Miristalkonium chloride

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 July 24, 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-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. For the definitive test, two mid-vessel samples (5 mL) of media were taken from the control and test vessels at 0 and 72 h (fresh media) and at 24 and 96 h (expired media) for analysis. At 1.65 and 8 mg/L, additional sets of samples (5 mL) were also taken from the vessels at four h following the death of all seven fish at each of these levels although these samples were not analysed as the data were not required. Two mid-vessel samples (5 mL) of media were also taken from the second test vessel at 1.65 mg/L on the day of preparation (fresh media) and after 24 h (expired). 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 in case further analysis was required. These duplicate samples were stored frozen, but it was not considered necessary to analyse these samples since the analytical recoveries obtained in the definitive test were considered acceptable. Thus, the reserve samples were discarded.

Vehicle:

yes

Remarks:

Water

Details on test solutions:

Test solution preparation
The test solutions were not prepared on the basis of the percent 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 for at least 20 h 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 was prepared by adding the required weight of test substance (nominal, 10, 10.5, 23.1, 50.9 or 112 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 and treated by ultrasound for between one and two minutes before being adjusted to volume. At 0.751 to 8 mg/L, the contents of the volumetric flask were poured into a test vessel containing diluent water (between 4 and 12 L). At 0.342 mg/L, an aliquot (479 mL) of the medium prepared at 10 mg/L (nominal) was added to diluent water (12 L) in a test vessel.

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 14.6ºC, pH values within the range 7.95 to 8.23, dissolved oxygen concentrations within the range 94 to 101% 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 00 and 02) equivalent to between 1 and 4% of the total wet-weight of fish in the holding tank. No food was given during the 20-h 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 12 July 2007; their mean total length was 5.63 cm and their mean wet weight was 1.71 g.

Diluent water
The water used to hold the fish and for the study was laboratory tap water, dechlorinated and softened by passage through a water purification system (Vivendi Water Systems; formerly Elga). It was passed through a high grade activated carbon filter to remove chlorine and any organic contaminants. A proportion of the supply then passed through a water softener before final reverse osmosis treatment to produce a highly purified water supply. The two grades of dechlorinated water were then remixed to give a supply with the desired water hardness. This water was then held in an intermediate tank where it was equilibrated to the test temperature and gently aerated before being supplied to the holding and test areas.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

170 to 182 mg/L as CaCO3

Test temperature:

14.5 to 14.6ºC

pH:

7.95 to 8.23

Dissolved oxygen:

94 to 101% air saturation value (ASV)

Nominal and measured concentrations:

0, 0.342, 0.751, 1.65, 3.64 and 8.00 mg/L

Details on test conditions:

Experimental design and test concentrations
The study comprised one range finding test, an analytical trial and a definitive test. Both of the toxicity tests were conducted under semi-static conditions, with daily renewal of the media. The range finding test employed nominal concentrations of 0.1, 1 and 10 mg/L and a diluent water control, each with three fish per vessel. The test media were individually prepared at each concentration by adding the test substance to diluent water without the aid of any auxiliary substances. After 96 h, all of the fish at 10 mg/L had died but no mortality occurred at 0.01 or 1 mg/L. Sub-lethal effects were exhibited by all of the fish exposed at 1 mg/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 1 mg/L. Analysis of a sample of this medium confirmed that the intended exposure concentration could be adequately achieved (94% of nominal). Based on the results of the range finding test, the definitive test employed the following nominal concentrations of ADBAC C12-C14: 0.342, 0.751, 1.65, 3.64 and 8 mg/L. A dilution medium control group was also included in the test. In the definitive test, seven fish were placed at random into each glass aquarium containing the prepared control or test media. Each vessel (25 x 25 x 45 cm; width x depth x length) contained 14 L of medium to a depth of 13 cm. This provided an initial static loading of 0.86 g bodyweight/L. Within four h of starting this test, all of the fish at 1.65 and 8 mg/L had died but only one fish had died at the interim level of 3.64 mg/L and an error in the formulation procedure was thought to have occurred. The following day, the results of analysis confirmed that the test media had been correctly formulated, so a second vessel was established at 1.65 mg/L with a new batch of seven fish taken from the same stock as those used to start the definitive test on the previous day and was run in parallel with the original vessels established for the definitive test. No mortality or sub-lethal effects were exhibited by the fish in the second (additional) vessel during the initial two and four h of exposure but all fish were dead at 24 h. Therefore, the deaths observed in the original vessel established at 1.65 mg/L had no impact on the test result and the definitive test was continued to 96 h. All data are presented in this report but only that from the second test vessel at 1.65 mg/L has been used in LC50 caculations.

Environmental conditions
Treatment and control groups were maintained at 15 ± 2ºC throughout the exposure period and constant to within ±1ºC 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.

Medium renewal
The fish were exposed to the control or test conditions for a period of 96 h with daily batch renewal of the media to ensure the maintenance of satisfactory environmental conditions and to maintain stable exposure levels.

Diluent water
The water used to hold the fish and for the study was laboratory tap water, dechlorinated and softened by passage through a water purification system (Vivendi Water Systems; formerly Elga). It was passed through a high grade activated carbon filter to remove chlorine and any organic contaminants. A proportion of the supply then passed through a water softener before final reverse osmosis treatment to produce a highly purified water supply. The two grades of dechlorinated water were then remixed to give a supply with the desired water hardness. This water was then held in an intermediate tank where it was equilibrated to the test temperature and gently aerated before being supplied to the holding and test areas.

Reference substance (positive control):

not specified

Key result

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

ca. 0.791 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95% Confidence interval: 0.53 & 1.15

Key result

Duration:

96 h

Dose descriptor:

EC100

Effect conc.:

ca. 1.65 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

ca. 0.342 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

The measured concentrations of test substance ranged between 89 and 108% of their nominal concentrations in samples of freshly prepared media, confirming that the intended exposure concentrations were achieved. After 24 h (in samples of expired media), the measured levels had been maintained, ranging between 81 and 100% of nominal except at the two lowest levels (0.342 and 0.751 mg/L) where the measured levels had decreased to 58 and 72% respectively, of their nominal values after 24 h. The intended exposure concentrations were adequately achieved and maintained. In addition, the test substance is known to be readily soluble and hydrolytically stable, and since the test vessels were pre-treated with test substance 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.

Treatment-related effects, which were observed at 0.751 mg/l and higher concentrations, consisted of effects on pigmentation, behaviour, respiration and co-ordination.

Sublethal observations / clinical signs:

Table 1: Cumulative mortality

Nominal test substance exposure concentrations  (mg/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
0.342	0	0	0	0	1	1	14
0.751	0	0	0	1	2	2	29
1.65^a	1	7	7	7	7	7	100
1.65^b	0	0	7	7	7	7	100
3.64	0	1	7	7	7	7	100
8.00	7	7	7	7	7	7	100
^aOriginal test vessel prepared at the start of the definitive test; data not used in this report. ^bSecond (additional) vessel prepared approximately 24 h after the start of the test.

Table 2: Key safety descriptor values derived from experiemental results

	Test substance (mg/L;nominal)
2-h LC50 value (95% Confidence Interval)	5.4 (3.64 & 8)
4-h LC50 value (95% Confidence Interval)	4.84 (3.64 & 6.17)
24-h LC50 value (95% Confidence Interval)	1.11 (0.751 & 1.65)
48-h LC50 value (95% Confidence Interval)	0.998 (0.0751 & 1.27)
72 & 96 h LC50 values (95% Confidence Interval)	0.791 (0.53 & 1.15)
100%mortality	1.65
No-observedeffectconcentration(NOEC)	0.342

The treatment-related effects were considered significant at 0.751 mg/L and higher concentrations and comprised effects on pigmentation, behaviour, respiration and co-ordination. The darkened pigmentation exhibited by one fish at 0.342 mg/L was not considered to be a significant treatment-related effect and this had no impact on either the validity or integrity of the study.

Validity criteria fulfilled:

yes

Conclusions:

Under study conditions, the 96 h LC50 value (95% Confidence interval) and NOEC value were determined to be 0.791 mg/L (0.53 & 1.15) and 0.342 mg/L respectively.

Executive summary:

A study was conducted to determine the acute toxicity of test substance, C14 ADBAC (purity: 99.1%), to rainbow trout (Oncorhynchus mykiss), according to the OECD Guideline 203 and EU method C.1. The study was performed under semi-static conditions, with a daily renewal of media. Groups of seven juvenile fish were exposed for 96 h to five concentrations of test substance nominal concentrations of 0.342, 0.751, 1.65, 3.64 and 8 mg/L, in diluent water. A control group of seven fish was placed into diluent water alone. Test solutions were renewed daily. 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 and were maintained between renewals of the media, the test results were expressed in terms of their nominal values. Observations of the fish were made after approximately 2, 4, 24, 48, 72 and 96 h of exposure. The 96 h LC50 and LC100 values were determined to be 0.791 mg/L (95% confidence interval: 0.53 & 1.15) and 1.65 mg/L respectively. The no observed effect concentration (NOEC) was determined to be 0.342 mg/L. Under study conditions, the 96 h LC50 value and NOEC value were determined to be 0.791 mg/L and 0.342 mg/L respectively (Jenkins, 2008).

Description of key information

Based on the study results, the 96 h LC50 value value of the test substance for the mortality in fish was determined to be 0.791 mg/L (nominal).

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

0.791 mg/L

Additional information

A study was conducted to determine the acute toxicity of test substance, C14 ADBAC (purity: 99.1%), to rainbow trout (Oncorhynchus mykiss), according to the OECD Guideline 203 and EU method C.1. The study was performed under semi-static conditions, with a daily renewal of media. Groups of seven juvenile fish were exposed for 96 h to five concentrations of test substance nominal concentrations of 0.342, 0.751, 1.65, 3.64 and 8 mg/L, in diluent water. A control group of seven fish was placed into diluent water alone. Test solutions were renewed daily. 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 and were maintained between renewals of the media, the test results were expressed in terms of their nominal values. Observations of the fish were made after approximately 2, 4, 24, 48, 72 and 96 h of exposure. The 96 h LC50 and LC100 values were determined to be 0.791 mg/L (95% confidence interval: 0.53 & 1.15) and 1.65 mg/L respectively. The no observed effect concentration (NOEC) was determined to be 0.342 mg/L. Under study conditions, the 96 h LC50 value and NOEC value were determined to be 0.791 mg/L and 0.342 mg/L respectively (Jenkins, 2008).