Fatty acids, rape-oil, hydrogenated, reaction products with diethylenetriamine, di-Me sulfate-quaternized

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

From October 16, 2017 to October 21, 2017

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

KL2 due to RA study

Justification for type of information:

Refer to section 13 for details on the read-across justification. The study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Remarks:

High-performance liquid chromatography (HPLC)

Details on sampling:

To demonstrate that nominal exposure concentrations were being achieved the concentrations of test substance in the test vessels were measured using the high-performance liquid chromatography method. At the start of the test additional sacrificial vessels with dispensed test solution were taken for whole sample analysis and at the end of the test, replicate vessels of the dilution water control and each test concentration were taken for whole sample analysis. The method of whole sample analysis was used due to the low solubility of the compound.

Vehicle:

yes

Remarks:

Elendt's M4 D. magna medium

Details on test solutions:

The study was run with a dilution water control and nominal exposure concentrations of 0.0625, 0.125, 0.25, 0.5 and 1.0 mg/L. A primary stock concentrate of test substance, with a nominal concentration of 10 mg/L, was prepared by adding a nominal 0.01 g of test substance to 1000 mL of culture medium. The resultant stock was observed to be clear and colourless and was used to prepare the test solutions. This was achieved by the direct addition of the appropriate amount of concentrate to dilution water in a volumetric flask. The control consisted of culture medium only. 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 h light: 8 h 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 h old, obtained from two culture vessels, were used for testing. The parent animals were 20 ± 1 d old and 23 ± 1 d old and had been maintained with a twice weekly renewal of reconstituted water medium since birth. The test organisms and the cultures from which they were obtained showed no evidence of disease before the test period.

Test type:

static

Water media type:

other: Elendt's M4 D. magna medium

Limit test:

no

Total exposure duration:

48 h

Test temperature:

20±1°C

pH:

7.72 to 8.08

Dissolved oxygen:

9.12 to 9.31 mg/L

Nominal and measured concentrations:

Control and nominal concentrations of 0.0625, 0.125, 0.25, 0.5 and 1.0 mg/L
Control and mean measured concentrations of 0.040, 0.077, 0.15, 0.27 and 0.55 mg/L

Details on test conditions:

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.

Reference substance (positive control):

no

Key result

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

ca. 0.43 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat. (total fraction)

Basis for effect:

mobility

Remarks on result:

other: Confidence interval: 0.45– 0.45 mg/L

Remarks:

Linear Interpolation (ICPIN)

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

ca. 0.21 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat. (total fraction)

Basis for effect:

mobility

Remarks on result:

other: Confidence interval: 0.21– 0.21 mg/L

Remarks:

Linear Interpolation (ICPIN)

Key result

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

ca. 0.27 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat. (total fraction)

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

ca. 0.15 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat. (total fraction)

Basis for effect:

mobility

Details on results:

Analytical data
The limit of quantification of Test substance in this study was 0.004 mg/L. All analytical values are quoted to two significant figures and percentages to the nearest integer. The measured concentrations at the start of the study were 71-79% of nominal and at the end were 37-48% of nominal. On the basis of the analytical data the mean measured concentrations were used for the calculation and reporting of results.

Biological data
Based on immobility compared to the control (p <0.05) the 48 h No Observed Effect Concentration (NOEC) was determined to be 0.043 mg/L and the Lowest Observed Effect Concentration (LOEC) was 0.097 mg/L. There was no immobility observed in the dilution water control. No other symptoms of toxicity were observed.

Analytical results

Nominal concentration of test substance (mg/L)	Measured concentration of test substance (mg/L)				Mean measured concentration (mg/L)	Mean measured concentration (%)
	0 h		48 h
	(mg/L)	% of nominal	(mg/L)	% of nominal
Control	<LOQ	-	<LOQ	-	-	-
0.0625	0.049	79	0.030	48	0.040	64
0.125	0.094^a	75	0.060^b	48	0.077	62
0.25	0.18	72	0.12	47	0.15	59
0.5	0.36	71	0.19	37	0.27	54
1.0	0.71	71	0.39	39	0.55	55

All measurements are quoted to 2 significant figures

^a          Mean of triplicate analyses: 0.093, 0.094, 0.096 mg/L

^b          Mean of triplicate analyses: 0.030, 0.032, 0.033 mg/L

The limit ofquantification (LOQ)in thisstudy was 0.004 mg/L. The analytical method LOQ was 0.002 mg/L but during analysis, all analytical samples were diluted x2, doubling the study LOQ. The values in Table 1 have been corrected for this dilution.

Daphnia magna response

Time (h)	Nominal concentration of test substance (mg/L)	Mean measured concentration of test substance (mg/L)	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
	0.0625	0.040	0	0	0	0	20	0	0
	0.125	0.077	0	0	0	0	20	0	0
	0.25	0.15	0	0	0	0	20	0	0
	0.5	0.27	0	0	0	0	20	0	0
	1.0	0.55	4	5	4	4	20	17	85
48	Control	-	0	0	0	0	20	0	0
	0.0625	0.040	0	0	0	0	20	0	0
	0.125	0.077	0	0	0	0	20	0	0
	0.25	0.15	0	0	0	0	20	0	0
	0.5	0.27	5	5	5	5	20	20	100
	1.0	0.55	5	5	5	5	20	20	100

Validity criteria

As no immobility or other signs of stress were observed in the control and dissolved oxygen concentration remained at ≥3 mg/L, this test has satisfied all OECD Guideline 202 validity criteria.

Validity criteria fulfilled:

yes

Conclusions:

Under study conditions, the 48 h EC50was determined to be 0.21 mg/L.

Executive summary:

A study was conducted to determine the acute toxicity of read across substance, 'di-C16-18-satd. and C18-24-unsatd. AEMIM-MS' to Daphnia magna, according to OECD Guideline 202, in compliance with GLP. Twenty test organisms were exposed to each nominal test substance concentrations of 0, 0.0625, 0.125, 0.25, 0.5 and 1.0 mg/L for 48 h under static conditions. The concentrations of test substance in the test vessels were measured using HPLC method of analysis. The mean measured concentrations of test substance were determined to be 0, 0.040, 0.077, 0.15, 0.27 and 0.55 mg/L respectively. Immobilisation was recorded at 24 and 48 h and compared with control values. Based on immobility compared to the control (p <0.05), the 48 h NOEC and LOEC were determined to be 0.15 and 0.27 mg/L (measured). No other symptoms of toxicity were observed. Based on the study results, the 48 h EC50 was calculated to be 0.21 mg/L (measured). As no immobility or other signs of stress were observed in the control and dissolved oxygen concentration remained at ≥3 mg/L, the test was considered to have satisfied all the validity criteria. Under study conditions, the 48 h EC50 and NOEC value of the read across substance in Daphnia magna was determined to be 0.21 and 0.15 mg/L (measured) respectively (Scymaris, 2017). Based on the results of the read across study, similar 48 h EC50 value can be expected for the test substance, 'di-C18-22 AAEMIM-MS', for short term toxicity to aquatic invertebrates.

Description of key information

Based on the results of the read across study and the study with the formulation containing the test substance, the 48 h EC50 value of the test substance for toxicity to aquatic invertebrates is considered to be 0.21 mg/L (measured).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.21 mg/L

Additional information

Study 1:

A study was conducted to determine the acute toxicity of read across substance, 'di-C16-18-satd. and C18-24 -unsatd. AEMIM-MS' to Daphnia magna, according to OECD Guideline 202, in compliance with GLP. Twenty test organisms were exposed to each nominal test substance concentrations of 0, 0.0625, 0.125, 0.25, 0.5 and 1.0 mg/L for 48 h under static conditions. The concentrations of test substance in the test vessels were measured using HPLC method of analysis. The mean measured concentrations of test substance were determined to be 0, 0.040, 0.077, 0.15, 0.27 and 0.55 mg/L respectively. Immobilisation was recorded at 24 and 48 h and compared with control values. Based on immobility compared to the control (p <0.05), the 48 h NOEC and LOEC were determined to be 0.15 and 0.27 mg/L (measured). No other symptoms of toxicity were observed. Based on the study results, the 48 h EC50 was calculated to be 0.21 mg/L (measured). As no immobility or other signs of stress were observed in the control and dissolved oxygen concentration remained at ≥3 mg/L, the test was considered to have satisfied all the validity criteria. Under study conditions, the 48 h EC50 and NOEC value of the test substance in Daphnia magna was determined to be 0.21 and 0.15 mg/L (measured) respectively (Scymaris, 2017).

Study 2: A study was conducted to determine the short-term toxicity potential of a formulation containing 39% of the test substance, 'di-C18 -22 AAEMIM-MS', to Daphnia magna according OECD guideline 202, in compliance with GLP. The test formulation was not soluble in water, therefore test water soluble fractions were prepared. Test solutions were stirred on magnetic stir plates at 22-23°C for 24 h 20 minutes, and allowed to sit for 1 h 10 minutes and then vacuum filtered and the filtrate was used for the testing.The loading rates used in the test were chosen based on the results of a 48 h range-finding test conducted previously. In this range finding test, nominal loading rates of 37.8, 193.4, 379.2, 3890.3, and 10012.1 mg/L were tested. There was 100% immobilisation observed at all loading rates. Therefore, much lower loading rates were tested in the definitive test, at nominal loading rates of 0.3, 0.6, 1.4, 2.9, and 5.7 mg test substance/L.At 24 h, an all-or-none response was observed, where there was no immobilisation at the two lowest loading rates tested (0.34 and 0.57 mg/L) and 100% immobilisation at the higher loading rates (≥1.49 mg/L). By 48 h, there were partial effects (mean 50% immobilisation) at these two lowest loading rates. No immobilised animal was reported in the control group. However, the test substance treated groups showed concentration-related immobilisation of the treated daphnids.The 24 h and 48 h EL50 estimate, based on nominal loading rates, were determined at 0.98 mg/L and 0.42 mg/L with 95% confidence limits of 0.57 and 1.49 mg/L and 0.28 – 0.54 mg/L, respectively. The pH, temperature and dissolved oxygen concentrations were measured in the test solutions at test initiation and completion. Under the study conditions, the 48 h EL50 of the test substance in Daphnia magna was determined to be 0.42 mg/L (i.e., equivalent to 0.16 mg a.i./L) (nominal) (Vizon, 2005).

Based on the available results, the 48 h EC50 value of 0.21 mg/L (measured) from the read across study, having higher active content, has been considered further for hazard/risk assessment.