Benzyltrimethylammonium hydroxide

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:

24 Sep 2018 - 24 Oct 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The substance can be produced through different process routes, yielding solutions in water or organic solvent (e.g. methanol or ethylene glycol).
It was initially unclear how to register the substance (mono-constituent or multi-constituent substance) and which manufactured substance to test to fulfil the REACH data requirements. After consultation with the ECHA helpdesk, the test program was started with the manufactured substance of the Lead registrant (solvent: methanol) in which the highest amount of solvent could be removed without causing degradation of the substance. This resulted in the selection of a solution of 56-57% BTMAOH in methanol as test substance.
During the course of the test program, and in order to aid meaningful risk assessment, after consultation with ECHA and upon ECHA's recommendation, it was considered to be more appropriate to test the water-based manufactured substance. As a consequence, some testing was performed with a BTMAOH solution in methanol, and some testing was performed with a BTMAOH solution in water.

The current entry reflects a test performed with a water-based test solution.

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

2004

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: All test concentrations and control
- Sampling method: 2.0 mL from the approximate centre of the test vessels at t=0 h and t=48 h.
- Sample storage conditions before analysis: Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility.
- At the end of the exposure period, the replicates were pooled at each concentration before sampling.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- The batch of test item tested was a clear and almost colourless liquid and completely soluble in test medium at the concentrations tested. A correction was made for the water content of the test item. All reported concentrations are based on the pure test item.
- Preparation of test solutions started with the highest concentration of 100 mg/L applying a 15-minute period of magnetic stirring to accelerate dissolution of the test item in test medium. After adjusting the pH from 10 to 7.9, the solution was used as the highest test concentration. Lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium. All test solutions were clear and colorless at the end of the preparation procedure.
- Controls: Test medium without test item or other additives

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: Straus 1820
- Source: In-house laboratory culture with a known history
- Age of parental stock: > 2 weeks
- Age at test initiation: <24 hours
- Validity of batch: Daphnids originated from a healthy stock, 2nd to 5th brood, showing no signs of stress such as mortality >20%, presence of males, ephippia or discoloured animals and there was no delay in the production of the first brood.

CULTIVATION
- Start of each batch: Approximately 250 newborn daphnids, i.e. less than 3 days old, were placed into 5 litres of M7 medium in an all-glass culture vessel.
- Maximum age of the cultures: 4 weeks
- Renewal of the cultures: After 7 days of cultivation, half of the medium twice a week.
- Temperature of medium: 18-22°C
- Feeding: Daily, a suspension of fresh water algae.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

180 mg/L (expressed as CaCO3)

Test temperature:

20°C

pH:

At start (t=0 h): 7.8-7.9
At end (t=48 h): 8.0-8.1

Dissolved oxygen:

At start (t=0 h): 9.0-9.1
At end (t=48 h): 8.7-8.8

Nominal and measured concentrations:

Nominal: 10, 18, 32, 56, 100 mg/L (corrected for water content) and control.
The measured concentrations were at 92-99% of the nominal concentrations throughout the test. Based on these results, the analytically confirmed nominal exposure concentrations were used to determine the effect parameters. See Table 1 in 'Any other information on results' for details on measured concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel: 60 mL, all-glass, containing 50 mL of test solution
- Aeration: no aeration
- Feeding: no feeding
- No. of organisms per vessel: 5 daphnids
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Introduction of daphnids: Within 15 minutes after preparation of the test solutions.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Adjusted ISO-medium, prepared with tap water purified by Reverse Osmosis and the following salts: CaCl2.2H2O: 211.5 mg/L, MgSO4.7H2O: 88.8 mg/L, NaHCO3: 46.7 mg/L, KCl: 4.2 mg/L
- Culture medium different from test medium: Yes (M7 medium)
- Intervals of water quality measurement: pH and dissolved oxygen: at the beginning and at the end of the test, for all concentrations and the control. Temperature of medium: continuously in a temperature control vessel, beginning at the start of the test.

OTHER TEST CONDITIONS
- Adjustment of pH: not during test (only during preparation of the highest test concentration solution).
- Photoperiod: 16 h photoperiod daily.

EFFECT PARAMETERS MEASURED
- Immobility (including mortality) at 24 h and at 48 h.

RANGE-FINDING STUDY
- Combined Limit/Range-Finding Test concentrations: 0.10, 1.0, 10, 100 mg/L (corrected for water content)
- Results used to determine the conditions for the definitive study: yes, expected EC50 between 10 and 100 mg/L

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (performed Nov 2018)

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

21 mg/L

Nominal / measured:

nominal

Conc. based on:

other: BTMAOH

Basis for effect:

mobility

Remarks on result:

other: 95% confidence interval: 17 - 25 mg/L

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

45 mg/L

Nominal / measured:

nominal

Conc. based on:

other: BTMAOH

Basis for effect:

mobility

Remarks on result:

other: 95% confidence interval: 39 - 53 mg/L

Details on results:

- Behavioural abnormalities: none observed in the Control group.
- Mortality of control: none
- No immobility was observed in the control and at the lowest test concentration throughout the test. A concentration-related increase of immobility was observed at nominally 18 mg/L and higher, resulting in complete immobility at the two highest test concentrations after 48 hours of exposure. See Table 2 in 'Any other information on results' for full details on immobility.
- All water quality parameters remained within the requirements as laid down in the study plan throughout the test.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- The 48h-EC50 was 0.40 mg/L with a 95% confidence interval between 0.33 and 0.48 mg/L.
- The responses in this reference test with K2Cr2O7 are within the ranges of the expected responses at the different concentrations, i.e. the 48h-EC50 was between 0.28 and 0.9 mg/L. Hence, the sensitivity of this batch of D. magna was in agreement with the historical data collected at the Test Facility.

Reported statistics and error estimates:

The 24 and 48h-EC50-values were calculated from the probits of the percentages of affected daphnids and the logarithms of the corresponding test item concentrations using the maximum likelihood estimation method.
ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used to perform the analysis.

Table 1: Final Test: Test Samples

Time of sampling [hours]	Concentration [mg/L]		Relative to nominal [%]	Relative to initial [%]
	Nominal	Analyzed
0	0	n.d.	n.a.
	10	9.21	92
	18	17.0	94
	32	31.5	98
	56	54.2	97
	100	92.6	93
48	0	n.d.	n.a.	n.a.
	10	9.46	95	103
	18	17.1	95	101
	32	31.7	99	101
	56	54.0	96	100
	100	94.0	94	101

n.d.Not detected.

n.a.Not applicable.

Table 2: Number of Introduced Daphnids and Incidence of Immobility in the Final Test 

Time (h)	Replicate	Test substance; Nominal conc. (mg/L)
		Control	10	18	32	56	100
0	A	5	5	5	5	5	5
	B	5	5	5	5	5	5
	C	5	5	5	5	5	5
	D	5	5	5	5	5	5
	Total introduced	20	20	20	20	20	20
24	A	0	0	0	0	4	5
	B	0	0	0	1	4	5
	C	0	0	0 (1)	0 (1)	4	5
	D	0	0	0	2 (1)	4	4 (1)
	Total immobilised	0	0	0	3	16	19
	Effect %	0	0	0	15	80	95
48	A	0	0	2	4	5	5
	B	0	0	3	4	5	5
	C	0	0	2	4	5	5
	D	0	0	2	4	5	5
	Total immobilised	0	0	9	16	20	20
	Effect %	0	0	45	80	100	100

( ) between brackets: number of daphnids observed trapped at the surface of the test solutions. These organisms were reimmersed into the respective solutions before recording of mobility.

Validity criteria fulfilled:

yes

Remarks:

See 'Overall remarks' for details on validity criteria

Conclusions:

The 48h-EC50 for Daphnia magna exposed to the test substance was 21 mg/L (corrected for water content), based on analytically confirmed nominal concentrations (95% confidence interval between 18 - 25 mg/L).

Executive summary:

In a 48 -h acute toxicity test conducted according to OECD guideline 202, Daphnia magna were exposed to the test substance at concentrations of 10, 18, 32, 56 and 100 mg/L (corrected for water content) and an untreated control (all with 4 replicates per concentration and 5 daphnids per replicate). A correction was made for the water content of the substance, i.e. all reported concentrations are based on the pure test substance.

Measured concentrations were at 92 -99% of the nominal concentrations throughout the test. Therefore, nominal concentrations were used to express the effect parameters.

No immobility was observed in the control and the lowest test concentration throughout the test. A concentration-related increase of immobility was observed at nominally 18 mg/L and higher, resulting in complete immobility at the two highest test concentrations at 48 h of exposure.

The 48h-EC₅₀ for immobility was 21 mg/L based on analytically confirmed nominal concentrations (95% confidence interval between 18 - 25 mg/L). The study met all validity criteria and is considered to be reliable without restrictions.

Description of key information

The 48h-EC50 for Daphnia magna exposed to the substance was 21 mg/L, based on analytically confirmed nominal concentrations (95% confidence interval between 18 - 25 mg/L).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

21 mg/L

Additional information

In a 48 -h acute toxicity test conducted according to OECD guideline 202, Daphnia magna were exposed to the substance at concentrations of 10, 18, 32, 56 and 100 mg/L and an untreated control (all with 4 replicates per concentration and 5 daphnids per replicate). A correction was made for the water content of the test item, i.e. all reported concentrations are based on BTMAOH.

Measured concentrations were at 92-99% of the nominal concentrations throughout the test. Therefore, nominal concentrations were used to express the effect parameters.

No immobility was observed in the control and the lowest test concentration throughout the test. A concentration-related increase of immobility was observed at nominally 18 mg/L and higher, resulting in complete immobility at the two highest test concentrations at 48 h of exposure.

The 48h-EC₅₀ for immobility was 21 mg/L based on analytically confirmed nominal concentrations (95% confidence interval between 18 - 25 mg/L). The study met all validity criteria and is considered to be reliable without restrictions.