2,2'-azobis[2-methylpropionamidine] 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:

2.5.-5.5.1999

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Due to missing analytical monitoring the observed effects cannot be unequivocally assigned to the test substance itself or its transformation products or a mixture of both

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

Guideline and version not stated in the report

Principles of method if other than guideline:

preliminary (screening) test only

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

no

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green alga
- Strain:
- Source (laboratory, culture collection): Insitute of Freshwater Ecology, Cumbria, UK
- Age of inoculum (at test initiation):
- Method of cultivation:

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

Test temperature:

23 ± 2 °C

pH:

7.2 - 7.7

Nominal and measured concentrations:

nominal: control, 0.01, 0.1, 1, 10, and 100 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL conical flasks
- Type (delete if not applicable): open / closed
- Material, size, headspace, fill volume: glass, 100 mL test culture
- Aeration:
- Type of flow-through (e.g. peristaltic or proportional diluter):
- Renewal rate of test solution (frequency/flow rate):
- Initial cells density: 1 x 10^4 cells /mL
- Control end cells density: 40.2 x 10^4 cells /mL
- No. of organisms per vessel: -
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2
- 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
- Adjustment of pH:
- Photoperiod: continuous illumination
- Light intensity and quality: 6 x 30 W "cool white" fluorescent tubes (7355 lux)
- Salinity (for marine algae): -

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: haemacytometer (Improved Neubauer)
- Chlorophyll measurement:
- Other:

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
- 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):

no

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Results with reference substance (positive control):

no

nominal concentration (mg/L)		Cell density per mL (x 10^4) at 72 hours	% inhibition
Control	R1	37.4	-
	R2	43.0
	mean	40.2
0.01	R1	37.3	0
	R2	47.9
	mean	42.6
0.1	R1	29.4	18
	R2	36.6
	mean	33
1	R1	9.50	71
	R2	13.5
	mean	11.5
10	R1	11.9	80
	R2	4.39
	mean	8.14
100	R1	1.00	98
	R2	1.00
	mean	1.00

R₁ - R₂ : Replicates 1 and 2

Validity criteria fulfilled:

not applicable

Conclusions:

Based on the experimental observations an effect concentration EC50 (72 h) = 0.5 mg/L was calculated. With view to the available information on the stability of the test substance a half-life of about 45 hours can be estimated indicating a fall below the 80 % threshold of the initial concentration. Therefore and in absence of analytical monitoring the observed effects can not be assigned unequivocally to 2,2'-azobis(2-amidinopropane)-dihydrochloride, its transformation products or a mixture of both. However, due to the precautionary principle the findings of the studies are considered in the classification and labellng and the hazard assessment.

Executive summary:

The effect of VAZO(R) 56 WSP on the growth of the unicellular green alga Selenastrum capricornutum was assessed under non-axenic conditions. The test concentrations were not verified by chemical analysis.

Duplicate algal cultures, with an initial cell density of 1*10⁴ ml^-1, were exposed to the test substance at nominal concentrations of 0.01, 0.1, 1,10, and 100 mg/L.

Cell densities were determined using a haemacytometer after 72 hours. The following values were derived from the data:

72 -hour EC50 value                                        : 0.50 mg/L (95% confidence limits 0.30 & 0.75 mg/L)

No-observed effect concentration (NOEC)       : 0.1 mg/L

Key value for chemical safety assessment

EC50 for freshwater algae:

0.5 mg/L

EC10 or NOEC for freshwater algae:

0.1 mg/L

Additional information

The study exposing freshwater algae at hand could not be fully validated due to missing analytical monitoring of the test substance. Based on existing data on stability of 2,2'-azobis(2 -amidinopropane)dihydrochloride in water it can be assumed that after 72 h less than 80 % of the starting concentration is available. Therefore, renewal of test substance is necessary to assure a constant exposure, e.g. semi-static test design. Since this is technically not possible, further testing on algae was not conducted. Instead, a semi-static study with Lemna minor was performed to examine the toxicity of 2,2'-azobis(2 -amidinopropane)dihydrochloride to aquatic plants. However, the calculated threshold from the algae study is considered for the classification of the substance and for the calculation of the PNECs providing for the findings of the study.