Reaction mass of N-2-hydroxyethylacetamide and N,O-diacetyl-2-aminoethanol

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 January 29, 2018 to February 01, 2018

Reliability:

2 (reliable with restrictions)

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:

no

Details on sampling:

No confirmatory analysis of the test solutions was performed as a validated analytical method could not be developed. One blank vessel (without algal inoculum) was incubated concurrently for each control and test concentration sampling occasion. Please refer to the statement from the test lab under the 'attached background material.

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 6.25, 12.5 25, 50 and 100 mg/L. A primary stock concentrate of test substance with a nominal concentration of 100 mg/L was prepared by weighing a nominal 0.2 g of test substance (weight: 0.20017 g) into a 2000 mL volumetric flask and made up to volume with AAP culture medium. The stock was then stirred for 11 minutes. The resultant stock was observed to be clear and colourless with no particles visible 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 AAP medium only. In all cases the final solutions contained nutrients. The 100 mg/L stock was used directly to fill the nominal 100 mg/L concentration test vessels. All test solutions were observed as clear and colourless after approximately 15 minutes stirring. 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 3-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. 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:

7.19 to 7.35

Nominal and measured concentrations:

0, 6.25 12.5, 25, 50 and 100 mg/L (nominal)
No confirmatory analysis of the test solutions was performed as a validated analytical method could not be developed.

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.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (total fraction)

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

other: EyC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (total fraction)

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

ca. 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (total fraction)

Basis for effect:

other: growth rate and biomass

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (total fraction)

Basis for effect:

other: growth rate and biomass

Results:

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 Bonferroni adjusted t test to identify significant differences (p<0.05) from the control. 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 growth rate/day (0-72 h)	Mean growth rate/d 95% Cl	Percentage ofcontrol (%)
Culture medium control	1.37	1.35-1.39	-
6.25	1.32*	1.23-1.40	96
12.5	1.33	1.29-1.37	97
25	1.34	1.31-1.38	98
50	1.34	1.27-1.42	98
100	1.35	1.29-1.40	98

* Significant difference (p <0.05) from the control. LOEC value not affected

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 (mg/L)
NOEC	100	-
LOEC	>100	-
ErC50	>100	N/A
ErC20	>100	N/A
ErC10	>100	N/A

Based on the growth rate, there was a significant difference (p <0.05) between the nominal 6.25 mg/L concentration and the control. However, as the growth rate was not significantly different (p <0.05) from the control in any of the higher concentrations, the LOEC was not affected

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. These cell particle densities were examined by one-way analysis of variance and a Bonferroni adjusted t test to identify significant differences (p<0.05) from the control.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 yield (0-72 h) (10^4cells/ml)	Mean yield 95% Cl (10^4cells/ml)	Percentage ofcontrol (%)
Culture medium control	34.9	32.6-37.2	-
6.25	30.0*	22.2-37.8	86
12.5	30.9	27.0-34.9	89
25	32.4	28.9-35.9	93
50	32.5	25.1-39.8	93
100	32.6	27.2-38.1	93

* Significant difference (p <0.05) from the control. LOEC value not affected

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 (mg/L)
NOEC	100	-
LOEC	>100	-
EyC50	>100	N/A
EyC20	>100	N/A
EyC10	>100	N/A

Based on the yield, there was a significant difference (p <0.05) between the nominal 6.25 mg/L concentration and the control. However, as the yield was not significantly different (p <0.05) from the control in any of the higher concentrations, the LOEC was not affected

Additional biological data

The microscopic observations, made at the end of the test, showed that compared to the control the algal cells sampled from all test solution concentrations appeared normal.

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 60.7 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 19%.

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 1.5%.

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

Validity criteria fulfilled:

yes

Conclusions:

Under the study conditions, 72 h ErC50, EyC50, NOEC and LOEC values for the test substance in freshwater green algae were determined to be >100, >100, 100 and >100 mg/L, respectively.

Executive summary:

A study was conducted to determine the acute toxicity of the test substance 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 test substance concentration were employed. Each replicate test vessel was inoculated with 0.296 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 0.585 × 104 cells/mL. This value was used for growth calculations. Three replicate algal cultures were exposed to test substance at nominal concentrations of 0, 6.25 12.5, 25, 50 and 100 mg/Lin AAP medium for 72 h. No confirmatory analysis of the test solutions was performed as a validated analytical method could not be developed. Algal cell particle densities (cell particles per unit volume measured as a surrogate for biomass) and growth rate were calculated for each replicate culture. ErC50 (for growth rate) and EyC50 (cell particle density) values were both found to be >100 mg/L. The NOEC and the LOEC were 100 mg/L and >100 mg/L, respectively, for both growth rate and cell particle densities. The study results fulfilled the all guideline required validity criteria. Under the study conditions, 72 h ErC50, EyC50, NOEC and LOEC values for the test substance in freshwater green algae were determined to be >100, >100, 100 and >100 mg/L, respectively (Scymaris, 2018).

Description of key information

Based on the study results, the 72 h ErC50 and NOEC values for the test substance for toxicity to freshwater green algae were determined to be >100 and 100 mg/L (nominal), respectively.

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

100 mg/L

Additional information

A study was conducted to determine the acute toxicity study of the test substance 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 0.296 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 0.585 × 104 cells/mL. This value was used for growth calculations. Three replicate algal cultures were exposed to test substance at nominal concentrations of 0,6.25 12.5, 25, 50 and 100 mg/Lin AAP medium for 72 h. No confirmatory analysis of the test solutions was performed as a validated analytical method could not be developed. Algal cell particle densities (cell particles per unit volume measured as a surrogate for biomass) and growth rate were calculated for each replicate culture. ErC50 value (for growth rate) and EyC50 value (cell particle density) were found to be >100 and >100 mg/L respectively. The no observed effect concentration (NOEC) and the lowest observed effect concentration (LOEC) values were found to be 100 mg/L and >100 mg/L, respectively, for both growth rate and cell particle densities. The study results were found to have fulfilled the all the Guideline required validity criteria. Under the study conditions, 72 h ErC50, EyC50, NOEC and LOEC values for the test substance with freshwater green algae, were determined to be >100, >100, 100 and >100 mg/L, respectively (Scymaris, 2018).