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

Toxicity to aquatic plants other than algae

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic plants other than algae

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29.11.2013-9.12.2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 221 (Lemna sp. Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 21.6 - 405.9 mg/L
- Sampling method: daily sampling at start (0h) and end (24h) of exposure period, cooled samples were transported on day 3 and day 7
- Sample storage conditions before analysis: in fridge at 5 ± 3 °C

Vehicle:

no

Test organisms (species):

Lemna minor

Details on test organisms:

TEST ORGANISM
- Common name: Duckweed
- Strain:
- Source (laboratory, culture collection): Federal Environment Agency, Berlin-Marienfelde, Germany
- Age of inoculum (at test initiation):
- Method of cultivation: Jungnickel agar medium

ACCLIMATION
- Acclimation period: 20 d before testing plant colonies were transfered to Steinberg medium
- Culturing media and conditions (same as test or not):
- Any deformed or abnormal cells observed: no

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

7 d

Test temperature:

22.4-23.7 °C

pH:

A maximal pH shift of 1.0 was observed in different test solutions during exposure intervals

Nominal and measured concentrations:

N: 400, 200, 100, 50, 25 mg/L
analytical monitoring showed recovery rates > 85 % for all concentrations during exposure period

Details on test conditions:

TEST SYSTEM
- Incubation chamber used: yes/no
- Test vessel: beaker covered with watch glass
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: glass, 150 mL, 50 mL headspace, 100 mL solution
- Type of cover: watch glass
- Aeration: no
- Agitation: no
- Type of flow-through (e.g. peristaltic or proportional diluter): -
- Renewal rate of test solution (frequency/flow rate): 24 h
- Control end cells density: -
- No. of colonies per vessel: -
- No. of fronds per colony: 9-11
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3 repl. for positive control, 6 repl. for negative control
- No. of vessels per vehicle control (replicates): -

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

SEDIMENT USED (for rooted aquatic vascular plants)
- Origin: -
- Textural classification (% sand, %silt and clay): -
- organic carbon (%): -
- Geographic location: -

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: no
- Photoperiod: 24 h
- Light intensity and quality: 91.3 µE/m2s ± 4.3% (Osram L36W/840, CoolWhite)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of frond number: computerized image analysis using Scanalyzer from LemnaTec, manual corrections, if necessary
- Determination of biomass: [dry weight / fresh
weight]
- Determination of frond area:
- Other:

RANGE-FINDING STUDY
- Test concentrations: -
- Results used to determine the conditions for the definitive study: -

Reference substance (positive control):

yes

Remarks:

3,5-DCP

Key result

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

7 774 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

frond number

Remarks on result:

other: 1603-2.5*10^8 mg/L

Duration:

7 d

Dose descriptor:

EC20

Effect conc.:

681.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

frond number

Remarks on result:

other: 354.5-21631 mg/L

Key result

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

190.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

frond number

Remarks on result:

other: 69.5-375.9 mg/L

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

frond number

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

frond number

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

781.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: frond area growth rate

Remarks on result:

other: 589.0-1237.6 mg/L

Duration:

7 d

Dose descriptor:

EC20

Effect conc.:

198.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: frond area growth rate

Remarks on result:

other: 162.9-233.2 mg/L

Key result

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

96.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: frond area growth rate

Remarks on result:

other: 65.6-123.7 mg/L

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: frond area growth rate

Key result

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: frond area growth rate

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

1 487.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

frond number

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

28.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

frond number

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

frond number

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

frond number

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: EC50 3 mg/L (target range: 2.2 - 3.8 mg/L (ISO 20079(2005))
- Other:

Reported statistics and error estimates:

Statistical differences between growth rate of the test and the control vessels were checked by performing Williams Multiple Sequential t-Test after Shapiro-Wilk's Test on normal distribution and Levene's Test on variance homogeneity.

Validity criteria fulfilled:

yes

Remarks:

growth rate of the frond number was 0.349 and therefore > 0.275 (validity criteria for study). Additional criteria (light intensity, temperature and reference control (3,5-DCP)) were also fulfiled.

Conclusions:

In a study according to OECD guideline 221 with Lemna minor an EC50(7d) 7777 mg/L and an EC10(7d) 190.8 mg/L were observed showing a low toxicity of the test item to aquatic plants.

Executive summary:

The Lemna growth inhibition test was performed as semi-static test with five nominal concentrations between 400 mg/L and 25 mg/L. The following results were obtained:

Growth rate (frond number)	Yield (frond number)
ErC50(7d): 7774 mg/L (95%-CI: 1603-2.5x10^8mg/L)	EyC50(7d): 1487.5 mg/L (95%-CI: n.d.)
ErC20(7d): 681.3 mg/L (95%-CI: 354.5-21631 mg/L)	EyC20(7d): 109.8 mg/L (95%-CI: n.d.)
ErC10(7d): 190.8 mg/L (95%-CI: 69.5-375.9 mg/L)	EyC10(7d): 28.1 mg/L (95%-CI: n.d.)
LOErC(7d): 50 mg/L NOErC(7d): 25 mg/L	LOEyC(7d): 50 mg/L
	NOEyC(7d): 25 mg/L
Growth rate (frond area)	Yield (frond area)
ErC50(7d): 781.6 mg/L (95%-CI: 589.0-1237.6 mg/L)	EyC50(7d): 233.5 mg/L (95%-CI: 72.5-359.8 mg/L)
ErC20(7d): 198.2 mg/L (95%-CI: 162.9-233.2 mg/L)	EyC20(7d): 68.1 mg/L (95%-CI: 33.5-98.4 mg/L)
ErC10(7d): 96.8 mg/L (95%-CI: 65.6-123.7 mg/L)	EyC10(7d): 35.7 mg/L (95%-CI: 11.9-59.6 mg/L)
LOErC(7d): 100 mg/L	LOEyC(7d): 50 mg/L
NOErC(7d): 50 mg/L	NOEyC(7d): 25 mg/L

n.d.: not determined due to mathematical reasons or inappropriate data

The analysis of the exposure concentration of the test item was performed with a valid method. The calibrated range of the method was 25 -400 mg/L. All measured test concentrations were in the range of 21.6 mg/L to 405 .9 mg/L and, accordingly, in the range of 86% of the lowest calibration standard to 101% of the highest calibration standard.

Description of key information

Key values were observed in OECD guideline study exposing Lemna minor for seven days under semi-static test conditions. With view to existing data on stability the test medium was renewed each day. Effect concentration of the frond number growth rate were selected for the assessment.

Key value for chemical safety assessment

EC50 for freshwater plants:

7 774 mg/L

EC10 or NOEC for freshwater plants:

190.8 mg/L

Additional information

Since the requested study using algae could not be conducted due to the instability of the substance in water and the limits in the test design of the OECD guideline 201, a semi-static study according to OECD guideline 221 exposing Lemna minor was performed. The concentration of the substance was renewed and analytically monitored daily. Based on the observable effects on the growth rate of Lemna minor a low toxicity of 2,2'-azobis (2 -amidinopropane)dihydrochloride to aquatic plants can be concluded.