1,1'-azobis(1-cyclohexanecarbonitrile)

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03.-07.12.18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

2006

Deviations:

yes

Remarks:

uncritical: stock solutions were autoclaved for 20 minutes, pre-culture was prepared 5 days before test start (validity criteria were observed)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

1,1'-azobis(1-cyclohexanecarbonitrile)

EC Number:

218-254-8

EC Name:

1,1'-azobis(1-cyclohexanecarbonitrile)

Cas Number:

2094-98-6

Molecular formula:

C14H20N4

IUPAC Name:

1,1'-azobis(1-cyclohexanecarbonitrile)

Test material form:

solid: granular

Sampling and analysis

Analytical monitoring:

yes

Test solutions

Vehicle:

no

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Green Algae - Chlorophytes
- Strain: 86.81
- Source (laboratory, culture collection): MBM Sciencebridge GmbH (Institut für Pflanzenphysiologie of Universität Göttingen), Jan 2006
- 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:

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

21.7 – 22.8 °C

pH:

7.6-8.3

Nominal and measured concentrations:

nominal concentration: 1 / 3.2 / 10 / 32 / 100 mg/L
geometric mean: 0.03 / 0.06 / 0.21 / 0.71 / 1.98 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: glass flasks
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: 45 ±1 mL
- Aeration:
- Type of flow-through (e.g. peristaltic or proportional diluter):
- Renewal rate of test solution (frequency/flow rate):
- Initial cells density: 2.4E3
- Control end cells density: 4.2E5
- 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: 5000 lux
- Salinity (for marine algae):

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: electronic particle counter (Beckman Coulter Z2)
- Chlorophyll measurement: -
- Other: -

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Justification for using less concentrations than requested by guideline: -
- Range finding study
- Test concentrations: not stated (non-GLP pre-test)
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

K2Cr2O7 (CAS No. 7778-50-9)

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.02 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.06 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.95 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 1.95 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no
- Unusual cell shape: no
- Colour differences: no
- Flocculation: no
- Adherence to test vessels: no
- Aggregation of algal cells: no
- Other:
- Any stimulation of growth found in any treatment: yes (@ nominal concentration: 1 mg/L)
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no

Results with reference substance (positive control):

72h EC50 0.79 mg/L

Any other information on results incl. tables

calculated cell numbers

nominal concentration mg/L	replicate	cell number/mL
		0 h	24 h	48 h	72 h
Blank control	1	2380	8200	33120	379600
	2	2380	5380	36020	420680
	3	2380	8120	45520	380580
	4	2380	9220	55620	414420
	5	2380	9980	68280	459980
	6	2380	9700	70740	459360
	mean	2380	8433	51550	419103
	SD	0	1679	16012	35666
1	1	2380	10600	77440	492680
	2	2380	10680	73500	477380
	3	2380	9580	66620	454700
	mean	2380	10287	72520	474920
	SD	0	613	5476	19109
3.2	1	2380	9380	48140	399620
	2	2380	8300	41340	324500
	3	2380	8460	33400	372520
	mean	2380	8713	40960	365547
	SD	0	583	7377	38042
10	1	2380	5120	29980	338340
	2	2380	8960	38140	332040
	3	2380	10760	58280	342060
	mean	2380	8280	42133	337480
	SD	0	2881	14566	5065
32	1	2380	11360	60860	330560
	2	2380	12180	73140	348840
	3	2380	12400	76740	362880
	mean	2380	11980	70247	347427
	SD	0	548	8326	16206
100	1	2380	11260	34640	37140
	2	2380	10720	42400	39740
	3	2380	10360	35320	35620
	mean	2380	10780	37453	37500
	SD	0	453	4297	2083

 

growth rate

nominal conc. in mg/L	replicate	growth rate [day^-1]
		24 h	48 h	72 h
Blank control	1	1.24	1.32	1.69
	2	0.82	1.36	1.72
	3	1.23	1.48	1.69
	4	1.35	1.58	1.72
	5	1.43	1.68	1.75
	6	1.41	1.70	1.75
	mean	1.25	1.52	1.72
	SD	0.23	0.16	0.03
1	1	1.49	1.74	1.78
	2	1.50	1.72	1.77
	3	1.39	1.67	1.75
	mean	1.46	1.71	1.77
	SD	0.06	0.04	0.01
3.2	1	1.37	1.50	1.71
	2	1.25	1.43	1.64
	3	1.27	1.32	1.68
	mean	1.30	1.42	1.68
	SD	0.07	0.09	0.04
10	1	0.77	1.27	1.65
	2	1.33	1.39	1.65
	3	1.51	1.60	1.66
	mean	1.20	1.42	1.65
	SD	0.39	0.17	0.01
32	1	1.56	1.62	1.64
	2	1.63	1.71	1.66
	3	1.65	1.74	1.68
	mean	1.62	1.69	1.66
	SD	0.05	0.06	0.02
100	1	1.55	1.34	0.92
	2	1.51	1.44	0.94
	3	1.47	1.35	0.90
	mean	1.51	1.38	0.92
	SD	0.04	0.06	0.02

 

daily growth rates

growth rates	days 0-1	days 1-2	days 2-3	CV of sectional daily growth rates	days 0-3
Replicate 1	1.237	1.396	2.439	39 %	1.691
Replicate 2	0.816	1.901	2.458	48 %	1.725
Replicate 3	1.227	1.724	2.124	27 %	1.692
Replicate 4	1.354	1.797	2.008	19 %	1.720
Replicate 5	1.433	1.923	1.908	16 %	1.755
Replicate 6	1.402	1.987	1.871	18 %	1.754
mean	1.245	1.788	2.135	28 %	1.723
SD	0.277	0.214	0.259		0.028
CV	18 %	12 %	12 %		2 %

 

Validity criteria

Parameter	Validity criteria	observed value	assessment
increase factor biomass	factor 16 in 72 h	176	valid
mean coefficient of variation of daily growth rates	max 35 %	28 %	valid
Coefficient of variation of average growth rate during the whole test period	max 7 %	2 %	valid

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Executive summary:

The study was performed using 5 concentrations ranging from 1 to 100 mg/L (nominal). Incubation time (test system Desmodesmus subspicatus)was 72 hours. The cell concentration of each replicate was determined by measuring the cell numbers every 24 hours with an electronic particle counter. Growth rate µ and the yield were determined from the cell number at the respective observation times.

Significant inhibition of algal growth was observed at the following concentrations:

3.2 – 100 mg/L (nominal concentrations)

 

At the beginning and at the end of the test, the content of the test item in the test solutions was determined using HPLC-UV-determination. Because of the poor solubility of the test item the measured concentrations lay between 1 % and 4 % of the nominal concentration during the test. Therefore, the determination of the results was based on the geometric mean of the measured concentrations.

 

The 72h-EC₅₀values of potassium dichromate (K₂Cr₂O₇, CAS No. 7778-50-9) were determined in a separate reference test. The values lay within the range of the laboratory (growth rate 0.73 - 1.10 mg/L, yield 0.21 – 0.66 mg/L).

 

The following results for the test item 1,1'-Azobis(cyclohexane-1-carbonitrile) were determined:

 

Table 3‑a         Results of the test item

Endpoint	NOEC	LOEC	EC10	EC50
Growth Rate	0.02 mg/L	0.06 mg/L	0.95 mg/L	> 1.85 mg/L
Yield	0.02 mg/L	0.06 mg/L	0.34 mg/L	0.94 mg/L