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

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From June 20, 2007 to August 08, 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

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-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.s., 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:

Samples were collected from each control and test vessel at 24 and 72 h (fresh) and 24 and 96 h (expired). The concentrations of test substance in the samples were measured using an LC-MS method of analysis.

Vehicle:

yes

Remarks:

Ethanol/Water

Details on test solutions:

Test solution preparation
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; therefore, throughout this report, the exposure concentrations and test results have been expressed in terms of active ingredient. The method of test solution preparation used during the definitive test was based on the results of a range finding test. As the test substance was known to adsorb, all glassware used in the test was pre-exposed to the appropriate concentration of the test substance overnight before use. The pre- conditioning media were discarded immediately before the test solutions were freshly prepared. On up to four occasions during the definitive test, the test medium at each concentration (nominal, 0.259, 0.570, 1.25, 2.76 or 6.07 mg a.i./L) was prepared by adding the required weight of test substance (nominal; 10, 14.1, 31, 68.2 or 150 mg) to diluent water (between ca. 0.5 and 3 l) in a volumetric flask (1, 2 or 5 l capacity). The contents of each flask were vigorously shaken before being adjusted to volume. At 0.570 to 6.07 mg a.i./L, the contents of the volumetric flask were poured into a test vessel containing diluent water (between 10 and 18 l). At 0.259 mg a.i./L, an aliquot (640 mL) of the medium prepared at 8.09 mg a.i./L (10 mg/L) was added to diluent water (18 l) in a test vessel and the final volume adjusted to 20 L.

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

Test species
Rainbow trout (Oncorhynchus mykiss). This test species has been selected based on its known sensitivity to changes in water quality, availability throughout the year and ease of maintenance.

Source
The fish were supplied by a commercial fish farm in the UK. They were reared at the farm from eggs that hatched in February 2007.

Acclimatisation
The stock of fish was obtained from the supplier on 18 June 2007 and they were held in an aerated supply of diluent water under flow-through conditions until use. During the 14-day period immediately before the definitive test, temperatures remained within the range 14.5 to 14.9°C, pH values within the range 7.57 to 7.98, dissolved oxygen concentrations within the range 93 to 96% air saturation value (ASV) and total hardness within the range 170 to 182 mg/l as CaCO3. Based on the laboratory measurements conducted nearest to the definitive study (4 May 2007) the light intensity of the holding area was within the range 288 and 457 lux. The fish were fed daily with an amount of commercial fish food (TROUW (UK) Ltd; Nutra Fry 02) equivalent to between 1 and 4% of the total wet-weight of fish in the holding tank. No food was given during the 21-hour period immediately before exposure or during the exposure period itself. No medication was given during the holding period, and <2% mortality occurred during the 14 days before the definitive test. The size of the fish used in the definitive test was determined by weighing and measuring a sample of ten fish taken at random from the holding tank on 26 July 2007; their mean total length was 6.37 cm and their mean wet weight was 2.62 g.

Environmental conditions
Treatment and control groups were maintained at 15 ± 2°C throughout the exposure period and constant to within ± 1°C during the study. The temperature of the water in the control vessel was continuously monitored during the study. Supplementary aeration was provided via narrow bore glass tubes. A photoperiod of 16 h light: 8 h dark was maintained, with 60 minute periods of subdued lighting at the beginning and end of each light phase. Daily records of temperature, pH and dissolved oxygen were kept for each control and test vessel together with measurements of total hardness for selected vessels at 0 h. The fish were not fed during the 96 h exposure period.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

Total hardness of control medium and the test medium at the highest concentration at the start of the test were 182 and 158 mg/L as CaCO3, respectively.

Test temperature:

Continuous monitoring of control vessel media temperature ranged between 13.8 and 14.8ºC.

pH:

7.65-8.20

Dissolved oxygen:

83-96% ASV (Percent air saturation value as a measure of dissolved oxygen concentration)

Conductivity:

Conductivity (20°C): 280 µS/cm

Nominal and measured concentrations:

0, 0.259, 0.570, 1.25, 2.76 and 6.07 mg a.i../L. Nominal values were used to determine the LC50.

Details on test conditions:

Fish was exposed to the control and test dilutions for 96 h under semi-static conditions (i.e. daily renewal of media). The prepared control and test dilutions were placed into the respective test vessels and gently aerated during the test to maintain dissolved oxygen concentrations above 60% air saturation value. For the definitive test, each exposure level and control comprised one replicate vessel. Fishes were not individually identified in this study, nor were they fed 24 h prior to the start of the study or during the study. The temperature of the test area was 15 ± 2ºC. A 16-h light: 8-h dark photoperiod was maintained with 60-minute transition periods. The fishes were inspected for mortalities or visible abnormalities after 24, 48, 72, and 96 h. Additional observations were made two and four h after the start of the definitive test. Criteria of death was the absence of respiratory movement and lack of response to external stimulation of the caudal peduncle.

Reference substance (positive control):

not specified

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

ca. 1.06 other: mg a.i./L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95% confidence limits of 0.731 and 1.53 mg a.i./L

Key result

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

ca. 0.259 other: mg a.i./L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality (fish)

Details on results:

Treatment-related effects, which were observed at 0.570 mg a.s./l and higher concentrations, consisted of effects on pigmentation, respiration and co-ordination.

Sublethal observations / clinical signs:

Table 1: Cumulative mortality

Nominal ADEBAC Exposure Concentrations (mg a.i./l)	Cumulative mortality (initial population = 7 fish/concentration)
	2 h	4 h	24 h	48 h	72 h	96 h	%
Control	0	0	0	0	0	0	0
0.259	0	0	0	0	0	0	0
0.570	0	0	0	0	0	1	14
1.25	0	0	0	0	3	4	57
2.76	0	0	7	7	7	7	100
6.07	7	7	7	7	7	7	100

 

Table 2: Mortality and observations

Based on the relationship between mortality and concentration the following values have been estimated:

	N-Alkyl(C12-C14)-N,N-dimethyl-N- ethylbenzylammonium chloride; ADEBAC (mg a.i./L; nominal)
2 & 4 h LC50 value (95% Confidence Interval)	4.09 (2.76 & 6.07)
24 & 48 h LC50 value (95% Confidence Interval)	1.86 (1.25 & 2.76)
72 h LC50 value (95% Confidence Interval)	1.35 (1.01 & 1.73)
96 h LC50 value (95% Confidence Interval)	1.06 (0.731 & 1.53)
100% mortality	2.76
No-observed effect concentration (NOEC)	0.259

Results

The measured concentrations of the test substance ranged between 73 and 93% of their nominal concentrations in samples of  freshly prepared media, except on one  occasion at the lowest concentration (0.259 mg a.i./l) where the measured level was only 63% of nominal. After 24 hours (in samples of expired media), the measured levels had been maintained, ranging between 63 and 90% of nominal (between 85 and 117% of their starting values). The intended exposure concentrations were adequately achieved and maintained between renewals of the media. In addition, the test substance is known to be readily soluble and hydrolytically stable, and since the test vessels were pre-treated with ADEBAC to prevent loss of the test substance 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 of the organisms to the nominal concentrations). Therefore, effect concentrations were determined based on nominal concentrations of the active ingredient, ADEBAC.

Validity criteria fulfilled:

yes

Conclusions:

Under study conditions, the 96 h LC50 and NOEC values for test substance with rainbow trout were found to be 1.06 mg a.i /L and 0.259 mg a.i./L (nominal) 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 Rainbow trout (Oncorhynchus mykiss) according to OECD Guideline 203 and EU Method C.1, in compliance with GLP. Seven juvenile fish were exposed to the test substance at concentrations of 0, 0.259, 0.570, 1.25, 2.76 and 6.07 mg a.i../L for 96 h under semi-static conditions. A control group of seven fish was placed into diluent water alone. The test vessels were pre-exposed to dilutions of the test substane to prevent loss of the test substance by adsorption/binding to the vessel walls. Test solutions were renewed daily. 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 were maintained between renewals of the media, the test results were expressed in terms of their nominal values. Mortality in the form of (i) absence of respiratory movement and (ii) absence of response to external stimulation of the caudal peduncle were observed at 2, 4, 24, 48, 72 and 96 h. In addition, subjective assessments were also made on the incidence and type of any sub-lethal effects compared with control fish. The 96 h LC50 value for test substance with rainbow trout was found to be 1.06 mg a.i /L, with 95% confidence limits of 0.731 and 1.53 mg a.i./L. Significant treatment-related sub-lethal effects on the fish were noted at 0.570 mg a.i./L and higher concentrations, giving a no observed effect concentration (NOEC) of 0.259 mg a.i./L as test substance. Under study conditions, the 96 h LC50 and NOEC values for test substance for toxicity to Rainbow trout were found to be 1.06 mg a.i /L (with 95% confidence limits of 0.731 and 1.53 mg a.i./L) and 0.259 mg a.i./L (nominal) respectively (Jenkins, 2008).

Description of key information

The 96 h LC50 and NOEC values for test substance, C12-14 ADEBAC, for toxicity to Rainbow trout were found to be 1.06 and 0.259 mg a.i./L (nominal) respectively.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

1.06 mg/L

Additional information

A study was conducted to determine the acute toxicity study of the test substance, C12-14 ADEBAC (active ingredient 80.93%), to the Rainbow trout (Oncorhynchus mykiss) according to OECD Guideline 203 and EU Method C.1, in compliance with GLP. Seven juvenile fish were exposed to the test substance at concentrations of 0, 0.259, 0.570, 1.25, 2.76 and 6.07 mg a.i../L for 96 h under semi-static conditions. A control group of seven fish was placed into diluent water alone. The test vessels were pre-exposed to dilutions of the test substane to prevent loss of the test substance by adsorption/binding to the vessel walls. Test solutions were renewed daily. 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 were maintained between renewals of the media, the test results were expressed in terms of their nominal values. Mortality in the form of(i) absence of respiratory movement and (ii) absence of response to external stimulation of the caudal peduncle were observed at 2, 4, 24, 48, 72 and 96 h. In addition, subjective assessments were also made on the incidence and type of any sub-lethal effects compared with control fish. The 96 h LC50 value for test substance with rainbow trout was found to be 1.06 mg a.i /L, with 95% confidence limits of 0.731 and 1.53 mg a.i./L. Significant treatment-related sub-lethal effects on the fish were noted at 0.570 mg a.i./L and higher concentrations, giving a no observed effect concentration (NOEC) of 0.259 mg a.i./L as test substance. Under study conditions, the 96 h LC50 and NOEC values for test substance for toxicity to Rainbow trout were found to be 1.06 mg a.i /L (with 95% confidence limits of 0.731 and 1.53 mg a.i./L) and 0.259 mg a.i./L (nominal) respectively (Jenkins, 2008).