Amides, coco, N,N-bis(hydroxyethyl); C10-12 and C18-unsatd. DEA

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 December 04, 2017 to December 11, 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

Remarks:

High-performance liquid chromatography mass spectrometry (HPLC/MS) method

Details on sampling:

To demonstrate that test vessels were dosed with nominal exposure concentrations of test substance, whole test vessels were analysed using the high-performance liquid chromatography mass spectrometry method. Samples for analysis at 0 h were taken from the excess test solutions and at 72 h from a single test replicate solution of the control and each test concentration.

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 0.625, 1.25, 2.5, 5.0 and 10 mg/L. A primary stock concentrate of test substance, with a nominal concentration of 100 mg/L, was prepared by weighing a nominal 0.1 g of test substance (actual weight: 0.09993 g) and making up to 1000 mL volume with the culture medium AAP in a volumetric flask. The stock was then stirred for 20 minutes. The resultant stock was observed to be a slightly hazy colourless solution with bubbles on the surface and was used to prepare the test solutions. This was achieved by adding the relevant volumes of the primary stock to AAP media and making up to 1000 mL volume 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 10 mg/L test solution was observed as having bubbles on the surface. The appropriate test solution (100 mL volume) was dispensed to each test and blank vessel.

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-day 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. The culture medium used for the test, and for the maintenance of cultures of the alga used as inoculum for the test was AAP-medium

Test type:

not specified

Water media type:

other: AAP-medium

Limit test:

no

Total exposure duration:

72 h

Test temperature:

22 ± 2ºC

pH:

7.17 to 8.32

Nominal and measured concentrations:

10 mg/L test concentration was selected as the highest concentration based on the toxicity demonstrated in a non-GLP range-finding study.
0, 0.625, 1.25, 2.5, 5.0 and 10 mg/L (Nominal)
0, 0.54, 1.2, 2.3, 4.6 and 8.8 mg/L (Mean measured)
a The nominal 0.097 mg/L test concentration was set up in error instead of 0.97 mg/L. As there was no significant effect at this concentration it was deemed not to affect the validity of the study.

Details on test conditions:

Appratus
The test vessels were glass conical flasks of 250 mL 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.:

ca. 2.9 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat. (total fraction)

Basis for effect:

growth rate

Remarks on result:

other: 95% Confidence interval: 2.5 - 3.3

Key result

Duration:

72 h

Dose descriptor:

other: EyC50

Effect conc.:

ca. 1.8 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat. (total fraction)

Basis for effect:

biomass

Remarks on result:

other: 95% Confidence interval: 1.5 - 1.9

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

ca. 1.25 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:

LOEC

Effect conc.:

ca. 2.5 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:

NOEC

Effect conc.:

ca. 0.625 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:

LOEC

Effect conc.:

ca. 1.25 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. 1.4 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat. (total fraction)

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval: 1.2-1.5

Key result

Duration:

72 h

Dose descriptor:

other: EyC10

Effect conc.:

ca. 0.9 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat. (total fraction)

Basis for effect:

biomass

Remarks on result:

other: 95% confidence interval: 0.73-1.1

Results:

Analytical data

The limits of quantification of test substance in this study were 0.02 mg/L for the 0 h time point samples and 0.2 mg/L for the 72 h time point samples. All analytical values were quoted to two significant figures and percentages to the nearest integer. The measured concentrations at the start of the study were 90-97% of nominal and at the end were 83-90% of nominal. Analytical calibrations were constructed using a minimum of 5 calibration levels, with a minimum R² value of 0.99. The maximum percentage difference from nominal concentration for standards at the LOQ is less than 30% and less than 20% at levels greater than the LOQ. On the basis of the analytical data the nominalconcentrations were used for the calculation and reporting of results. Please find attached method validation report "Analytical Method Validation in Algal media" in the below 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 EC₅₀, EC₂₀ and EC₁₀ values with their associated confidence intervals were subsequently calculated using the Linear Interpolation method (ICPIN).

The mean growth rates, are given in below table together with the rates expressed as percentages of the control.

Nominal concentration of test substance (mg/L)	Mean measured concentration of test substance (mg/L)	Mean growth rate/day (0-72 h)	Mean growth rate/day 95% Cl	Percentage ofcontrol (%)
Culture medium control	0	1.48	1.47-1.50	-
0.625	0.54	1.50	1.48-1.51	101
1.25	1.2	1.40	1.31-1.50	95
2.5	2.3	0.825 *	0.727-0.923	56
5.0	4.6	0.340 *	0.0710-0.609	23
10	8.8	0.403 *	0.307-0.498	27

* Significant differences (p <0.05) from the control

All biological measurements are quoted to 3 decimal places and percentages to the nearest integer.

The results obtained from these growth rate analyses, based on mean measured test concentrations, were as follows:

	Test substance (mg/L)	95% Confidence Limits
NOEC	1.25	N/A
LOEC	2.5	N/A
ErC50	2.9	2.5-3.3
ErC20	1.7	1.6-1.8
ErC10	1.4	1.2-1.5

Yield

The 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. The EC₅₀, EC₂₀ and EC₁₀ values with their associated confidence intervals were subsequently calculated using the Linear Interpolation method (ICPIN).

The yield mean values are shown in below table, together with the yields expressed as percentages of the control.

Nominal concentration of test substance (mg/L)	Mean measured concentration of test substance (mg/L)	Mean yield (0-72 h) (10^4cells/ml)	Mean yield (0-72 h) 95% CI	Percentage ofcontrol (%)
Culture medium control	0	44.5	42.2-46.9	-
0.625	0.54	46.5	44.6-48.4	104
1.25	1.2	35.2 *	25.5-44.9	79
2.5	2.3	5.77 *	3.95-7.58	13
5.0	4.6	0.983 *	-0.229-2.20	2
10	8.8	1.24 *	0.713-1.77	3

* Significant differences (p <0.05) from the control

Mean yield values are quoted to 3 significant figures and percentages to the nearest integer.

The results obtained from these statistical analysis, based on mean measured test concentrations, were as follows:

	Test substance (mg/L)	95% Confidence Limits
NOEC	0.625	N/A
LOEC	1.25	N/A
EyC50	1.8	1.5-1.9
EyC20	1.2	0.83-1.5
EyC10	0.90	0.73-1.1

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 nominal 0.625 and 1.25 mg/L test concentrations appeared normal. Algal cells from the nominal 2.5 mg/L concentration appeared deformed and enlarged. Algal cells from the nominal 5.0 and 10 mg/L concentrations appeared deformed.

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 (measured as a surrogate for biomass) was 85.6 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 8%.

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 in this test, was calculated to be 1.1%.

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, EyC50, ErC10, EyC10, NOErC, NOEyC, LOErC and LOEyC values of the test substance with freshwater green algae, were determined to be 2.9, 1.8, 1.4, 0.9, 1.25, 0.625, 2.5 and 1.25 mg/L (measured), respectively.

Executive summary:

A study was conducted to determine the acute toxicity study of the test substance, C10-12 and C18-unsatd. DEA, to freshwater green algae (Pseudokirchneriella subcapitata), according to OECD Guideline 201, in compliance with GLP. Six replicates of the culture medium control and triplicates of each concentration of the test substance were employed. Each replicate test vessel was inoculated with 0.892 mL of the inoculum culture to give a nominal cell density of 0.5 × 104 cells/mL. One 100 mL volume of Coulter electrolyte, inoculated in the same manner, had a cell density of 0.527 × 104 cells/mL. This value was used for growth calculations. Three replicate algal cultures were exposed to test substance at nominal concentrations of 0, 0.625, 1.25, 2.5, 5.0 and 10 mg/L in AAP medium for 72 h. The exposure levels of test substance in aqueous samples of test media were monitored using a HPLC/MS method of analysis. Based on whole sample extraction (dissolved and undissolved), the measured concentration of the test substances were found to be 0, 0.54, 1.2, 2.3, 4.6 and 8.8 mg/L. The test results were expressed in terms 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, ErC10 values (for growth rate) and EyC50, EyC10 values (Cell particle density) were found to be 2.9, 1.4 and 1.8, 0.9 mg/L respectively. The NOEC and the LOEC values were found to be 1.25 mg/L and 2.5 mg/L, respectively, for growth rate and 0.625 mg/L and 1.25 mg/L, respectively, for biomass. All validity criteria were considered to be met. Under the study conditions, the 72 h ErC50, EyC50, ErC10, EyC10, NOErC, NOEyC, LOErC and LOEyC values of the test substance with freshwater green algae, were determined to be 2.9, 1.8, 1.4, 0.9, 1.25, 0.625, 2.5 and 1.25 mg/L (measured), respectively (Scymaris, 2018).

Description of key information

Based on the study results, the 72 h ErC50, EyC50, ErC10, EyC10, NOErC, NOEyC, LOErC and LOEyC values of the test substance with freshwater green algae, were determined to be 2.9, 1.8, 1.4, 0.9, 1.25, 0.625, 2.5 and 1.25 mg/L (measured), respectively.

Key value for chemical safety assessment

EC50 for freshwater algae:

2.9 mg/L

EC10 or NOEC for freshwater algae:

1.4 mg/L

Additional information

A study was conducted to determine the acute toxicity study of the test substance, C10-12 and C18-unsatd. DEA, to freshwater green algae (Pseudokirchneriella subcapitata), according to OECD Guideline 201, in compliance with GLP. Six replicates of the culture medium control and triplicates of each concentration of the test substance were employed. Each replicate test vessel was inoculated with 0.892 mL of the inoculum culture to give a nominal cell density of 0.5 × 104 cells/mL. One 100 mL volume of Coulter electrolyte, inoculated in the same manner, had a cell density of 0.527 × 104 cells/mL. This value was used for growth calculations. Three replicate algal cultures were exposed to test substance at nominal concentrations of 0, 0.625, 1.25, 2.5, 5.0 and 10 mg/L in AAP medium for 72 h. The exposure levels of test substance in aqueous samples of test media were monitored using a HPLC/MS method of analysis. Based on whole sample extraction (dissolved and undissolved), the measured concentration of the test substances were found to be 0, 0.54, 1.2, 2.3, 4.6 and 8.8 mg/L. The test results were expressed in terms 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, ErC10 values (for growth rate) and EyC50, EyC10 values (Cell particle density) were found to be 2.9, 1.4 and 1.8, 0.9 mg/L respectively. The NOEC and the LOEC values were found to be 1.25 mg/L and 2.5 mg/L, respectively, for growth rate and 0.625 mg/L and 1.25 mg/L, respectively, for biomass. All validity criteria were considered to be met. Under the study conditions, the 72 h ErC50, EyC50, ErC10, EyC10, NOErC, NOEyC, LOErC and LOEyC values of the test substance with freshwater green algae, were determined to be 2.9, 1.8, 1.4, 0.9, 1.25, 0.625, 2.5 and 1.25 mg/L (measured), respectively (Scymaris, 2018).