N-(2-hydroxyethyl)-N-[2-[(1-oxooctyl)amino]ethyl]-β-alanine

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:

2007-06-05 to 2007-06-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

2006

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: all test concentrations and the control
- Frequency: at t=0 h, t=24 h and t=72 h
- Volume: 2 mL
- Sample storage conditions before analysis: Samples were stored in a freezer until analysis

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Preparation of test solutions started with highest test concentrations of 100 mg a.i./L (combined limit/range-finding test) or 220 mg a.i./L (final test). No special treatment other than a 15-minute magnetic stirring period was necessary to completely dissolve the test substance in the test
medium. The lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium. The final test solutions were all clear and colourless
- Controls: Test medium without test substance or other additives

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green alga
- Strain: NIVA CHL 1
- Source (laboratory, culture collection): in-house laboratory culture
- Age of inoculum (at test initiation): 4 days
- Method of cultivation: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C. Light intensity was 60 to 120 µE/m²/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm.

ACCLIMATION
- Culturing media and conditions (same as test or not): same as test

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

24 mg/L expressed as CaCO3 and the pH: 8.1 ± 0.2.

Test temperature:

22.7°C at the start of the test. During the exposure period the temperature measured in the incubator was maintained between 22.1 and 22.8°C.

pH:

control: 8.1 (t=0h), 7.9 (t=72h)
4.6 mg/L: 8.2 (t=0h), 7.9 (t=72h)
10 mg/L: 8.2 (t=0h), 7.9 (t=72h)
22 mg/L: 8.0 (t=0h), 7.9 (t=72h)
46 mg/L: 7.9 (t=0h), 7.9 (t=72h)
100 mg/L: 7.7 (t=0h), 7.9 (t=72h)
220 mg/L: 7.4 (t=0h), 7.8 (t=72h)

Nominal and measured concentrations:

nominal: 0, 4.6, 10, 22, 46, 100 and 220 mg/L
Analysis of the samples taken during the final test showed that measured concentrations were stable and in agreement with nominal throughout the 72-hour test period (90-97%). The lowest test group showed a slightly lower recovery between 82 and 86% relative to nominal.

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Material, size, headspace, fill volume: 100 mL, all-glass, containing 50 mL of test solution
- Aeration: continuous shaking
- Initial cells density: 1E04 cells/mL
- Control end cells density: 142.4E04 cells/mL
- No. of organisms per vessel: 1E04
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- 1 extra replicate of each concentration with algae for sampling after 24 hours.
- 2 replicates of the highest concentration without algae

GROWTH MEDIUM
- Standard medium used: yes (M2; according to the OECD 201 Guideline)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:Milli-Ro water (tap-water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA)
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: Continuously
- Light intensity and quality: TLD-lamps of the type ‘Cool-white’ of 30 Watt, with a light intensity within the range of 83 to 85 µE/m²/s.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: At the beginning of the test, cells were counted using a microscope and a counting chamber.
Thereafter cell densities were determined by spectrophotometric measurement of samples at 720 nm using a Varian Cary 50 single beam spectrophotometer with immersion probe (pathlength =20 mm). Algal medium was used as blank.

TEST CONCENTRATIONS
- Spacing factor for test concentrations final test: 2.2
- Range finding study: yes, with 48 h exposure period
- Test concentrations range-finding test: 0, 0.1, 1, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: The expected EC50 for growth rate reduction exceeded a nominal active
ingredient concentration of 100 mg/L. The expected EC50 for yield inhibition was between active ingredient concentrations of 10 and 100 mg/L.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

< 4.6 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.62 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: 95% c.i. 0.32 - 1.21 a.i. mg/L

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

65 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: 95% c.i. 42 - 99 mg a.i./L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

< 4.6 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

5.2 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

biomass

Remarks on result:

other: 95% c.i. 4.7 - 5.7 mg a.i./L

Details on results:

- Exponential growth in the control (for algal test): yes
- Growth rates were increasingly reduced with increasing concentrations, however the percentages reduction only slightly increased at concentrations of 22 mg a.i./L and higher with a maximum obtained reduction of 53%. Statistically significant reduction of growth rate was found in all concentrations tested
- Inhibition of yield increased with increasing concentration of the test item reaching a plateau value at 22 mg a.i./L. Statistically significant inhibition of yield was found at all test concentrations

Results with reference substance (positive control):

- Results with reference substance valid: Yes
- 72 h ErC50: 1.9 mg/L (95% c.i. 1.3 - 2.7 mg/L)
historical ranges: 72 h ErC50: 0.82 to 2.3 mg/L

Mean cell densities (x 1E04 cells/mL) during the final test:

Concentration Amphopropionate C8 (mg a.i./L)	Exposure time (hours)
	0	24	48	72
control	1.0	5.4	23.6	142.4
4.6	1.0	4.8	14.9	79.5
10	1.0	4.6	11.0	26.7
22	1.0	4.4	8.9	19.1
46	1.0	4.4	6.5	13.4
100	1.0	4.7	6.5	10.1
220	1.0	5.4	8.2	11.6

Validity criteria fulfilled:

yes

Conclusions:

The NOEC, EC10 and EC50 of Amphopropionate C8 to algae based on growth rate were < 4.6 mg a.i./L, 0.62 mg a.i./L (95% c.i. 0.32 - 1.21 a.i. mg/L) and 65 mg a.i./L (95% c.i. 42-99 mg/L). The NOEC and EC50 of Amphopropionate C8 to algae based on cell yield were < 4.6 mg a.i./L and 5.2 mg a.i./L (95% c.i. 4.7-5.7 mg/L).

Executive summary:

In a 72 hour toxicity study, the cultures of Pseudokirchneriella subcapitata, strain NIVA CHL 1 were exposed to Amphopropionate C8 (50.6% a.i.) at nominal concentrations of 0 (control), 4.6, 10, 22, 46, 100 and 220 mg/L under static conditions in accordance with OECD guideline 201 (adopted March 23, 2006) and EU Method C.3 (1992). Analysis of the samples taken during the final test showed that measured concentrations were stable and in agreement with nominal throughout the 72-hour test period (90-97%). The lowest test group showed a slightly lower recovery between 82 and 86% relative to nominal.

The NOEC, EC10 and EC50 of Amphopropionate C8 to algae based on growth rate were < 4.6 mg a.i./L, 0.62 mg a.i./L (95% c.i. 0.32 - 1.21 a.i. mg/L) and 65 mg a.i./L (95% c.i. 42-99 mg/L). The NOEC and EC50 of Amphopropionate C8 to algae based on cell yield were < 4.6 mg a.i./L and 5.2 mg a.i./L (95% c.i. 4.7-5.7 mg/L).

 

Results Synopsis

 

Test Organism: Pseudokirchneriella subcapitata

Test Type: Static

 

72hr NOErC: < 4.6 mg a.i./L

72 hr ErC50: 65 mg a.i./L                    95% c.i. 42 -99 mg/L

72hr NOEyC: < 4.6mg a.i./L

72 hr EyC50: 5.2 mg a.i./L                 95% c.i. 4.7 -5.7 mg/L

Endpoint(s) Effected:  growth rate, yield

Description of key information

NOEC(growth rate) <4.6 mg a.i./L; ErC10 = 0.62 mg a.i./L (95% c.i. 0.32 - 1.21 a.i. mg/L); ErC50 65 mg a.i./L (95% c.i. 42-99 mg/L); OECD TG 201, Pseudokirchneriella subcapitata, GLP, RL1

Key value for chemical safety assessment

EC50 for freshwater algae:

65 mg/L

EC10 or NOEC for freshwater algae:

0.62 mg/L

Additional information

In a 72 hour toxicity study, the cultures of Pseudokirchneriella subcapitata, strain NIVA CHL 1 were exposed to Amphopropionate C8 (50.6% a.i.) at nominal concentrations of 0 (control), 4.6, 10, 22, 46, 100 and 220 mg/L under static conditions in accordance with OECD guideline 201 (adopted March 23, 2006) and EU Method C.3 (1992). Analysis of the samples taken during the final test showed that measured concentrations were stable and in agreement with nominal throughout the 72-hour test period (90-97%). The lowest test group showed a slightly lower recovery between 82 and 86% relative to nominal.

The NOEC, EC10 and EC50 of Amphopropionate C8 to algae based on growth rate were < 4.6 mg a.i./L, 0.62 mg a.i./L (95% c.i. 0.32 - 1.21 a.i. mg/L) and 65 mg a.i./L (95% c.i. 42-99 mg/L). The NOEC and EC50 of Amphopropionate C8 to algae based on cell yield were < 4.6 mg a.i./L and 5.2 mg a.i./L (95% c.i. 4.7-5.7 mg/L).