[No public or meaningful name is available]

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

effects on growth of green algae

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 2009 - November 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2-Amino-5-cyano-N,3-dimethylbenzamide

Cas Number:

890707-29-6

Molecular formula:

C10H11N3O

IUPAC Name:

2-Amino-5-cyano-N,3-dimethylbenzamide

Test material form:

solid: particulate/powder

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Samples of control, solvent control and all test item concentrations (1.2, 2.6, 5.6, 12.4, 27.3 and 60 mg/L) were collected at 0 h and 72 h for analytical determination.

Test solutions

Vehicle:

yes

Remarks:

Dimethyl formamide (maximum dimethyl formamide concentration in test substance stock solution (60 mg/L) was less than 0.1 mL/L).

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A stock solution of test item (60 mg/L) was prepared by dissolving 53 mg of test item in 88 μL of dimethyl formamide and to this 883 mL filtered OECD TG 201 nutrient medium was added. The stock solution was kept in the sonicator for 60 minutes. After sonication, pH was adjusted to 8.10 using 0.1N NaOH solution. The test item stock solution was clear and colorless with no insoluble material present.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Green alga
- Strain: Pseudokirchneriella subcapitata
- Source: Primary culture supplied by Inst. Plant Physiology, University of Göttingen, Germany and maintained in the laboratory by periodical subculturing (once a week).

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

22.7-23.5°C

pH:

6.85-8.13

Nominal and measured concentrations:

Nominal concentrations: 1.2, 2.6, 5.6, 12.4, 27.3 and 60 mg/L. Mean measured concentrations: 1.131, 2.483, 5.266, 11.716, 25.810 and 57.210 mg/L

Details on test conditions:

TEST SYSTEM
- flasks were shaken continuously at approximately 100 rpm
- Test vessel: 250 mL Erlenmeyer flasks
- Type: closed with sterile non-absorbent cotton plugs
- Fill volume: 100 mL
- Initial cells density: 10000 cells/mL
- Control end cells density: 1241667
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes, OECD TG 201 medium

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: One day prior to inoculation, 4.5 L of culture medium was prepared by adding a known volume of each nutrient stock solution into a sterile beaker. The OECD TG 201 medium was then brought to 4.5 L volume with deionised water and the pH was adjusted to 8.13 using 0.1N NaOH solution. After the measurement of pH, the OECD TG 201 medium was sterilized through 0.2 μm pore size sterile membrane filters under aseptic conditions. On inoculation day, the pH of the culture medium was checked and measured as pH 8.07.
- Intervals of water quality measurement: pH was measured in each control and treatment group at test initiation and termination.

OTHER TEST CONDITIONS
- Photoperiod: continuous cool-white fluorescent lighting
- Light intensity and quality: 6550 to 6854 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Healthy cell counts were recorded approximately 24, 48 and 72 hours after test initiation.

TEST CONCENTRATIONS
- Range finding study: yes
- Test concentrations: Nominal concentrations: 0 (control), solvent control (0.1 mL/L N,N-dimethyl formamide), 0.1, 1.0, 10, 60 and 120 mg/L.

Reference substance (positive control):

no

Remarks:

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

26.727 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

cell number

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 57.21 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

5.266 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks:

cell number

Effect concentrations 4

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

2.483 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks:

cell number

Any other information on results incl. tables

Table1. Summary of algal growth inhibition following exposure of Pseudokirchneriella subcapitata to test substance for 72 hours

 

Mean measured conc. (mg/L)	% inhibition relative to solvent control
	Cell biomass	Growth rate
Solvent control (0.0)	-	-
1.131	0.5830	0.5035
2.483	1.6761	1.4189
5.266	4.5183*	4.0278*
11.716	5.6114*	5.2178*
25.810	6.4859*	6.0874*
57.210	21.3525*	43.4814*

 

*Significantly different from the control (Bonferroni ‘t’ test, alpha = 0.05)

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The reductions in healthy cell count and growth rate indicate a dose-dependent response for increasing concentrations of the test item.

Executive summary:

The effect of the test substance on the green alga Pseudokirchneriella subcapitata (formerly known as Selenastrum capricornumtum) was determined in a static, 72 hour test without test medium renewal. The test was conducted according to OECD Guideline for the Testing of Chemicals: 201 (2006) and EEC C.3.

Treatments consisted of an untreated control, solvent control, and six nominal test concentrations of 1.2, 2.6, 5.6, 12.4, 27.3 and 60 mg/L. For the definitive (dose response) test, the organisms were exposed for 72 hours (3 days) without test medium renewal. The concentrations of the test substance in test solutions 1.131, 2.483, 5.266, 11.716, 25.810, and 57.210 mg/L were measured at 0 and 72 hours. The recovery of measured concentrations were 95.85-97.32% of the nominal concentrations.

The EC50 and NOEC for Pseudokirchneriella subcapitata were based on mean measured concentrations and healthy cell count (biomass) and growth rate.

The 72 hr EbC50 based on healthy cell count was 26.727 mg/L and the NOEC was 2.483 mg /L.

The 72 hr ErC50 based on growth rate was 57.210 mg/L and the NOEC was 2.483 mg/L.