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

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From September 04, 2017 to September 09, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

To demonstrate that test vessels were dosed with nominal exposure concentrations of test substance, whole test vessels were analysed for both dissolved and undissolved test substance using the high-performance liquid chromatography mass spectrometry method. At the start of the test, additional sacrificial test vessels were used for sampling and at the end of the test one replicate of the dilution water control and each test concentration was sampled. The method of whole sample analysis was used due to the low solubility of the compound.

Vehicle:

yes

Remarks:

AAP-medium

Details on test solutions:

Preparation of test solutions
The study was run with a culture medium control and nominal exposure concentrations of 0.004, 0.01, 0.04, 0.11, 0.33 and 1.0 mg/L. A primary stock concentrate of Quaternium 91, with a nominal concentration of 10 mg/L, was prepared by weighing a nominal 0.01 g of test substance and making up to 1000 mL with the culture medium AAP in a volumetric flask. The stock was sonicated in an ultrasonic bath for 3 h and left to stir over night. The resultant stock was observed to be a hazy colourless solution and was used to prepare the test solutions. This was achieved by adding the relevant volumes of the primary stock to 1000 mL of stirring AAP media in a volumetric flask. The control consisted of culture medium only. In all cases the final solutions contained nutrients. The test solutions were all observed to be clear and colourless. The appropriate test solution (100 mL volume) was dispensed to each test and blank vessel.

1 mg/L test concentration was selected as the highest concentration based on a non-GLP range finding study.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

The test species was the unicellular green alga Pseudokirchneriella subcapitata strain CCAP 278/4 from laboratory cultures maintained under axenic conditions. A 4 d old culture of the alga in the exponential growth phase was used as inoculum for the test. The culture was grown in the medium, and under the environmental conditions, described for the test.

Test type:

not specified

Water media type:

other: AAP-medium

Limit test:

no

Total exposure duration:

72 h

Test temperature:

22 ± 2ºC

pH:

7.23 to 7.42

Nominal and measured concentrations:

1 mg/L highest concentration (Based on non-GLP range finding study)
0, 0.004, 0.01, 0.04, 0.11, 0.33 and 1.0 mg/L (nominal)
0, 0.0024, 0.0070, 0.017, 0.060 and 0.15 mg/L (measured) [Based on whole sample extraction (dissolved and undissolved test substance)]

Details on test conditions:

Appratus
The test vessels were glass conical flasks of 250mL nominal capacity closed with foam bungs. Each flask contained 100 mL of test solution. The cultures were incubated at 22±2°C (the nominal test temperature), under continuous "cool-white" illumination of approximately 6000 lux, with nominal orbital shaking at 160 rpm.

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

other: ErC50

Effect conc.:

> 0.15 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat. (total fraction)

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

other: NOECr

Effect conc.:

ca. 0.06 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat. (total fraction)

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

other: LOECr

Effect conc.:

ca. 0.15 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat. (total fraction)

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

other: EyC50

Effect conc.:

ca. 0.123 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat. (total fraction)

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

other: NOECy

Effect conc.:

ca. 0.017 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat. (total fraction)

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

other: LOECy

Effect conc.:

ca. 0.06 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat. (total fraction)

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

other: ErC10

Effect conc.:

ca. 0.082 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat. (total fraction)

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

other: EyC10

Effect conc.:

ca. 0.04 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat. (total fraction)

Basis for effect:

biomass

Results:

Analytical data

The limit of quantification of test substance in this study was 0.0020 mg/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 analytical results for the 0.004 mg/L concentration at 0 and 72 h were below the limit of quantification (LOQ). Where values were <LOQ, 0 (zero) was used to calculate the mean measured concentration. On the basis of the analytical data the mean measuredconcentrations were used for the calculation and reporting of results. Please refer to the Analytical method validation in algal media document in attached background material section.

Biological data

Algal cell particle densities (cell particles per unit volume) were measured as a surrogate for biomass.

Growth rates

The growth rate (0 to 72 h) was calculated for each replicate culture. The growth rates were examined by one-way analysis of variance and a two‑sample t test to identify significant differences (p<0.05) between the control and solvent control. There was no significant effect (p=0.1737) between the control and solvent control. Therefore, the solvent control was compared to the treatments by one-way analysis of variance and Bonferroni adjusted t test to identify significant differences (p<0.05). The results obtained from these growth rate analyses, based on mean measured test concentrations, were as follows:

	Test substance (mg/L)
NOEC	0.06
LOEC	0.15
ErC50	>0.15
ErC20	0.120
ErC10	0.0816

Yield

This response is defined as the biomass at the end of the test minus the starting biomass. For the purposes of calculation, the cell particle density count (cell particles per unit volume) is an acceptable surrogate for biomass. These cell particle densities were examined by one-way analysis of variance and two-sample t test to identify significant differences (p<0.05) from the control and solvent control. There was no significant effect (p=0.1781) between the control and solvent control. Therefore, the solvent control was compared to the treatments by one-way analysis of variance and Bonferroni adjusted t test to identify significant differences (p<0.05). The EC₅₀, EC₂₀and EC₁₀values with their associated confidence intervals were subsequently calculated using the Linear Interpolation method.The results obtained from these statistical analysis, based on mean measured test concentrations, were as follows:

	Test substance (mg/L)
NOEC	0.017
LOEC	0.06
EyC50	0.123
EyC20	0.0684
EyC10	0.0401

Additional biological data

The microscopic observations, made at the end of the test, showed that compared to the control the algal cells sampled from the 0.004, 0.01, 0.04, 0.11 and 0.33 mg/L test concentrations appeared normal. Cells sampled from 1.0 mg/L test concentration appeared small.

Validity criteria

The validity criteria specified in the OECD 201 guideline are;

1) To achieve ≥16 fold exponential increase in biomass in the control replicates within the 72 h test period. In this test cell particle density increase was 38 over the 72 h for the control.

2) The mean coefficients of variation for control replicate sectional (daily) specific growth rates must not exceed 35% and in this test, was determined to be 31%.

3) The replicate coefficient of variation of average specific growth rates during the whole test period in the control replicate cultures must not exceed 7% and was calculated as 91%.

Based on the study results, it was concluded that the study has fulfilled validity criteria.

Validity criteria fulfilled:

yes

Conclusions:

Under the study conditions, the 72 h ErC50, ErC10, EyC50, EyC10, NOECr, NOECy LOECr and LOECy values of the test substance, di-C18-22 AAEMIM-MS, for toxicity to freshwater green algae, were determined to be >0.15, 0.0816, 0.0123, 0.0401, 0.06, 0.017, 0.15 and 0.06 mg/L (measured), respectively.

Executive summary:

A study was conducted to determine the acute toxicity study of the test substance, ‘di-C18-22 AAEMIM-MS’ (active: 100%), to freshwater green algae (Pseudokirchneriella subcapitata), according to OECD Guideline 201, in compliance with GLP. Six replicates of the culture medium control and solvent control and triplicates of each concentration of the test substance were employed. Three replicate algal cultures, with a nominal cell density of approximately 0.5 x 10⁴cells/mL, were exposed to test substance at nominal concentrations of 0.004, 0.01, 0.04, 0.11, 0.33 and 1.0 mg/L in AAP medium for 72 h. One 100 mL volume of Coulter electrolyte, inoculated in the same manner, had a cell density of 0.629 × 10⁴cells/mL and was used for growth calculations. The exposure levels of test substance in aqueous samples of test media were monitored using a HPLC method of analysis. Based on whole sample extraction (dissolved and undissolved), the measured concentration of the test substances were found to be 0, 0.0024, 0.0070, 0.017, 0.060 and 0.15 mg/L. The test results were expressed in terms of measured concentration of test substance. Algal cell particle densities (cell particles per unit volume measured as a surrogate for biomass) and growth rate were calculated for each replicate culture. The ErC50 and ErC10 values for growth rate were found to be >0.15 and 0.0816 mg/L respectively, whereas, EyC50 and EyC10 values for cell particle density was found to be 0.123 and 0.0401 mg/L respectively. The NOEC and the LOEC values for growth rate were found to be 0.06 mg/L and 0.15 mg/L, respectively and for cell particle densities, they were found to be 0.017 and 0.06 mg/L, respectively. The study was considered to have met all the validity criteria. Under the study conditions, the 72 h ErC50, ErC10, EyC50, EyC10, NOECr, NOECy LOECr and LOECy values of the test substance, di-C18-22 AAEMIM-MS, for toxicity to freshwater green algae, were determined to be >0.15, 0.0816, 0.0123, 0.0401, 0.06, 0.017, 0.15 and 0.06 mg/L (measured), respectively (Scymaris, 2017).

Description of key information

Based on the results of the study, the 72 h ErC50, ErC10, EyC50, EyC10, NOECr, NOECy LOECr and LOECy values of the test substance, di-C18-22 AAEMIM-MS, for toxicity to freshwater green algae, were determined to be >0.15, 0.0816, 0.0123, 0.0401, 0.06, 0.017, 0.15 and 0.06 mg/L (measured), respectively.

Key value for chemical safety assessment

EC50 for freshwater algae:

0.15 mg/L

EC10 or NOEC for freshwater algae:

0.082 mg/L

Additional information

A study was conducted to determine the acute toxicity study of the test substance, ‘di-C18-22 AAEMIM-MS’ (active: 100%), to freshwater green algae (Pseudokirchneriella subcapitata), according to OECD Guideline 201, in compliance with GLP. Six replicates of the culture medium control and solvent control and triplicates of each concentration of the test substance were employed. Three replicate algal cultures, with a nominal cell density of approximately 0.5 x 10⁴cells/mL, were exposed to test substance at nominal concentrations of 0.004, 0.01, 0.04, 0.11, 0.33 and 1.0 mg/L in AAP medium for 72 h. One 100 mL volume of Coulter electrolyte, inoculated in the same manner, had a cell density of 0.629 × 10⁴cells/mL and was used for growth calculations. The exposure levels of test substance in aqueous samples of test media were monitored using a HPLC method of analysis. Based on whole sample extraction (dissolved and undissolved), the measured concentration of the test substances were found to be 0, 0.0024, 0.0070, 0.017, 0.060 and 0.15 mg/L. The test results were expressed in terms of measured concentration of test substance. Algal cell particle densities (cell particles per unit volume measured as a surrogate for biomass) and growth rate were calculated for each replicate culture. The ErC50 and ErC10 values for growth rate were found to be >0.15 and 0.0816 mg/L respectively, whereas, EyC50 and EyC10 values for cell particle density was found to be 0.123 and 0.0401 mg/L respectively. The NOEC and the LOEC values for growth rate were found to be 0.06 mg/L and 0.15 mg/L, respectively and for cell particle densities, they were found to be 0.017 and 0.06 mg/L, respectively. The study was considered to have met all the validity criteria. Under the study conditions, the 72 h ErC50, ErC10, EyC50, EyC10, NOECr, NOECy LOECr and LOECy values of the test substance, di-C18-22 AAEMIM-MS, for toxicity to freshwater green algae, were determined to be >0.15, 0.0816, 0.0123, 0.0401, 0.06, 0.017, 0.15 and 0.06 mg/L (measured), respectively (Scymaris, 2017).