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Ecotoxicological information

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

Constituent 1
Chemical structure
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 concentrationsopen allclose all
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
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
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
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/Lreplicatecell number/mL
0 h24 h48 h72 h
Blank control12380820033120379600
22380538036020420680
32380812045520380580
42380922055620414420
52380998068280459980
62380970070740459360
mean 2380843351550419103
SD016791601235666
1123801060077440492680
223801068073500477380
32380958066620454700
mean23801028772520474920
SD0613547619109
3.212380938048140399620
22380830041340324500
32380846033400372520
mean2380871340960365547
SD0583737738042
1012380512029980338340
22380896038140332040
323801076058280342060
mean2380828042133337480
SD02881145665065
32123801136060860330560
223801218073140348840
323801240076740362880
mean23801198070247347427
SD0548832616206
10012380112603464037140
22380107204240039740
32380103603532035620
mean2380107803745337500
SD045342972083

 


growth rate



























































































































































































































nominal conc. in mg/Lreplicategrowth rate [day-1]
24 h 48 h72 h
Blank control11.241.321.69
20.821.361.72
31.231.481.69
41.351.581.72
51.431.681.75
61.411.701.75
mean1.251.521.72
SD0.230.160.03
111.491.741.78
21.501.721.77
31.391.671.75
mean1.461.711.77
SD0.060.040.01
3.211.371.501.71
21.251.431.64
31.271.321.68
mean1.301.421.68
SD0.070.090.04
1010.771.271.65
21.331.391.65
31.511.601.66
mean1.201.421.65
SD0.390.170.01
3211.561.621.64
21.631.711.66
31.651.741.68
mean1.621.691.66
SD0.050.060.02
10011.551.340.92
21.511.440.94
31.471.350.90
mean1.511.380.92
SD0.040.060.02

 


daily growth rates





















































































growth ratesdays 0-1days 1-2days 2-3CV of sectional daily growth ratesdays 0-3
Replicate 11.2371.3962.43939 %1.691
Replicate 20.8161.9012.45848 %1.725
Replicate 31.2271.7242.12427 %1.692
Replicate 41.3541.7972.00819 %1.720
Replicate 51.4331.9231.90816 %1.755
Replicate 61.4021.9871.87118 %1.754
mean1.2451.7882.13528 %1.723
SD0.2770.2140.259 0.028
CV18 %12 %12 % 2 %

 


Validity criteria





























ParameterValidity criteriaobserved valueassessment
increase factor biomassfactor 16 in 72 h176valid
mean coefficient of variation of daily growth ratesmax 35 %28 %valid
Coefficient of variation of average growth rate during the whole test periodmax 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