{[(2,4 and 2,5 and 2,6)-dimethylphenyl and 2-ethylphenyl]amino}{[(2,4 and 2,5 and 2,6)-dimethylphenyl and 2-ethylphenyl]amino}methaniminium 3-[(4-anilinophenyl)diazenyl]benzenesulfonate

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 2014-08-05 to 2014-08-22

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The reliability is rated 2 because the study followed a standard guideline of reference (OECD 201), which describes a procedure designed to evaluate this endpoint. The results were reviewed for reliability and assessed as valid. However, the study was not conducted under GLP conditions.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

GLP compliance:

no

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0, 0.61, 1.2, 2.2, 4.2 and 8 mg/L for the main test (% v/v saturated solution)
- Sample storage conditions before analysis: stored at -25 +/- 5 °C before analysis

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method of preparation: Saturation of the experimental solution:
100 mg of test item was dispersed in 1 liters test medium
Stirring for 24 hours.
Filtration through a PTFE of 0.22 μm to remove undissolved test item
Obtention of a 100% v/v saturated solution.
Series of dilution to give further test concentration for the range finding study and the main test.

- Controls: control group maintained under identical conditions but not exposed to the test item
- Vehicle: none
- Evidence of undissolved material (e.g. precipitate, surface film, etc): no

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchnerella subcapitata (Selenastrum capricornutum)
- Strain: CCAP278/2
- Source (laboratory, culture collection): SAMS Research Services Ltd, Scotland
- Age of inoculum (at test initiation): culture in exponential growth phase
- Method of cultivation: medium LC-Oligo (pH 7.1+/-0.1), temperature 21-24°C, light: 16h light/8h dark, ca. 4500lux, bubbling aeration

ACCLIMATION
- Acclimation period: 3 days
- 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:

no data

Test temperature:

21-24°C

pH:

Stock solution 100 mg/L : 7,6.
Test solution :
concentration mg/L 8.0 4.2 2.2 1.2 0.61 control
pH t0 7.6 7.6 7.7 7.7 7.7 7.9
pH t72 7.8 7.8 7.7 7.7 7.8 8.3

Dissolved oxygen:

no data

Salinity:

not applicable

Nominal and measured concentrations:

measured concentrations: 0.2, 0.5, 1.07, 2.12, and 4.0 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: flasks of 30 mL
- Type: closed
- Material, size, headspace, fill volume: test volume 20 mL
- Aeration: none
- Renewal rate of test solution: none (static mode)
- Initial cells density: ca. 10^4 cells/mL from pre-culture

GROWTH MEDIUM
- Standard medium used: yes (from OECD guideline 2011)

OTHER TEST CONDITIONS
- Photoperiod: constant (24h/24h)
- Light intensity and quality: daylight-type (400-700 nm), approximately 7500 lux (55 microeinstein/m2/s)

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.9
- Test concentrations: 8 mg/L, 4.2 mg/L, 2.2 mg/L, 1.2 mg/L, and 0.61 mg/L
- Results used to determine the conditions for the definitive study:
Percentages of growth rate inhibition (% of stock solution at 100 mg/L)
100%: 100%
32%: 100%
10%: 100%
3.2%: 77%
1%: 13%
0.32%: 9%

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (K2Cr2O7)

Duration:

72 h

Dose descriptor:

other: ErC

Effect conc.:

ca. 1.25 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL 1.20 - 1.29

Duration:

72 h

Dose descriptor:

other: ErC20

Effect conc.:

ca. 0.81 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL 0.77 - 0.86

Duration:

72 h

Dose descriptor:

other: ErC10

Effect conc.:

ca. 0.63 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL 0.59 - 0.69

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

ca. 0.2 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes

Results with reference substance (positive control):

- Results with reference substance valid? yes
- ErC50: 1.05 mg/L ; consistent with previous results of laboratory and between 0.92 and 1.46 mg/L - acceptable range of sensitivity of algae P. subcapitata as defined by NF EN ISO 8692 : 2012

Reported statistics and error estimates:

CErx% calculated using the Hill Equation (macro Regtox-ev6.6.2xls)
NOEC calculated using the T-test of Bonferroni (Toxcalc software)

Validation criteria:

- control mean growth rate at 72h > 0.92 j^-1 (2.0 j^-1)

- control solution growth rate variation coefficient < 7% (0.29%)

- variation of pH in control solutions after 72h < 1.5 units (+0.4 unit)

- reference substance results valid

Validity criteria fulfilled:

yes

Conclusions:

Under the experimental conditions,
72h - ErC50 = 1.25 mg/L
72h - NOEC = 0.20 mg/L

Executive summary:

A study was performed to assess the acute toxicity of the test item on the growth of the green alga Pseudokirchneriella subcapitata.

The method followed was designed to be compatible with OECD Guidelines for Testing of Chemicals (2006) No. 201, "Frechwater Alga and Cyanobacteria, Growth Inhibilition Test".

The median effective concentrations (72h-ErC50) and the non-observed effect concentration (72h-NOEC) values at 72 hrs with regard to the growth rate were determined. The following concentrations were tested: 0.2, 0.5, 1.07, 2.12, and 4.0 mg/L (measured concentrations).

Each concentration and the controls were inoculated with approximately 10000 cells per mL. The test was carried out over 72 hours at a temperature of 21 -24°C and constantly stirred (250 rev/min). Each vessel concentration was tested 3 times. 6 controls were run with the same experimental condition. Algal concentration was counted using a Malassez counting chamber/ Validation experiments and the pre cultivation ensured that algae were of appropriate vialibility and susceptibility.

Under the experimental conditions:     

* 72h - ErC50 = 1.25 mg/L (1.20 - 1.29 mg/L)

* 72h - ErC20 = 0.81 mg/L (0.77 - 0.86 mg/L)

* 72h - ErC10 = 0.63 mg/L (0.59 - 0.69 mg/L)

* 72h - NOEC = 0.20 mg/L

Description of key information

LC₅₀(72h) for freshwater algae (Pseudokirchneriella subcapitata): 1.25 mg/L (OECD TG 201; static)

Key value for chemical safety assessment

EC50 for freshwater algae:

1.25 mg/L

EC10 or NOEC for freshwater algae:

0.2 mg/L

Additional information

The EC50 (72 hours) was 1.25 mg/L (measured concentration) with 95% confidence limits of 1.20 - 1.29 mg/L.

The EC10 (72 hours) was 0.63 mg/L (measured concentration) with 95% confidence limits of 0.59 - 0.69 mg/L.

The NOEC (72 hours) was 0.2 mg/L.