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

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07.02.12-15.02.12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Laboratory culture: sludge was taken from ESN (Stadtentsorgung Neustadt) STP D-67435 NW-Lachen-Speyerdorf
- Method of cultivation:
- Preparation of inoculum for exposure:
- Pretreatment: sludge was filtrated, washed with tap water and afterwards resuspended in tap water. Sludge was aerated during the whole period from sampling until usage in test. Sludge was fed daily with 50 mL/L synthetic sewage feed.
- Initial biomass concentration: 1.25 g suspended solids/L, respectively 1.51 g suspended solids/L, dry matter in the test

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Hardness:

1.08 mmol

Test temperature:

16.9 - 18.8 °C (pre-test)
18.9 - 19.7 °C (main study)

pH:

8.18

Nominal and measured concentrations:

pre-test: 1000, 100, 10, 1 mg/L (nominal)
main study: 3200, 1000, 320, 100, 32, 10 mg/L (nominal)

Details on test conditions:

TEST SYSTEM
- Test vessel: beakers
- Type (delete if not applicable): open / closed
- Material, size, headspace, fill volume: glass, 500 mL
- Aeration: purified air, using Pasteur pipettes, flow approx. 0.75 L/min
- Type of flow-through (e.g. peristaltic or proportional diluter): -
- Renewal rate of test solution (frequency/flow rate): -
- No. of organisms per vessel: 250 mL inoculum
- No. of vessels per concentration (replicates): positive control: one replicate(/concentration; pre-test: one replicate (lower concentrations), five replicates (highest concentrations); main study: two replicates/treatment
- No. of vessels per control (replicates): two replicates before and two after measuring positive control and test item, respectively
- No. of vessels per vehicle control (replicates): -
- Biomass loading rate:

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: tap water
- Total organic carbon: 0.5 mg/l
- Particulate matter:
- Metals:< 0.007 mg/L
- Pesticides: < LOD
- Chlorine: 12 mg/L
- Alkalinity: not stated
- Ca/mg ratio: 7
- Conductivity: 223 µS/cm
- Culture medium different from test medium:
- Intervals of water quality measurement:

OTHER TEST CONDITIONS
- Adjustment of pH: -
- Photoperiod: -
- Light intensity: -

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : inhibition of respiration rate

TEST CONCENTRATIONS
- Spacing factor for test concentrations:
- Justification for using less concentrations than requested by guideline:
- Range finding study
- Test concentrations:1000, 100, 10, 1 mg/L (nominal)
- Results used to determine the conditions for the definitive study:

Reference substance (positive control):

yes

Remarks:

3,5 DCP (CAS# 591-35-5)

Key result

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: 95% CI 3-13 mg/L

Duration:

3 h

Dose descriptor:

other: EC20

Effect conc.:

28 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: 95% CI 14-44 mg/L

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

360 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: 95% CI 280-510 mg/L

Results with reference substance (positive control):

pre-test: positve control (3,5-DCP) 7.8 mg/L
main test: positive control (3,5-DCP) 11.3 mg/L

Reported statistics and error estimates:

linear regression

Vessel No	content	concentration in mg/L	O2consumption in mg//L*min)	O2consumption in mg//L*h)	Inhibition in %	pH
1	control	0	0.5680	34.078	0	8.3
2	control	0	0.5909	35.452	0	8.2
3	positive control	5.0	0.4870	29.221	24.9	8.2
4	positive control	10.1	0.3492	20.950	46.2	8.2
5	positive control	20.2	0.1937	11.624	70.1	8.4
6	positive control	40.4	0.1176	7.059	81.9	8.4
7	control	0	0.6507	39.044	0	8.3
8	control	0	0.6829	40.971	0	8.2
9	test item	3201.2	0.1721	10.325	73.5	8.3
10	test item	3200.2	0.1927	11.560	70.3	8.3
11	test item	1000.2	0.2320	13.918	64.2	8.4
12	test item	1000.4	0.2350	14.102	63.8	8.3
13	test item	320.4	0.3145	18.872	51.5	8.3
14	test item	321.0	0.3072	18.433	52.6	8.3
15	test item	100.4	0.3919	23.517	39.6	8.3
16	test item	98.8	0.4042	24.254	37.7	8.3
17	test item	31.4	0.5397	32.380	16.8	8.2
18	test item	33.0	0.5313	31.879	18.1	8.2
19	test item	10.2	0.6529	39.174	-0.7	8.2
20	test item	11.2	0.6462	38.774	0.4	8.2
21	control	0	0.6979	41.875	0	8.2
22	control	0	0.7010	42.061	0	8.2

Validity criteria fulfilled:

yes

Remarks:

The results of the positive controls were within the stated range of tests with 3,5-DCP. Furthermore the CV of the oxygen control were less than 30 % and the O2 uptake of the blanks was more than 20 mg O2/ g activated sludge.

Conclusions:

Based on experimental observations the effect concentrations EC10(3h) = 7 mg/L and EC50(3h)= 360 mg/L were calculated. All validity criteria were fulfilled and hence the study is considered adequate for regulatory purposes.

Executive summary:

Two valid experiments were performed. In the pre-test, the test item was tested using four concentrations (ranging from 1000 to 1 mg/L nominal concentration). Duration of the test was three hours. Activated sludge was used as inoculum. It was taken from a domestic sewage treatment plant and washed before usage. The dry matter was determined as 2.5 g suspended solids/, giving a concentration of 1.25 g suspended solids/L in the test.

3,5 -dichlorophenol was used as positive control. Four concentrations were tested; an EC50 of 7.8 mg/L (95% confidence interval: < 12 mg/L) was determined, which lies within the range of 2 -25 mg/L.

As inhibition > 20% was observed, a study plan amendment was issued and a main study was performed. The main study was performed likewise using six concentrations, ranging from 3200 to 10 mg/L nominal concentration. the dry matter of the activated sludge was determined as 3.02 g suspended solids/L, giving a concentration of 1.51 g suspended solids/L in the test. The EC50 of the positive control was determined as 11.3 mg/L (95% confidence interval: 8.6 - 14.0 mg/L), which lies within the range of 2 -25 mg/L.

The following results for 2,2'-azobis(2 -amidinopropane)-dihydrochloride (= 2,2'-azobis[2 -methylpropionamidine] dihydrochloride) were determined:

3h EC10 = 7 mg/L (95% confidence interval: 3 -13 mg/L)

3h EC20 = 28 mg/L (95% confidence interval: 14 -44 mg/L)

3h EC50 = 360 mg/L (95% confidence interval: 280 -510 mg/L)

Description of key information

Key value is based on experimental results observed in an OECD 209 guideline study.

Key value for chemical safety assessment

EC50 for microorganisms:

360 mg/L

EC10 or NOEC for microorganisms:

7 mg/L