Amides, C16-C18 (even) , N,N'-ethylenebis

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:

15 Nov - 18 Nov 2005

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

GLP compliance:

yes

Remarks:

according to the Japanese GLP Standard

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0, 100 mg/L
- Sampling method: test solution for analysis was separately prepared for analysis at the initiation of the test. After exposure, same volume of test solution was sampled directly from each test vessel and cetrifuged (3000 rpm, 10 min) to get rid of alga.
- Sample storage conditions before analysis: analysis was conducted immediately after sampling, no storage

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: test item is immiscible with water and therefore a WAF was prepared by adding the test item in dilution water. After 30 minutes sonication, test water was stirred (750 rpm) for 24 hours at 23± 2 °C. Test water was filtered with No. 2 filter paper.
- Controls: dilution water
- Evidence of undissolved material (e.g. precipitate, surface film, etc): test water was colorless after preparation

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Strain: ATCC22662
- Source: American Type Culture Collection
- Method of cultivation: on rotary shaker

ACCLIMATION
- Acclimation period: 5 days (11/11/2005 - 15/11/2005)
- Culturing media and conditions (same as test or not): same as test

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

22.0 - 22.7 (23 ± 2 °C)

pH:

7.7 - 9.9

Nominal and measured concentrations:

nominal concentration: 100 mg/L
measured concentration: 0.053 (at the initiation), 0.00619 (after exposure) mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 300 mL Erlenmeyer flask with an air-permeable stopper
- Type: open
- Material, fill volume: glass, 100 mL
- Aeration: no
- Initial cells density: 1000 cells/mL
- Control end cells density: 2883100 ± 120600
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: not stated
- Intervals of water quality measurement: Determination and observation: pH (0 and 72 hour), temperature (0, 24, 48. 72 h) and apperance of test solutions (after preparation) were determined.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: not adjusted
- Photoperiod: continuously
- Light intensity and quality: 4000 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: counting chamber; electronic particle counter; after 0, 24, 48 and 72 h.

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: control, 10.0, 50.0, 100 mg/L
- Results used to determine the conditions for the definitive study: growth inhibition rate: 10.0 mg/L: -8%, 50.0 mg/L: -11%, 100 mg/L: -40%

Reference substance (positive control):

yes

Remarks:

potassium dichromate (reagent grade)

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 39.8 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Remarks:

refers to N N'-ethylene-bis-octadecanamide

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

> 39.8 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Remarks:

refers to N N'-ethylene-bis-octadecanamide

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.006 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

> 0.006 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): not observed
- Unusual cell shape: not observed
- Flocculation: not observed
- Adherence to test vessels: not observed
- Aggregation of algal cells: not observed

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EbC50: 0.430 ± 0.0698 mg/L (n=17)
- Other: this test is performed every 6 months.

Reported statistics and error estimates:

The EC50 values and associated 95% confidence limits could not be determined statistically because the test was conducted at one concentration level. The NOEC were determined by Student's t-testm subsequent to F test for homogeneity of variances. Statistical analyses were performed using Yukms Statlight #3 software (Yukms Corp., Tokyo) and all tests of significant were at α=0.05.

Table 1. Measured Concentration of the Test Substance in Test Cultures

Loading Rate (*1) (mg/L)	Measured Concentration (mg/L) (Percent of 0hr)
	0 Hour	72 Hour
Control	<0.002	<0.00002
100	0.053	0.00619 (12)

(*1): The ratio of test substance to water (in mg/L) used in the preparation of the WAF.

Table 2. Cell Densities of Pseudokirchneriella subcapitata during the 72 -Hour Exposure

Loading Rate [Measured Conc. at 0 hr] (mg/L)	Cell Densities (cells/mL)
		0 Hour	24 Hour	48 Hour	72 Hour
Control	Average	10000	82800	756900	2883100
	SD	0	11900	123400	120600
100 [0.053]	Average	1000	83000	778900	3413100
	SD	0	16300	138300	70200

Table 3: Validity criteria for OECD 201.

Criterion from the guideline	Outcome	Validity criterion fulfilled
The biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72-hour test period.	>16	yes
The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72-hour tests) in the control cultures must not exceed 35%	<35%	yes
The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7% in tests with Pseudokirchneriella subcapitata and Desmodesmus subspicatus. For other less frequently tested species, the value should not exceed 10%.	<7%	yes

Validity criteria fulfilled:

yes

Remarks:

Please refer to table 3 in section "Any other information on results incl. tables"

Description of key information

The substance is not toxic to aquatic algae.

Additional information

The toxicity of the substance to the freshwater algae Pseudokircheneriella subcapitata was investigated in a limit test following the OECD Guideline 201. A water accommodated fraction (WAF) with a nominal loading rate of 100 mg/L was prepared. The test concentration was verified by LC/MS. Effects on growth rate of Pseudokircheneriella subcapitata were not observed up to the highest attainable concentration. Therefore, an EC50 (72 h) > 0.053 mg/L (measured) and a NOEC (72 h) ≥ 0.053 mg/L (measured) was determined.

Based on the available study results, the substance is not considered toxic to aquatic algae.