Quaternary ammonium compounds, C12-14-alkyl[(ethylphenyl)methyl]dimethyl, chlorides

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From May 22, 2007 to July 30, 2007

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

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Chemical name:N-Alkyl(C12-C14)-N,N-dimethyl-N-ethylbenzylammonium chloride (ADEBAC)
Lot number:7226376
Expiry date:21 February 2008
Purity: 80.93% N-Alkyl(C12-C14)-N,N-dimethyl-N-ethylbenzylammonium chloride (ADEBAC), 9% ethanol, 1.6% free amine + Amine HCL
Appearance:Clear yellow liquid
Storage conditions:Room temperature in the dark
Stability: The a.i., ADEBAC, is hydrolytically and photolytically stable under the conditions of this study and related quaternary ammonium compounds have been shown to be stable in aqueous, alcohol and alcohol/aqueous solutions for extended periods, e.g. at least seven years under standard laboratory conditions

Analytical monitoring:

yes

Details on sampling:

The concentrations of test substance in samples of the aqueous test media were measured during the definitive test using a LC-MS method of analysis. Two samples (5 mL) were taken from the freshly-prepared control and test media. After 48 h, the contents of the test vessels from each group were pooled and further samples (5 mL) were taken for analysis. The samples were placed in polypropylene tubes containing an aliquot (5 mL) of methanol containing formic acid (0.2%). On each occasion, one of the samples was analysed and the other was stored frozen in case further analysis was required. It was not considered necessary to analyse these reserve samples since the results obtained for the original samples were considered acceptable. Thus, these reserve samples were discarded.

Vehicle:

yes

Remarks:

Water/Alcohol

Details on test solutions:

The test substance provided was a stock solution of 80% test substance (nominally) in water/alcohol. Test solutions were prepared on the basis of the test substance as provided. However, this study was designed to determine the effects of the active ingredient (test substance); therefore, throughout this report, the exposure concentrations and test results have been expressed in terms of active ingredient (test substance). The following method of test solution preparation used during the definitive test was based on the results of a range finding test and an analytical trial. To minimise any loss of test substance by adsorption, all glassware used in the study was pre- conditioned to the test substance at an appropriate concentration overnight before use. The pre-conditioning media were discarded immediately before the test solutions were freshly prepared. For the definitive test, an aqueous stock solution (nominal, 8.09 mg a.i./L) was prepared by adding the test substance (10 mg) directly to the dilution medium (ca. 0.5 L) in a volumetric flask (1 L). The contents of this flask were shaken vigorously before it was adjusted to volume with dilution medium (1 L). Aliquots (0.625, 1.25, 2.5, 5 or 10 mL/L) of this stock solution were diluted with dilution medium to provide the test media.

Test organisms (species):

Daphnia magna

Details on test organisms:

Stock cultures of Daphnia magna were maintained in glass vessels containing between 500 and 1500 mL of Elendt M4 culture medium in a temperature-controlled laboratory at nominally 20 ± 2ºC. A photoperiod of 16 h light: 8 h dark was maintained, with 60-minute transition periods of subdued lighting at the beginning and end of each light phase. Original Dapnhia broodstock was obtained from the Institute National de Recherche Chimique Appliqué (IRChA), France. 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 parthenogenesis.The day before the start of the study, all juvenile Daphnia were removed from the laboratory cultures. The following morning, juveniles produced by the gravid (egg-bearing) adult Daphnia were removed from the culture vessels and held in a separate holding vessel; these animals, which were less than 24 h old, were used in the test.

Dilution medium
The test organisms were maintained and the tests conducted in Elendt M4 medium. The medium was prepared in deionised water produced by reverse osmosis.

Test type:

static

Water media type:

other: Elendt M4 medium

Limit test:

no

Total exposure duration:

48 h

Hardness:

The total hardness and alkalinity of the batch of Elendt M4 medium used were 232 and 45 mg/L as CaCO3, respectively.

Test temperature:

20.2 - 21.2°C

pH:

7.49-7.81

Dissolved oxygen:

as 97-101% Air saturation value (ASV)

Nominal and measured concentrations:

0.00506, 0.0101, 0.0202, 0.0405 and 0.0809 mg a.i./L nominal test substance

Details on test conditions:

Experimental design and test concentrations
The study comprised a range finding test, an analytical trial and a definitive test, all conducted under static (no renewal) conditions. The range finding test employed nominal concentrations of 0.000809, 0.00809, 0.0809 and 0.809 mg a.i./L in which the test substance was dispersed in the dilution medium. A dilution medium control group was also included in the test. Ten Daphnia, equally divided between two replicate vessels, were employed for each control and test group. After 48 h, all of the Daphnia exposed at 0.0809 and 0.809 mg a.i./L were immobile and no adverse effects were noted at 0.000809 or 0.00809 mg a.i./L. No results were obtained for samples taken during the range finding test due to a problem with the original analytical method. Following the development of a suitable analytical method, an analytical trial was performed in which a batch of test medium was prepared at nominal 0.00809 mg a.i./L. Analysis of a sample of this medium confirmed that the intended exposure concentration could be adequately achieved (80% of nominal). Based on the results of the range finding test, the definitive test employed nominal test substance concentrations of 0.00506, 0.0101, 0.0202, 0.0405 and 0.0809 mg a.i./L and a control group.

Reference substance (positive control):

not specified

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

ca. 0.015 other: mg a.i./L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mobility

Remarks on result:

other:

Remarks:

(95% confidence limits of 0.0139 and 0.0202 mg a.i./L).

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

ca. 0.01 other: mg a.i./L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mobility

Key result

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

ca. 0.018 other: mg a.i./L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mobility

Remarks on result:

other:

Remarks:

(95% confidence limits of 0.0165 and 0.0208 mg a.i./L)

Key result

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

ca. 0.041 other: mg a.i./L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mobility

Details on results:

Results
At the start of the test, the measured concentrations of test substance in samples of the aqueous test media ranged between 78 and 85% of their nominal values. At 48 h, in samples of expired media, the measured levels at 0.0101 to 0.0809 mg a.i./L ranged between 40 and 78% of their nominal values (between 47 and 100% of their starting concentrations), with the greatest losses occurring at the lower concentrations. At the lowest test level (0.00506 mg a.i./L), the measured concentration had decreased to below the limit of detection of the analytical method (0.002 mg a.i./L). The intended exposure concentrations were achieved. In addition, the test substance is known to be readily soluble and is hydrolytically stable, and since the test vessels were pre-treated with test substance to prevent loss due to absorption/binding to the vessel walls, any reduction of test substance concentration observed during the exposure period was attributed to absorption onto the test organism (i.e. representative of exposure to the nominal concentrations). Therefore, effect concentrations (EC50 values) were determined based on nominal concentrations of the active ingredient, test substance.

Immobility

Observations of the Daphnia in each control and test vessel were made after 24 and 48 h. Based on these findings the following values have been estimated:

Table 1: Cumulative immobilisation

Nominal exposure concentrations (mg a.i./L) test substance	Cumulative numbers of immobileDaphnia
	24 hours						48 hours
	R1	R2	R3	R4	total	%	R1	R2	R3	R4	total	%
Control	0	0	0	0	0	0	0	0	0	0	0	0
0.00506	0	0	0	0	0	0	0	0	0	0	0	0
0.0101	0	0	0	0	0	0	0	0	0	0	0	0
0.0202	4	2	4	3	13	65	5	4	4	5	18	90
0.0405	5	5	5	5	20	100	5	5	5	5	20	100
0.0809	5	5	5	5	20	100	5	5	5	5	20	100
Rn        :          replicate number.

 

 

Table 2: Key dose descriptor values

	Test substance (mg a.i./L; nominal)
24 h EC50 values (95% Confidence Interval)	0.0179 (0.0165 & 0.0208)
48 h EC50 values (95% Confidence Interval)	0.0154 (0.0139 & 0.0202)
100% immobility	0.0405
No-observed effect concentration (NOEC)	0.0101

Validity criteria fulfilled:

yes

Conclusions:

Under study conditions, the 48 h EC50 and NOEC values for test substance with Daphnia magna were found to be 0.0154 mg a.i./L (95% confidence limits of 0.0139 and 0.0202 mg a.i./L) and 0.0101 mg a.i./L respectively.

Executive summary:

A study was conducted to determine the acute toxicity study of the test substance, C12-14 ADEBAC (active: 80.93%), to the Daphnia magna according to OECD Guideline 202 and EU Method C.2, in compliance with GLP. Daphnia magna were exposed to the test substance at nominal concentrations of 0.00506, 0.0101, 0.0202, 0.0405 and 0.0809 mg a.i./L for 48 h under static conditions. The numbers of mobile, immobile and floating Daphnia were counted approximately 24 and 48 h after the start of the study.The exposure levels of the test substance in aqueous samples of the test media were monitored using a LC-MS method of analysis. Since the intended exposure concentrations were substantially acheived and maintained, the test results were expressed in terms of their nominal values. The 48 h EC50 of test substance for the immobilisation of Daphnia magna was determined to be at 0.0154 mg a.i./L (95% confidence limits of 0.0139 and 0.0202 mg a.i./L) and the NOEC was at 0.0101 mg a.i./L. 100% immobilisation was observed at 0.0405 mg a.i./L. No adverse effects were observed in the control replicates during the study.Under study conditions, the48 h EC50 and NOEC values for the test substance with Daphnia magna were determined to be0.0154 mg a.i./L (95% confidence limits of 0.0139 and 0.0202 mg a.i./L) and 0.0101 mg a.i./L respectively (Jenkins, 2008).

Description of key information

The 48 h EC50 and NOEC values for the test substance, C12-14 ADEBAC, with Daphnia magna were determined to be 0.0154 and 0.0101 mg a.i./L respectively (nominal).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.015 mg/L

Additional information

A study was conducted to determine the acute toxicity study of the test substance, C12-14 ADEBAC (purity: 80.93%), to the Daphnia magna according to OECD Guideline 202 and EU Method C.2, in compliance with GLP. Daphnia magna were exposed to the test substance at nominal concentrations of 0.00506, 0.0101, 0.0202, 0.0405 and 0.0809 mg a.i./L for 48 h under static conditions. The numbers of mobile, immobile and floating Daphnia were counted approximately 24 and 48 h after the start of the study. The exposure levels of the test substance in aqueous samples of the test media were monitored using a LC-MS method of analysis. Since the intended exposure concentrations were substantially acheived, the test results were expressed in terms of their nominal values. The 48 h EC50 of test substance for the immobilisation of Daphnia magna was determined to be at 0.0154 mg a.i./L (95% confidence limits of 0.0139 and 0.0202 mg a.i./L) and the NOEC was at 0.0101 mg a.i./L. 100% immobilisation was observed at 0.0405 mg a.i./L. No adverse effects were observed in the control replicates during the study. Under study conditions, the 48 h EC50 and NOEC values for the test substance with Daphnia magna were determined to be 0.0154 mg a.i./L (95% confidence limits of 0.0139 and 0.0202 mg a.i./L) and 0.0101 mg a.i./L respectively (Jenkins, 2008).