Benzododecinium chloride

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Vehicle:

not specified

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

23 +/- 2°C

pH:

7.8-9.7

Nominal and measured concentrations:

0, 0.0032, 0.010, 0.032, 0.10, 0.32, 1.0 mg/L nominal test substance concentrations
0, 0.0019, 0.0044, 0.016, 0.054, 0.16 and 1 mg/L measured test substance concentration

Details on test conditions:

- Exposure system: shaking culture (100 rpm)
- Test solution volume: 100 mL (OECD medium) per vessel
- No. of vessels per concentration (replicates): 3
- Initial cells density: 10000 cells/mL
- Lighting: 4000 lux, continuous

Reference substance (positive control):

not specified

Key result

Duration:

72 h

Dose descriptor:

other: ErC50

Effect conc.:

ca. 0.068 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval: 0.061 to 0.074 mg/L

Key result

Duration:

72 h

Dose descriptor:

other: EbC50

Effect conc.:

ca. 0.044 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% confidence interval: 0.039 to 0.049 mg/L

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

ca. 0.032 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

ca. 0.01 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Results

For detailed result tables, kindly refer to the attached background material section of the IUCLID.

Validity criteria fulfilled:

not specified

Conclusions:

Under study conditions, after 72 h EbC50 and ErC50 values of the test substance for inhibition of growth or growth rate in algae were determined to be 0.044 and 0.068 mg/L (nominal), respectively. The NOEC values for biomass and growth rate were determined to be 0.01 mg/L and 0.032 mg/L respectively.

Executive summary:

A study was conducted to determine the toxicity study of the test substance, C12 ADBAC (purity: >99%), to freshwater green algae, Pseudokirchneriella subcapitata, according to OECD Guideline 201, in compliance with GLP. Three replicate algal cultures, with a nominal initial cell density of approximately 1 x 10⁴cells/mL, were exposed to test substance at nominal concentrations of 0, 0.0032, 0.010, 0.032, 0.10, 0.32, 1.0 mg/L in OECD algal medium for 72 h, under static conditions. The exposure levels of test substance in aqueous samples of test media were monitored using a fluorometry method of analysis. The measured concentrations of the test substance were determined to be 0, 0.0019, 0.0044, 0.016, 0.054, 0.16 and 1 mg/L. The test results were expressed in terms of their nominal values. Cell numbers were counted daily to monitor growth. The test results were expressed in terms of the area under the growth curve and average specific growth rate. The 72 h ErC50 and NOEC values (95% Confidence Intervals) for average specific growth rate were found to be 0.068 (0.061 & 0.074) and 0.032 mg/L (nominal), whereas, 72 h EbC50 and NOEC values (95% Confidence Intervals) for area under the growth curve were found to be 0.044 (0.039 & 0.049) and 0.010 mg/L (nominal) respectively. Under study conditions, after 72 h EbC50 and ErC50 values of the test substance for inhibition of growth or growth rate in algae were determined to be 0.044 and 0.068 mg/L (nominal), respectively. The NOEC values for biomass and growth rate were determined to be 0.01 mg/L and 0.032 mg/L respectively (NITE, 2001).

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

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

Adequacy of study:

supporting study

Study period:

2001

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 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Samples from the controls and the test flasks containing the different test substance concentrations were taken after 0, 23, 47 and 71h. 5 mL samples of cultures were added to 10 mL of Ultima Gold XR scintillation liquid.

Vehicle:

yes

Details on test solutions:

- For the range-finding test, an amount of 20.9 mg test substance was dissolved in 100 mL of algal medium. Dilutions were then prepared in medium so as to yield a test substance concentrations series of 0, 10, 104, 1,040 and 10,400 µg/L.
- For the growth inhibition test, an amount of 13.7 mg test substance was dissolved in 500 mL algal medium. Dilutions were the prepared in medium to yield a nominal test substance concentration series of 0, 3.7, 13, 24, 43, 137 and 435 µg/L.
- For the 14C-spiking stock solution, an amount of 10 µL of 14C-labelled test substance solution (containing 47760 Bq) was added to 10 mL ultrapure water.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

The fresh water green alga Selenastrum capricornutum (CCAP 278/4)
The culture was supplied by CCAP, The Freshwater Biological Association, the Ferry House, Far Sawrey, Ambleside, Cumbria LA22 OLP, England.

A pre-culture of algae in exponential growth phase was prepared as per the OECD guideline no. 201 using medium as described in the OECD guideline.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

22 - 23 °C (average = 22.3°C)

pH:

8.2-8.3 (in the absence of algal cell)
8.2-8.6 (in the presence of algal cell)

Nominal and measured concentrations:

Nominal test concentrations: 0, 3.7, 13, 24, 43, 136 and 431 µg a.i./L.
Measured concentrations:

Details on test conditions:

An algal inoculum was added to each flask to achieve nominal density of 10,000 cells/mL. Nominal test concentrations were 0, 3.7, 13, 24, 43, 136 and 431 µg a.i./L. These concentrations refer to the total of labelled and non-labelled test substance and after addition of algal substance. Flasks were continuously shaken at 100 rpm. The test was carried out at 22.3°C average, and a pH of 8.2. Illumination was 60-120 µmol s-1.m-2 using fluorescent lights. Algal cell densities were determined in the treatments and control at 0, 23, 47 and 71h. The pH was measured in all cultures after 71h. All other parameters were also within the guideline recommendations.
Test was carried out in triplicate with six controls containing algae only and a single background series containing test substance without algae. Algal densities and biovolume were determined after 0, 23, 47 and 71h. The measured values were corrected for the background values in appropriate blanks. The mean values were used for further calculations.
The algal growth was determined by electronic particle counting after 0, 23, 47 and 71h incubation. The test fulfilled the validity criteria of sufficient growth and a minimum increase of test medium pH. The effect values were calculated using a statistic parametric model.

Reference substance (positive control):

yes

Remarks:

The test system used is checked with reference substances (K2Cr2O7 and/or 3,5-dicholorophenol) on an approximately yearly basis

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

49 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: 40 - 61; 95% confidence limits

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

14 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

biomass

Remarks on result:

other: 11 - 22; 95% confidence limits

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

<= 1.2 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: growth rate inhibition was only 3.1% at this level which was considered negligible

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.009 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): Microscopic inspection of morphology of algal cells in pre-culture at the start of the test revealed normal cells. At the end of the test in the cultures treated with nominal test concentrations of 24 and 43 µg/L, small pale cells were observed. At higher test substance concentrations, no algae were found.
- Flocculation: No
- Adherence to test vessels: No
- Any stimulation of growth found in any treatment: No

For further details on results, please refer to the attachment under 'Attached background material'

Results with reference substance (positive control):

The test system used is checked with reference substances (K2Cr2O7 and/or 3,5-dicholorophenol) on an approximately yearly basis. The result of these reference tests demonstrate a constant quality of the test system during an extended period of at least 15 years. The resulting EC50 values are also comparable to the mean values found in international ring tests.

Reported statistics and error estimates:

Parametric model

- The range-finding test revealed that inhibiting effects could be expected at nominal test substance concentrations higher than 10 µg/L.

- The control growth rate (0.065/h) is higher than the minimum cell multiplication factor of 16 during a three d test given in the guideline (corresponding to a growth rate of 0.038/h).

- The highest pH increase in the test medium was 0.4, which is well below the limit given in the guideline.

- The test therefore met the validity criteria of the guideline.

Validity criteria fulfilled:

yes

Conclusions:

Based on the results of the read across study, the 72h ErC50, ErC10, EbC50 and EbC10 were determined to be at 49, 9, 14 and <1.2 µg a.i./L respectively. The 72 h NOEC was ≤1.2 µg a.i./L

Executive summary:

A study was conducted to determine the toxicity to aquatic algae of the read across substance, C12-16 ADBAC (49-51% active; radiolabeled purity: 99.7%) according to OECD Guideline 201 and EU Method C.3, in compliance with GLP. Selenastrum capricornutum were exposed for 72 h at nominal concentrations of 0, 3.7, 13, 24, 43, 136 and 431 µg a.i./L under static conditions. Concentrations were analytically determined at the start of the test and after 23, 47 and 72 h. Algal growth was measured by electronic particle counting after 0, 23, 47 and 72 h. The read across substance concentrations were found to be relatively stable during the test and therefore the mean measured read across substance concentrations of 0, 1.2, 5.1, 11, 22, 98 and 382 µg/L were used for calculation of effect parameters. The test fulfilled the validity criteria of sufficient growth and minimum increase of test medium pH. Statistical assessment of data by application of parametric model indicated a significant effect on the growth rate only. Under the study conditions, the 72h ErC50, ErC10, EbC50 and EbC10 were determined to be at 49, 9, 14 and <1.2 µg a.i./L respectively. The 72 h NOEC was ≤1.2 µg a.i./L (Mayer, 2001).

Reference 3

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Name of test material (as cited in study report): Alkyldimethylbenzylammonium chloride (ADBAC); (i.e., benzyl C12-16 alkyl dimethyl ammonium chloride)
- Physical state: slightly yellowish liquid
- Analytical purity: 49-51% (average 50%)
- Impurities (identity and concentrations): Alkyldimethylbenzylammonium chloride in water
- Storage condition of test material: Room temperature
- Radiochemical purity (if radiolabelling): 99.7%
- Specific activity (if radiolabelling): 25 mCi/mmol
- Locations of the label (if radiolabelling): [14C]-alkyldimethylbenzylammonium chloride

Analytical monitoring:

yes

Details on sampling:

Samples from the controls and the test flasks containing the different test substance concentrations were taken after 0, 23, 47 and 71h. 5 mL samples of cultures were added to 10 mL of Ultima Gold XR scintillation liquid.

Vehicle:

yes

Details on test solutions:

- For the range-finding test, an amount of 20.9 mg test substance was dissolved in 100 mL of algal medium. Dilutions were then prepared in medium so as to yield a test substance concentrations series of 0, 10, 104, 1,040 and 10,400 µg/L.
- For the growth inhibition test, an amount of 13.7 mg test substance was dissolved in 500 mL algal medium. Dilutions were the prepared in medium to yield a nominal test substance concentration series of 0, 3.7, 13, 24, 43, 137 and 435 µg/L.
- For the 14C-spiking stock solution, an amount of 10 µL of 14C-labelled test substance solution (containing 47760 Bq) was added to 10 mL ultrapure water.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

The fresh water green alga Selenastrum capricornutum (CCAP 278/4)
The culture was supplied by CCAP, The Freshwater Biological Association, the Ferry House, Far Sawrey, Ambleside, Cumbria LA22 OLP, England.

A pre-culture of algae in exponential growth phase was prepared as per the OECD guideline no. 201 using medium as described in the OECD guideline.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

22 - 23 °C (average = 22.3°C)

pH:

8.2-8.3 (in the absence of algal cell)
8.2-8.6 (in the presence of algal cell)

Nominal and measured concentrations:

Nominal test concentrations: 0, 3.7, 13, 24, 43, 136 and 431 µg a.i./L.
Measured concentrations:

Details on test conditions:

An algal inoculum was added to each flask to achieve nominal density of 10,000 cells/mL. Nominal test concentrations were 0, 3.7, 13, 24, 43, 136 and 431 µg a.i./L. These concentrations refer to the total of labelled and non-labelled test substance and after addition of algal substance. Flasks were continuously shaken at 100 rpm. The test was carried out at 22.3°C average, and a pH of 8.2. Illumination was 60-120 µmol s-1.m-2 using fluorescent lights. Algal cell densities were determined in the treatments and control at 0, 23, 47 and 71h. The pH was measured in all cultures after 71h. All other parameters were also within the guideline recommendations.
Test was carried out in triplicate with six controls containing algae only and a single background series containing test substance without algae. Algal densities and biovolume were determined after 0, 23, 47 and 71h. The measured values were corrected for the background values in appropriate blanks. The mean values were used for further calculations.
The algal growth was determined by electronic particle counting after 0, 23, 47 and 71h incubation. The test fulfilled the validity criteria of sufficient growth and a minimum increase of test medium pH. The effect values were calculated using a statistic parametric model.

Reference substance (positive control):

yes

Remarks:

The test system used is checked with reference substances (K2Cr2O7 and/or 3,5-dicholorophenol) on an approximately yearly basis

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

49 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: 40 - 61; 95% confidence limits

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

14 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

biomass

Remarks on result:

other: 11 - 22; 95% confidence limits

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

<= 1.2 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: growth rate inhibition was only 3.1% at this level which was considered negligible

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.009 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): Microscopic inspection of morphology of algal cells in pre-culture at the start of the test revealed normal cells. At the end of the test in the cultures treated with nominal test concentrations of 24 and 43 µg/L, small pale cells were observed. At higher test substance concentrations, no algae were found.
- Flocculation: No
- Adherence to test vessels: No
- Any stimulation of growth found in any treatment: No

For further details on results, please refer to the attachment under 'Attached background material'

Results with reference substance (positive control):

The test system used is checked with reference substances (K2Cr2O7 and/or 3,5-dicholorophenol) on an approximately yearly basis. The result of these reference tests demonstrate a constant quality of the test system during an extended period of at least 15 years. The resulting EC50 values are also comparable to the mean values found in international ring tests.

Reported statistics and error estimates:

Parametric model

- The range-finding test revealed that inhibiting effects could be expected at nominal test substance concentrations higher than 10 µg/L.

- The control growth rate (0.065/h) is higher than the minimum cell multiplication factor of 16 during a three d test given in the guideline (corresponding to a growth rate of 0.038/h).

- The highest pH increase in the test medium was 0.4, which is well below the limit given in the guideline.

- The test therefore met the validity criteria of the guideline.

Validity criteria fulfilled:

yes

Conclusions:

Under the study conditions, the 72h ErC50, ErC10, EbC50 and EbC10 were 49, 9, 14 and <1.2 µg a.i./L respectively. The 72 h NOEC was ≤1.2 µg a.i./L

Executive summary:

A study was conducted to determine the toxicity to aquatic algae of the test substance, C12-16 ADBAC (49-51% active; radiolabeled purity: 99.7%) according to OECD Guideline 201 and EU Method C.3, in compliance with GLP. Selenastrum capricornutum were exposed for 72 h at nominal concentrations of 0, 3.7, 13, 24, 43, 136 and 431 µg a.i./L under static conditions. Concentrations were analytically determined at the start of the test and after 23, 47 and 72 h. Algal growth was measured by electronic particle counting after 0, 23, 47 and 72 h. The test substance concentrations were found to be relatively stable during the test and therefore the mean measured test substance concentrations of 0, 1.2, 5.1, 11, 22, 98 and 382 µg/L were used for calculation of effect parameters. The test fulfilled the validity criteria of sufficient growth and minimum increase of test medium pH. Statistical assessment of data by application of parametric model indicated a significant effect on the growth rate only. Under the study conditions, the 72h ErC50, ErC10, EbC50 and EbC10 were 49, 9, 14 and <1.2 µg a.i./L respectively. The 72 h NOEC was ≤1.2 µg a.i./L (Mayer, 2001).       

Description of key information

The 72 h ErC50 and NOEC values of the test substance, which was determined at 68 and 32 µg/L (nominal), based on effects on growth rate, has been considered further for hazard and risk assessment.

Key value for chemical safety assessment

EC50 for freshwater algae:

68 µg/L

EC10 or NOEC for freshwater algae:

32 µg/L

Additional information

Study 1: A study was conducted to determine the toxicity study of the test substance, C12 ADBAC (purity: >99%), to freshwater green algae, Pseudokirchneriella subcapitata, according to OECD Guideline 201, in compliance with GLP. Three replicate algal cultures, with a nominal initial cell density of approximately 1 x 10⁴cells/mL, were exposed to test substance at nominal concentrations of 0, 0.0032, 0.010, 0.032, 0.10, 0.32, 1.0 mg/L in OECD algal medium for 72 h, under static conditions. The exposure levels of test substance in aqueous samples of test media were monitored using a fluorometry method of analysis. The measured concentrations of the test substance were determined to be 0, 0.0019, 0.0044, 0.016, 0.054, 0.16 and 1 mg/L. The test results were expressed in terms of their nominal values. Cell numbers were counted daily to monitor growth. The test results were expressed in terms of the area under the growth curve and average specific growth rate. The 72 h ErC50 and NOEC values (95% Confidence Intervals) for average specific growth rate were found to be 0.068 (0.061 & 0.074) and 0.032 mg/L (nominal), whereas, 72 h EbC50 and NOEC values (95% Confidence Intervals) for area under the growth curve were found to be 0.044 (0.039 & 0.049) and 0.010 mg/L (nominal) respectively. Under study conditions, after 72 h EbC50 and ErC50 values of the test substance for inhibition of growth or growth rate in algae were determined to be 0.044 and 0.068 mg/L ( (or 44 or 68 µg/L; nominal), respectively. The NOEC values for biomass and growth rate were determined to be 0.01 mg/L (or 10 µg/L) and 0.032 mg/L (or 32 µg/L) respectively (NITE, 2001).

Study 2: A study was conducted to determine the toxicity to aquatic algae of the read across substance, C12-16 ADBAC (49-51% active; radiolabeled purity: 99.7%) according to OECD Guideline 201 and EU Method C.3, in compliance with GLP. Selenastrum capricornutum were exposed for 72 h at nominal concentrations of 0, 3.7, 13, 24, 43, 136 and 431 µg a.i./L under static conditions. Concentrations were analytically determined at the start of the test and after 23, 47 and 72 h. Algal growth was measured by electronic particle counting after 0, 23, 47 and 72 h. The read across substance concentrations were found to be relatively stable during the test and therefore the mean measured read across substance concentrations of 0, 1.2, 5.1, 11, 22, 98 and 382 µg/L were used for calculation of effect parameters. The test fulfilled the validity criteria of sufficient growth and minimum increase of test medium pH. Statistical assessment of data by application of parametric model indicated a significant effect on the growth rate only. Under the study conditions, the 72h ErC50, ErC10, EbC50 and EbC10 were determined to be at 49, 9, 14 and <1.2 µg a.i./L respectively. The 72 h NOEC was ≤1.2 µg a.i./L (Mayer, 2001).

The available information with test or read across substance suggests that algae is not the most sensitive species. Although a direct comparison of the target substance is not possible with the read across substance due to its UVCB nature, generally their 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 ErC50 and NOEC values derived for the test substance has been taken forward for hazard and risk assessment.