Hexadecyltrimethylammonium methyl sulphate

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 April 04, 2017 to April 06, 2017

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:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

At the start of the test the excess test solutions were sampled and at the end of the test one replicate of the dilution water control and each test concentration was sampled.

Vehicle:

no

Details on test solutions:

Glass beakers of 250 mL nominal capacity were used as test vessels, with four replicates per test concentration. Each vessel contained 200 mL of test solution providing a depth of approximately 60 mm. The beakers were covered with loose fitting glass lids. The positions of the treatments were randomly allocated within the test area. The test was initiated by the addition of 5 impartially selected D. magna, in <2.0 mL of dilution water, to each test vessel. Each treatment contained a total of 20 D. magna.

This study was run with a dilution water control and nominal exposure concentrations of 5.6, 10, 18, 32, 56 and 100 μg/L. Primary stock concentrate of the test substance with a nominal concentration of 10 mg/L, was prepared by adding a nominal 10 mg of test substance to 1000 mL of dilution water. The concentrate was then placed in an ultrasonic bath for approximately 1 hour. The resultant stock was observed to be a clear and colourless solution and was used to prepare the test solutions. All test solutions were clear and colourless.

Test organisms (species):

Daphnia magna

Details on test organisms:

The test organism was the freshwater crustacean, Daphnia magna, obtained from continuous laboratory cultures held at Scymaris.
The stock cultures of D. magna were maintained in a reconstituted water medium, the same as the test dilution water, at a temperature of 20 ± 2 °C. The cultures were maintained in 2 L glass vessels with a working volume of 1.6 L. A photoperiod of 16 hours light:8 hours dark, with 20 minute transition periods was provided.
The D. magna cultures were fed on a mixed algae diet of Chlorella vulgaris, strain CCAP 211/12 and Pseudokirchneriella subcapitata, strain CCAP 278/4. The D. magna cultures were fed daily ad libitum depending on age and density of the culture. Culture conditions were such that the D. magna reproduction was by diploid parthenogenesis. D. magna <24 hours old, obtained from a single culture vessel, were used for testing. The parent animals were 14 ± 1 day old and had been maintained with a twice weekly renewal of reconstituted water medium since birth. The test organisms and the culture from which they were obtained showed no evidence of disease before the test period.

Test type:

static

Water media type:

other: reconstituted water medium

Limit test:

no

Total exposure duration:

48 h

Test temperature:

20 ± 1 °C

pH:

In control (begining of the experiment): 7.40
In 82 μg/L solution (begining of the experiment): 7.56

In control (48 h): 7.67; 7.81
In 82 μg/L solution (48 h): 7.92; 7.99

Dissolved oxygen:

In control (begining of the experiment): 9.04 mg/L
In 82 μg/L solution (begining of the experiment): 8.92; 8.81 mg/L

In control (48 h): 8.91 mg/L
In 82 μg/L solution (48 h): 8.86; 8.79 mg/L

Conductivity:

Conductivity 615 (μS/cm)

Nominal and measured concentrations:

Control and nominal concentrations of 5.6, 10, 18, 32, 56 and 100 μg/L.
Control and mean measured concentrations of 7.1, 4.7, 9.4, 16, 40 and 82 μg/L.

Details on test conditions:

Observation of effects
An assessment of the response of the D. magna was made 24 and 48 hours after the commencement of the test. Each D. magna was viewed by eye and was defined as affected if showing no whole-body movement, relative to the water, within a period of 15 seconds even if movement of individual appendages was visible. D. magna so affected were termed immobile. Any overt symptoms of toxicity were also recorded. The median effect concentration (EC50) was defined as the concentration resulting in 50% immobilisation of the D. magna in the time period specified. The number of D. magna immobilised in the replicates of the control and each exposure concentration were summed for each time period. The 24 and 48 hour EC50 values and their 95% confidence limits were calculated using CETIS . The Lowest Observed Effect Concentration (LOEC) was defined as the lowest tested concentration at which is observed to have a harmful effect on immobilisation when compared to the control (p <0.05). However, all test concentrations above the LOEC must have a harmful effect equal to or greater than the LOEC. The No Observed Effect Concentration (NOEC) was defined as the concentration immediately below the LOEC.

Physical and chemical parameters
Measurements were undertaken at the beginning and end of the test for pH and dissolved oxygen concentration using calibrated meters. At the start of the test the dissolved oxygen concentration and pH of the control and highest exposure concentration were measured using the excess solution remaining after filling the test vessels. At the end of the test, the dissolved oxygen concentration and pH of two replicates of the dilution water control and highest exposure concentration were measured. Temperature values were continually recorded using a Max/Min thermometer checked against a mercury in glass thermometer. Current, maximum and minimum temperatures were recorded daily, in an additional test vessel.

Reference substance (positive control):

no

Key result

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

ca. 34.46 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% confidence interval: 28.92 – 43.32 μg/L

Remarks:

equivalent to 30.59 μg a.i./L

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

ca. 25 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% confidence interval: 19.46 – 28.79 μg/L

Remarks:

equivalent to 22.19 μg a.i./L

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

ca. 16 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: equivalent to 14.2 μg a.i./L

Key result

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

ca. 40 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: equivalent to 35.5 μg a.i./L

Details on results:

There was no immobility observed in the dilution water control. No other symptoms of toxicity were observed.

Results

Analytical data

The limit of quantification of test substance in this study was 2.0 µg/L. All analytical values are quoted to two significant figures and percentages to the nearest integer. On the basis of the analytical data the mean measured concentrations were used for the calculation and reporting of results.

The numbers of D. magna immobilised after 24 and 48 hours were recorded. The results obtained (based on mean measured concentrations of test substance) were:

Time	EC50	95% confidence limits	Calculation method
24 hours	34.46 µg/L	28.92 – 43.32 µg/L	Linear Interpolation (ICPIN)
48 hours	25 µg/L	19.46 – 28.79 µg/L	Linear Interpolation (ICPIN)

The EC100 is defined as the lowest test concentration that caused 100% immobilisation within the period of the test, therefore, 48 hours EC100 = 40 µg/L. Based on immobility compared to the control (p <0.05) the 48 hour No Observed Effect Concentration (NOEC) was determined to be 16 µg/L and the Lowest Observed Effect Concentration (LOEC)  was  40 µg/L. There was no immobility observed in the dilution water control. No other symptoms of toxicity were observed.

Physical and chemical data

Dissolved oxygen concentrations ranged from 8.79 to 9.04 mg/L and the pH values ranged from 7.40 to 7.99. Temperature values taken at 0, 24 and 48 hours were 20.8, 20.6 and 20.8 deg C. During the study the temperature ranged from 19.9 – 21.9 deg C. This temperature range deviated from the ± 1oC prescribed in the study plan and test guideline however has not affected the outcome of the study.

Analytical results 

Nominal concentration (µg/L)	Measured concentration (µg/L)				Mean measured concentration(µg/L)	Mean measured concentration(%)
	0 hour		48 hour
	(µg/L)	% of nominal	(µg/L)	% of nominal
Control	<LOQ	-	<LOQ	-	<LOQ	-
5.6	5.0	90	9.1	163	7.1	126
10	6.9a	69	2.5b	25	4.7	47
18	13	72	5.7	32	9.4	52
32	13	41	19	59	16	50
56	38	68	41	73	40	71
100	81	81	82	82	82	82

All measurements are quoted to 2 significant figures

A Mean of triplicate analyses: 6.7, 7.3, 6.7µg/L

B Mean of triplicate analyses: 2.5, 2.5, 2.5µg/L

The limit of quantification (LOQ) in the study was 2.0 µg/L

Daphnia magna response

Time (hour)	Mean measured concentration	Number immobilised per replicate				Total number tested	Total number immobilised	Percentage immobilised (%)
		A	B	C	D
24	Control	0	0	0	0	20	0	0
	7.1	0	0	0	0	20	0	0
	4.7	0	0	0	0	20	0	0
	9.4	0	0	0	0	20	0	0
	16	0	0	0	0	20	0	0
	40	4	4	2	3	20	13	65
	82	5	4	5	5	20	19	95
48	Control	0	0	0	0	20	0	0
	7.1	0	0	0	0	20	0	0
	4.7	0	0	0	0	20	0	0
	9.4	0	0	0	0	20	0	0
	16	1	0	2	1	20	4	20
	40	5	5	5	5	20	20	100
	82	5	5	5	5	20	20	100

Validity criteria

The OECD 202 Test Guideline details the following performance criteria for the test validity:

- In the control, no more than 10% of the daphnids should have been immobilised or show other signs of stress;

- The dissolved oxygen concentration at the end of the test should be  3.0 mg/L in control and test vessels.

- As no immobility or other signs of stress were observed in the control and dissolved oxygen concentration remained at greather than equal to 3 mg/L this test has satisfied all validity criteria.

Validity criteria fulfilled:

yes

Conclusions:

Under the study conditions, the 48 h EC50, NOEC and LOEC for the test substance were determined to be 25, 16 and 40 μg/L, respectively (equivalent to 22.19, 14.2 and 35.5 μg a.i./L).

Executive summary:

A study was conducted to determine the acute toxicity of the test substance, C16 TMA-MS, (88.75% active) to Daphnia magna according to OECD Guideline 202, in compliance with GLP. The study was performed using nominal concentrations of the test substance 5.6, 10, 18, 32, 56 and 100 μg/L (mean measured concentrations of 7.1, 4.7, 9.4, 16, 40 and 82 μg/L). Glass beakers of 250 mL nominal capacity were used as test vessels, with four replicates per test concentration. Each vessel contained 200 mL of test solution providing a depth of approximately 60 mm. The beakers were covered with loose fitting glass lids. The test was initiated by the addition of 5 impartially selected D. magna, in <2.0 mL of dilution water, to each test vessel. Each treatment contained a total of 20 D. magna. Test solutions were analytically verified by liquid chromatography method, thus toxicity results are concerning measured values. Under the study conditions, the 48 h EC50, NOEC and LOEC for the test substance were determined to be 25, 16 and 40 μg/L, respectively (equivalent to 22.19, 14.2 and 35.5 μg a.i./L) (Scymaris, 2017).

Description of key information

Based on the study results, the 48 h EC50 value of 25 μg/L (measured; equivalent to 22.19 μg a.i./L) for toxicity to daphnia has been considered further for hazard/risk assessment.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC50

Effect concentration:

22.19 µg/L

Additional information

A study was conducted to determine the acute toxicity of the test substance, C16 TMA-MS, (88.75% active) to Daphnia magna according to OECD Guideline 202, in compliance with GLP. The study was performed using nominal concentrations of the test substance5.6, 10, 18, 32, 56 and 100 μg/L (mean measured concentrations of 7.1, 4.7, 9.4, 16, 40 and 82 μg/L).Glass beakers of 250 mL nominal capacity were used as test vessels, with four replicates per test concentration. Each vessel contained 200 mL of test solution providing a depth of approximately 60 mm. The beakers were covered with loose fitting glass lids. The test was initiated by the addition of 5 impartially selected D. magna, in <2.0 mL of dilution water, to each test vessel. Each treatment contained a total of 20 D. magna. Test solutions were analytically verified by liquid chromatography method, thus toxicity results are concerning measured values.Under the study conditions, the 48 h EC50, NOEC and LOEC for the test substance were determined to be 25, 16 and 40 μg/L (measured), respectively (equivalent to 22.19, 14.2 and 35.5 μg a.i./L) (Scymaris, 2017).