Quaternary ammonium compounds, C12-14-alkyltrimethyl, Me sulfates

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 March 13, 2018 to March 19, 2018

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

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Remarks:

HPLC method

Vehicle:

yes

Remarks:

water

Details on test solutions:

A stock solution containing 1.0 mg/L test substance in dilution water was prepared. The lower concentrations to be tested were prepared by dilution of this stock solution with dilution water.

Test organisms (species):

Daphnia magna

Details on test organisms:

- Species: Daphnia magna
- Authority: STRAUS
- Strain: Berlin
- Sex: female
- Age: between 0 and 24 hours
- Origin: Umweltbundesamt Berlin
- Breeding: In-house breeding since 27. September 2007

Selection of the test system was made following the proposal of the guidelines.

Animal Husbandry
Daphnia magna is bred in the LAUS GmbH throughout the year. The animals are kept for the use in toxicity tests. They multiply by parthenogenesis, thus being genetically identi-cal. The husbandry is performed similar to the method described in the OECD guideline, following SOP 115 002 01 („Zucht und Hälterung von Daphnia magna STRAUS“), Version 12 from 02. Feb. 2015.
Vessels: preserving glasses, nominal volume 2 L
Medium: M4-Medium (recipe of ELENDT)
Food: unicellular green algae (Desmodesmus subspicatus)
Medium renewal: twice a week
Photo period : 16/8 hours, using neon tubes
Temperature 20 ± 2°C

Dilution water specification:
Parameter Concentration in mg/L
CaCl2*2H2O 293.80
MgSO4*7H2O 123.30
NaHCO3 64.80
KCl 5.80
Resulting hardness in mmol/L: 2.502
Resulting hardness in mg CaCO3/L: 250
Deviations from the nominal weighted loads were less than 5%. Exact values ae recorded in the raw data. After preparation, the dilution water was aerated and the pH was meas-ured. The pH was 7.8.

Test type:

static

Water media type:

freshwater

Total exposure duration:

48 h

Hardness:

250 mg CaCO3/L

Test temperature:

19.6 - 20.8°C

pH:

7.8 to 7.9

Dissolved oxygen:

8.5 mg/L

Nominal and measured concentrations:

Nominal concentrations: 10, 22, 46, 100 and 220 µg/L
Measured concentrations at 0 h: 4.34, 14.43, 28.83, 61.71 and 132.29 µg/L
Measured concentrations at 48 h: 1.48, 5.33, 14.66, 39.95 and 75.79 µg/L
Geometric mean measured concentration: 2.5, 8.8, 20.6, 49.7 and 100.7 µg/L

Reference substance (positive control):

yes

Remarks:

Potassium dichromate K2Cr2O7 (CAS No. 7778-50-9)

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.05 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

mobility

Details on results:

The study was performed using 5 concentrations ranging from 10 to 220 µg/L (nominal). For each test concentration, 20 Daphnia were exposed to the test substance for 48 hours in a static test system. After 24 and 48 hours, the immobilised Daphnia were counted. The highest tested concentration showed toxicity of 50% immobilisation. None of the an-imals was immobilised in the blank control. Potassium dichromate K2Cr2O7 (CAS No. 7778-50-9) was used as positive control in a current reference study to assure that the test conditions are reliable.

At the beginning and at the end of the test, the content of the test substance in the test solutions was determined using HPLC-LC-MS-MS-determination. The concentrations determined at the start of the test were between 43 and 66% of the nominal concentration. At the end of the test the determined concentrations were between 15 and 40% of the nominal concentration. During validation the test substance was stable under test condi-tions. Because of the very low tested concentrations in this study, it might be possible that the measurable amount of test substance was biased by the presence of the test organ-ism (ingestion or adsorption of test substance onto the Daphnia). Therefore, the determi-nation of the biological results was based on the geometric mean of the measured con-centrations.

Results with reference substance (positive control):

The 24h-EC50i value was determined as 1.6 mg/L. The value was within the requested range for the 24h-EC50 between 0.6 - 2.1 mg/L of potassium dichromate K2Cr2O7 as speci-fied in the OECD guideline. Since the fits showed sufficient statistical correspondence of the data with the dose-response-equation, this reference study can be considered valid.

Reported statistics and error estimates:

Statistical calculation using toxrat® professional 3.2.1

In the blank control, none of the Daphnia was immobilised (see table below).

Nominal Concentration in µg/L	Immobility 24 hours					Immobility 48 hours
	absolute				in %	absolute				in %
Blank control	0	0	0	0	0	0	0	0	0	0
10	0	0	0	0	0	0	0	0	0	0
22	0	0	0	0	0	0	0	0	0	0
46	0	0	0	0	0	0	0	0	0	0
100	0	0	0	0	0	0	0	0	0	0
220	0	1	0	2	15	3	2	3	2	50

pH and O₂-values

Nominal Concentration in µg/L	pH		O2-Concentration in mg/L
	0 h	48 h	0 h	48 h
Blank control	7.8	7.8	8.6	8.4
10	7.8	7.8	8.6	8.5
22	7.8	7.9	8.6	8.4
46	7.9	7.9	8.6	8.4
100	7.8	7.8	8.6	8.3
220	7.8	7.8	8.6	8.4

Analytical Determinations:

The measured concentrations at the beginning of the test were between 43 and 66% of the nominal concentration and after 48 hours the measured concentrations were between 15 and 40% of the nominal concentration. During validation the test substance was stable under test conditions. Because of the very low tested concentrations in this study, it might be possible that the measurable amount of test substance was biased by the presence of the test organism (ingestion or adsorption of test substance onto the Daphnia). Therefore, the determination of the biological results was based on the geometric mean of the measured concentration. Geometric mean is calculated by multiplication of the n participating concentrations and taking the n^throot. Since measured concentrations at the end of the test were not usable (faulty QC sample stock solution), new QC samples were prepared and stored together with the samples till the next measurement.

Measured Concentrations

Nominal Concentration Test Substance	Measured concentration t = 0 h	Measured concentration t = 48 h	Measured concentration as % of nominal concentration t = 0 h	Measured concentration as % of nominal concentration t = 48 h
µg/L	µg/L	µg/L	%	%
Blank control	0.41	0.3	--	--
10	4.34	1.48	43	15
22	14.43	5.33	66	24
46	28.83	14.66	63	32
100	61.71	39.95	62	40
220	132.29	75.79	60	34

LOQ (Limit of quantification) = 0.19 µg/L

Geometric Mean

Nominal Concentration Test Substance	Geometric Mean of measured concentrations
µg/L	µg/L
Blank control	--
10	2.5
22	8.8
46	20.6
100	49.7
220	100.1

The biological results are presented in the following table:

Biological Results Test Substance (based on geometric mean of measured concentrations)

Parameter	Value	95%-confidence interval
24h EC50	n.d.	n.d.
48h EC50	100.1 µg/L	n.d.
48h NOEC	49.7 µg/L	--
48h LOEC	100.1 µg/L	--

n.d. = not determined due to mathematical reasons

Validity:

1)   Immobilisation in the controls may not exceed 10 %.
2) Immobilisation in the controls was 0 %.

3) The concentration of dissolved oxygen at the end of the test must be at least 3 mg/L. The lowest concentration of dissolved oxygen at the end of the test was 8.3 mg/L.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the study, the 48 h EC50 and NOEC were determined to be at 0.1001 mg a.i./L and 0.0497 mg a.i./L (nominal) respectively.

Executive summary:

A study was conducted to determine the short-term toxicity of the test substance to aquatic invertebrates according to OECD Guideline 202 and EU Method C.2, in compliance with GLP. Daphnia magna were exposed in a static test to nominal concentrations of 0, 10, 22, 46, 100 and 220 µg/L test substance, using 20 daphnids per concentration. Mobility was monitored throughout the experiment. Analytical dose verification was performed at the beginning and at the end of the test. The content of the test substance in the test solutions was determined using HPLC-LC-MS-MS-determination. The concentrations determined at the start of the test were between 43 and 66% of the nominal concentration. At the end of the test the determined concentrations were between 15 and 40% of the nominal concentration. During validation, the test substance was found to be stable under test conditions. Because of the very low tested concentrations in this study, it might be possible that the measurable amount of test substance was biased by the presence of the test organism (ingestion or adsorption of test substance onto the Daphnia). Therefore, the determination of the biological results was based on the geometric mean of the measured concentrations. The result of the test is considered valid as all validity criteria are met. Under the conditions of the study, the 48 h EC50 was determined to be at 0.1001 mg a.i./L (nominal) (Wigh, 2018).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.1 mg/L

Additional information

A study was conducted to determine the short-term toxicity of the test substance to aquatic invertebrates according to OECD Guideline 202 and EU Method C.2, in compliance with GLP. Daphnia magna were exposed in a static test to nominal concentrations of 0, 10, 22, 46, 100 and 220 µg/L test substance, using 20 daphnids per concentration. Mobility was monitored throughout the experiment. Analytical dose verification was performed at the beginning and at the end of the test. The content of the test substance in the test solutions was determined using HPLC-LC-MS-MS-determination. The concentrations determined at the start of the test were between 43 and 66% of the nominal concentration. At the end of the test the determined concentrations were between 15 and 40% of the nominal concentration. During validation, the test substance was found to be stable under test conditions. Because of the very low tested concentrations in this study, it might be possible that the measurable amount of test substance was biased by the presence of the test organism (ingestion or adsorption of test substance onto the Daphnia). Therefore, the determination of the biological results was based on the geometric mean of the measured concentrations. The result of the test is considered valid as all validity criteria are met. Under the conditions of the study, the 48 h EC50 was determined to be at 0.1001 mg a.i./L (nominal) (Wigh, 2018).