on the results of the read across study, the 3 h EC20, EC50 and EC80 were determined to be 3.4, 7.75 and 17.8 mg a.i./L respectively. The 3 h NOEC was established at 1.6 mg a.i./L.

Executive summary:

A study was conducted to determine the toxicity to microorganisms of the read across substance, C12 -16 ADBAC (49 -51% active) according to OECD Guideline 209, in compliance with GLP. A mixture of activated sludge, synthetic sewage feed and a range of concentrations of the test or reference substance were prepared in 1 L glass beakers. The mixtures were aerated and, after an incubation period of 3 h at a temperature between 21.9 and 23°C, the decrease in the oxygen concentration in the mixtures was recorded during a period of 10 min. The inhibitory effect of the read across substance at a particular concentration was expressed as a percentage of the mean respiration rates of the controls. The substance was tested at nominal concentrations of 0, 1, 3.2, 10, 32, 100, 320 and 1000 mg/L (equivalent to ca. 0, 0.5, 1.6, 5.0, 16, 50, 160 and 500 mg a.i./L). No analytical dose determination was performed. A control test with reference substance 3,5-dichlorophenol yielded an EC50 value of 5 mg/L, which is within the range of prescribed by guideline. The validity criteria of the guideline were fulfilled. Under the study conditions, the 3 h EC20, EC50 and EC80 were determined to be 3.4, 7.75 and 17.8 mg a.i./L respectively. The 3 h NOEC was established at 1.6 mg a.i./L (Mayer, 2001).

Reference 4

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From May 12, 2000 to June 20, 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Physical state: Pale yellow soloured liquid
- Analytical purity: 49 to 51%
- Lot/batch No.: K 9225906
- Storage condition of test material: Room temperature

Analytical monitoring:

no

Details on test solutions:

A stock solution of the test was prepared by dissolving 2.0008 g of the test substance in 1,000 mL of BOD dilution water.
A stock solution of the reference substance was prepared according to OECD guideline no 209.

The pH of the sludge suspension was found to be 7.1 before use. Control mixtures were prepared by combining 16 mL of synthetic sewage feed with the appropriate volumes of the test substance stock solution and dilution water to a volume of 300 mL in one litre glass beaker.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Laboratory culture: Municipal oxidation ditch, which is used to treat the domestic sewage (Hazerwoude Dorp, the Netherlands)
- Preparation of inoculum for exposure: Approximately 20 litres of activated sludge was taken from the municipal oxidation ditch, which is used to treat the domestic sewage. The sample was centrifuged and the supernatant discarded. The activated sludge was washed thrice by centrifugation and re-suspension in tap water twice and once in dilution water. The dry weight of the sludge suspension was determined. The mixed liquor suspended solids content was found to be 7.1 g/L.
- Initial biomass concentration: The dry weight was checked prior to use in the test and was found at a mixed liquor suspended solids level of 3.7 g/L.

Test type:

semi-static

Water media type:

freshwater

Total exposure duration:

3 h

Test temperature:

21.9 - 23°C

pH:

7.6 - 7.8

Nominal and measured concentrations:

Nominal concentrations: 0, 3.2, 10, 32, 100, 320 and 1,000 mg/L

Details on test conditions:

A stock solution of the test was prepared by dissolving 2.0008 g of the test substance in 1,000 mL of BOD dilution water.
A stock solution of the reference substance was prepared according to OECD guideline no 209.

The pH of the sludge suspension was found to be 7.1 before use. Control mixtures were prepared by combining 16 mL of synthetic sewage feed with the appropriate volumes of the test substance stock solution and dilution water to a volume of 300 mL in one litre glass beaker.

The test was started by adding 200 mL of sludge suspension to the mixtures with and without test substance. The incubation of mixtures was started at the intervals of 12 min and the first and last sample were controls without the test substance. The mixtures were aerated vigorously and incubated at the temperature between 21.9 and 23°C for 3h. After 3h, a sample of each mixture was poured into a BOD bottle of and stirred vigorously. An oxygen electrode (connected to a WTW OXI 2000 meter) was inserted into the sample and oxygen concentration was recorded every minute during a period of about 10 min.

Simultaneously, three concentrations (5, 12 and 30 mg/L) of the reference substance 3,5-dichlorophenol were tested. Each concentration of reference was diluted with dilution water to 284 mL, mixed with 16 mL of synthetic sewage feed and 200 mL sludge suspension.

The BOD dilution water was prepared according to NEN Standard No. 6634.
The synthetic sewage feed was prepared according to OECD guideline no. 209.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

7.75 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: 95% confidence limits of 6.35 and 9.5 mg a.i./L

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

1.6 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: calculated

Details on results:

The EC50 of the test substance for activated sludge was 7.75 mg a.i./L with 95% confidence limits of 6.35 and 9.5 mg a.i./L. The EC20 and EC80 were 3.4 and 17.8 mg a.i./L, respectively. The NOEC of the test substance was found to be 1.6 mg a.i./L.

Results with reference substance (positive control):

- Results with reference substance valid: yes
- Relevant effect levels: The 3-h EC50 of reference substance was found to be 5 mg/L, which is in the range prescribed by OECD guideline (range of 5-30 mg/L).

The respiration rates of two controls of the test were found to be 13.69 and 14.75 mg O2/g/h, respectively. The difference between the two values amounted to 3.8% being within the validity criterion (15%) of OECD guideline. Further, the 3-h EC50 of reference substance was found to be 5 mg/L, which is in the range prescribed by OECD guideline (range of 5-30 mg/L).

Validity criteria fulfilled:

yes

Conclusions:

Under the study conditions, the 3 h EC20, EC50 and EC80 were determined to be 3.4, 7.75 and 17.8 mg a.i./L respectively. The 3 h NOEC was established at 1.6 mg a.i./L.

Executive summary:

A study was conducted to determine the toxicity to microorganisms of the test substance, C12 -16 ADBAC (49 -51% active) according to OECD Guideline 209, in compliance with GLP. A mixture of activated sludge, synthetic sewage feed and a range of concentrations of the test or reference substance were prepared in 1 L glass beakers. The mixtures were aerated and, after an incubation period of 3 h at a temperature between 21.9 and 23°C, the decrease in the oxygen concentration in the mixtures was recorded during a period of 10 min. The inhibitory effect of the test substance at a particular concentration was expressed as a percentage of the mean respiration rates of the controls. The substance was tested at nominal concentrations of 0, 1, 3.2, 10, 32, 100, 320 and 1000 mg/L (equivalent to ca. 0, 0.5, 1.6, 5.0, 16, 50, 160 and 500 mg a.i./L). No analytical dose determination was performed. A control test with reference substance 3,5-dichlorophenol yielded an EC50 value of 5 mg/L, which is within the range of prescribed by guideline. The validity criteria of the guideline were fulfilled. Under the study conditions, the 3 h EC20, EC50 and EC80 were determined to be 3.4, 7.75 and 17.8 mg a.i./L respectively. The 3 h NOEC was established at 1.6 mg a.i./L (Mayer, 2001).

Description of key information

The 21 h EC50 value of the test substance, which was determined at 7.03 mg/L or 7030 µg/L (nominal) based on the growth inhibition of Pseudomonas aeruginosa, has been considered further for hazard and risk assessment.

Key value for chemical safety assessment

EC50 for microorganisms:

7.03 mg/L

Additional information

Study 1: A study was conducted to determine the effect of the test substance C12 ADBAC (purity not specified) on the growth inhibition of Tetrahymena pyriformis and Pseudomonas aeruginosa according to DIN 38412. For Tetrahymena pyriformis, the ciliate suspensions (5 × 10E4 cells /mL) were in the ratio 9:1 with dilution levels of the chemical (dissolved in demineralized water) and filled into microtiter plates (100 μL / well, 8 Wells per dilution step of the test substance). The plates were initially incubated for 18 h at 25°C, then after adding a 0.5% solution of 3- (4,5-dimethylthiazol-2-yl) 2.5 Diphenyl-tetrazolium bromide (MTT) incubated for a further 3 h. For Pseudomonas aeruginosa, as per DIN 38412 part 8 modified method the bacterial suspensions (10E8 cfu/mL) were in the ratio 9:1 with dilution levels of the chemical (dissolved in deficiency medium) and in microtitration platesfilled (100 μL / well, 8 wells per dilution stage of test substance). The plates were first 18 h at 37°C incubated, then after addition of a 0.5% solution of 3 -(4,5-dimethylthiazol-2 -yl) 2,5 -diphenyl-tetrazolium bromide (MTT) incubated for a further 3 h. Cytotoxicity was determined photometrically at 550 nm wavelength, by measuring the conversion of MTT to formazan. Under the study conditions, the 21 h EC50 of test substance to Pseudomonas aeruginosa and Tetrahymena pyriformis were determined to be 7.103 and 53.88 mg/L respectively (Huber, 1994).

Study 2: A study was conducted to determine the effect of test substance, C12 ADBAC (purity not specified), on the growth inhibition of sewage micro-organism according to test methods used in the UK (Anon, 1985) and US (Alsopet al., 1980). An inoculum of sewage microorganism was incubated in nutrient broth and test substance was added in different concentration (no further details). The growth rate of this culture was determined by measurement of turbidity increase of the solution with time at a wavelength of 530 nm. The toxicity of test substances was plotted as a function of concentration and the EC50 value was obtained (concentration causing a 50% reduction in growth rate). This test was performed at two different laboratories and the EC50 values determined were 3.2 and 7.5 mg/L. There was an acceptable correlation between the results obtained by these two laboratories. Under the study conditions, the EC50 of test substance was determined to be 3.2 to 7.5 mg/L (ECETOC, 1986). 

Study 3: A study was conducted to determine the effect of test substance, C12 ADBAC (purity not specified), on the respiratory (oxygen uptake) inhibition of pure cultures of nitrifying micro-organisms according to OECD Guideline 209. The test was performed at three different laboratories and the EC50 values determined were 10, 16 and 24 mg/L respectively. There was an acceptable correlation between the results obtained by these two laboratories. Under the study conditions, the 3 h EC50 of test substance was determined to range from 10-24 mg/L (ECETOC, 1986)

The effect value selected for the test substance is further supported by the results from the study with the read across substance, C12 -16 ADBAC in activated sludge presented below:

Study 4: A study was conducted to determine the toxicity to microorganisms of the read across substance, C12 -16 ADBAC (49 -51% active) according to OECD Guideline 209, in compliance with GLP. A mixture of activated sludge, synthetic sewage feed and a range of concentrations of the test or reference substance were prepared in 1 L glass beakers. The mixtures were aerated and, after an incubation period of 3 h at a temperature between 21.9 and 23°C, the decrease in the oxygen concentration in the mixtures was recorded during a period of 10 min. The inhibitory effect of the read across substance at a particular concentration was expressed as a percentage of the mean respiration rates of the controls. The substance was tested at nominal concentrations of 0, 1, 3.2, 10, 32, 100, 320 and 1000 mg/L (equivalent to ca. 0, 0.5, 1.6, 5.0, 16, 50, 160 and 500 mg a.i./L). No analytical dose determination was performed. A control test with reference substance 3,5-dichlorophenol yielded an EC50 value of 5 mg/L, which is within the range of prescribed by guideline. The validity criteria of the guideline were fulfilled. Under the study conditions, the 3 h EC20, EC50 and EC80 were determined to be 3.4, 7.75 and 17.8 mg a.i./L respectively. The 3 h NOEC was established at 1.6 mg a.i./L (Mayer, 2001).

Although a direct comparison of the target substance is not possible with the read across substance due to its UVCB nature, generally the results support the fact that the toxicity of the quaternary ammonium compounds increase with carbon chain length until C14 carbon atoms, after which the trend was reported to be opposite (Nicaet al., 2017). Therefore, preferably the EC50 value derived for the test substance at 7.03 mg/L or 7030 µg/L (nominal), based on the growth inhibition ofPseudomonas aeruginosa, has been considered further for hazard and risk assessment.