2,2'-[azobis(1-methylethylidene)]bis[4,5-dihydro-1H-imidazole dihydrochloride

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:

03/11-06/11/14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: freshly stock solution 80.0 mg/L, dilution
- Eluate:
- Differential loading:
- Controls:
- Chemical name of vehicle (organic solvent, emulsifier or dispersant):
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)):
- Evidence of undissolved material (e.g. precipitate, surface film, etc.):

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name:
- Strain: HINDÁK, SAG 61.81
- Source (laboratory, culture collection): Pflanzenphysiologisches Institut der Universität Göttingen, Nikolausberger Str. 18
- Age of inoculum (at test initiation):
- Method of cultivation: on Z-Agar (every month), 24 h light

ACCLIMATION
- Acclimation period:
- Culturing media and conditions (same as test or not):
- Any deformed or abnormal cells observed:

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

0.24 mmol Ca+Mg/L

Test temperature:

22 °C

pH:

7.62-8.43

Nominal and measured concentrations:

60.0 - 27.3 - 12.4 - 5.64 - 2.56 - 1.16 mg/L (nominal)
38.8 - 18.3 - 8.44 - 3.66 - 1.60 - 0.762 mg/L (geometric measured mean)

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer-Flask
- Type (delete if not applicable): closed (cotton wool plugs)
- Material, size, headspace, fill volume: glass, 250 mL,100 mL
- Aeration:
- Type of flow-through (e.g. peristaltic or proportional diluter):
- Renewal rate of test solution (frequency/flow rate):
- Initial cells density: 7085 cells/mL
- Control end cells density:
- No. of organisms per vessel:
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): -

GROWTH MEDIUM
- Standard medium used: yes
- Detailed composition if non-standard medium was used:

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
- Total organic carbon:
- Particulate matter:
- Metals:
- Pesticides:
- Chlorine:
- Alkalinity:
- Ca/mg ratio:
- Conductivity:
- Culture medium different from test medium:
- Intervals of water quality measurement:

OTHER TEST CONDITIONS
- Sterile test conditions: yes/no
- Adjustment of pH:
- Photoperiod:
- Light intensity and quality:
- Salinity (for marine algae):

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter]
- Chlorophyll measurement:
- Other:

TEST CONCENTRATIONS
- Spacing factor for test concentrations:
- Justification for using less concentrations than requested by guideline:
- Range finding study
- Test concentrations:
- Results used to determine the conditions for the definitive study:

Reference substance (positive control):

yes

Remarks:

K2Cr2O7

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

< 0.762 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

2.13 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

13.5 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Results with reference substance (positive control):

ErC50 = 0.749 (0.715-0.788 mg/L (valid range: 0.821 ± 0.388)

Validity criteria fulfilled:

yes

Conclusions:

In this study, Azo Initiator VA044 was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata over the period of 72 hours. The EC50-value for inhibition of specific growth rate (ErC50) after 72 hours was 13.5 mg test item/L, the ErC20- and ErC10-values were determined to be 2.13 and < 0.762 mg test item/L, respectively. All effect values are based on geometric mean measured concentrations of the test item.

Executive summary:

The toxicity of Azo Initiator VA044 to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 and Council Regulation (EC) No. 440/2008/ method C.3 from 2014 -11 -03 to 2014 -11 -06 at Dr. U.Noack-Laboratorien in 31157 Sarstedt, Germany. The aim of the study was to assess the effects on the growth rate over a period of 72 hours.

The test item Azo Initiator VA044 is a white powder and has a water solubility of 347 g/L at 25 °C. The stock solution of 80.0 mg/L was freshly prepared with dilution water. Further test concentrations were prepared by diluting the stock solution with dilution water. The tested solutions were slightly yellow with precipitation.

The study was conducted under static conditions with an intial cell density of 7085 cells/mL and reduced light intensity dur to the light sensitivity of the test item. Based on a preliminary range finding test, five concentratin levels were tested in a geometrical series with a dilution factor of 2.2 (nominal): 1.16 - 2.56 - 5.64 - 12.4 - 27.3 - 60 mg/L, corresponding a geometric mean measured test item concentrations of 0.762 - 1.60 - 3.66 - 8.44 - 18.3 - 38.8 mg/L. Three replicates were tested for each test item concentration and six replicates for the control. The environmental conditions were within the acceptable limits.

All test concentrations of Azo Initiator VA044 and the control were analytically verified by LC-MS/MS at the start and the and of the exposure. The measured concentrations of VA044 at the start of the exposure were in the range of 94 - 109 % of the nominal test item concentrations and 39 - 45 % at the end of the exposure.

All effect values are based on the geometric mean measured concentrations of the test item Azo Initiator VA044. The growth inhibition results of the test item are summarized below:

ErC10 < 0.762 mg/L

ErC20 2.13 (1.58 -2.77) mg/L

ErC50 13.5 (11.4 -16.1) mg/L

Description of key information

Based on the results of the available study exposing Pseudokirchneriella subcapitata for 72 hours an ErC50 -value of 13.5 mg/L and an ErC10 -value < 0.762 mg/L were determined. The short-term value is used as point of departure for the PNEC calculations.

Key value for chemical safety assessment

EC50 for freshwater algae:

13.5 mg/L

EC10 or NOEC for freshwater algae:

0.762 mg/L