Miristalkonium chloride

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 23, 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

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-benzylammonium chloride (ADBAC C12-C14)
Lot number: 612212
Expiry date: 21 December 2009
Purity: 99.1% C14 ADBAC, 0.6% Free amine, 0.4% Amine hydrochloride
Appearance: White powder
Storage conditions: Room temperature
Stability: The a.i., ADBAC, is hydrolytically and photolytically stable under the conditions of this study and has been shown to be stable in aqueous, alcohol and alcohol/aqueous solutions for extended periods, e.g. at least five 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

Details on test solutions:

The test solutions were not prepared on the basis of the percent active ingredient. The following method of test solution preparation used during the definitive test was based on the results of a formulation trial and range finding test. 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 10 mg/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 per L) of this stock solution were diluted with dilution medium to provide the test media.

Test organisms (species):

Daphnia magna

Details on test organisms:

Test organism
Daphnia magna used in this study were cultured in-house and were obtained from a strain originating from the Institute National de Recherche Chimique Appliqué (IRChA), France. This species has been selected based on its known sensitivity to changes in water quality, availability throughout the year and ease of maintenance. Stock cultures of Daphnia magna were maintained in glass vessels containing approximately 0.8 L of Elendt M4 culture medium in a temperature-controlled laboratory at nominally 20 ± 2°C. 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. Based on the laboratory measurements conducted nearest to the definitive test (3 May 2007) the light intensity of the culture area was 272 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 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 (Appendix 2). 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

Test temperature:

19.5-21.1°C

pH:

7.54-7.95

Dissolved oxygen:

101-103% ASV (air saturation value)

Nominal and measured concentrations:

0, 0.00625, 0.0125, 0.025, 0.05 and 0.1 mg/L

Details on test conditions:

Experimental design and test concentrations
The study comprised of a range finding test, two analytical trials and a definitive test, all conducted under static (no renewal) conditions. The range finding test employed nominal concentrations of 0.001, 0.01, 0.1 and 1 mg/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 0.1 and 1 mg/L were immobile and no adverse effects were noted at 0.001 or 0.01 mg/L. No results were obtained for samples taken during the range finding test due to a problem with the original analytical method. Two analytical trials were performed, the first of which was invalid due to analytical problems. Following the development of a suitable analytical method, a second trial was performed in which a batch of test medium was prepared at nominal 0.01 mg/L. Analysis of a sample of this medium confirmed that the intended exposure concentration could be adequately achieved (87% of nominal). Based on the results of the range finding test, the definitive test employed nominal ADBAC C12-C14 concentrations of 0.00625, 0.0125, 0.025, 0.05 and 0.1 mg/L and a control group.

Reference substance (positive control):

not specified

Key result

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

ca. 0.021 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% Confidence interval: 0.0175 & 0.0239 mg/L

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

ca. 0.016 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% Confidence interval: 0.0125 & 0.0181 mg/L

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

ca. 0.013 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

ca. 0.025 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

At the start of the test, the measured concentrations of test substance in samples of the aqueous test media ranged between 89 and 108% of their nominal values. In samples of expired media, at 48 h, levels ranged between 29 and 85% of their nominal values (between 27 and 96% of their starting concentrations), with the greatest losses occurring at the lower concentrations. The intended exposure concentrations were achieved. 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 avoid loss due to binding to the vessel walls, the reduction in 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.

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

Test substance exposure concentrations (mg a.i./L)	Cumulative numbers of immobile Daphnia
	24 h						48 h
	R1	R2	R3	R4	total	%	R1	R2	R3	R4	total	%
Control	0	0	0	0	0	0	0	0	0	0	0	0
0.00625	0	0	0	0	0	0	0	0	0	0	0	0
0.0125	0	0	1	0	1	5	0	0	1	1	2	10
0.025	1	5	4	5	15	75	5	5	5	5	20	100
0.05	5	5	5	5	20	100	5	5	5	5	20	100
0.1	5	5	5	5	20	100	5	5	5	5	20	100
Rn        :          replicate number.

Table 2: Key derived safety descriptor values

	Test substance (mg/L; nominal)
24 h EC50  value (95% Confidence Interval)	0.0207 (0.0175 & 0.0239)
48 h EC50 value (95% Confidence Interval)	0.0164 (0.0125 & 0.0181)
100% immobility	0.025
No-observed effect concentration (NOEC)	0.0125

Validity criteria fulfilled:

yes

Conclusions:

Under study conditions, 48 h EC50 and NOEC values of the test substance for the immobilization of Daphnia magna were determined to be 0.0164 mg/L (95% confidence interval: 0.0125 and 0.0181 mg/L) and 0.0125 mg/L respectively.

Executive summary:

A study was conducted to determine the acute toxicity of test substance, C14 ADBAC (purity: 99.1%), to Daphnia magna under static exposure conditions, according to the OECD Guideline 202 and EU method C.2, in compliance with GLP. Groups of twenty Daphnia, less than 24 h old, were exposed for 48 h to test substance at nominal concentrations of 0.00625, 0.0125, 0.025, 0.05 and 0.1 mg/L in Elendt M4 medium. The control group comprised twenty Daphnia in Elendt M4 medium alone. The test vessels were pre-exposed to dilutions of test substance to prevent loss of the test substance by absorption/binding to the vessel walls. The exposure levels of test substance in aqueous samples of test media were monitored using a LC-MS method of analysis. Since the intended exposure concentrations were substantially achieved, the test results were expressed in terms of their nominal values. Observations of the Daphnia in each control and test vessel were made after approximately 24 and 48 h. Based on the study observations, the 48 h EC50 and EC100 of test substance for the immobilisation of Daphnia magna was determined to be 0.0164 mg/L (95% confidence limits of 0.0125 and 0.0181 mg/L) and 0.025 mg/L respectively. The no-observed effect concentration (NOEC) of test substance with Daphnia magna was determined to be 0.0125 mg/L. Under study conditions, 48 h EC50 and NOEC values of the test substance for the immobilization of Daphnia magna were determined to be 0.0164 mg/L (95% confidence interval: 0.0125 and 0.0181 mg/L) and 0.0125 mg/L respectively (Jenkins, 2008).

Description of key information

Based on the study results, the 48 h EC50 of test substance for the immobilisation of Daphnia magna was determined to be 0.0164 mg/L (nominal).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.016 mg/L

Additional information

A study was conducted to determine the acute toxicity of test substance, C14 ADBAC (purity: 99.1%), to Daphnia magna under static exposure conditions, according to the OECD Guideline 202 and EU method C.2, in compliance with GLP. Groups of twenty Daphnia, less than 24 h old, were exposed for 48 h to test substance at nominal concentrations of 0.00625, 0.0125, 0.025, 0.05 and 0.1 mg/L in Elendt M4 medium. The control group comprised twenty Daphnia in Elendt M4 medium alone. The test vessels were pre-exposed to dilutions of test substance to prevent loss of the test substance by absorption/binding to the vessel walls. The exposure levels of test substance in aqueous samples of test media were monitored using a LC-MS method of analysis. Since the intended exposure concentrations were substantially achieved, the test results were expressed in terms of their nominal values. Observations of the Daphnia in each control and test vessel were made after approximately 24 and 48 h. Based on the study observations, the 48 h EC50 and EC100 of test substance for the immobilisation of Daphnia magna was determined to be 0.0164 mg/L (95% confidence limits of 0.0125 and 0.0181 mg/L) and 0.025 mg/L respectively. The no-observed effect concentration (NOEC) of test substance with Daphnia magna was determined to be 0.0125 mg/L. Under study conditions, 48 h EC50 and NOEC values of the test substance for the immobilization of Daphnia magna were determined to be 0.0164 mg/L (95% confidence interval: 0.0125 and 0.0181 mg/L) and 0.0125 mg/L respectively (Jenkins, 2008).