Benzododecinium chloride

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2017

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

not specified

GLP compliance:

not specified

Remarks:

It was laboratory research study and therefore GLP compliance is not mandatory.

Analytical monitoring:

yes

Remarks:

High-performance liquid chromatography (HPLC)

Details on sampling:

Chemical analyses of exposure medium contamination were determined using HPLC method. All the measurements provided favorable basic conditions for further toxicity tests. To assess contamination accuracy, analyses were performed for two samples with the lowest and the highest concentrations at the beginning and end of the test according to OECD Guideline 202. The results showed that measured concentrations varied generally less than 5% for DDBAC from the nominal concentrations. As the Guideline describes, if evidence is available to demonstrate that the concentration of the test substance has been satisfactorily maintained within 20% of the nominal or measured initial concentration throughout the test, then the results can be based on nominal or measured
initial values. So, all calculations were based on nominal concentrations in the current study in order to simplify calculations.

Vehicle:

yes

Remarks:

Water

Details on test solutions:

Tests substance was diluted with distilled water to yield 45% stock solution (450,000 mg/L).

Test organisms (species):

Daphnia magna

Details on test organisms:

The D. magna was the pure strain introduced from the Research Center for Pesticide Environmental Toxicology (Beijing, China). Glass beakers (2-L capacity) were kept with ground water from Tai’an, Shandong Province, China (Site: 36°15′17″N, 117°06′15″E) with pH of 6–9, hardness of 140–250 mg/L and the dissolved oxygen concentration is more than 4.0 mg/L even at the end of the test (valid according to OECD Guideline 202). The D. magna were fed daily with the algae
Scenedesmus obliquus at a concentration of 3 × 105 cells/ml. The tested D. magna were younger than 24 h old.

Test type:

static

Water media type:

other: ground water

Limit test:

no

Total exposure duration:

48 h

Hardness:

140–250 mg/L

Test temperature:

19.5-21.1°C

pH:

6 to 9

Dissolved oxygen:

>4 mg/L

Nominal and measured concentrations:

0, 0.001, 0.005, 0.01, 0.05 and 0.1 mg/L nominal test substance concentrations

Details on test conditions:

The acute toxicity test was carried out in accordance with OECD 202 guideline to determine the mobility of D. magna. The test substance was diluted with groundwater in toxicity test. After the pre-test, five or more concentrations and a control treatment with only ground water were designed in the independent
toxicity test. All of the concentrations of the DDBAC used in the official tests were 0.001, 0.005, 0.01, 0.05 and 0.1 mg/L, respectively. Ten neonatal D. magna were added in 50 mL of dilution. Test organisms were not fed during the 48 h-toxicity testing. The test vessels were maintained at 20±1 °C and a photoperiod of 16:8 h (L:D) until the end of experiment. The examined endpoint was immobilisation, which was defined as the inability to swim after 15 s of gentle agitation. The measurements were repeated in triplicate with 30 D. magna for each concentration of chemicals. The pH, hardness and dissolved oxygen concentration were measured before and every 24 h after chemical exposure.

Reference substance (positive control):

not specified

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

ca. 0.007 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% Confidence interval: 0.00309 & 0.0121 mg/L

Details on results:

At the end of experiments, controls in each treatment were observed no more than 10% of the D. magna be immobilized. The pH values of dilutions were 7.5±0.5 while the hardness was kept at 140–250 mg/L and the dissolved oxygen concentrations were more than 4.0 mg/L. All the mentioned performance criteria were favorable for the acute tests to be valid according to OECD Guideline 202 on “Daphnia sp., Acute Immobilisation Test and Reproduction Test”.

Reported statistics and error estimates:

The EC50 values were calculated by the probit regression of the Daphnia immobilisation rates against the log10 values of chemical concentrations. All of the statistical analyses were performed using the SPSS statistical package (ver.17.0, USA). Its regression equation was: Y = 0.827X + 1.802, R2 = 0.922.

Validity criteria fulfilled:

yes

Conclusions:

Under the study conditions, 48 h EC50 value of the test substance for the immobilization of Daphnia magna was determined to be 0.00661 mg/L (95% confidence interval: 0.00309 and 0.0121 mg/L).

Executive summary:

A study was conducted to determine the acute toxicity of the test substance, C12 ADBAC (purity not specified), toDaphnia magnaunder static, according to the OECD Guideline 202. Group of thirty daphnia in ground water were exposed to the test substance concentrations at 0, 0.001, 0.005, 0.01, 0.05 and 0.1 mg/L for 48 h. Test organisms were not fed during the 48 h-toxicity testing. The examined endpoint was immobilisation, which was defined as the inability to swim after 15 s of gentle agitation. The pH, hardness and dissolved oxygen concentration were measured before and every 24 h after chemical exposure. Chemical analyses of exposure medium contamination were determined using HPLC method. The results showed that measured concentrations varied generally less than 5% for DDBAC from the nominal concentrations. Therefore, all calculations were based on nominal concentrations in the current study in order to simplify calculations. The EC50 values were calculated by the probit regression of the Daphnia immobilisation rates against the log10 values of chemical concentrations. The 48 h EC50 for the test substance was determined to be 0.00661 mg/L (95% confidence interval: 0.00309 and 0.0121 mg/L). At the end of experiments, the pH values of dilutions were 7.5±0.5 while the hardness was kept at 140–250 mg/L; the dissolved oxygen concentrations were more than 4.0 mg/L.The controls in each treatment showed no more than 10% of immobility of D. magna. All the mentioned performance criteria were favorable for the acute tests to be valid according to OECD Guideline 202. Under the study conditions, the 48 h EC50 value of the test substance for the immobilization of Daphnia magna was determined to be 0.00661 mg/L (95% confidence interval: 0.00309 and 0.0121 mg/L) (Li, 2018).

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

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 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Vehicle:

yes

Remarks:

Water

Test organisms (species):

Daphnia magna

Details on test organisms:

- Sex: Female
- Age at study initiation: Juvenile (Less than 24 hours after birth)

Test type:

semi-static

Water media type:

other: Elendt M4 medium

Limit test:

no

Total exposure duration:

48 h

Test temperature:

20 +/- 1 deg C

pH:

8.1 - 8.5

Dissolved oxygen:

8.1 - 8.2 mg/L

Nominal and measured concentrations:

0, 0.010, 0.018, 0.032, 0.056, 0.10 mg/L nominal test substance concentrations
0, 0.006, 0.011, 0.024, 0.035, 0.081 mg/L geomatric mean measured test substance concentrations

Details on test conditions:

- Renewal rate of test solution: total amount of test solution after 24 hours
- Test solution volume: 100 mL per vessel
- No. of vessels per concentration (replicates): 4
- No. of organisms per vessel: 20 per concentration (5 per vessel)
- Ligthing: 16 hours light / 8 hours dark

Reference substance (positive control):

not specified

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

ca. 0.032 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% Confidence interval: 0.028 & 0.034 mg/L

For results tables and figure, kindly refer to the attached material section of the IUCLID.

Validity criteria fulfilled:

yes

Conclusions:

Under the study conditions, 48 h EC50 value of the test substance for the immobilization of Daphnia magna was determined to be 0.032 mg/L (95% confidence interval: 0.028 & 0.034) (measured).

Executive summary:

A study was conducted to determine the acute toxicity of test substance, C12 ADBAC (purity: >99%), to Daphnia magna, according to the OECD Guideline 202, in compliance with GLP. The study was performed under semi-static conditions, with a daily renewal of medium. Groups of 20 juvenile fish were exposed for 48 h to five concentrations of test substance nominal concentrations of 0, 0.010, 0.018, 0.032, 0.056, 0.10 mg/L, in Elendt M4 medium. A control group of 20 fish was placed into Elendt M4 medium alone. Test solution was renewed daily. The exposure levels of test substance in aqueous samples of test media were monitored using a Fluorometry. The geomatric mean measured concentrations of test substance were determined to be 0, 0.006, 0.011, 0.024, 0.035, 0.081 mg/L. The 48 h EC50 and EC100 values were determined to be 0.032 mg/L (95% confidence interval: 0.028 & 0.034) and 0.081 mg/L (measured) respectively. The no observed effect concentration (NOEC) was determined to be 0.024 mg/L (measured). No mortality was observed in control group fish and dissolved oxygen concentration was 8.1 to 8.2. Under the study conditions, the 48 h EC50 value (95% Confidence interval) and NOEC value were determined to be 0.032 mg/L (95% confidence interval: 0.028 & 0.034) and 0.024 mg/L measured concentrations (geometric mean), respectively (NITE, 2001).

Reference 3

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

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

Adequacy of study:

supporting study

Study period:

From February 22, 2007 to February 24, 2007

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

KL2 due to RA

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

yes

Remarks:

but no impact on the study as the validity criteria for the study were met

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

yes

Remarks:

but no impact on the study as the validity criteria for the study were met

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1010 (Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids)

Deviations:

yes

Remarks:

but no impact on the study as the validity criteria for the study were met

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Name of test material (as cited in study report):ADBAC (i.e., benzyl C12-16 alkyl dimethyl ammonium chloride)
- Physical state: Clear liquid
- Analytical purity: 51%
- Lot/batch No.: DEGE108913
- Expiration date of the lot/batch: April 2007
- Stability under test conditions: Hydrolytically and photolytically stable under the conditions of this study and under standard laboratory conditions.
- Storage condition of test material: Ambient temperature
- Other: The test substance is known to be readily soluble, hydrolytically stable and has a very low vapour pressure.

Analytical monitoring:

yes

Details on sampling:

Two samples (5 mL) were taken from the freshly-prepared control and test media. After 48h, the contents of the test vessels from each group were pooled and further samples (5 mL) were taken for analysis. The samples were taken using a pipette and placed in glass vials; both of these items were pre-treated with a silanising agent (DMDCS) before use.

On each occasion, one of the samples was analysed and the other was stored in case further analysis was required. The reserve samples were stored frozen; it was not considered necessary to analyse these samples since the results obtained for the original samples were considered acceptable. Thus, the reserve samples were discarded.

Details on test solutions:

Preparation of test solutions:
The test organisms were maintained and the tests were conducted in softened Elendt M4 medium. The medium was prepared in deionised water produced by reverse osmosis. A stock solution for preparation of the test substance concentrations was prepared by diluting an aliquot of the test substance (19.6 mg) in the dilution medium (0.5 L) in a volumetric flask (1L). The contents of this flask were shaken vigorously before it was adjusted to volume with dilution medium. Aliquots (0.7, 1.2, 1.9, 3.1 or 5.5mL) of this stock solution were diluted with dilution medium to provide the test media.
- Mean water quality parameters, including temperature, pH, dissolved oxygen, total hardness and alkalinity remained within acceptable limits throughout the study.

Monitoring of the test substance concentration:
Duplicate samples of each freshly prepared control and test media were collected and the concentration of the test substance measured using an LC-MS method of analysis. The measured concentrations of the test substance ranged between 80 and 97% of their nominal concentrations at the start of the test confirming that the intended exposure concentrations were achieved, so test results are expressed in terms of their nominal values. After 48h, the measured levels ranged between 30 and 42% of nominal. The test substance is known to be readily soluble, hydrolytically stable, and has a very low vapour pressure. In addition, the test vessels were pre-treated with the test substance to prevent loss of the test substance to absorption/binding to the vessel walls. Therefore, the reduction of test substance concentration observed during the exposure period was attributed to absorption onto the test organism (i.e. representative of exposure of the organisms to the nominal concentrations) and the EC50 value was determined using nominal concentrations.

Test organisms (species):

Daphnia magna

Details on test organisms:

Original Daphnia broodstock was obtained from the Institute National de Recherche Chimique Appliqué (IRChA), France. Stock cultures of Daphnia magna were maintained in glass vessels containing approximately 0.8L of Elendt M4 culture medium in a temperature-controlled laboratory at nominally 20 ± 2ºC. A photoperiod of 16h light: 8h dark was maintained, with 60-min transition periods of subdued lighting at the beginning and end of each light phase. The light intensity of the test area was 611 lux.

- The culture medium was renewed three times each week.

-Cultures were fed daily with a suspension of the unicellular green algae, Pseudokirchneriella subcapitata, to provide nominally 0.1 to 0.2 mg carbon per daphnid, per day, except during the initial three days when a slightly lower ration was given (0.05 to 0.08 mg carbon per daphnid).
- Culture conditions ensure that the stock animals reproduce by pathenogenesis.
- The day before the start of the study, all juvenile Daphnia were removed from the laboratory cultures. The following morning, juveniles produced by gravid adult Daphnia were removed from the culture vessels and held in a separate holding vessel; these animals, which were less than 24h old, were used in the test.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test temperature:

20 ± 1ºC

pH:

7.44-7.73

Dissolved oxygen:

99-104% air saturation value

Nominal and measured concentrations:

Nominal concentration: 0.007, 0.012, 0.019, 0.031, 0.052 mg a.i./L.

Details on test conditions:

Twenty juvenile daphnids (<24 h old) were exposed in each control and test group at the following nominal concentrations: 0.007, 0.012, 0.019, 0.031, and 0.052 mg a.i./L. The Daphnia were placed at random into glass vessels, four replicates of five animals per vessel, each containing approximately 150 mL of medium (resulting in a loading rate of 30 mL medium per organism). Each vessel was loosely covered with a watch glass. The temperature of the test area was 20 ± 1ºC. A 16-hour light: 8-hour dark photoperiod was maintained, with 60-minute periods of subdued lighting at the beginning and end of each phase. No supplementary aeration was employed and no feed was given during the exposure period.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

0.023 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mobility

Remarks on result:

other: 0.019-0.24 mg a.i./L

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.016 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mobility

Remarks on result:

other: 0.0144-0.0177 mg a.i./L

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.012 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

0.031 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mobility

Details on results:

Test substance analysis:
The measured concentration of the test substance ranged between 80-97% of their nominal concentrations at the start of the test confirming that the intended exposure concentrations were achieved, so test results are expressed in terms of their nominal values. After 48h, the measured levels ranged between 30 and 42% of nominal. The test substance is known to be readily soluble, hydrolytically stable and has a very low vapour pressure. In addition, the test vessels were pre-treated with the test substance to prevent loss of the test substance to absorption/binding to the vessel walls. Therefore, the reduction of test substance concentration observed during the exposure period was attributed to absorption onto the test organism (i.e., representative of exposure of the organism to the nominal concentrations) and the EC50 value was determined using nominal concentrations.
Immobility: See the above results under 'Effect concentrations' and the table under 'any other information on results incl. tables'.

Results with reference substance (positive control):

The results from the most recent test performed prior to this study indicated that the reference substance 4h EC50 to Daphnia magna was 0.52 mg/L. This was within the range typically obtained in the laboratory (0.3 to 0.8 mg/L)

Reported statistics and error estimates:

Statistical analysis was performed using the SAFEStat LD50 application, SAS 8.2 (SAS Institute, 1999). Test results were expressed in terms of the nominal concentrations. The “no observed effect concentration” (NOEC) was derived by direct inspection of the data on the immobility of the animals. An incidence rate of more than 10% was considered to be significant.

Table 1. Immobilisation data

Concentration (mg a.i./l)	Percent Immobile at:
	24 h	48 h
0.0	0	0
0.007	0	0
0.012	0	5
0.019	15	85
0.031	100	100
0.052	100	100

For further details on all measurements, please refer to the attachment under 'Attached bakground material'.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the study, the 48h EC50 value of the test substance for the immobilisation of Daphnia magna was 0.016 mg a.i./L and the 48h NOEC was 0.012 mg a.i./L.

Executive summary:

A study was conducted to determine the short-term toxicity of the test substance, C12 ADBAC (51% active) to Daphnia magna according to OECD Guideline 202, EU Method C.2 and US EPA OPPTS 850.1010, in compliance with GLP. Twenty juvenile daphnids (<24 h old) were exposed in each control and test group at the following nominal concentrations: 0.007, 0.012, 0.019, 0.031, and 0.052 mg a.i./L under static conditions. Duplicate samples of each freshly prepared control and test media were collected and the concentration of test substance was measured using an LC-MS method of analysis. The measured concentrations ranged between 80 and 97% of their nominal concentrations at the start of the test confirming that the intended exposure concentrations were achieved, so test results are expressed in terms of their nominal values. After 48 hours, the measured levels ranged between 30 and 42% of nominal. The test substance is known to be readily soluble, hydrolytically stable, and has a very low vapour pressure. In addition, the test vessels were pre-treated with the test substanance to prevent loss of the test substance to absorption/binding to the vessel walls. Therefore, the reduction of test substance concentration observed during the exposure period was attributed to absorption onto the test organism (i.e. representative of exposure of the organisms to the nominal concentrations) and the EC50 value was therefore determined using nominal concentrations. Observations (for mobility) of the Daphnia were made after approximately 24 and 48 h. The “no observed effect concentration” (NOEC) was derived by direct inspection of the data on the immobility of the animals. An incidence rate of more than 10% was considered to be significant. Under the conditions of the study, the 48 h EC50 was 0.016 mg a.i./L and the 48 h NOEC was 0.012 mg a.i./L (Jenkins, 2007).

Reference 4

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From February 22, 2007 to February 24, 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

yes

Remarks:

but no impact on the study as the validity criteria for the study were met

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

yes

Remarks:

but no impact on the study as the validity criteria for the study were met

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1010 (Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids)

Deviations:

yes

Remarks:

but no impact on the study as the validity criteria for the study were met

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Name of test material (as cited in study report):ADBAC (i.e., benzyl C12-16 alkyl dimethyl ammonium chloride)
- Physical state: Clear liquid
- Analytical purity: 51%
- Lot/batch No.: DEGE108913
- Expiration date of the lot/batch: April 2007
- Stability under test conditions: Hydrolytically and photolytically stable under the conditions of this study and under standard laboratory conditions.
- Storage condition of test material: Ambient temperature
- Other: The test substance is known to be readily soluble, hydrolytically stable and has a very low vapour pressure.

Analytical monitoring:

yes

Details on sampling:

Two samples (5 mL) were taken from the freshly-prepared control and test media. After 48h, the contents of the test vessels from each group were pooled and further samples (5 mL) were taken for analysis. The samples were taken using a pipette and placed in glass vials; both of these items were pre-treated with a silanising agent (DMDCS) before use.

On each occasion, one of the samples was analysed and the other was stored in case further analysis was required. The reserve samples were stored frozen; it was not considered necessary to analyse these samples since the results obtained for the original samples were considered acceptable. Thus, the reserve samples were discarded.

Details on test solutions:

Preparation of test solutions:
The test organisms were maintained and the tests were conducted in softened Elendt M4 medium. The medium was prepared in deionised water produced by reverse osmosis. A stock solution for preparation of the test substance concentrations was prepared by diluting an aliquot of the test substance (19.6 mg) in the dilution medium (0.5 L) in a volumetric flask (1L). The contents of this flask were shaken vigorously before it was adjusted to volume with dilution medium. Aliquots (0.7, 1.2, 1.9, 3.1 or 5.5mL) of this stock solution were diluted with dilution medium to provide the test media.
- Mean water quality parameters, including temperature, pH, dissolved oxygen, total hardness and alkalinity remained within acceptable limits throughout the study.

Monitoring of the test substance concentration:
Duplicate samples of each freshly prepared control and test media were collected and the concentration of the test substance measured using an LC-MS method of analysis. The measured concentrations of the test substance ranged between 80 and 97% of their nominal concentrations at the start of the test confirming that the intended exposure concentrations were achieved, so test results are expressed in terms of their nominal values. After 48h, the measured levels ranged between 30 and 42% of nominal. The test substance is known to be readily soluble, hydrolytically stable, and has a very low vapour pressure. In addition, the test vessels were pre-treated with the test substance to prevent loss of the test substance to absorption/binding to the vessel walls. Therefore, the reduction of test substance concentration observed during the exposure period was attributed to absorption onto the test organism (i.e. representative of exposure of the organisms to the nominal concentrations) and the EC50 value was determined using nominal concentrations.

Test organisms (species):

Daphnia magna

Details on test organisms:

Original Daphnia broodstock was obtained from the Institute National de Recherche Chimique Appliqué (IRChA), France. Stock cultures of Daphnia magna were maintained in glass vessels containing approximately 0.8L of Elendt M4 culture medium in a temperature-controlled laboratory at nominally 20 ± 2ºC. A photoperiod of 16h light: 8h dark was maintained, with 60-min transition periods of subdued lighting at the beginning and end of each light phase. The light intensity of the test area was 611 lux.

- The culture medium was renewed three times each week.

-Cultures were fed daily with a suspension of the unicellular green algae, Pseudokirchneriella subcapitata, to provide nominally 0.1 to 0.2 mg carbon per daphnid, per day, except during the initial three days when a slightly lower ration was given (0.05 to 0.08 mg carbon per daphnid).
- Culture conditions ensure that the stock animals reproduce by pathenogenesis.
- The day before the start of the study, all juvenile Daphnia were removed from the laboratory cultures. The following morning, juveniles produced by gravid adult Daphnia were removed from the culture vessels and held in a separate holding vessel; these animals, which were less than 24h old, were used in the test.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test temperature:

20 ± 1ºC

pH:

7.44-7.73

Dissolved oxygen:

99-104% air saturation value

Nominal and measured concentrations:

Nominal concentration: 0.007, 0.012, 0.019, 0.031, 0.052 mg a.i./L.

Details on test conditions:

Twenty juvenile daphnids (<24 h old) were exposed in each control and test group at the following nominal concentrations: 0.007, 0.012, 0.019, 0.031, and 0.052 mg a.i./L. The Daphnia were placed at random into glass vessels, four replicates of five animals per vessel, each containing approximately 150 mL of medium (resulting in a loading rate of 30 mL medium per organism). Each vessel was loosely covered with a watch glass. The temperature of the test area was 20 ± 1ºC. A 16-hour light: 8-hour dark photoperiod was maintained, with 60-minute periods of subdued lighting at the beginning and end of each phase. No supplementary aeration was employed and no feed was given during the exposure period.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

0.023 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mobility

Remarks on result:

other: 0.019-0.24 mg a.i./L

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.016 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mobility

Remarks on result:

other: 0.0144-0.0177 mg a.i./L

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.012 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

0.031 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mobility

Details on results:

Test substance analysis:
The measured concentration of the test substance ranged between 80-97% of their nominal concentrations at the start of the test confirming that the intended exposure concentrations were achieved, so test results are expressed in terms of their nominal values. After 48h, the measured levels ranged between 30 and 42% of nominal. The test substance is known to be readily soluble, hydrolytically stable and has a very low vapour pressure. In addition, the test vessels were pre-treated with the test substance to prevent loss of the test substance to absorption/binding to the vessel walls. Therefore, the reduction of test substance concentration observed during the exposure period was attributed to absorption onto the test organism (i.e., representative of exposure of the organism to the nominal concentrations) and the EC50 value was determined using nominal concentrations.
Immobility: See the above results under 'Effect concentrations' and the table under 'any other information on results incl. tables'.

Results with reference substance (positive control):

The results from the most recent test performed prior to this study indicated that the reference substance 4h EC50 to Daphnia magna was 0.52 mg/L. This was within the range typically obtained in the laboratory (0.3 to 0.8 mg/L)

Reported statistics and error estimates:

Statistical analysis was performed using the SAFEStat LD50 application, SAS 8.2 (SAS Institute, 1999). Test results were expressed in terms of the nominal concentrations. The “no observed effect concentration” (NOEC) was derived by direct inspection of the data on the immobility of the animals. An incidence rate of more than 10% was considered to be significant.

Table 1. Immobilisation data

Concentration (mg a.i./l)	Percent Immobile at:
	24 h	48 h
0.0	0	0
0.007	0	0
0.012	0	5
0.019	15	85
0.031	100	100
0.052	100	100

For further details on all measurements, please refer to the attachment under 'Attached bakground material'.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the study, the 48h EC50 value of the test substance for the immobilisation of Daphnia magna was 0.016 mg a.i./L and the 48h NOEC was 0.012 mg a.i./L.

Executive summary:

A study was conducted to determine the short-term toxicity of the test substance, C12 -16 ADBAC (51% active) to Daphnia magna according to OECD Guideline 202, EU Method C.2 and US EPA OPPTS 850.1010, in compliance with GLP. Twenty juvenile daphnids (<24 h old) were exposed in each control and test group at the following nominal concentrations: 0.007, 0.012, 0.019, 0.031, and 0.052 mg a.i./L under static conditions. Duplicate samples of each freshly prepared control and test media were collected and the concentration of test substance was measured using an LC-MS method of analysis. The measured concentrations ranged between 80 and 97% of their nominal concentrations at the start of the test confirming that the intended exposure concentrations were achieved, so test results are expressed in terms of their nominal values. After 48 hours, the measured levels ranged between 30 and 42% of nominal. The test substance is known to be readily soluble, hydrolytically stable, and has a very low vapour pressure. In addition, the test vessels were pre-treated with the test substance to prevent loss of the test substance to absorption/binding to the vessel walls. Therefore, the reduction of test substance concentration observed during the exposure period was attributed to absorption onto the test organism (i.e. representative of exposure of the organisms to the nominal concentrations) and the EC50 value was therefore determined using nominal concentrations. Observations (for mobility) of the Daphnia were made after approximately 24 and 48 h. The “no observed effect concentration” (NOEC) was derived by direct inspection of the data on the immobility of the animals. An incidence rate of more than 10% was considered to be significant. Under the conditions of the study, the 48 h EC50 was 0.016 mg a.i./L and the 48 h NOEC was 0.012 mg a.i./L (Jenkins, 2007).

Description of key information

The 48 h EC50 value of the test substance, which was determined at 32 µg/L (measured, geometric mean), based on effects on mobility, has been considered further for hazard and risk assessment.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

32 µg/L

Additional information

Study 1: A study was conducted to determine the acute toxicity of test substance, C12 ADBAC (purity: >99%), to Daphnia magna, according to the OECD Guideline 202, in compliance with GLP. The study was performed under semi-static conditions, with a daily renewal of medium. Groups of 20 juvenile fish were exposed for 48 h to five concentrations of test substance nominal concentrations of 0, 0.010, 0.018, 0.032, 0.056, 0.10 mg/L, in Elendt M4 medium. A control group of 20 fish was placed into Elendt M4 medium alone. Test solution was renewed daily. The exposure levels of test substance in aqueous samples of test media were monitored using a Fluorometry. The geomatric mean measured concentrations of test substance were determined to be 0, 0.006, 0.011, 0.024, 0.035, 0.081 mg/L. The 48 h EC50 and EC100 values were determined to be 0.032 mg/L (95% confidence interval: 0.028 & 0.034) and 0.081 mg/L (measured) respectively. The no observed effect concentration (NOEC) was determined to be 0.024 mg/L (measured). No mortality was observed in control group fish and dissolved oxygen concentration was 8.1 to 8.2. Under the study conditions, the 48 h EC50 value (95% Confidence interval) and NOEC value were determined to be 0.032 mg/L (95% confidence interval: 0.028 & 0.034) and 0.024 mg/L measured concentrations (geometric mean), respectively (NITE, 2001).

Study 2: A study was conducted to determine the acute toxicity of the test substance, C12 ADBAC (purity not specified), toDaphnia magnaunder static, according to the OECD Guideline 202. Group of thirty daphnia in ground water were exposed to the test substance concentrations at 0, 0.001, 0.005, 0.01, 0.05 and 0.1 mg/L for 48 h. Test organisms were not fed during the 48 h-toxicity testing. The examined endpoint was immobilisation, which was defined as the inability to swim after 15 s of gentle agitation. The pH, hardness and dissolved oxygen concentration were measured before and every 24 h after chemical exposure. Chemical analyses of exposure medium contamination were determined using HPLC method. The results showed that measured concentrations varied generally less than 5% for DDBAC from the nominal concentrations. Therefore, all calculations were based on nominal concentrations in the current study in order to simplify calculations. The EC50 values were calculated by the probit regression of the Daphnia immobilisation rates against the log10 values of chemical concentrations. The 48 h EC50 for the test substance was determined to be 0.00661 mg/L (95% confidence interval: 0.00309 and 0.0121 mg/L). At the end of experiments, the pH values of dilutions were 7.5±0.5 while the hardness was kept at 140–250 mg/L; the dissolved oxygen concentrations were more than 4.0 mg/L.Thecontrols in each treatment showed no more than 10% of immobility of D. magna. All the mentioned performance criteria were favorable for the acute tests to be valid according to OECD Guideline 202. Under the study conditions, the 48 h EC50 value of the test substance for the immobilization of Daphnia magna was determined to be 0.00661 mg/L (95% confidence interval: 0.00309 and 0.0121 mg/L) (Li, 2018).

(Li, 2018).

As expected, the presence of higher suspended particles in the ground water together with the strong adsorption potential of the test substance, results in EC50 values, which are 10 -fold lower than the study conducted with guideline-compliant test media. Therefore, given the uncertainty around the use of ground water as a test media and its value for hazard or classification and labelling, the 48 h EC50 value of 0.032 mg/L has been considered further for hazard and risk assessment.

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 Daphnia magna (presented below), which were determined to be in the same range:

Study3: A study was conducted to determine the short-term toxicity of the test substance, C12 -16 ADBAC (51% active) to Daphnia magna according to OECD Guideline 202, EU Method C.2 and US EPA OPPTS 850.1010, in compliance with GLP. Twenty juvenile daphnids (<24 h old) were exposed in each control and test group at the following nominal concentrations: 0.007, 0.012, 0.019, 0.031, and 0.052 mg a.i./L under static conditions. Duplicate samples of each freshly prepared control and test media were collected and the concentration of test substance was measured using an LC-MS method of analysis. The measured concentrations ranged between 80 and 97% of their nominal concentrations at the start of the test confirming that the intended exposure concentrations were achieved, so test results are expressed in terms of their nominal values. After 48 hours, the measured levels ranged between 30 and 42% of nominal. The test substance is known to be readily soluble, hydrolytically stable, and has a very low vapour pressure. In addition, the test vessels were pre-treated with the test substance to prevent loss of the test substance to absorption/binding to the vessel walls. Therefore, the reduction of test substance concentration observed during the exposure period was attributed to absorption onto the test organism (i.e. representative of exposure of the organisms to the nominal concentrations) and the EC50 value was therefore determined using nominal concentrations. Observations (for mobility) of the Daphnia were made after approximately 24 and 48 h. The “no observed effect concentration” (NOEC) was derived by direct inspection of the data on the immobility of the animals. An incidence rate of more than 10% was considered to be significant. Under the conditions of the study, the 48 h EC50 was 0.016 mg a.i./L and the 48 h NOEC was 0.012 mg a.i./L (Jenkins, 2007).